Patent Application
20150291971
The Sequence Listing associated with this application is filed in electronic format via EFS-Web and hereby incorporated by reference into the specification in its entirety. The name of the text file containing the Sequence Listing is Sequence_Listing—074016—0079—01. The size of the text file is 33,696 KB, and the text file was created on Jun. 26, 2015.
This invention relates generally to a method for producing a transgenic cell with increased gamma-aminobutyric acid (GABA) content as compared to a corresponding non-transformed wild type cell.
In particular, this invention relates to plant cells and plants with increased gamma-aminobutyric acid (GABA) content as compared to a corresponding non-transformed wild type.
The invention also deals with methods of producing and screening for and breeding such plant cells or plants.
Gamma-aminobutyric acid is used to enhance growth of specified plants, prevent development of powdery mildew on grapes, and suppress certain other plant diseases. Humans and animals normally ingest and metabolize gamma-aminobutyric acid in variable amounts. Gamma-aminobutyric acid was registered (licensed for sale) as growth enhancing pesticidal active ingredient in 1998. Gamma-aminobutyric acid is an important signal which helps to regulate mineral availability in plants. Minerals support the biochemical pathways governing growth and reproduction as well as the pathways that direct plant's response to a variety of biotic and abiotic stresses. Mineral needs are especially high during times of stress and at certain stages of plant growth. Gamma-aminobutyric acid levels in plants naturally increase at these times.
Gamma-Aminobutyric acid (GABA), a nonprotein amino acid, is often accumulated in plants following environmental stimuli that can also cause ethylene production. Exogenous GABA causes up to a 14-fold increase in the ethylene production rate after about 12 h. GABA causes increases in ACC synthase mRNA accumulation, ACC levels, ACC oxidase mRNA levels and in vitro ACC oxidase activity. Possible roles of GABA as a signal transducer are suggested, see Plant Physiol.115(1):129-35(1997).
Gamma-aminobutyric acid (GABA), a four-carbon non-protein amino acid, is a significant component of the free amino acid pool in most prokaryotic and eukaryotic organisms. In plants, stress initiates a signal-transduction pathway, in which increased cytosolic Ca2+ activates Ca2+/calmodulin-dependent glutamate decarboxylase activity and GABA synthesis. Elevated H+ and substrate levels can also stimulate glutamate decarboxylase activity. GABA accumulation probably is mediated primarily by glutamate decarboxylase. Experimental evidence supports the involvement of GABA synthesis in pH regulation, nitrogen storage, plant development and defense, as well as a compatible osmolyte and an alternative pathway for glutamate utilization, see Trends Plant Sci. 4(11):446-452(1999).
Rapid GABA accumulation in response to wounding may play a role in plant defense against insects (Ramputh and Brown, Plant Physiol. 111(1996): 1349-1352). The development of gamma aminobutyrate (GABA) as a potential control agent in plant-invertebrate pest systems has been reviewed in Shelp et al., Canadien Journal of Botany (2003) 81, 11, 1045-1048. The authors describe that available evidence indicates that GABA accumulation in plants in response to biotic and abiotic stresses is mediated via the activation of glutamate decarboxylase. More applied research, based on the fact that GABA acts as an inhibitory neurotransmitter in invertebrate pests, indicates that ingested GABA disrupts nerve functioning and causes damage to oblique-banded leafroller larvae, and that walking or herbivory by tobacco budworm and oblique-banded leafroller larvae stimulate GABA accumulation in soybean and tobacco, respectively. In addition, elevated levels of endogenous GABA in genetically engineered tobacco deter feeding by tobacco budworm larvae and infestation by the northern root-knot nematode. Therefore the author concluded that genetically engineered crop species having high GABA-producing potential may be an alternative strategy to chemical pesticides for the management of invertebrate pests.
During angiosperm reproduction, pollen grains form a tube that navigates through female tissues to the micropyle, delivering sperm to the egg. In vitro, GABA stimulates pollen tube growth.
Much of the recent work on GABA in plants has concentrated on its metabolic role (Fait et al., Trends in Plant Sci., Vol. 13, Nr. 1, pp 14-19, 2007) and on stress/pest-associated and signalling roles (Bouche et al., Trends in Plant Sci., Vol. 9, Nr. 3, pp 110-115, 2004).
Accumulation of GABA in plant tissues and transport fluids are responses to many abiotic stresses (Allan et al., J Exp Bot, Vol. 59, No. 9, pp. 2555-2564, 2008). Beuve et al. (in PCE, 27, 1035-1046, 2004) found that nitrate influx and GABA were positively correlated in short- and long-term experiments and that exogenous GABA supply to the roots induced a significant increase of BnNrt2 (Nitrate transporter) mRNA expression.
A further approach was the use of GABA for stimulation of plant growth by applying GABA to plants foliage, stems and/or roots in a 1 to 5000 ppm GABA solution, preferrably together with a readily metabolized carbon source (organic acids, amino acids, simple carbohydrates, and mixtures of organic acids amino acids and simple carbohydrates).
Even though the role of GABA in the cell is not yet understood and the action mechanisms not yet clarified, due to these physiological roles and agrobiotechnological potential of GABA there is a need to identify genes of enzymes and other proteins involved in GABA metabolism.
Especially there is a need to generate mutants or transgenic plant lines with which to modify the GABA content in plants in order to enhance the plant yield traits.
Accordingly, in a first embodiment, the invention relates to a method for producing a transgenic cell with increased gamma-aminobutyric acid (GABA) content as compared to a corresponding non-transformed wild type cell by increasing or generating one or more activities selected from the group consisting of: 60S ribosomal protein, ABC transporter permease protein, acetyltransferase, acyl-carrier protein, At4g32480-protein, At5g16650-protein, ATP-binding protein, Autophagy-related protein, auxin response factor, auxin transcription factor, b1003-protein, b1522-protein, b2739-protein, b3646-protein, B4029-protein, Branched-chain amino acid permease, calcium-dependent protein kinase, cytochrome c oxidase subunit VIII, elongation factor Tu, Factor arrest protein, fumarylacetoacetate hydrolase, geranylgeranyl pyrophosphate synthase, glucose dehydrogenase, glycosyl transferase, harpin-induced family protein, homocitrate synthase, hydrolase, isochorismate synthase, MFS-type transporter protein, microsomal beta-keto-reductase, polygalacturonase, protein phosphatase, pyruvate kinase, Sec-independent protein translocase subunit, serine protease, thioredoxin, thioredoxin family protein, transcriptional regulator, ubiquinone biosynthesis monooxygenase, and YHR213W-protein.
Accordingly, in one embodiment, the method according to the invention relates to a method, which comprises:
providing a non-human cell or organism, a microorganism, a non-human animal, animal tissue or animal cell, preferably a plant cell, a plant tissue a plant; and
increasing or generating one or more activities selected from the group consisting of: 60S ribosomal protein, ABC transporter permease protein, acetyltransferase, acyl-carrier protein, At4g32480-protein, At5g16650-protein, ATP-binding protein, Autophagy-related protein, auxin response factor, auxin transcription factor, b1003-protein, b1522-protein, b2739-protein, b3646-protein, B4029-protein, Branched-chain amino acid permease, calcium-dependent protein kinase, cytochrome c oxidase subunit VIII, elongation factor Tu, Factor arrest protein, fumarylacetoacetate hydrolase, geranylgeranyl pyrophosphate synthase, glucose dehydrogenase, glycosyl transferase, harpin-induced family protein, homocitrate synthase, hydrolase, isochorismate synthase, MFS-type transporter protein, microsomal beta-keto-reductase, polygalacturonase, protein phosphatase, pyruvate kinase, Sec-independent protein translocase subunit, serine protease, thioredoxin, thioredoxin family protein, transcriptional regulator, ubiquinone biosynthesis monooxygenase, and YHR213W-protein., e.g. conferring an increase of GABA in said organism; and
growing said non-human cell or organism, a microorganism, a non-human animal, animal tissue or animal cell, preferably a plant cell, a plant tissue a plant under conditions which permit the production of the increased GABA content
and optionally the GABA synthesized by the organism is recovered or isolated.
In a further embodiment the invention provides a method for producing a transgenic cell with increased gamma-aminobutyric acid (GABA) content as compared to a corresponding non-transformed wild type cell comprising at least one of the steps selected from the group consisting of:
In a further embodiment the invention provides a method for producing a transgenic cell with increased gamma-aminobutyric acid (GABA) content as compared to a corresponding non-transformed wild type cell wherein the expression of at least one nucleic acid molecule comprising a nucleic acid molecule selected from the group consisting of:
is increased or generated.
In a further embodiment the invention provides a method for producing a transgenic cell with increased gamma-aminobutyric acid (GABA) content as compared to a corresponding non-transformed wild type cell, wherein the transgenic cell is a plant cell, a plant or a part thereof with increased gamma-aminobutyric acid (GABA) content as compared to a corresponding non-transformed wild type.
In a further embodiment the invention provides a method for producing a transgenic cell with increased gamma-aminobutyric acid (GABA) content as compared to a corresponding non-transformed wild type cell wherein the transgenic plant cell, a plant or a part thereof is derived from a monocotyledonous plant, a dicotyledonous plant or a gymnosperm plant.
In a further embodiment the invention provides a method for producing a transgenic cell with increased gamma-aminobutyric acid (GABA) content as compared to a corresponding non-transformed wild type cell wherein the transgenic plant is selected from the group consisting of maize, wheat, rye, oat, triticale, rice, barley, soybean, peanut, cotton, oil seed rape, including canola and winter oil seed rape, corn, manihot, pepper, sunflower, flax, borage, safflower, linseed, primrose, rapeseed, turnip rape, tagetes, solanaceous plants, potato, tobacco, eggplant, tomato, Vicia species, pea, alfalfa, coffee, cacao, tea, Salix species, oil palm, coconut, perennial grass, forage crops and Arabidopsis thaliana.
In a further embodiment the invention provides an isolated nucleic acid molecule comprising a nucleic acid molecule selected from the group consisting of:
and
In a further embodiment the invention provides a nucleic acid molecule, whereby the nucleic acid molecule according to (a) to (k) is at least in one or more nucleotides different from the sequence depicted in column 5 or 7 of table I A and preferably encodes a protein which differs at least in one or more amino acids from the protein sequences depicted in column 5 or 7 of table II A.
In a further embodiment the invention provides a nucleic acid construct which confers the expression of the above described nucleic acid molecule, comprising one or more regulatory elements.
In a further embodiment the invention provides a vector comprising said nucleic acid molecule or said nucleic acid.
In a further embodiment the invention provides a host cell, which has been transformed stably or transiently with said vector, said nucleic acid molecule or said nucleic acid construct and which shows due to the transformation an increased gamma-aminobutyric acid (GABA) content as compared to a corresponding non-transformed wild type.
In a further embodiment the invention provides a process for producing a polypeptide, wherein the polypeptide is expressed in said host nucleus or host cell as mentioned above.
In a further embodiment the invention provides a polypeptide produced by the process as mentioned above or encoded by the nucleic acid molecule as mentioned above whereby the polypeptide distinguishes over the sequence as shown in table II A by one or more amino acids
In a further embodiment the invention provides an antibody, which binds specifically to the polypeptide produced by the process as mentioned above or encoded by the nucleic acid molecule as mentioned above whereby the polypeptide distinguishes over the sequence as shown in table II A by one or more amino acids.
In a further embodiment the invention provides a cell nucleus, cell, plant cell nucleus, plant cell plant tissue, propagation material, pollen, progeny, harvested material or a plant comprising the nucleic acid molecule as depicted above or the host nucleus or the host cell as depicted above.
In a further embodiment the invention provides a transgenic plant cell nucleus, transgenic plant cell, transgenic plant or part thereof as described above derived from a monocotyledonous plant or a dicotyledonous plant.
In a further embodiment the invention provides the transgenic plant cell nucleus, transgenic plant cell, transgenic plant or part thereof as mentioned above, wherein the corresponding plant is selected from the group consisting of corn (maize), wheat, rye, oat, triticale, rice, barley, soybean, peanut, cotton, oil seed rape, including canola and winter oil seed rape, manihot, pepper, sunflower, flax, borage, safflower, linseed, primrose, rapeseed, turnip rape, tagetes, solanaceous plants comprising potato, tobacco, egg-plant, tomato; Vicia species, pea, alfalfa, coffee, cacao, tea, Salix species, oil palm, coconut, perennial grass, forage crops and Arabidopsis thaliana.
Preferably the transgenic plant cell nucleus, transgenic plant cell, transgenic plant or part thereof of is selected from the group consisting of corn, soy, oil seed rape (including canola and winter oil seed rape), cotton, wheat and rice.
In a further embodiment the invention provides a transgenic plant comprising one or more of plant cell nuclei or plant cells, progeny, seed or pollen or produced by a transgenic plant as mentioned above.
In a further embodiment the invention provides a transgenic plant, transgenic plant cell nucleus, transgenic plant cell, plant comprising one or more of such transgenic plant cell nuclei or plant cells, progeny, seed or pollen derived from or produced by a transgenic plant a described above, wherein said transgenic plant, transgenic plant cell nucleus, transgenic plant cell, plant comprising one or more of such transgenic plant cell nuclei or plant cells, progeny, seed or pollen is genetically homozygous for a transgene conferring increased yield as compared to a corresponding non-transformed wild type plant cell, a transgenic plant or a part thereof.
In a further embodiment the invention provides a process for the identification of a compound conferring an increased gamma-aminobutyric acid (GABA) content as compared to a corresponding non-transformed wild type, comprising the steps:
In a further embodiment the invention provides a method for the production of an agri-cultural composition comprising the steps of the method described above and formulating the compound identified above in a form acceptable for an application in agriculture.
In a further embodiment the invention provides a composition comprising the nucleic acid molecule of the invention, the polypeptide of the invention, said nucleic acid construct, said vector, the compound mentioned above, the antibody of the invention, and optionally an agricultural acceptable carrier.
In a further embodiment the invention provides an isolated polypeptide as depicted in table II, preferably table II B which is selected from Arabidopsis thaliana, Azotobacter vinelandii, Brassica napus, Escherichia coli, Physcomitrella patens, Saccharomyces cerevisiae, Synechocystis sp., and/or Thermus thermophilus.
In a further embodiment the invention provides the use of a nucleic acid molecule encoding a polypeptide with the activity selected from the group consisting of 60S ribosomal protein, ABC transporter permease protein, acetyltransferase, acyl-carrier protein, At4g32480-protein, At5g16650-protein, ATP-binding protein, Autophagy-related protein, auxin response factor, auxin transcription factor, b1003-protein, b1522-protein, b2739-protein, b3646-protein, B4029-protein, Branched-chain amino acid permease, calcium-dependent protein kinase, cytochrome c oxidase subunit VIII, elongation factor Tu, Factor arrest protein, fumarylacetoacetate hydrolase, geranylgeranyl pyrophosphate synthase, glucose dehydrogenase, glycosyl transferase, harpin-induced family protein, homocitrate synthase, hydrolase, isochorismate synthase, MFS-type transporter protein, microsomal beta-keto-reductase, polygalacturonase, protein phosphatase, pyruvate kinase, Sec-independent protein translocase subunit, serine protease, thioredoxin, thioredoxin family protein, transcriptional regulator, ubiquinone biosynthesis monooxygenase, and YHR213W-protein for preparing a cell, preferably plant cell with increased gamma-aminobutyric acid (GABA) content as compared to a corresponding non-transformed wild type.
In a further embodiment the invention provides the use of a nucleic acid molecule encoding a polypeptide with the activity selected from the group consisting of 60S ribosomal protein, ABC transporter permease protein, acetyltransferase, acyl-carrier protein, At4g32480-protein, At5g16650-protein, ATP-binding protein, Autophagy-related protein, auxin response factor, auxin transcription factor, b1003-protein, b1522-protein, b2739-protein, b3646-protein, B4029-protein, Branched-chain amino acid permease, calcium-dependent protein kinase, cytochrome c oxidase subunit VIII, elongation factor Tu, Factor arrest protein, fumarylacetoacetate hydrolase, geranylgeranyl pyrophosphate synthase, glucose dehydrogenase, glycosyl transferase, harpin-induced family protein, homocitrate synthase, hydrolase, isochorismate synthase, MFS-type transporter protein, microsomal beta-keto-reductase, polygalacturonase, protein phosphatase, pyruvate kinase, Sec-independent protein translocase subunit, serine protease, thioredoxin, thioredoxin family protein, transcriptional regulator, ubiquinone biosynthesis monooxygenase, and YHR213W-protein as markers for selection of plants or plant cells with increased gamma-aminobutyric acid (GABA) content as compared to a corresponding non-transformed wild type.
In a further embodiment the method according to the invention is used to produce a transgenic plant cell, a plant or a part thereof with increased gamma-aminobutyric acid (GABA) content as compared to a corresponding non-transformed wild type which is derived from a monocotyledonous plant, a dicotyledonous plant or a gymnosperm plant.
The present invention provides methods for producing transgenic plant cells or plants with increased gamma-aminobutyric acid (GABA) content as compared to a corresponding non-transformed wild type and which can show increased tolerance to environmental stress and/or increased yield and/or biomass production as compared to a corresponding (non-transformed) wild type or starting plant cell by increasing or generating one or more of said activities mentioned above.
The present invention provides methods for producing transgenic plant cells or plants with increased gamma-aminobutyric acid (GABA) content as compared to a corresponding non-transformed wild type and with an increased abiotic stress resistance as compared to a corresponding (non-transformed) wild type or starting plant cell by increasing or generating one or more of said activities mentioned above.
The present invention provides methods for producing transgenic plant cells or plants with increased gamma-aminobutyric acid (GABA) content as compared to a corresponding non-transformed wild type and with an increased nitrate influx as compared to a corresponding (non-transformed) wild type or starting plant cell by increasing or generating one or more of said activities mentioned above.
The present invention provides methods for producing transgenic plant cells or plants with increased gamma-aminobutyric acid (GABA) content as compared to a corresponding non-transformed wild type and with an increased plant growth as compared to a corresponding (non-transformed) wild type or starting plant cell by increasing or generating one or more of said activities mentioned above.
Gamma-aminobutyric acid enhances nutrient uptake by roots and leaves so that plant nutrient levels are higher than those achieved by using nutrients alone. When plants are stressed and nutrient uptake is limited, gamma-aminobutyric acid can facilitates nutrient utilization, thereby enhancing growth during stress and/or under sub-optimal growing and cultering conditions of plants.
Accordingly, in one embodiment, the present invention provides a method for producing a plant with increased yield as compared to a corresponding wild type plant comprising at least the following step: increasing or generating one or more activities selected from the group consisting of 60S ribosomal protein, ABC transporter permease protein, acetyltransferase, acyl-carrier protein, At4g32480-protein, At5g16650-protein, ATP-binding protein, Autophagy-related protein, auxin response factor, auxin transcription factor, b1003-protein, b1522-protein, b2739-protein, b3646-protein, B4029-protein, Branched-chain amino acid permease, calcium-dependent protein kinase, cytochrome c oxidase subunit VIII, elongation factor Tu, Factor arrest protein, fumarylacetoacetate hydrolase, geranylgeranyl pyrophosphate synthase, glucose dehydrogenase, glycosyl transferase, harpin-induced family protein, homocitrate synthase, hydrolase, isochorismate synthase, MFS-type transporter protein, microsomal beta-keto-reductase, polygalacturonase, protein phosphatase, pyruvate kinase, Sec-independent protein translocase subunit, serine protease, thioredoxin, thioredoxin family protein, transcriptional regulator, ubiquinone biosynthesis monooxygenase, and YHR213W-protein.
“Yield” as described herein refers in one embodiment to harvestable yield of a plant. The yield of a plant can depend on the specific plant/crop of interest as well as its intended application (such as food production, feed production, processed food production, biofuel, biogas or alcohol production, or the like) of interest in each particular case. Thus, in one embodiment, yield is calculated as harvest index (expressed as a ratio of the weight of the respective harvestable parts divided by the total biomass), harvestable parts weight per area (acre, squaremeter, or the like); and the like.
Preferably, the preferred enhanced or improved yield characteristics of a plant described herein according to the present invention can be achieved in the absence or presence of stress conditions.
The meaning of “yield” is, thus, mainly dependent on the crop of interest and the inteded application, and it is understood, that the skilled person will understand in each particular case what is meant from the circumstances of the description.
For the purposes of the description of the present invention, enhanced or increased “yield” refers to one or more yield parameters selected from the group consisting of biomass yield, dry biomass yield, aerial dry biomass yield, underground dry biomass yield, freshweight biomass yield, aerial freshweight biomass yield, underground freshweight biomass yield; enhanced yield of harvestable parts, either dry or freshweight or both, either aerial or underground or both; enhanced yield of crop fruit, either dry or freshweight or both, either aerial or underground or both; and preferably enhanced yield of seeds, either dry or freshweight or both, either aerial or underground or both.
The term “yield” as used herein generally refers to a measurable product from a plant, particularly a crop.
Yield and yield increase (in comparison to an origin or wild-type plant) can be measured in a number of ways. It is understood that a skilled person will be able to apply the correct meaning in view of the particular embodiments, the particular crop concerned and the specific purpose or application
In one embodiment, an increase in yield refers to increased biomass yield and/or an increased seed yield.
In one embodiment, “yield” refers to biomass yield, e.g. to dry weight biomass yield and/or freshweight biomass yield. Biomass yield refers to the aerial or underground parts of a plant, depending on the specific circumstances (test conditions, specific crop of interest, application of interest, and the like). In one embodiment, biomass yield refers to the aerial and underground parts. Biomass yield may be calculated as freshweight, dry weight or a moisture adjusted basis. Biomass yield may be calculated on a per plant basis or in relation to a specific area (e.g. biomass yield per acre/square meter/or the like).
In other embodiment, “yield” refers to seed yield which can be measured by one or more of the following parameters: number of seeds or number of filled seeds (per plant or per area (acre/square meter/or the like)); seed filling rate (ratio between number of filled seeds and total number of seeds); number of flowers per plant; seed biomass or total seeds weight (per plant or per area (acre/square meter/or the like); thousand kernel weight (TKW; extrapolated from the number of filled seeds counted and their total weight; an increase in TKW may be caused by an increased seed size, an increased seed weight, an increased embryo size, and/or an increased endosperm). Other parameters allowing to measure seed yield are also known in the art. Seed yield may be determined on a dry weight or on a fresh weight basis, or typically on a moisture adjusted basis, e.g. at 15.5 percent moisture.
Said increased yield in accordance with the present invention can typically be achieved by enhancing or improving, in comparison to an origin or wild-type plant, one or more yield-related traits of the plant. Such yield-related traits of a plant the improvement of which results in increased yield comprise, without limitation, the increase of the intrinsic yield capacity of a plant, improved nutrient use efficiency, and/or increased stress tolerance.
Accordingly, in one embodiment, the yield-related trait conferring an increase of the plant's yield is an increase of the intrinsic yield capacity of a plant and can be, for example, manifested by improving the specific (intrinsic) seed yield (e.g. in terms of increased seed/grain size, increased ear number, increased seed number per ear, improvement of seed filling, improvement of seed composition, embryo and/or endosperm improvements, or the like); modification and improvement of inherent growth and development mechanisms of a plant (such as plant height, plant growth rate, pod number, pod position on the plant, number of internodes, incidence of pod shatter, efficiency of nodulation and nitrogen fixation, efficiency of carbon assimilation, improvement of seedling vigour/early vigour, enhanced efficiency of germination (under stressed or non-stressed conditions), improvement in plant architecture, cell cycle modifications, photosynthesis modifications, various signalling pathway modifications, modification of transcriptional regulation, modification of translational regulation, modification of enzyme activities, and the like); and/or the like.
Accordingly, in one embodiment, the yield-related trait conferring an increase of the plant's yield is an improvement or increase of stress tolerance of a plant and can be for example manifested by improving or increasing a plant's tolerance against stress, particularly abiotic stress. In the present application, abiotic stress refers generally to abiotic environmental conditions a plant is typically confronted with, including conditions which are typically referred to as “abiotic stress” conditions including, but not limited to, drought (tolerance to drought may be achieved as a result of improved water use efficiency), heat, low temperatures and cold conditions (such as freezing and chilling conditions), salinity, osmotic stress, shade, high plant density, mechanical stress, oxidative stress, and the like.
Accordingly, in one embodiment, said increased yield in accordance with the present invention can typically be achieved by enhancing or improving, in comparison to a non-transformed starting or wild-type plant, one or more yield-related traits of a plant. Such yield-related traits of a plant of which results in increased yield comprise, without limitation, the increase of the intrinsic yield capacity of a plant, improved nutrient use efficiency, and/or increased stress tolerance, for example an increased drought tolerance and/or low temperature tolerance.
In one embodiment the abiotic stress resistance and/or tolerance refers to water stress resistance, especially under conditions of transient and repetitive abiotic stress, preferably cycling drought.
Thus, in one embodiment of the present invention, an increased plant yield is mediated by increasing the “nutrient use efficiency of a plant”, e.g. by improving the nutrient use efficiency of nutrients including, but not limited to, phosphorus, potassium, and nitrogen.
An increased nutrient use efficiency is in one embodiment an enhanced nitrogen uptake, assimilation, accumulation or utilization. These complex processes are associated with absorption, translocation, assimilation, and redistribution of nitrogen in the plant.
For example, there is a need for plants that are capable to a more efficiently nitrogen uptake so that less nitrogen is required for growth and therefore resulting in the improved level of yield under nitrogen deficiency or nitrogen limiting conditions. Further, higher yields may be obtained with current or standard levels of nitrogen supply or uptake.
Accordingly, in one embodiment of the present invention, plant yield is increased by increasing nitrogen uptake of a plant or a part thereof. Thus, it is a further object of this invention to provide a plant, which shows an enhanced nitrogen uptake, and/or exhibit, under conditions of limited nitrogen supply, an increased yield, as compared to a corresponding wild type plant.
Accordingly, in one embodiment, the present invention relates to a method for increasing the yield, comprising the following steps:
(a) measuring the N content in the soil, and
(b) determining, whether the N-content in the soil is optimal or suboptimal for the growth of an origin or wild type plant, e.g. a crop, and
(c1) growing the plant of the invention in said soil, if the N-content is suboptimal for the growth of the origin or wild type plant, or
(c2) growing the plant of the invention in the soil and comparing the yield with the yield of a standard, an origin or a wild type plant and selecting and growing the plant, which shows the highest yield, if the N-content is optimal for the origin or wild type plant.
In a further embodiment of the present invention, plant yield is increased by increasing the plant's stress tolerance(s).
Generally, the term “increased tolerance to stress” can be defined as survival of plants, and/or higher yield production, under stress conditions as compared to a non-transformed wild type or starting plant.
During its life-cycle, a plant is generally confronted with a diversity of environmental conditions. Any such conditions, which may, under certain circumstances, have an impact on plant yield, are herein referred to as “stress” condition. Environmental stresses may generally be divided into biotic and abiotic (environmental) stresses. Unfavourable nutrient conditions are sometimes also referred to as “environmental stress”. The present invention does also contemplate solutions for this kind of environmental stress, e.g. referring to increased nutrient use efficiency.
In a preferred embodiment of the present invention, plant yield is increased by increasing the abiotic stress tolerance(s) of a plant or a part thereof.
For the purposes of the description of the present invention, the terms “enhanced tolerance to abiotic stress”, “enhanced resistance to abiotic environmental stress”, “enhanced tolerance to environmental stress”, “improved adaptation to environmental stress” and other variations and expressions similar in its meaning are used interchangeably and refer, without limitation, to an improvement in tolerance to one or more abiotic environmental stress(es) as described herein and as compared to a corresponding origin or wild type plant or a part thereof.
The term abiotic stress tolerance(s) refers for example low temperature tolerance, drought tolerance, heat tolerance, salt stress tolerance and others.
Stress tolerance in plants like low temperature, drought, heat and salt stress tolerance can have a common theme important for plant growth, namely the availability of water. Plants are typically exposed during their life cycle to conditions of reduced environmental water content. The protection strategies are similar to those of chilling tolerance.
Accordingly, in one embodiment of the present invention, said yield-related trait relates to an increased water use efficiency of the plant of the invention and/or an increased tolerance to drought conditions of the plant of the invention.
In one embodiment of the present invention drought stress means any environmental stress which leads to a lack of water in plants or reduction of water supply to plants, including a secondary stress by low temperature and/or salt, and/or a primary stress during drought or heat, e.g. desiccation etc.
In accordance with the present invention, in one embodiment, increased tolerance to drought conditions can be determinated and quantified according to the following method:
Transformed plants are grown individually in pots in a growth chamber (York Industriekälte GmbH, Mannheim, Germany). Germination is induced. In case the plants are Arabidopsis thaliana sown seeds are kept at 4° C., in the dark, for 3 days in order to induce germination. Subsequently conditions are changed for 3 days to 20° C./6° C. day/night temperature with a 16/8 h day-night cycle at 150 μE/m2s. Subsequently the plants are grown under standard growth conditions. In case the plants are Arabidopsis thaliana, the standard growth conditions are: photoperiod of 16 h light and 8 h dark, 20° C., 60% relative humidity, and a photon flux density of 200 μE. Plants are grown and cultured until they develop leaves. In case the plants are Arabidopsis thaliana they are watered daily until they were approximately 3 weeks old. Starting at that time drought was imposed by withholding water. After the non-transformed wild type plants show visual symptoms of injury, the evaluation starts and plants are scored for symptoms of drought symptoms and biomass production comparison to wild type and neighbouring plants for 5-6 days in succession.
In one embodiment increased drought resistance refers to resistance to drought cycles, meaning alternating periods of drought and re-watering. repetitive stress is applied to plants without leading to desiccation.
In the present invention, enhanced tolerance to cycling drought may, for example and preferably, be determined according to the following method:
Transformed plants are grown in pots in a growth chamber (e.g. York, Mannheim, Germany). In case the plants are Arabidopsis thaliana soil is prepared as 1:1 (v/v) mixture of nutrient rich soil (GS90, Tantau, Wansdorf, Germany) and quarz sand. Pots (6 cm diameter) are filled with this mixture and placed into trays. Water is added to the trays to let the soil mixture take up appropriate amount of water for the sowing procedure (day 1) and subsequently seeds of transgenic A. thaliana plants and their wild-type controls are sown in pots. Then the filled tray is covered with a transparent lid and transferred into a precooled (4° C.-5° C.) and darkened growth chamber. Stratification is established for a period of 3 days in the dark at 4° C.-5° C. or, alternatively, for 4 days in the dark at 4° C. Germination of seeds and growth is initiated at a growth condition of 20° C., 60% relative humidity, 16 h photoperiod and illumination with fluorescent light at 200 μmol/m2s. Covers are removed 7-8 days after sowing. BASTA selection can be done at day 10 or day 11 (9 or 10 days after sowing) by spraying pots with plantlets from the top. In the standard experiment, a 0.07% (v/v) solution of BASTA concentrate (183 g/I glufosinate-ammonium) in tap water is sprayed once or, alternatively, a 0.02% (v/v) solution of BASTA is sprayed three times. The wild-type control plants are sprayed with tap water only (instead of spraying with BASTA dissolved in tap water) but are otherwise treated identically. Plants are individualized 13-14 days after sowing by removing the surplus of seedlings and leaving one seedling in soil. Transgenic events and wild-type control plants are evenly distributed over the chamber.
The water supply throughout the experiment is limited and plants are subjected to cycles of drought and re-watering. Watering is carried out at day 1 (before sowing), day 14 or day 15, day 21 or day 22, and, finally, day 27 or day 28. For measuring biomass production, plant fresh weight is determined one day after the final watering (day 28 or day 29) by cutting shoots and weighing them. Besides weighing, phenotypic information is added in case of plants that differ from the wild type control. Plants are in the stage prior to flowering and prior to growth of inflorescence when harvested. Significance values for the statistical significance of the biomass changes are calculated by applying the ‘student's’ t test (parameters: two-sided, unequal variance).
Accordingly, in one embodiment of the invention, the increased cold resistance manifests in an biomass increase of the transgenic plant of the invention compared to a wild type control under the stress condition of cycling drought.
Accordingly, in one embodiment, the present invention relates to a method for increasing the yield, comprising the following steps:
(a) determining, whether the water supply in the area for planting is optimal or sub-optimal for the growth of an origin or wild type plant, e.g. a crop, and/or determining the visual symptoms of injury of plants growing in the area for planting; and
(b1) growing the plant of the invention in said soil, if the water supply is suboptimal for the growth of an origin or wild type plant or visual symptoms for drought can be found at a standard, origin or wild type plant growing in the area; or
(b2) growing the plant of the invention in the soil and comparing the yield with the yield of a standard, an origin or a wild type plant and selecting and growing the plant, which shows the highest yield, if the water supply is optimal for the origin or wild type plant.
Visual symptoms of injury stating for one or any combination of two, three or more of the following features:
a) wilting
b) leaf browning
c) loss of turgor, which results in drooping of leaves or needles stems, and flowers,
d) drooping and/or shedding of leaves or needles,
e) the leaves are green but leaf angled slightly toward the ground compared with controls,
f) leaf blades begun to fold (curl) inward,
g) premature senescence of leaves or needles,
h) loss of chlorophyll in leaves or needles and/or yellowing.
In a further embodiment of the present invention, said yield-related trait of the plant of the invention is an increased tolerance to heat conditions of said plant.
In-another embodiment of the present invention, said yield-related trait of the plant of the invention is an increased low temperature tolerance of said plant, e.g. comprising freezing tolerance and/or chilling tolerance.
Low temperatures impinge on a plethora of biological processes. They retard or inhibit almost all metabolic and cellular processes. The response of plants to low temperature is an important determinant of their ecological range. The problem of coping with low temperatures is exacerbated by the need to prolong the growing season beyond the short summer found at high latitudes or altitudes.
Most plants have evolved adaptive strategies to protect themselves against low temperatures. Generally, adaptation to low temperature may be divided into chilling tolerance, and freezing tolerance.
Chilling tolerance is naturally found in species from temperate or boreal zones and allows survival and an enhanced growth at low but non-freezing temperatures. Species from tropical or subtropical zones are chilling sensitive and often show wilting, chlorosis or necrosis, slowed growth and even death at temperatures around 10° C. during one or more stages of development. Accordingly, improved or enhanced “chilling tolerance” or variations thereof refers herein to improved adaptation to low but non-freezing temperatures around 10° C., preferably temperatures between 1 to 18° C., more preferably 4-14° C., and most preferred 8 to 12° C.; hereinafter called “chilling temperature”.
Freezing tolerance allows survival at near zero to particularly subzero temperatures. It is believed to be promoted by a process termed cold-acclimation which occurs at low but non-freezing temperatures and provides increased freezing tolerance at subzero temperatures. In addition, most species from temperate regions have life cycles that are adapted to seasonal changes of the temperature. For those plants, low temperatures may also play an important role in plant development through the process of stratification and vernalisation. It becomes obvious that a clear-cut distinction between or definition of chilling tolerance and freezing tolerance is difficult and that the processes may be overlapping or interconnected.
Improved or enhanced “freezing tolerance” or variations thereof refers herein to improved adaptation to temperatures near or below zero, namely preferably temperatures below 4° C., more preferably below 3 or 2° C., and particularly preferred at or below 0 (zero) ° C. or below −4° C., or even extremely low temperatures down to −10° C. or lower; hereinafter called “freezing temperature.
“Improved adaptation” to environmental stress like e.g. freezing and/or chilling temperatures refers herein to an improved plant performance resulting in an increased yield, particularly with regard to one or more of the yield related traits as defined in more detail above.
Accordingly, the plant of the invention may in one embodiment show an early seedling growth after exposure to low temperatures in comparison to an chilling-sensitive wild type or origin, improving in a further embodiment seed germination rates. The process of seed germination strongly depends on environmental temperature and the properties of the seeds determine the level of activity and performance during germination and seedling emergence when being exposed to low temperature. The method of the invention further provides in one embodiment a plant which show under chilling condition an reduced delay of leaf development.
In one embodiment the method of the invention relates to a production of a tolerant major crop, e.g. corn (maize), bean, rice, soy bean, cotton, tomato, banana, cucumber or potato because most major crops are chilling-sensitive.
In the present invention, enhanced tolerance to low temperature may, for example and preferably, be determined according to the following method:
Transformed plants are grown in pots in a growth chamber (e.g. York, Mannheim, Germany). In case the plants are Arabidopsis thaliana seeds thereof are sown in pots containing a 3.5:1 (v:v) mixture of nutrient rich soil (GS90, Tantau, Wansdorf, Germany) and sand. Plants are grown under standard growth conditions. In case the plants are Arabidopsis thaliana, the standard growth conditions are: stratification is established for a period of 3 days in the dark at 4° C.-5° C.; germination of seeds and growth at a photoperiod of 16 h light, optionally fluorescent light at 150-200 μmol/m2s, and 8 h dark, 20° C., 60% relative humidity, and a photon flux density of 200 μmol/m2s. BASTA selection can be done at day 9 after sowing by spraying pots with plantlets from the top. Therefore, a 0.07% (v/v) solution of BASTA concentrate (183 g/I glufosinate-ammonium) in tap water is sprayed. The wild-type control plants are sprayed with tap water only (instead of spraying with BASTA dissolved in tap water) but are otherwise treated identically. Plants are grown and cultured. In case the plants are Arabidopsis thaliana they are watered every second day. After 9 to 10 days or after 12-13 days, the plants are individualized. Cold (e.g. chilling at 11-12° C.) is applied 14 days or 14-16 days after sowing until the end of the experiment. After a total growth period of 29 to 31, or 35-37 days the plants are harvested and rated by the fresh weight of the arial parts of the plants, in the case of Arabidopsis preferably the rossettes.
Accordingly, in one embodiment, the present invention relates to a method for increasing yield, comprising the following steps:
(a) determining, whether the temperature in the area for planting is optimal or sub-optimal for the growth of an origin or wild type plant, e.g. a crop; and
(b1) growing the plant of the invention in said soil; if the temperature is suboptimal low for the growth of an origin or wild type plant growing in the area; or
(b2) growing the plant of the invention in the soil and comparing the yield with the yield of a standard, an origin or a wild type plant and selecting and growing the plant, which shows the highest yield; if the temperature is optimal for the origin or wild type plant.
In one embodiment of the invention, the term “abiotic stress” encompass even the absence of substantial abiotic stress. In the present invention, the biomass increase may, for example and preferably, be determined according to the following method:
Transformed plants are grown in pots in a growth chamber (e.g. York, Mannheim, Germany). In case the plants are Arabidopsis thaliana seeds thereof are sown in pots containing a 3.5:1 (v:v) mixture of nutrient rich soil (GS90, Tantau, Wansdorf, Germany) and optionally quarz sand.
Plants are grown under standard growth conditions.
Pots are filled with soil mixture and placed into trays. Water is added to the trays to let the soil mixture take up appropriate amount of water for the sowing procedure. In case the plants are Arabidopsis thaliana the seeds for transgenic A. thaliana plants and their non-trangenic wild-type controls are sown in pots (6 cm diameter). Then the filled tray is covered with a transparent lid and transferred into a precooled (4° C.-5° C.) and darkened growth chamber. Stratification is established for a period of 3-4 days in the dark at 4° C.5° C. Germination of seeds and growth is initiated at a growth condition of 20° C., 60% relative humidity, 16 h photoperiod and illumination with fluorescent light at approximately 170 μmol/m2s. Covers are removed 7-8 days after sowing. BASTA selection is done at day 10 or day 11 (9 or 10 days after sowing) by spraying pots with plantlets from the top. In the standard experiment, a 0.07% (v/v) solution of BASTA concentrate (183 g/I glufosinate-ammonium) in tap water is sprayed once or, alternatively, a 0.02% (v/v) solution of BASTA is sprayed three times. The wild-type control plants are sprayed with tap water only (instead of spraying with BASTA dissolved in tap water) but are otherwise treated identically. Plants are individualized 13-14 days after sowing by removing the surplus of seedlings and leaving one seedling in soil. Transgenic events and wild-type control plants are evenly distributed over the chamber.
Watering is carried out every two days after removing the covers in a standard experiment or, alternatively, every day. For measuring biomass performance, plant fresh weight was determined at harvest time (24-29 days after sowing) by cutting shoots and weighing them. Plants are in the stage prior to flowering and prior to growth of inflorescence when harvested. Transgenic plants are compared to the non-transgenic wild-type control plants harvested at the same day. Significance values for the statistical significance of the biomass changes can be calculated by applying the ‘student's’ t test (parameters: two-sided, unequal variance).
Biomass production can be measured by weighing plant rosettes. Biomass increase can be calculated as ratio of average weight for transgenic plants compared to average weight of wild type control plants from the same experiment.
In a further embodiment of the present invention, yield-related trait may also be increased salinity tolerance (salt tolerance), tolerance to osmotic stress, increased shade tolerance, increased tolerance to a high plant density, increased tolerance to mechanical stresses, and/or increased tolerance to oxidative stress.
Accordingly, in one embodiment of the present invention, yield is increased by improving one or more of the yield-related traits as defined herein.
Thus, the present invention provides a method for producing a transgenic plant showing an increased nutrient use efficiency as compared to a corresponding origin or wild type plant, by increasing or generating one or more activities selected from the group consisting of 60S ribosomal protein, ABC transporter permease protein, acetyltransferase, acyl-carrier protein, At4g32480-protein, At5g16650-protein, ATP-binding protein, Autophagy-related protein, auxin response factor, auxin transcription factor, b1003-protein, b1522-protein, b2739-protein, b3646-protein, B4029-protein, Branched-chain amino acid permease, calcium-dependent protein kinase, cytochrome c oxidase subunit VIII, elongation factor Tu, Factor arrest protein, fumarylacetoacetate hydrolase, geranylgeranyl pyrophosphate synthase, glucose dehydrogenase, glycosyl transferase, harpin-induced family protein, homocitrate synthase, hydrolase, isochorismate synthase, MFS-type transporter protein, microsomal beta-keto-reductase, polygalacturonase, protein phosphatase, pyruvate kinase, Sec-independent protein translocase subunit, serine protease, thioredoxin, thioredoxin family protein, transcriptional regulator, ubiquinone biosynthesis monooxygenase, and YHR213W-protein (“activities”).
In other embodiments, the present invention provides a method for producing a plant showing an increased stress resistance, particularly abiotic stress resistance, as compared to a corresponding origin or wild type plant, by increasing or generating one or more said activities.
In another embodiment, the abiotic stress resistance achieved in accordance with the methods of the present invention, and shown by the transgenic plant of the invention; is increased low temperature tolerance, particularly increased tolerance to chilling.
Thus, in one further embodiment of the present invention, a method is provided for producing a transgenic plant; progenies, seeds, and/or pollen derived from such plant; each showing an increased nitrogen uptake and an increased low temperature tolerance, particularly chilling tolerance, as compared to a corresponding non-transformed wild type plant cell or plant, by increasing or generating one or more of said activities.
Furthermore, in one embodiment, the present invention provides a transgenic plant showing one or more increased yield-related trait as compared to a corresponding non-transformed origin or wild type plant cell or plant, by increasing or generating one or more activities selected from the above mentioned group of activities.
Further, the present invention relates to method for producing a plant with increased yield as compared to a corresponding wild type plant comprising at least one of the steps selected from the group consisting of:
(i) increasing or generating the activity of a polypeptide comprising a polypeptide, a consensus sequence or at least one polypeptide motif as depicted in column 5 or 7 of table II or of table IV, respectively;
(ii) increasing or generating the activity of an expression product of a nucleic acid molecule comprising a polynucleotide as depicted in column 5 or 7 of table I, and
(iii) increasing or generating the activity of a functional equivalent of (i) or (ii).
In one embodiment, the increase or generation of said one or more activities is conferred by one or more nucleic acid sequences comprising a polynucleotide selected from the group as shown in table I, column 5 or 7. Accordingly, the increase or generation of said one or more activities is for example conferred by one or more expression products of said nucleic acid molecule, e.g. proteins. Accordingly, in the present invention described above, the increase or generation of said one or more activities is for example conferred by one or more protein(s) each comprising a polypeptide selected from the group as depicted in table II, column 5 and 7.
Thus, in one embodiment, the present invention provides a method for producing a plant showing increased yield as compared to a corresponding origin or wild type plant, by increasing or generating one or more activities selected from the group consisting of 60S ribosomal protein, ABC transporter permease protein, acetyltransferase, acyl-carrier protein, At4g32480-protein, At5g16650-protein, ATP-binding protein, Autophagy-related protein, auxin response factor, auxin transcription factor, b1003-protein, b1522-protein, b2739-protein, b3646-protein, B4029-protein, Branched-chain amino acid permease, calcium-dependent protein kinase, cytochrome c oxidase subunit VIII, elongation factor Tu, Factor arrest protein, fumarylacetoacetate hydrolase, geranylgeranyl pyrophosphate synthase, glucose dehydrogenase, glycosyl transferase, harpin-induced family protein, homocitrate synthase, hydrolase, isochorismate synthase, MFS-type transporter protein, microsomal beta-keto-reductase, polygalacturonase, protein phosphatase, pyruvate kinase, Sec-independent protein translocase subunit, serine protease, thioredoxin, thioredoxin family protein, transcriptional regulator, ubiquinone biosynthesis monooxygenase, and YHR213W-protein, which is conferred by one or more nucleic acid sequences comprising a polynucleotide selected from the group as shown in table I, column 5 or 7 or by one or more proteins each comprising a polypeptide encoded by one or more nucleic acid sequences selected from the group as shown in table I, column 5 or 7. or by one or more protein(s) each comprising a polypeptide selected from the group as depicted in table II, column 5 and 7. As mentioned, the increase yield can be mediated by one or more yield-related traits. Thus, the method of the invention relates to the production of a plant showing said one or more yield-related traits.
Thus, the present invention provides a method for producing a plant showing an increased nutrient use efficiency, e.g. nitrogen uptake, increased stress resistance particularly abiotic stress resistance, increased water use efficiency, and/or an increased stress resistance, particularly abiotic stress resistance, particular low temperature tolerance or draught tolerance or an increased intrinsic yield.
Further, the present invention relates to a method for producing a plant with increased yield as compared to a corresponding origin or wild type transgenic plant, which comprises
(a) increasing or generating, in a plant cell nucleus, a plant cell, a plant or a part thereof, one or more activities selected from the group consisting of 60S ribosomal protein, ABC transporter permease protein, acetyltransferase, acyl-carrier protein, At4g32480-protein, At5g16650-protein, ATP-binding protein, Autophagy-related protein, auxin response factor, auxin transcription factor, b1003-protein, b1522-protein, b2739-protein, b3646-protein, B4029-protein, Branched-chain amino acid permease, calcium-dependent protein kinase, cytochrome c oxidase subunit VIII, elongation factor Tu, Factor arrest protein, fumarylacetoacetate hydrolase, geranylgeranyl pyrophosphate synthase, glucose dehydrogenase, glycosyl transferase, harpin-induced family protein, homocitrate synthase, hydrolase, isochorismate synthase, MFS-type transporter protein, microsomal beta-keto-reductase, polygalacturonase, protein phosphatase, pyruvate kinase, Sec-independent protein translocase subunit, serine protease, thioredoxin, thioredoxin family protein, transcriptional regulator, ubiquinone biosynthesis monooxygenase, and YHR213W-protein; and
(b) cultivating or growing the plant cell, the plant or the part thereof under conditions which permit the development of the plant cell, the plant or the part thereof; and
(c) recovering a plant showing increased yield as compared to a corresponding non-transformed origin or wild type plant;
(d) and optionally, selecting the plant or a part thereof, showing increased yield as compared to a corresponding non-transformed wild type plant cell, a transgenic plant or a part thereof which shows visual symptoms of deficiency and/or death.
It was further an object of the present invention to provide a plant cell and/or a plant with enhanced tolerance to abiotic environmental stress and/or showing under conditions of abiotic environmental stress an increased yield, as compared to a corresponding non-transformed wild type or starting plant cell and/or plant.
It was found that this object is achieved by providing a cell, plant cell and/or plant according to the present invention described herein.
In one embodiment of the present invention, these traits are achieved by a process for an enhanced tolerance to abiotic environmental stress in a cell, preferably from a photosynthetic active organism, preferably a plant, as compared to a corresponding (non-transformed) wild type or starting photosynthetic active organism.
In a further embodiment, “enhanced tolerance to abiotic environmental stress” in a photosynthetic active organism means that the photosynthetic active organism, preferably a plant, when confronted with abiotic environmental stress conditions as mentioned above, e.g. like low temperature conditions including chilling and freezing temperatures or drought, exhibits an enhanced yield, e.g. a yield as mentioned above, e.g. a seed yield or biomass yield, as compared to a corresponding (non-transformed) wild type or starting photosynthetic active organism.
In an embodiment thereof, the term “enhanced tolerance to abiotic environmental stress” in a photosynthetic active organism means that the photosynthetic active organism, preferably a plant, when confronted with abiotic environmental stress conditions like low temperature conditions including chilling and freezing temperatures, exhibits an enhanced dry biomass yield as compared to a corresponding non-transformed wild type photosynthetic active organism.
In an embodiment thereof, the term “enhanced tolerance to abiotic environmental stress” in a photosynthetic active organism means that the photosynthetic active organism, preferably a plant, when confronted with abiotic environmental stress conditions like low temperature conditions including chilling and freezing temperatures, exhibits an enhanced aerial dry biomass yield as compared to a corresponding non-transformed wild type photosynthetic active organism.
In an embodiment thereof, the term “enhanced tolerance to abiotic environmental stress” in a photosynthetic active organism means that the photosynthetic active organism, preferably a plant, when confronted with abiotic environmental stress conditions like low temperature conditions including chilling and freezing temperatures, exhibits an enhanced underground dry biomass yield as compared to a corresponding non-transformed wild type photosynthetic active organism.
In another embodiment thereof, the term “enhanced tolerance to abiotic environmental stress” in a photosynthetic active organism means that the photosynthetic active organism, preferably a plant, when confronted with abiotic environmental stress conditions like low temperature conditions including chilling and freezing temperatures, exhibits an enhanced fresh weight biomass yield as compared to a corresponding non-transformed wild type photosynthetic active organism.
In an embodiment thereof, the term “enhanced tolerance to abiotic environmental stress” in a photosynthetic active organism means that the photosynthetic active organism, preferably a plant, when confronted with abiotic environmental stress conditions like low temperature conditions including chilling and freezing temperatures, exhibits an enhanced aerial fresh weight biomass yield as compared to a corresponding non-transformed wild type photosynthetic active organism.
In an embodiment thereof, the term “enhanced tolerance to abiotic environmental stress” in a photosynthetic active organism means that the photosynthetic active organism, preferably a plant, when confronted with abiotic environmental stress conditions like low temperature conditions including chilling and freezing temperatures, exhibits an enhanced underground fresh weight biomass yield as compared to a corresponding non-transformed wild type photosynthetic active organism.
In another embodiment thereof, the term “enhanced tolerance to abiotic environmental stress” in a photosynthetic active organism means that the photosynthetic active organism, preferably a plant, when confronted with abiotic environmental stress conditions like low temperature conditions including chilling and freezing temperatures, exhibits an enhanced yield of harvestable parts of a plant as compared to a corresponding non-transformed wild type photosynthetic active organism.
In an embodiment thereof, the term “enhanced tolerance to abiotic environmental stress” in a photosynthetic active organism means that the photosynthetic active organism, preferably a plant, when confronted with abiotic environmental stress conditions like low temperature conditions including chilling and freezing temperatures, exhibits an enhanced yield of dry harvestable parts of a plant as compared to a corresponding non-transformed wild type photosynthetic active organism.
In an embodiment thereof, the term “enhanced tolerance to abiotic environmental stress” in a photosynthetic active organism means that the photosynthetic active organism, preferably a plant, when confronted with abiotic environmental stress conditions like low temperature conditions including chilling and freezing temperatures, exhibits an enhanced yield of dry aerial harvestable parts of a plant as compared to a corresponding non-transformed wild type photosynthetic active organism.
In an embodiment thereof, the term “enhanced tolerance to abiotic environmental stress” in a photosynthetic active organism means that the photosynthetic active organism, preferably a plant, when confronted with abiotic environmental stress conditions like low temperature conditions including chilling and freezing temperatures, exhibits an enhanced yield of underground dry harvestable parts of a plant as compared to a corresponding non-transformed wild type photosynthetic active organism.
In another embodiment thereof, the term “enhanced tolerance to abiotic environmental stress” in a photosynthetic active organism means that the photosynthetic active organism, preferably a plant, when confronted with abiotic environmental stress conditions like low temperature conditions including chilling and freezing temperatures, exhibits an enhanced yield of fresh weight harvestable parts of a plant as compared to a corresponding non-transformed wild type photosynthetic active organism.
In an embodiment thereof, the term “enhanced tolerance to abiotic environmental stress” in a photosynthetic active organism means that the photosynthetic active organism, preferably a plant, when confronted with abiotic environmental stress conditions like low temperature conditions including chilling and freezing temperatures, exhibits an enhanced yield of aerial fresh weight harvestable parts of a plant as compared to a corresponding non-transformed wild type photosynthetic active organism.
In an embodiment thereof, the term “enhanced tolerance to abiotic environmental stress” in a photosynthetic active organism means that the photosynthetic active organism, preferably a plant, when confronted with abiotic environmental stress conditions like low temperature conditions including chilling and freezing temperatures, exhibits an enhanced yield of underground fresh weight harvestable parts of a plant as compared to a corresponding non-transformed wild type photosynthetic active organism.
In a further embodiment, the term “enhanced tolerance to abiotic environmental stress” in a photosynthetic active organism means that the photosynthetic active organism, preferably a plant, when confronted with abiotic environmental stress conditions like low temperature conditions including chilling and freezing temperatures, exhibits an enhanced yield of the crop fruit as compared to a corresponding non-transformed wild type photosynthetic active organism.
In an embodiment thereof, the term “enhanced tolerance to abiotic environmental stress” in a photosynthetic active organism means that the photosynthetic active organism, preferably a plant, when confronted with abiotic environmental stress conditions like low temperature conditions including chilling and freezing temperatures, exhibits an enhanced yield of the fresh crop fruit as compared to a corresponding non-transformed wild type photosynthetic active organism.
In an embodiment thereof, the term “enhanced tolerance to abiotic environmental stress” in a photosynthetic active organism means that the photosynthetic active organism, preferably a plant, when confronted with abiotic environmental stress conditions like low temperature conditions including chilling and freezing temperatures, exhibits an enhanced yield of the dry crop fruit as compared to a corresponding non-transformed wild type photosynthetic active organism.
In an embodiment thereof, the term “enhanced tolerance to abiotic environmental stress” in a photosynthetic active organism means that the photosynthetic active organism, preferably a plant, when confronted with abiotic environmental stress conditions like low temperature conditions including chilling and freezing temperatures, exhibits an enhanced grain dry weight as compared to a corresponding non-transformed wild type photosynthetic active organism.
In a further embodiment, the term “enhanced tolerance to abiotic environmental stress” in a photosynthetic active organism means that the photosynthetic active organism, preferably a plant, when confronted with abiotic environmental stress conditions like low temperature conditions including chilling and freezing temperatures, exhibits an enhanced yield of seeds as compared to a corresponding non-transformed wild type photosynthetic active organism.
In an embodiment thereof, the term “enhanced tolerance to abiotic environmental stress” in a photosynthetic active organism means that the photosynthetic active organism, preferably a plant, when confronted with abiotic environmental stress conditions like low temperature conditions including chilling and freezing temperatures, exhibits an enhanced yield of fresh weight seeds as compared to a corresponding non-transformed wild type photosynthetic active organism.
In an embodiment thereof, the term “enhanced tolerance to abiotic environmental stress” in a photosynthetic active organism means that the photosynthetic active organism, preferably a plant, when confronted with abiotic environmental stress conditions like low temperature conditions including chilling and freezing temperatures, exhibits an enhanced yield of dry seeds as compared to a corresponding non-transformed wild type photosynthetic active organism.
In another embodiment of the present invention, these traits are achieved by a process for an increased yield under conditions of environmental stress, particularly abiotic environmental stress, in a photosynthetic active organism, preferably a plant, as compared to a corresponding (non-transformed) wild type or starting photosynthetic active organism.
In one embodiment thereof, the term “increased yield” means that the photosynthetic active organism, especially a plant, exhibits an increased yield, e.g. exhibits an increased growth rate, under conditions of abiotic environmental stress, compared to the corresponding wild-type photosynthetic active organism.
An increased growth rate may be reflected inter alia by or confers an increased biomass production of the whole plant, or an increased biomass production of the aerial parts of a plant, or by an increased biomass production of the underground parts of a plant, or by an increased biomass production of parts of a plant, like stems, leaves, blossoms, fruits, and/or seeds.
In an embodiment thereof, increased yield includes higher fruit yields, higher seed yields, higher fresh matter production, and/or higher dry matter production.
In another embodiment thereof, the term “increased yield” means that the photosynthetic active organism, preferably plant, exhibits a prolonged growth under conditions of abiotic environmental stress, as compared to the corresponding non-transformed wild type photosynthetic active organism. A prolonged growth comprises survival and/or continued growth of the photosynthetic active organism, preferably plant, at the moment when the non-transformed wild type photosynthetic active organism shows visual symptoms of deficiency and/or death.
In another embodiment thereof, the term “increased yield” means that the photosynthetic active organism, preferably plant, exhibits an increased gamma-aminobutyric acid (GABA) content as compared to a corresponding non-transformed wild type.
In another preferred embodiment a photosynthetic active organism, especially a plant, shows increased yield under conditions of abiotic environmental stress, e.g. a plant, shows an enhanced tolerance to abiotic environmental stress or another yield-related trait.
In another embodiment this invention fulfills the need to identify new, unique genes capable of conferring an increased yield, e.g. an enhanced tolerance to abiotic environmental stress or another yield-related trait, to photosynthetic active organism, preferably plants, upon expression or over-expression of endogenous and/or exogenous genes.
In another embodiment thereof this invention fulfills the need to identify new, unique genes capable of conferring an increased yield, e.g. an enhanced tolerance to abiotic environmental stress or another yield-related trait, to photosynthetic active organism, preferably plants, upon expression or over-expression of endogenous genes.
In another embodiment thereof this invention fulfills the need to identify new, unique genes capable of conferring an increased yield, e.g. an enhanced tolerance to abiotic environmental stress or another yield-related trait, to photosynthetic active organism, preferably plants, upon expression or over-expression of exogenous genes.
In another embodiment this invention fulfills the need to identify new, unique genes capable of conferring an enhanced tolerance to abiotic environmental stress in combination with an increase of yield to photosynthetic active organism, preferably plants, upon expression or over-expression of endogenous and/or exogenous genes.
Accordingly, the present invention relates to a method for producing a for example transgenic photosynthetic active organism or a part thereof, or a plant cell, a plant or a part thereof e.g. for the generation of such a plant, with increased yield, e.g. with an increased yield-related trait, for example, increased nutrient use efficiency, increased intrinsic yield capacity, and/or increased stress tolerance, preferably water stress resistance, especially under conditions of transient and repetitive abiotic stress, preferably cycling drought and/or low temperature tolerance and/or another increased yield-related trait as compared to a corresponding for example non-transformed wild type photosynthetic active organism or a part thereof, or a plant cell, a plant or a part thereof, which comprises
(a) increasing or generating one or more activities selected from the group consisting of 60S ribosomal protein, ABC transporter permease protein, acetyltransferase, acyl-carrier protein, At4g32480-protein, At5g16650-protein, ATP-binding protein, Autophagy-related protein, auxin response factor, auxin transcription factor, b1003-protein, b1522-protein, b2739-protein, b3646-protein, B4029-protein, Branched-chain amino acid permease, calcium-dependent protein kinase, cytochrome c oxidase subunit VIII, elongation factor Tu, Factor arrest protein, fumarylacetoacetate hydrolase, geranylgeranyl pyrophosphate synthase, glucose dehydrogenase, glycosyl transferase, harpin-induced family protein, homocitrate synthase, hydrolase, isochorismate synthase, MFS-type transporter protein, microsomal beta-keto-reductase, polygalacturonase, protein phosphatase, pyruvate kinase, Sec-independent protein translocase subunit, serine protease, thioredoxin, thioredoxin family protein, transcriptional regulator, ubiquinone biosynthesis monooxygenase, and YHR213W-protein in a photosynthetic active organism or a part thereof, e.g. a plant cell, a plant or a part thereof, and
(b) growing the photosynthetic active organism or a part thereof, e.g. a plant cell, a plant or a part thereof under conditions which permit the development of a photosynthetic active organism or a part thereof, preferably a plant cell, a plant or a part thereof, with increased yield, e.g. with an increased yield-related trait, for example enhanced tolerance to abiotic environmental stress, increased nutrient use efficiency, increased drought tolerance and/or another increased yield-related trait as compared to a corresponding, e.g. non-transformed, wild type photosynthetic active organism or a part thereof, preferably a plant cell, a plant or a part thereof.
In an embodiment the present invention relates to a method for producing a, e.g. transgenic, photosynthetic active organism or a part thereof, preferably a plant cell, a plant or a part thereof with increased yield, e.g. with an increased yield-related trait, for example enhanced tolerance to abiotic environmental stress, increased nutrient use efficiency, increased drought tolerance and/or another increased yield-related trait as compared to a corresponding e.g. non-transformed wild type photosynthetic active organism or a part thereof, preferably a plant cell, a plant or a part thereof, which comprises
(a) increasing or generating one or more activities selected from the group consisting of: 60S ribosomal protein, ABC transporter permease protein, acetyltransferase, acyl-carrier protein, At4g32480-protein, At5g16650-protein, ATP-binding protein, Autophagy-related protein, auxin response factor, auxin transcription factor, b1003-protein, b1522-protein, b2739-protein, b3646-protein, B4029-protein, Branched-chain amino acid permease, calcium-dependent protein kinase, cytochrome c oxidase subunit VIII, elongation factor Tu, Factor arrest protein, fumarylacetoacetate hydrolase, geranylgeranyl pyrophosphate synthase, glucose dehydrogenase, glycosyl transferase, harpin-induced family protein, homocitrate synthase, hydrolase, isochorismate synthase, MFS-type transporter protein, microsomal beta-keto-reductase, polygalacturonase, protein phosphatase, pyruvate kinase, Sec-independent protein translocase subunit, serine protease, thioredoxin, thioredoxin family protein, transcriptional regulator, ubiquinone biosynthesis monooxygenase, and YHR213W-protein in a photosynthetic active organism or a part thereof, preferably a plant cell, a plant or a part thereof,
(b) growing the photosynthetic active organism or a part thereof, preferably a plant cell, a plant or a part thereof together with e.g. non-transformed wildtype photosynthetic active organism or a part thereof, preferably a plant, under conditions of abiotic environmental stress
(c) selecting the photosynthetic active organism or a part thereof, preferably a plant cell, a plant or a part thereof, with increased yield, e.g. with an increased yield-related trait, for example enhanced tolerance to abiotic environmental stress, increased nutrient use efficiency, increased drought tolerance and/or another increased yield-related trait, as compared to a corresponding, e.g non-transformed, wild type photosynthetic active organism or a part thereof, preferably a plant cell, a plant or a part thereof, after the, e.g. non-transformed, wild type photosynthetic active organism or a part thereof, preferably a plant cell, a plant or a part thereof, show visual symptoms of deficiency and/or death.
Climate and culturing conditions for plants can be classified into mega-environments according to the one used by CIMMYT to guide its breeding programmes in wheat and maize.
A mega-environment is a broad, not necessarily contiguous geographic area with similar biotic and abiotic stresses and cropping system requirements. In fact a mega-environment is defined by crop production factors (temperature, rainfall, sunlight, latitude, elevation, soil characteristics, and diseases), consumer preferences (the color of the grain and how it would be used), and wheat growth habit.
For CIMMYT researchers identified six mega-environments for spring wheats and three each for facultative and winter wheat.
Such mega-enviroments are feasible for every plant species including crops.
In one embodiment the present invention provides a transgenic plant cell, a plant or a part thereof with increased yield under sub-optimal growing conditions as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof.
Such sub-optimal growing conditions can be for example mega-enviromentals with low rainfall, as for example the wheat mega-enviroments ME1, ME4, ME4A, ME4B, ME4C, ME5, ME5B, ME6, ME6B, ME9, ME12 or the respective mega-environment for the specific plant species.
Such mega-environments are feasible for every sub-optimal growth condition, temperature or nutrient disposability.
In order to compare the yield of plants of the same species in correlation with environment conditions the parameter of yield potential is significant. Yield potential is defined as the yield of a plant when grown in environments to which it is adapted, with nutrients and water non-limiting and with pests, diseases, weeds, lodging, and other stresses effectively controlled. In this embodiment “Yield” refers to the mass of product at final harvest.
Under field conditions the yield potential will not be achieved. Nevertheless, it is a parameter which defines the optimal cultivating conditions in an mega-environment because only under optimal conditions the yield potential will be achieved.
In one embodiment sub-optimal growing condition is any condition which does not correspond to the respective condition where the yield potential can be achieved.
In one embodiment optimal growth conditions, including nutrient disposability, are conditions selected from the group consisting of:
climatic and environmental conditions, including nutrient disposability as they were predominantly in the last 50 25, 20, 15, 10 or 5 years over a period of 3, 6, 12 month or a cultivation period in the mega-enviroments known as Wheatbelt Region in Western Australia, corn belt in the U.S.A. (comprising at least one of the states of Iowa, Indiana, Ill., Ohio, South Dakota, Nebraska, Kansas, Minnesota, Wisconsin, Michigan, Missouri and Kentucky),
climatic and environmental conditions as they were predominantly in the last 50 25, 20, 15, 10 or 5 years over a period of 3, 6, 12 month or a cultivation period in the mega-environments as mentioned for maize and wheat by CIMMYT.
In one embodiment the invention relates to a method for increasing the yield per acre or per cultivated area comprising the steps:
performing a analysis of environmental conditions to measure the level of nutrients (including water) available in the soil or rainfall per cultivating cycle,
comparing the result with the value of the respective condition with the value under optimal growing condition,
cultivating a plant of the respective class/genera according to the invention in case at east one measured condition deviates for 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100% or more from the value under optimal growing condition.
In one embodiment the invention relates to a method for increasing the yield per acre in mega environments comprising the steps:
performing a soil analysis to measure the level of nutrients available in the soil, comparing the result with the value necessarily for achieving the yield potential of a class/genera of a plant,
cultivating a plant of the respective class/genera according to the invention in case at east one nutrient is limited.
In one embodiment of the invention relates to a method for increasing the yield per acre in mega environments comprising the steps:
measuring the precipitation over a time period of at least one plant generation,
comparing with the value for achieving the yield potential of a class/genera of a plant, cultivating a plant of the respective class/genera according to the invention in case the precipitation is decreased.
In one embodiment of the invention relates to a method for increasing the yield per acre in mega environments comprising the steps:
measuring the time periods between the rainfalls over a time period of at least one plant generation,
comparing with the value for achieving the yield potential of a class/genera of a plant and cultivating a plant of the respective class/genera according to the invention in case the dry season is increased.
Comprises/comprising and grammatical variations thereof when used in this specification are to be taken to specify the presence of stated features, integers, steps or components or groups thereof, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.
In accordance with the invention, the term “plant cell” or the term “organism” as understood herein relates always to a plant cell or a organelle thereof, preferably a plastid, more preferably chloroplast.
As used herein, “plant” is meant to include not only a whole plant but also a part thereof i.e., one or more cells, and tissues, including for example, leaves, stems, shoots, roots, flowers, fruits and seeds.
Surprisingly it was found, that the transgenic expression of a protein as shown in table II, column 3 in a plant such as Arabidopsis thaliana C24 for example, conferred transgenic a plant cell, a plant or a part thereof with increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof.
Accordingly, in one embodiment, in case the activity of the Saccharomyces cerevisiae nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 42 or polypeptide SEQ ID NO.: 43, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 42 or polypeptide SEQ ID NO.: 43, respectively is increased or generated or if the activity “Factor arrest protein” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Arabidopsis thaliana nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 654 or polypeptide SEQ ID NO.: 655, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 654 or polypeptide SEQ ID NO.: 655, respectively is increased or generated or if the activity “transcriptional regulator” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Arabidopsis thaliana nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 706 or polypeptide SEQ ID NO.: 707, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 706 or polypeptide SEQ ID NO.: 707, respectively is increased or generated or if the activity “protein phosphatase” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Arabidopsis thaliana nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 751 or polypeptide SEQ ID NO.: 752, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 751 or polypeptide SEQ ID NO.: 752, respectively is increased or generated or if the activity “pyruvate kinase” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Arabidopsis thaliana nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 1156 or polypeptide SEQ ID NO.: 1157, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 1156 or polypeptide SEQ ID NO.: 1157, respectively is increased or generated or if the activity “thioredoxin family protein” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Arabidopsis thaliana nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 1510 or polypeptide SEQ ID NO.: 1511, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 1510 or polypeptide SEQ ID NO.: 1511, respectively is increased or generated or if the activity “harpin-induced family protein” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Arabidopsis thaliana nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 1598 or polypeptide SEQ ID NO.: 1599, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 1598 or polypeptide SEQ ID NO.: 1599, respectively is increased or generated or if the activity “glycosyl transferase” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Arabidopsis thaliana nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 1670 or polypeptide SEQ ID NO.: 1671, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 1670 or polypeptide SEQ ID NO.: 1671, respectively is increased or generated or if the activity “auxin response factor” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Arabidopsis thaliana nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 1874 or polypeptide SEQ ID NO.: 1875, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 1874 or polypeptide SEQ ID NO.: 1875, respectively is increased or generated or if the activity “At4g32480-protein” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Arabidopsis thaliana nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 1936 or polypeptide SEQ ID NO.: 1937, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 1936 or polypeptide SEQ ID NO.: 1937, respectively is increased or generated or if the activity “calcium-dependent protein kinase” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Arabidopsis thaliana nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 2492 or polypeptide SEQ ID NO.: 2493, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 2492 or polypeptide SEQ ID NO.: 2493, respectively is increased or generated or if the activity “At5g16650-protein” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Azotobacter vinelandii nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 2553 or polypeptide SEQ ID NO.: 2554, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 2553 or polypeptide SEQ ID NO.: 2554, respectively is increased or generated or if the activity “elongation factor Tu” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Azotobacter vinelandii nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 3408 or polypeptide SEQ ID NO.: 3409, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 3408 or polypeptide SEQ ID NO.: 3409, respectively is increased or generated or if the activity “ABC transporter permease protein” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Azotobacter vinelandii nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 3564 or polypeptide SEQ ID NO.: 3565, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 3564 or polypeptide SEQ ID NO.: 3565, respectively is increased or generated or if the activity “hydrolase” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Azotobacter vinelandii nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 3728 or polypeptide SEQ ID NO.: 3729, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 3728 or polypeptide SEQ ID NO.: 3729, respectively is increased or generated or if the activity “fumarylacetoacetate hydrolase” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Escherichia coli nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 4068 or polypeptide SEQ ID NO.: 4069, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 4068 or polypeptide SEQ ID NO.: 4069, respectively is increased or generated or if the activity “glucose dehydrogenase” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Escherichia coli nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 4176 or polypeptide SEQ ID NO.: 4177, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 4176 or polypeptide SEQ ID NO.: 4177, respectively is increased or generated or if the activity “serine protease” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Escherichia coli nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 4364 or polypeptide SEQ ID NO.: 4365, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 4364 or polypeptide SEQ ID NO.: 4365, respectively is increased or generated or if the activity “ATP-binding protein” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Escherichia coli nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 4717 or polypeptide SEQ ID NO.: 4718, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 4717 or polypeptide SEQ ID NO.: 4718, respectively is increased or generated or if the activity “isochorismate synthase” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Escherichia coli nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 4864 or polypeptide SEQ ID NO.: 4865, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 4864 or polypeptide SEQ ID NO.: 4865, respectively is increased or generated or if the activity “MFS-type transporter protein” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Escherichia coli nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 4903 or polypeptide SEQ ID NO.: 4904, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 4903 or polypeptide SEQ ID NO.: 4904, respectively is increased or generated or if the activity “b1003-protein” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Escherichia coli nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 4909 or polypeptide SEQ ID NO.: 4910, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 4909 or polypeptide SEQ ID NO.: 4910, respectively is increased or generated or if the activity “b1522-protein” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Escherichia coli nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 4954 or polypeptide SEQ ID NO.: 4955, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 4954 or polypeptide SEQ ID NO.: 4955, respectively is increased or generated or if the activity “b2739-protein” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Escherichia coli nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 5121 or polypeptide SEQ ID NO.: 5122, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 5121 or polypeptide SEQ ID NO.: 5122, respectively is increased or generated or if the activity “b3646-protein” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Escherichia coli nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 5319 or polypeptide SEQ ID NO.: 5320, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 5319 or polypeptide SEQ ID NO.: 5320, respectively is increased or generated or if the activity “B4029-protein” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Escherichia coli nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 5387 or polypeptide SEQ ID NO.: 5388, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 5387 or polypeptide SEQ ID NO.: 5388, respectively is increased or generated or if the activity “acetyltransferase” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Physcomitrella patens nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 5458 or polypeptide SEQ ID NO.: 5459, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 5458 or polypeptide SEQ ID NO.: 5459, respectively is increased or generated or if the activity “acyl-carrier protein” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Synechocystis sp. nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 6041 or polypeptide SEQ ID NO.: 6042, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 6041 or polypeptide SEQ ID NO.: 6042, respectively is increased or generated or if the activity “geranylgeranyl pyrophosphate synthase” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Thermus thermophilus nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 6469 or polypeptide SEQ ID NO.: 6470, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 6469 or polypeptide SEQ ID NO.: 6470, respectively is increased or generated or if the activity “Sec-independent protein translocase subunit” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Thermus thermophilus nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 6739 or polypeptide SEQ ID NO.: 6740, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 6739 or polypeptide SEQ ID NO.: 6740, respectively is increased or generated or if the activity “homocitrate synthase” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Saccharomyces cerevisiae nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 7510 or polypeptide SEQ ID NO.: 7511, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 7510 or polypeptide SEQ ID NO.: 7511, respectively is increased or generated or if the activity “polygalacturonase” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Saccharomyces cerevisiae nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 7633 or polypeptide SEQ ID NO.: 7634, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 7633 or polypeptide SEQ ID NO.: 7634, respectively is increased or generated or if the activity “thioredoxin” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Brassica napus nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 53 or polypeptide SEQ ID NO.: 54, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 53 or polypeptide SEQ ID NO.: 54, respectively is increased or generated or if the activity “pyruvate kinase” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Saccharomyces cerevisiae nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 7137 or polypeptide SEQ ID NO.: 7138, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 7137 or polypeptide SEQ ID NO.: 7138, respectively is increased or generated or if the activity “microsomal beta-keto-reductase” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Saccharomyces cerevisiae nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 7208 or polypeptide SEQ ID NO.: 7209, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 7208 or polypeptide SEQ ID NO.: 7209, respectively is increased or generated or if the activity “Branched-chain amino acid permease” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Saccharomyces cerevisiae nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 7274 or polypeptide SEQ ID NO.: 7275, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 7274 or polypeptide SEQ ID NO.: 7275, respectively is increased or generated or if the activity “ubiquinone biosynthesis monooxygenase” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Saccharomyces cerevisiae nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 7489 or polypeptide SEQ ID NO.: 7490, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 7489 or polypeptide SEQ ID NO.: 7490, respectively is increased or generated or if the activity “YHR213W-protein” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Saccharomyces cerevisiae nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 8239 or polypeptide SEQ ID NO.: 8240, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 8239 or polypeptide SEQ ID NO.: 8240, respectively is increased or generated or if the activity “60S ribosomal protein” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Saccharomyces cerevisiae nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 8397 or polypeptide SEQ ID NO.: 8398, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 8397 or polypeptide SEQ ID NO.: 8398, respectively is increased or generated or if the activity “Autophagy-related protein” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Saccharomyces cerevisiae nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 8227 or polypeptide SEQ ID NO.: 8228, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 8227 or polypeptide SEQ ID NO.: 8228, respectively is increased or generated or if the activity “cytochrome c oxidase subunit VIII” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
Accordingly, in one embodiment, in case the activity of the Saccharomyces cerevisiae nucleic acid molecule or a polypeptide comprising the nucleic acid SEQ ID NO.: 8423 or polypeptide SEQ ID NO.: 8424, respectively is increased or generated, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 8423 or polypeptide SEQ ID NO.: 8424, respectively is increased or generated or if the activity “Branched-chain amino acid permease” is increased or generated in an plant cell, plant or part thereof an increased GABA content as compared to a corresponding non-transformed wild type plant cell, a plant or a part thereof is conferred.
In a further embodiment, an increased tolerance to abiotic environmental stress and/or increased yield related trait, in particular increased low temperature tolerance, compared to a corresponding non-modified, e.g. a non-transformed, wild type plant is conferred if the activity of a polypeptide comprising the polypeptide shown in SEQ ID NO. 2493, or encoded by a nucleic acid molecule comprising the nucleic acid molecule shown in SEQ ID NO. 2492, or a homolog of said nucleic acid molecule or polypeptide, is increased or generated. For example, the activity of a corresponding nucleic acid molecule or a polypeptide derived from Arabidopsis thaliana is increased or generated, preferably comprising the nucleic acid molecule shown in SEQ ID NO. 2492 or polypeptide shown in SEQ ID NO. 2493, respectively, or a homolog thereof. E.g. an increased tolerance to abiotic environmental stress, in particular increased low temperature tolerance, compared to a corresponding non-modified, e.g. a non-transformed, wild type plant is conferred if the activity “At5g16650-protein” or if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, depicted in table I, II or IV, column 7, respective same line as SEQ ID NO.: 2492 or SEQ ID NO.: 2493, respectively, is increased or generated in a plant or part thereof. Preferably, the increase occurs cytoplasmic.
Particularly, an increase of yield from 1.05-fold to 1.075-fold, for example plus at least 100% thereof, under conditions of low temperature is conferred compared to a corresponding non-modified, e.g. non-transformed, wild type plant.
In a further embodiment, an increased tolerance to abiotic environmental stress and/or increased yield related trait, in particular increased low temperature tolerance, compared to a corresponding non-modified, e.g. a non-transformed, wild type plant is conferred if the activity of a polypeptide comprising the polypeptide shown in SEQ ID NO. 7138, or encoded by a nucleic acid molecule comprising the nucleic acid molecule shown in SEQ ID NO. 7137, or a homolog of said nucleic acid molecule or polypeptide, is increased or generated. For example, the activity of a corresponding nucleic acid molecule or a polypeptide derived from Saccharomyces cerevisiae is increased or generated, preferably comprising the nucleic acid molecule shown in SEQ ID NO. 7137 or polypeptide shown in SEQ ID NO. 7138, respectively, or a homolog thereof. E.g. an increased tolerance to abiotic environmental stress, in particular increased low temperature tolerance, compared to a corresponding non-modified, e.g. a non-transformed, wild type plant is conferred if the activity “microsomal beta-keto-reductase” or if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, depicted in table I, II or IV, column 7, respective same line as SEQ ID NO.: 7137 or SEQ ID NO.: 7138, respectively, is increased or generated in a plant or part thereof. Preferably, the increase occurs cytoplasmic.
Particularly, an increase of yield from 1.05-fold to 1.068-fold, for example plus at least 100% thereof, under conditions of low temperature is conferred compared to a corresponding non-modified, e.g. non-transformed, wild type plant.
In a further embodiment, an increased tolerance to abiotic environmental stress and/or increased yield related trait, in particular increased low temperature tolerance, compared to a corresponding non-modified, e.g. a non-transformed, wild type plant is conferred if the activity of a polypeptide comprising the polypeptide shown in SEQ ID NO. 7209, or encoded by a nucleic acid molecule comprising the nucleic acid molecule shown in SEQ ID NO. 7208, or a homolog of said nucleic acid molecule or polypeptide, is increased or generated. For example, the activity of a corresponding nucleic acid molecule or a polypeptide derived from Saccharomyces cerevisiae is increased or generated, preferably comprising the nucleic acid molecule shown in SEQ ID NO. 7208 or polypeptide shown in SEQ ID NO. 7209, respectively, or a homolog thereof. E.g. an increased tolerance to abiotic environmental stress, in particular increased low temperature tolerance, compared to a corresponding non-modified, e.g. a non-transformed, wild type plant is conferred if the activity “Branched-chain amino acid permease” or if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, depicted in table I, II or IV, column 7, respective same line as SEQ ID NO.: 7208 or SEQ ID NO.: 7209, respectively, is increased or generated in a plant or part thereof. Preferably, the increase occurs cytoplasmic.
Particularly, an increase of yield from 1.05-fold to 1.206-fold, for example plus at least 100% thereof, under conditions of low temperature is conferred compared to a corresponding non-modified, e.g. non-transformed, wild type plant.
In a further embodiment, an increased tolerance to abiotic environmental stress and/or increased yield related trait, in particular increased low temperature tolerance, compared to a corresponding non-modified, e.g. a non-transformed, wild type plant is conferred if the activity of a polypeptide comprising the polypeptide shown in SEQ ID NO. 8240, or encoded by a nucleic acid molecule comprising the nucleic acid molecule shown in SEQ ID NO. 8239, or a homolog of said nucleic acid molecule or polypeptide, is increased or generated. For example, the activity of a corresponding nucleic acid molecule or a polypeptide derived from Saccharomyces cerevisiae is increased or generated, preferably comprising the nucleic acid molecule shown in SEQ ID NO. 8239 or polypeptide shown in SEQ ID NO. 8240, respectively, or a homolog thereof. E.g. an increased tolerance to abiotic environmental stress, in particular increased low temperature tolerance, compared to a corresponding non-modified, e.g. a non-transformed, wild type plant is conferred if the activity “60S ribosomal protein” or if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, depicted in table I, II or IV, column 7, respective same line as SEQ ID NO.: 8239 or SEQ ID NO.: 8240, respectively, is increased or generated in a plant or part thereof. Preferably, the increase occurs cytoplasmic.
Particularly, an increase of yield from 1.05-fold to 1.230-fold, for example plus at least 100% thereof, under conditions of low temperature is conferred compared to a corresponding non-modified, e.g. non-transformed, wild type plant.
In a further embodiment, an increased tolerance to abiotic environmental stress and/or increased yield related trait, in particular increased low temperature tolerance, compared to a corresponding non-modified, e.g. a non-transformed, wild type plant is conferred if the activity of a polypeptide comprising the polypeptide shown in SEQ ID NO. 8424, or encoded by a nucleic acid molecule comprising the nucleic acid molecule shown in SEQ ID NO. 8423, or a homolog of said nucleic acid molecule or polypeptide, is increased or generated. For example, the activity of a corresponding nucleic acid molecule or a polypeptide derived from Saccharomyces cerevisiae is increased or generated, preferably comprising the nucleic acid molecule shown in SEQ ID NO. 8423 or polypeptide shown in SEQ ID NO. 8424, respectively, or a homolog thereof. E.g. an increased tolerance to abiotic environmental stress, in particular increased low temperature tolerance, compared to a corresponding non-modified, e.g. a non-transformed, wild type plant is conferred if the activity “Branched-chain amino acid permease” or if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, depicted in table I, II or IV, column 7, respective same line as SEQ ID NO.: 8423 or SEQ ID NO.: 8424, respectively, is increased or generated in a plant or part thereof. Preferably, the increase occurs cytoplasmic.
Particularly, an increase of yield from 1.05-fold to 1.206-fold, for example plus at least 100% thereof, under conditions of low temperature is conferred compared to a corresponding non-modified, e.g. non-transformed, wild type plant.
In a further embodiment, an increased tolerance to abiotic environmental stress and/or increased yield related trait, in particular increased intrinsic yield, compared to a corresponding non-modified, e.g. a non-transformed, wild type plant is conferred if the activity of a polypeptide comprising the polypeptide shown in SEQ ID NO. 7209, or encoded by a nucleic acid molecule comprising the nucleic acid molecule shown in SEQ ID NO. 7208, or a homolog of said nucleic acid molecule or polypeptide, is increased or generated. For example, the activity of a corresponding nucleic acid molecule or a polypeptide derived from Saccharomyces cerevisiae is increased or generated, preferably comprising the nucleic acid molecule shown in SEQ ID NO. 7208 or polypeptide shown in SEQ ID NO. 7209, respectively, or a homolog thereof. E.g. an increased tolerance to abiotic environmental stress and/or increased yield related trait, in particular increased intrinsic yield, compared to a corresponding non-modified, e.g. a non-transformed, wild type plant is conferred if the activity “Branched-chain amino acid permease” or if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, depicted in table I, II or IV, column 7, respective same line as SEQ ID NO.: 7208 or SEQ ID NO.: 7209, respectively, is increased or generated in a plant or part thereof. Preferably, the increase occurs cytoplasmic. Particularly, an increase of yield from 1.05-fold to 1.522-fold, for example plus at least 100% thereof, under standard conditions, e.g. in the absence of nutrient deficiency and/or stress conditions is conferred compared to a corresponding control, e.g. an non-modified, e.g. non-transformed, wild type plant.
E.g. an increased tolerance to abiotic environmental stress and/or increased yield related trait, in particular increased intrinsic yield, compared to a corresponding non-modified, e.g. a non-transformed, wild type plant is conferred if the activity “Branched-chain amino acid permease” or if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, depicted in table I, II or IV, column 7, respective same line as SEQ ID NO.: 7208 or SEQ ID NO.: 7209, respectively, is increased or generated in a plant or part thereof. Preferably, the increase occurs plastidic. Particularly, an increase of yield from 1.05-fold to 1.232-fold, for example plus at least 100% thereof, under standard conditions, e.g. in the absence of nutrient deficiency and/or stress conditions is conferred compared to a corresponding control, e.g. an non-modified, e.g. non-transformed, wild type plant.
In a further embodiment, an increased tolerance to abiotic environmental stress and/or increased yield related trait, in particular increased intrinsic yield, compared to a corresponding non-modified, e.g. a non-transformed, wild type plant is conferred if the activity of a polypeptide comprising the polypeptide shown in SEQ ID NO. 8240, or encoded by a nucleic acid molecule comprising the nucleic acid molecule shown in SEQ ID NO. 8239, or a homolog of said nucleic acid molecule or polypeptide, is increased or generated. For example, the activity of a corresponding nucleic acid molecule or a polypeptide derived from Saccharomyces cerevisiae is increased or generated, preferably comprising the nucleic acid molecule shown in SEQ ID NO. 8239 or polypeptide shown in SEQ ID NO. 8240, respectively, or a homolog thereof. E.g. an increased tolerance to abiotic environmental stress and/or increased yield related trait, in particular increased intrinsic yield, compared to a corresponding non-modified, e.g. a non-transformed, wild type plant is conferred if the activity “60S ribosomal protein” or if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, depicted in table I, II or IV, column 7, respective same line as SEQ ID NO.: 8239 or SEQ ID NO.: 8240, respectively, is increased or generated in a plant or part thereof. Preferably, the increase occurs cytoplasmic. Particularly, an increase of yield from 1.05-fold to 1.546-fold, for example plus at least 100% thereof, under standard conditions, e.g. in the absence of nutrient deficiency and/or stress conditions is conferred compared to a corresponding control, e.g. an non-modified, e.g. non-transformed, wild type plant.
In a further embodiment, an increased tolerance to abiotic environmental stress and/or increased yield related trait, in particular increased intrinsic yield, compared to a corresponding non-modified, e.g. a non-transformed, wild type plant is conferred if the activity of a polypeptide comprising the polypeptide shown in SEQ ID NO. 8398, or encoded by a nucleic acid molecule comprising the nucleic acid molecule shown in SEQ ID NO. 8397, or a homolog of said nucleic acid molecule or polypeptide, is increased or generated. For example, the activity of a corresponding nucleic acid molecule or a polypeptide derived from Saccharomyces cerevisiae is increased or generated, preferably comprising the nucleic acid molecule shown in SEQ ID NO. 8397 or polypeptide shown in SEQ ID NO. 8398, respectively, or a homolog thereof. E.g. an increased tolerance to abiotic environmental stress and/or increased yield related trait, in particular increased intrinsic yield, compared to a corresponding non-modified, e.g. a non-transformed, wild type plant is conferred if the activity “Autophagy-related protein” or if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, depicted in table I, II or IV, column 7, respective same line as SEQ ID NO.: 8397 or SEQ ID NO.: 8398, respectively, is increased or generated in a plant or part thereof. Preferably, the increase occurs cytoplasmic. Particularly, an increase of yield from 1.05-fold to 1.399-fold, for example plus at least 100% thereof, under standard conditions, e.g. in the absence of nutrient deficiency and/or stress conditions is conferred compared to a corresponding control, e.g. an non-modified, e.g. non-transformed, wild type plant.
In a further embodiment, an increased tolerance to abiotic environmental stress and/or increased yield related trait, in particular increased intrinsic yield, compared to a corresponding non-modified, e.g. a non-transformed, wild type plant is conferred if the activity of a polypeptide comprising the polypeptide shown in SEQ ID NO. 8424, or encoded by a nucleic acid molecule comprising the nucleic acid molecule shown in SEQ ID NO. 8423, or a homolog of said nucleic acid molecule or polypeptide, is increased or generated. For example, the activity of a corresponding nucleic acid molecule or a polypeptide derived from Saccharomyces cerevisiae is increased or generated, preferably comprising the nucleic acid molecule shown in SEQ ID NO. 8423 or polypeptide shown in SEQ ID NO. 8424, respectively, or a homolog thereof. E.g. an increased tolerance to abiotic environmental stress and/or increased yield related trait, in particular increased intrinsic yield, compared to a corresponding non-modified, e.g. a non-transformed, wild type plant is conferred if the activity “Branched-chain amino acid permease” or if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, depicted in table I, II or IV, column 7, respective same line as SEQ ID NO.: 8423 or SEQ ID NO.: 8424, respectively, is increased or generated in a plant or part thereof. Preferably, the increase occurs cytoplasmic. Particularly, an increase of yield from 1.05-fold to 1.522-fold, for example plus at least 100% thereof, under standard conditions, e.g. in the absence of nutrient deficiency and/or stress conditions is conferred compared to a corresponding control, e.g. an non-modified, e.g. non-transformed, wild type plant. E.g. an increased tolerance to abiotic environmental stress and/or increased yield related trait, in particular increased intrinsic yield, compared to a corresponding non-modified, e.g. a non-transformed, wild type plant is conferred if the activity “Branched-chain amino acid permease” or if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, depicted in table I, II or IV, column 7, respective same line as SEQ ID NO.: 8423 or SEQ ID NO.: 8424, respectively, is increased or generated in a plant or part thereof. Preferably, the increase occurs plastidic. Particularly, an increase of yield from 1.05-fold to 1.232-fold, for example plus at least 100% thereof, under standard conditions, e.g. in the absence of nutrient deficiency and/or stress conditions is conferred compared to a corresponding control, e.g. an non-modified, e.g. non-transformed, wild type plant.
In a further embodiment, an increased tolerance to abiotic environmental stress and/or increased yield related trait, in particular increased drought resistance, preferably cycling drought, compared to a corresponding non-modified, e.g. a non-transformed, wild type plant is conferred if the activity of a polypeptide comprising the polypeptide shown in SEQ ID NO. 7209, or encoded by a nucleic acid molecule comprising the nucleic acid molecule shown in SEQ ID NO. 7208, or a homolog of said nucleic acid molecule or polypeptide, is increased or generated. For example, the activity of a corresponding nucleic acid molecule or a polypeptide derived from Saccharomyces cerevisiae is increased or generated, preferably comprising the nucleic acid molecule shown in SEQ ID NO. 7208 or polypeptide shown in SEQ ID NO. 7209, respectively, or a homolog thereof. E.g. an increased tolerance to abiotic environmental stress and/or increased yield related trait, in particular increased resistance to drought, preferably cycling drought, compared to a corresponding non-modified, e.g. a non-transformed, wild type plant is conferred if the activity “Branched-chain amino acid permease” or if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, depicted in table I, II or IV, column 7, respective same line as SEQ ID NO.: 7208 or SEQ ID NO.: 7209, respectively, is increased or generated in a plant or part thereof. Preferably, the increase occurs plastidic. Particularly, an increase of yield from 1.05-fold to 1.351-fold, for example plus at least 100% thereof, is conferred compared to a corresponding control, e.g. an non-modified, e.g. non-transformed, wild type plant.
In a further embodiment, an increased tolerance to abiotic environmental stress and/or increased yield related trait, in particular increased drought resistance, preferably cycling drought, compared to a corresponding non-modified, e.g. a non-transformed, wild type plant is conferred if the activity of a polypeptide comprising the polypeptide shown in SEQ ID NO. 8424, or encoded by a nucleic acid molecule comprising the nucleic acid molecule shown in SEQ ID NO. 8423, or a homolog of said nucleic acid molecule or polypeptide, is increased or generated. For example, the activity of a corresponding nucleic acid molecule or a polypeptide derived from Saccharomyces cerevisiae is increased or generated, preferably comprising the nucleic acid molecule shown in SEQ ID NO. 8423 or polypeptide shown in SEQ ID NO. 8424, respectively, or a homolog thereof. E.g. an increased tolerance to abiotic environmental stress and/or increased yield related trait, in particular increased resistance to drought, preferably cycling drought, compared to a corresponding non-modified, e.g. a non-transformed, wild type plant is conferred if the activity “Branched-chain amino acid permease” or if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, depicted in table I, II or IV, column 7, respective same line as SEQ ID NO.: 8423 or SEQ ID NO.: 8424, respectively, is increased or generated in a plant or part thereof. Preferably, the increase occurs plastidic.
Particularly, an increase of yield from 1.05-fold to 1.351-fold, for example plus at least 100% thereof, is conferred compared to a corresponding control, e.g. an non-modified, e.g. non-transformed, wild type plant.
For the purposes of the invention, as a rule the plural is intended to encompass the singular and vice versa.
Unless otherwise specified, the terms “polynucleotides”, “nucleic acid” and “nucleic acid molecule” are interchangeably in the present context. Unless otherwise specified, the terms “peptide”, “polypeptide” and “protein” are interchangeably in the present context. The term “sequence” may relate to polynucleotides, nucleic acids, nucleic acid molecules, peptides, polypeptides and proteins, depending on the context in which the term “sequence” is used. The terms “gene(s)”, “polynucleotide”, “nucleic acid sequence”, “nucleotide sequence”, or “nucleic acid molecule(s)” as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. The terms refer only to the primary structure of the molecule.
Thus, the terms “gene(s)”, “polynucleotide”, “nucleic acid sequence”, “nucleotide sequence”, or “nucleic acid molecule(s)” as used herein include double- and single-stranded DNA and/or RNA. They also include known types of modifications, for example, methylation, “caps”, substitutions of one or more of the naturally occurring nucleotides with an analog. Preferably, the DNA or RNA sequence comprises a coding sequence encoding the herein defined polypeptide.
A “coding sequence” is a nucleotide sequence, which is transcribed into an RNA, e.g. a regulatory RNA, such as a miRNA, a ta-siRNA, cosuppression molecule, an RNAi, a ribozyme, etc. or into a mRNA which is translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5′-terminus and a translation stop codon at the 3′-terminus. A coding sequence can include, but is not limited to mRNA, cDNA, recombinant nucleotide sequences or genomic DNA, while introns may be present as well under certain circumstances.
As used in the present context a nucleic acid molecule may also encompass the untranslated sequence located at the 3′ and at the 5′ end of the coding gene region, for example at least 500, preferably 200, especially preferably 100, nucleotides of the sequence upstream of the 5′ end of the coding region and at least 100, preferably 50, especially preferably 20, nucleotides of the sequence downstream of the 3′ end of the coding gene region. In the event for example the antisense, RNAi, snRNA, dsRNA, siRNA, miRNA, ta-siRNA, cosuppression molecule, ribozyme etc. technology is used coding regions as well as the 5′- and/or 3′-regions can advantageously be used.
However, it is often advantageous only to choose the coding region for cloning and expression purposes.
“Polypeptide” refers to a polymer of amino acid (amino acid sequence) and does not refer to a specific length of the molecule. Thus, peptides and oligopeptides are included within the definition of polypeptide. This term does also refer to or include posttranslational modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
The term “Table I” used in this specification is to be taken to specify the content of Table I A and Table I B. The term “Table II” used in this specification is to be taken to specify the content of Table II A and Table II B. The term “Table I A” used in this specification is to be taken to specify the content of Table I A. The term “Table I B” used in this specification is to be taken to specify the content of Table I B. The term “Table II A” used in this specification is to be taken to specify the content of Table II A. The term “Table II B” used in this specification is to be taken to specify the content of Table II B. In one preferred embodiment, the term “Table I” means Table I B. In one preferred embodiment, the term “Table II” means Table II B.
The terms “comprise” or “comprising” and grammatical variations thereof when used in this specification are to be taken to specify the presence of stated features, integers, steps or components or groups thereof, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.
In accordance with the invention, a protein or polypeptide has the “activity of an protein as shown in table II, column 3” if its de novo activity, or its increased expression directly or indirectly leads to and confers an increased GABA content as compared to a corresponding non-transformed wild type and the protein has the above mentioned activities of a protein as shown in table II, column 3. Throughout the specification the activity or preferably the biological activity of such a protein or polypeptide or an nucleic acid molecule or sequence encoding such protein or polypeptide is identical or similar if it still has the biological or enzymatic activity of a protein as shown in table II, column 3, or which has at least 10% of the original enzymatic activity, preferably 20%, particularly preferably 30%, most particularly preferably 40% in comparison to a protein as shown in table II, column 3 or 5.
The terms “increased”, “rised”, “extended”, “enhanced”, “improved” or “amplified” relate to a corresponding change of a property in a plant, an organism, a part of an organism such as a tissue, seed, root, leave, flower etc. or in a cell and are interchangeable. Preferably, the overall activity in the volume is increased or enhanced in cases if the increase or enhancement is related to the increase or enhancement of an activity of a gene product, independent whether the amount of gene product or the specific activity of the gene product or both is increased or enhanced or whether the amount, stability or translation efficacy of the nucleic acid sequence or gene encoding for the gene product is increased or enhanced.
The terms “increase” relate to a corresponding change of a property an organism or in a part of a plant, an organism, such as a tissue, seed, root, leave, flower etc. or in a cell. Preferably, the overall activity in the volume is increased in cases the increase relates to the increase of an activity of a gene product, independent whether the amount of gene product or the specific activity of the gene product or both is increased or generated or whether the amount, stability or translation efficacy of the nucleic acid sequence or gene encoding for the gene product is increased.
Under “change of a property” it is understood that the activity, expression level or amount of a gene product or the metabolite content is changed in a specific volume relative to a corresponding volume of a control, reference or wild type, including the de novo creation of the activity or expression.
The terms “increase” include the change of said property in only parts of the subject of the present invention, for example, the modification can be found in compartment of a cell, like a organelle, or in a part of a plant, like tissue, seed, root, leave, flower etc. but is not detectable if the overall subject, i.e. complete cell or plant, is tested.
Accordingly, the term “increase” means that the specific activity of an enzyme as well as the amount of a compound or metabolite, e.g. of a polypeptide, a nucleic acid molecule of the invention or an encoding mRNA or DNA, can be increased in a volume.
The terms “wild type”, “control” or “reference” are exchangeable and can be a cell or a part of organisms such as an organelle like a chloroplast or a tissue, or an organism, in particular a plant, which was not modified or treated according to the herein described process according to the invention. Accordingly, the cell or a part of organisms such as an organelle like a chloroplast or a tissue, or an organism, in particular a plant used as wild type, control or reference corresponds to the cell, organism, plant or part thereof as much as possible and is in any other property but in the result of the process of the invention as identical to the subject matter of the invention as possible. Thus, the wild type, control or reference is treated identically or as identical as possible, saying that only conditions or properties might be different which do not influence the quality of the tested property.
Preferably, any comparison is carried out under analogous conditions. The term “analogous conditions” means that all conditions such as, for example, culture or growing conditions, water content of the soil, temperature, humidity or surrounding air or soil, assay conditions (such as buffer composition, temperature, substrates, pathogen strain, concentrations and the like) are kept identical between the experiments to be compared.
The “reference”, “control”, or “wild type” is preferably a subject, e.g. an organelle, a cell, a tissue, an organism, in particular a plant, which was not modified or treated according to the herein described process of the invention and is in any other property as similar to the subject matter of the invention as possible. The reference, control or wild type is in its genome, transcriptome, proteome or metabolome as similar as possible to the subject of the present invention. Preferably, the term “reference-” “control-” or “wild type-”-organelle, -cell, -tissue or -organism, in particular plant, relates to an organelle, cell, tissue or organism, in particular plant, which is nearly genetically identical to the organelle, cell, tissue or organism, in particular plant, of the present invention or a part thereof preferably 95%, more preferred are 98%, even more preferred are 99.00%, in particular 99.10%, 99.30%, 99.50%, 99.70%, 99.90%, 99.99%, 99.999% or more. Most preferable the “reference”, “control”, or “wild type” is a subject, e.g. an organelle, a cell, a tissue, an organism, which is genetically identical to the organism, cell or organelle used according to the process of the invention except that the responsible or activity conferring nucleic acid molecules or the gene product encoded by them are amended, manipulated, exchanged or introduced according to the inventive process.
In case, a control, reference or wild type differing from the subject of the present invention only by not being subject of the process of the invention can not be provided, a control, reference or wild type can be an organism in which the cause for the modulation of an activity conferring the increased GABA content as compared to a corresponding non-transformed wild type or expression of the nucleic acid molecule of the invention as described herein has been switched back or off, e.g. by knocking out the expression of responsible gene product, e.g. by antisense inhibition, by inactivation of an activator or agonist, by activation of an inhibitor or antagonist, by inhibition through adding inhibitory antibodies, by adding active compounds as e.g. hormones, by introducing negative dominant mutants, etc. A gene production can for example be knocked out by introducing inactivating point mutations, which lead to an enzymatic activity inhibition or a destabilization or an inhibition of the ability to bind to cofactors etc.
Accordingly, preferred reference subject is the starting subject of the present process of the invention. Preferably, the reference and the subject matter of the invention are compared after standardization and normalization, e.g. to the amount of total RNA, DNA, or Protein or activity or expression of reference genes, like housekeeping genes, such as ubiquitin, actin or ribosomal proteins.
The increase or modulation according to this invention can be constitutive, e.g. due to a stable permanent transgenic expression or to a stable mutation in the corresponding endogenous gene encoding the nucleic acid molecule of the invention or to a modulation of the expression or of the behavior of a gene conferring the expression of the polypeptide of the invention, or transient, e.g. due to an transient transformation or temporary addition of a modulator such as a agonist or antagonist or inducible, e.g. after transformation with a inducible construct carrying the nucleic acid molecule of the invention under control of a inducible promoter and adding the inducer, e.g. tetracycline or as described herein below.
The increase in activity of the polypeptide amounts in a cell, a tissue, a organelle, an organ or an organism or a part thereof preferably to at least 5%, preferably to at least 20% or at to least 50%, especially preferably to at least 70%, 80%, 90% or more, very especially preferably are to at least 200%, 300% or 400%, most preferably are to at least 500% or more in comparison to the control, reference or wild type. In one embodiment the term increase means the increase in amount in relation to the weight of the organism or part thereof (w/w).
In one embodiments the increase in activity of the polypeptide amounts in an organelle such as a plastid.
The specific activity of a polypeptide encoded by a nucleic acid molecule of the present invention or of the polypeptide of the present invention can be tested as described in the examples. In particular, the expression of a protein in question in a cell, e.g. a plant cell in comparison to a control is an easy test and can be performed as described in the state of the art.
The term “increase” includes, that a compound or an activity is introduced into a cell or a subcellular compartment or organelle de novo or that the compound or the activity has not been detectable before, in other words it is “generated”.
Accordingly, in the following, the term “increasing” also comprises the term “generating” or “stimulating”. The increased activity manifests itself in an increased GABA content as compared to a corresponding non-transformed wild type plant cell, plant or part thereof.
The sequence of Ymr052w from Saccharomyces cerevisiae, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as Factor arrest protein.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “Factor arrest protein” from Saccharomyces cerevisiae or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “Factor arrest protein”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of At1g43850 from Arabidopsis thaliana, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as transcriptional regulator.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “transcriptional regulator” from Arabidopsis thaliana or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “transcriptional regulator”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of At2g28890 from Arabidopsis thaliana, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as protein phosphatase.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “protein phosphatase” from Arabidopsis thaliana or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “protein phosphatase”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of At3g04050 from Arabidopsis thaliana, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as pyruvate kinase.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “pyruvate kinase” from Arabidopsis thaliana or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “pyruvate kinase”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of At3g08710 from Arabidopsis thaliana, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as thioredoxin family protein.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “thioredoxin family protein” from Arabidopsis thaliana or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “thioredoxin family protein”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of At3g11650 from Arabidopsis thaliana, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as harpin-induced family protein.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “harpin-induced family protein” from Arabidopsis thaliana or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “harpin-induced family protein”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of At3g27540 from Arabidopsis thaliana, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as glycosyl transferase.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “glycosyl transferase” from Arabidopsis thaliana or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “glycosyl transferase”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of At3g61830 from Arabidopsis thaliana, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as auxin response factor.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “auxin response factor” from Arabidopsis thaliana or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “auxin response factor”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of At4g32480 from Arabidopsis thaliana, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as At4g32480-protein.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “At4g32480-protein” from Arabidopsis thaliana or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “At4g32480-protein”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of At4g35310 from Arabidopsis thaliana, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as calcium-dependent protein kinase.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “calcium-dependent protein kinase” from Arabidopsis thaliana or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “calcium-dependent protein kinase”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of At5g16650 from Arabidopsis thaliana, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as At5g16650-protein.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “At5g16650-protein” from Arabidopsis thaliana or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “At5g16650-protein”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of AvinDRAFT—2344 from Azotobacter vinelandii, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as elongation factor Tu.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “elongation factor Tu” from Azotobacter vinelandii or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “elongation factor Tu”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of AvinDRAFT—2521 from Azotobacter vinelandii, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as ABC transporter permease protein.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “ABC transporter permease protein” from Azotobacter vinelandii or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “ABC transporter permease protein”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of AvinDRAFT—5103 from Azotobacter vinelandii, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as hydrolase.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “hydrolase” from Azotobacter vinelandii or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “hydrolase”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of AvinDRAFT—5292 from Azotobacter vinelandii, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as fumarylacetoacetate hydrolase.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “fumarylacetoacetate hydrolase” from Azotobacter vinelandii or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “fumarylacetoacetate hydrolase”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of B0124 from Escherichia coli, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as glucose dehydrogenase.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “glucose dehydrogenase” from Escherichia coli or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “glucose dehydrogenase”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of B0161 from Escherichia coli, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as serine protease.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “serine protease” from Escherichia coli or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “serine protease”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of B0449 from Escherichia coli, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as ATP-binding protein.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “ATP-binding protein” from Escherichia coli or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “ATP-binding protein”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of B0593 from Escherichia coli, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as isochorismate synthase.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “isochorismate synthase” from Escherichia coli or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “isochorismate synthase”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of B0898 from Escherichia coli, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as MFS-type transporter protein.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “MFS-type transporter protein” from Escherichia coli or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “MFS-type transporter protein”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of B1003 from Escherichia coli, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as b1003-protein.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “b1003-protein” from Escherichia coli or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “b1003-protein”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of B1522 from Escherichia coli, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as b1522-protein.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “b1522-protein” from Escherichia coli or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “b1522-protein”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of B2739 from Escherichia coli, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as b2739-protein.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “b2739-protein” from Escherichia coli or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “b2739-protein”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of B3646 from Escherichia coli, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as b3646-protein.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “b3646-protein” from Escherichia coli or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “b3646-protein”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of B4029 from Escherichia coli, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as B4029-protein.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “64029-protein” from Escherichia coli or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “B4029-protein”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of B4256 from Escherichia coli, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as acetyltransferase.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “acetyltransferase” from Escherichia coli or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “acetyltransferase”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of C_PP034008079R from Physcomitrella patens, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as acyl-carrier protein.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “acyl-carrier protein” from Physcomitrella patens or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “acyl-carrier protein”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of Slr0739 from Synechocystis sp., e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as geranylgeranyl pyrophosphate synthase.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “geranylgeranyl pyrophosphate synthase” from Synechocystis sp. or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “geranylgeranyl pyrophosphate synthase”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of TTC0019 from Thermus thermophilus, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as Sec-independent protein translocase subunit.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “Sec-independent protein translocase subunit” from Thermus thermophilus or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “Sec-independent protein translocase subunit”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of TTC1550 from Thermus thermophilus, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as homocitrate synthase.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “homocitrate synthase” from Thermus thermophilus or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “homocitrate synthase”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of Yjr153w from Saccharomyces cerevisiae, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as polygalacturonase.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “polygalacturonase” from Saccharomyces cerevisiae or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “polygalacturonase”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of Ylr043c from Saccharomyces cerevisiae, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as thioredoxin.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “thioredoxin” from Saccharomyces cerevisiae or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “thioredoxin”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of 51340801_CANOLA from Brassica napus, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as pyruvate kinase.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “pyruvate kinase” from Brassica napus or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “pyruvate kinase”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of Ybr159w from Saccharomyces cerevisiae, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as microsomal beta-keto-reductase.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “microsomal beta-keto-reductase” from Saccharomyces cerevisiae or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “microsomal beta-keto-reductase”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of YDR046C from Saccharomyces cerevisiae, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as Branched-chain amino acid permease.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “Branched-chain amino acid permease” from Saccharomyces cerevisiae or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “Branched-chain amino acid permease”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of YGR255C from Saccharomyces cerevisiae, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as ubiquinone biosynthesis monooxygenase.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “ubiquinone biosynthesis monooxygenase” from Saccharomyces cerevisiae or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “ubiquinone biosynthesis monooxygenase”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of YHR213W from Saccharomyces cerevisiae, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as YHR213W-protein.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “YHR213W-protein” from Saccharomyces cerevisiae or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “YHR213W-protein”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of YPL249C-A from Saccharomyces cerevisiae, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as 60S ribosomal protein.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “60S ribosomal protein” from Saccharomyces cerevisiae or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “60S ribosomal protein”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of YPR185W from Saccharomyces cerevisiae, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as Autophagy-related protein.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “Autophagy-related protein” from Saccharomyces cerevisiae or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “Autophagy-related protein”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of Ylr395c from Saccharomyces cerevisiae, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as cytochrome c oxidase subunit VIII.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “cytochrome c oxidase subunit VIII” from Saccharomyces cerevisiae or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a “cytochrome c oxidase subunit VIII”, preferably it is the molecule of section (a) or (b) of this paragraph.
The sequence of YDR046C—2 from Saccharomyces cerevisiae, e.g. as shown in column 5 of Table I, [sequences from Saccharomyces cerevisiae has been published in Goffeau et al., Science 274 (5287), 546-547, 1996, sequences from Escherichia coli has been published in Blattner et al., Science 277 (5331), 1453-1474 (1997), and its activity is published described as Branched-chain amino acid permease.
Accordingly, in one embodiment, the process of the present invention comprises increasing or generating the activity of a gene product with the activity of a “Branched-chain amino acid permease” from Saccharomyces cerevisiae or its functional equivalent or its homolog, e.g. the increase of
as mentioned herein, for the an increased GABA content as compared to a corresponding non-transformed wild type as mentioned.
Accordingly, in one embodiment, the molecule which activity is to be increased in the process of the invention is the gene product with an activity of described as a
“Branched-chain amino acid permease”, preferably it is the molecule of section (a) or (b) of this paragraph.
It was further observed that increasing or generating the activity of a gene shown in Table XII, e.g. a nucleic acid molecule derived from the nucleic acid molecule shown in Table XII in A. thaliana conferred increased stress tolerance, e.g. increased low temperature tolerance, compared to the wild type control. Thus, in one embodiment, a nucleic acid molecule indicated in Table XII or its homolog as indicated in Table I or the expression product is used in the method of the present invention to increase stress tolerance, e.g. increase low temperature, of a plant compared to the wild type control.
It was further observed that increasing or generating the activity of a gene shown in Table XI, e.g. a nucleic acid molecule derived from the nucleic acid molecule shown in Table XI in A. thaliana conferred increased stress tolerance, e.g. increased cycling drought tolerance, compared to the wild type control. Thus, in one embodiment, a nucleic acid molecule indicated in Table XI or its homolog as indicated in Table I or the expression product is used in the method of the present invention to increase stress tolerance, e.g. increase cycling drought tolerance, of a plant compared to the wild type control.
It was further observed that increasing or generating the activity of a gene shown in Table X, e.g. a nucleic acid molecule derived from the nucleic acid molecule shown in Table X in A. thaliana conferred increase in intrinsic yield, e.g. increased biomass under standard conditions, e.g. increased biomass under non-deficiency or non-stress conditions, compared to the wild type control. Thus, in one embodiment, a nucleic acid molecule indicated in Table X or its homolog as indicated in Table I or the expression product is used in the method of the present invention to increase intrinsic yield, e.g. to increase yield under standard conditions, e.g. increase biomass under non-deficiency or non-stress conditions, of the plant compared to the wild type control.
Surprisingly, it was observed that a increasing or generating of at least one gene conferring an activity selected from the group consisting of: 60S ribosomal protein, ABC transporter permease protein, acetyltransferase, acyl-carrier protein, At4g32480-protein, At5g16650-protein, ATP-binding protein, Autophagy-related protein, auxin response factor, auxin transcription factor, b1003-protein, b1522-protein, b2739-protein, b3646-protein, B4029-protein, Branched-chain amino acid permease, calcium-dependent protein kinase, cytochrome c oxidase subunit VIII, elongation factor Tu, Factor arrest protein, fumarylacetoacetate hydrolase, geranylgeranyl pyrophosphate synthase, glucose dehydrogenase, glycosyl transferase, harpin-induced family protein, homocitrate synthase, hydrolase, isochorismate synthase, MFS-type transporter protein, microsomal beta-keto-reductase, polygalacturonase, protein phosphatase, pyruvate kinase, Sec-independent protein translocase subunit, serine protease, thioredoxin, thioredoxin family protein, transcriptional regulator, ubiquinone biosynthesis monooxygenase, and YHR213W-protein or of a gene comprising a nucleic acid sequence described in column 5 of Table I in Arabidopsis thaliana conferred an increased GABA content as compared to a corresponding non-transformed wild type.
It was observed that increasing or generating the activity of a gene product with the activity of a “Factor arrest protein” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 42 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 12.35-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “transcriptional regulator” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 654 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 5.47-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “protein phosphatase” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 706 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 12.21-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “pyruvate kinase” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 751 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 26.89-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “thioredoxin family protein” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 1156 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 3.64-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “harpin-induced family protein” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 1510 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 3.21-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “glycosyl transferase” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 1598 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 4.27-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “auxin response factor” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 1670 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 16.46-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “At4g32480-protein” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 1874 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 7.44-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “calcium-dependent protein kinase” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 1936 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 5.40-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “At5g16650-protein” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 2492 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 3.07-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “elongation factor Tu” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 2553 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 6.42-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “ABC transporter permease protein” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 3408 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 1.99-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “hydrolase” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 3564 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 10.13-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “fumarylacetoacetate hydrolase” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 3728 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 14.56-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “glucose dehydrogenase” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 4068 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 4.07-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “serine protease” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 4176 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 16.31-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “ATP-binding protein” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 4364 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 15.36-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “isochorismate synthase” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 4717 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 3.59-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “MFS-type transporter protein” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 4864 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 175.83-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “b1003-protein” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 4903 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 9.49-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “b1522-protein” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 4909 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 22.61-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “b2739-protein” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 4954 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 14.55-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “b3646-protein” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 5121 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 3.02-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “B4029-protein” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 5319 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 77.37-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “acetyltransferase” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 5387 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 3.19-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “acyl-carrier protein” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 5458 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 3.02-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “geranylgeranyl pyrophosphate synthase” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 6041 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 3.55-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “Sec-independent protein translocase subunit” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 6469 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 7.25-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “homocitrate synthase” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 6739 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 2.93-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “polygalacturonase” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 7510 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 6.77-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “thioredoxin” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 7633 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 2.10-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “pyruvate kinase” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 53 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 3.22-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “microsomal beta-keto-reductase” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 7137 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 2.23-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “Branched-chain amino acid permease” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 7208 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 48.39-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “ubiquinone biosynthesis monooxygenase” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 7274 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 31.94-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “YHR213W-protein” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 7489 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 7.79-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “60S ribosomal protein” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 8239 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 6.64-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “Autophagy-related protein” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 8397 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 47.89-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “cytochrome c oxidase subunit VIII” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 8227 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 131.19-fold as shown in the Examples.
It was observed that increasing or generating the activity of a gene product with the activity of a “Branched-chain amino acid permease” encoded by a gene comprising the nucleic acid sequence SEQ ID NO.: 8423 in Arabidopsis thaliana conferred an increased yield, preferably an increase GABA content compared with the wild type control between 1.1% and 48.39-fold as shown in the Examples.
Thus, according to the method of the invention for an increased GABA content in a plant cell, plant or a part thereof compared to a control or wild type can be achieved.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 43, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 42 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 42 or polypeptide SEQ ID NO.: 43, respectively is increased or generated or if the activity “Factor arrest protein” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 655, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 654 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 654 or polypeptide SEQ ID NO.: 655, respectively is increased or generated or if the activity “transcriptional regulator” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 707, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 706 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 706 or polypeptide SEQ ID NO.: 707, respectively is increased or generated or if the activity “protein phosphatase” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 752, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 751 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 751 or polypeptide SEQ ID NO.: 752, respectively is increased or generated or if the activity “pyruvate kinase” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 1157, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 1156 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 1156 or polypeptide SEQ ID NO.: 1157, respectively is increased or generated or if the activity “thioredoxin family protein” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 1511, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 1510 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 1510 or polypeptide SEQ ID NO.: 1511, respectively is increased or generated or if the activity “harpin-induced family protein” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 1599, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 1598 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 1598 or polypeptide SEQ ID NO.: 1599, respectively is increased or generated or if the activity “glycosyl transferase” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 1671 or preferably SEQ ID NO: 8590, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 1670 or preferably SEQ ID NO: 8589 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 1670 or polypeptide SEQ ID NO.: 1671, respectively is increased or generated or if the activity “auxin response factor” or “auxin transcription factor” resp. is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 1875, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 1874 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 1874 or polypeptide SEQ ID NO.: 1875, respectively is increased or generated or if the activity “At4g32480-protein” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 1937, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 1936 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 1936 or polypeptide SEQ ID NO.: 1937, respectively is increased or generated or if the activity “calcium-dependent protein kinase” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 2493, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 2492 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 2492 or polypeptide SEQ ID NO.: 2493, respectively is increased or generated or if the activity “At5g16650-protein” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 2554, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 2553 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 2553 or polypeptide SEQ ID NO.: 2554, respectively is increased or generated or if the activity “elongation factor Tu” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 3409, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 3408 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 3408 or polypeptide SEQ ID NO.: 3409, respectively is increased or generated or if the activity “ABC transporter permease protein” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 3565, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 3564 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 3564 or polypeptide SEQ ID NO.: 3565, respectively is increased or generated or if the activity “hydrolase” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 3729, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 3728 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 3728 or polypeptide SEQ ID NO.: 3729, respectively is increased or generated or if the activity “fumarylacetoacetate hydrolase” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 4069, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 4068 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 4068 or polypeptide SEQ ID NO.: 4069, respectively is increased or generated or if the activity “glucose dehydrogenase” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 4177, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 4176 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 4176 or polypeptide SEQ ID NO.: 4177, respectively is increased or generated or if the activity “serine protease” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 4365, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 4364 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 4364 or polypeptide SEQ ID NO.: 4365, respectively is increased or generated or if the activity “ATP-binding protein” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 4718, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 4717 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 4717 or polypeptide SEQ ID NO.: 4718, respectively is increased or generated or if the activity “isochorismate synthase” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 4865, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 4864 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 4864 or polypeptide SEQ ID NO.: 4865, respectively is increased or generated or if the activity “MFS-type transporter protein” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 4904, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 4903 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 4903 or polypeptide SEQ ID NO.: 4904, respectively is increased or generated or if the activity “b1003-protein” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 4910, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 4909 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 4909 or polypeptide SEQ ID NO.: 4910, respectively is increased or generated or if the activity “b1522-protein” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 4955, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 4954 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 4954 or polypeptide SEQ ID NO.: 4955, respectively is increased or generated or if the activity “b2739-protein” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 5122, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 5121 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 5121 or polypeptide SEQ ID NO.: 5122, respectively is increased or generated or if the activity “b3646-protein” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 5320, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 5319 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 5319 or polypeptide SEQ ID NO.: 5320, respectively is increased or generated or if the activity “B4029-protein” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 5388, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 5387 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 5387 or polypeptide SEQ ID NO.: 5388, respectively is increased or generated or if the activity “acetyltransferase” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 5459, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 5458 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 5458 or polypeptide SEQ ID NO.: 5459, respectively is increased or generated or if the activity “acyl-carrier protein” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 6042, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 6041 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 6041 or polypeptide SEQ ID NO.: 6042, respectively is increased or generated or if the activity “geranylgeranyl pyrophosphate synthase” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 6470, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 6469 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 6469 or polypeptide SEQ ID NO.: 6470, respectively is increased or generated or if the activity “Sec-independent protein translocase subunit” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 6740, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 6739 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 6739 or polypeptide SEQ ID NO.: 6740, respectively is increased or generated or if the activity “homocitrate synthase” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 7511, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 7510 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 7510 or polypeptide SEQ ID NO.: 7511, respectively is increased or generated or if the activity “polygalacturonase” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 7634, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 7633 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 7633 or polypeptide SEQ ID NO.: 7634, respectively is increased or generated or if the activity “thioredoxin” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 54, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 53 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 53 or polypeptide SEQ ID NO.: 54, respectively is increased or generated or if the activity “pyruvate kinase” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 7138, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 7137 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 7137 or polypeptide SEQ ID NO.: 7138, respectively is increased or generated or if the activity “microsomal beta-keto-reductase” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 7209, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 7208 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 7208 or polypeptide SEQ ID NO.: 7209, respectively is increased or generated or if the activity “Branched-chain amino acid permease” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 7275, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 7274 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 7274 or polypeptide SEQ ID NO.: 7275, respectively is increased or generated or if the activity “ubiquinone biosynthesis monooxygenase” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 7490, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 7489 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 7489 or polypeptide SEQ ID NO.: 7490, respectively is increased or generated or if the activity “YHR213W-protein” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 8240, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 8239 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 8239 or polypeptide SEQ ID NO.: 8240, respectively is increased or generated or if the activity “60S ribosomal protein” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 8398, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 8397 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 8397 or polypeptide SEQ ID NO.: 8398, respectively is increased or generated or if the activity “Autophagy-related protein” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 8228, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 8227 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 8227 or polypeptide SEQ ID NO.: 8228, respectively is increased or generated or if the activity “cytochrome c oxidase subunit VIII” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
Accordingly, in one embodiment, in case the activity of a polypeptide according to the polypeptide SEQ ID NO.: 8424, or encoded by a nucleic acid molecule comprising the nucleic acid SEQ ID NO.: 8423 or a homolog of said nucleic acid molecule or polypeptide, e.g. if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, as depicted in Table I, II or IV, column 7 in the respective same line as the nucleic acid molecule SEQ ID NO.: 8423 or polypeptide SEQ ID NO.: 8424, respectively is increased or generated or if the activity “Branched-chain amino acid permease” is increased or generated in an organism, preferably an increased GABA content as compared with the wild type is conferred in said organism.
The term “expression” refers to the transcription and/or translation of a codogenic gene segment or gene. As a rule, the resulting product is an mRNA or a protein. However, expression products can also include functional RNAs such as, for example, antisense, nucleic acids, tRNAs, snRNAs, rRNAs, RNAi, siRNA, ribozymes etc. Expression may be systemic, local or temporal, for example limited to certain cell types, tissues organs or organelles or time periods.
In one embodiment, the process of the present invention comprises one or more of the following steps
a) stabilizing a protein conferring the increased expression of a protein encoded by the nucleic acid molecule of the invention or of the polypeptid of the invention having the herein-mentioned activity selected from the group consisting of 60 S ribosomal protein, ABC transporter permease protein, acetyltransferase, acyl-carrier protein, At4g32480-protein, At5g16650-protein, ATP-binding protein, Autophagy-related protein, auxin response factor, auxin transcription factor, b1003-protein, b1522-protein, b2739-protein, b3646-protein, B4029-protein, Branched-chain amino acid permease, calcium-dependent protein kinase, cytochrome c oxidase subunit VIII, elongation factor Tu, Factor arrest protein, fumarylacetoacetate hydrolase, geranylgeranyl pyrophosphate synthase, glucose dehydrogenase, glycosyl transferase, harpin-induced family protein, homocitrate synthase, hydrolase, isochorismate synthase, MFS-type transporter protein, microsomal beta-keto-reductase, polygalacturonase, protein phosphatase, pyruvate kinase, Sec-independent protein translocase subunit, serine protease, thioredoxin, thioredoxin family protein, transcriptional regulator, ubiquinone biosynthesis monooxygenase, and YHR213W-protein and conferring an increased GABA content as compared to a corresponding non-transformed wild type;
b) stabilizing a mRNA conferring the increased expression of a protein encoded by the nucleic acid molecule of the invention or its homologs or of a mRNA encoding the polypeptide of the present invention having the herein-mentioned activity selected from the group consisting of 60S ribosomal protein, ABC transporter permease protein, acetyltransferase, acyl-carrier protein, At4g32480-protein, At5g16650-protein, ATP-binding protein, Autophagy-related protein, auxin response factor, auxin transcription factor, b1003-protein, b1522-protein, b2739-protein, b3646-protein, B4029-protein, Branched-chain amino acid permease, calcium-dependent protein kinase, cytochrome c oxidase subunit VIII, elongation factor Tu, Factor arrest protein, fumarylacetoacetate hydrolase, geranylgeranyl pyrophosphate synthase, glucose dehydrogenase, glycosyl transferase, harpin-induced family protein, homocitrate synthase, hydrolase, isochorismate synthase, MFS-type transporter protein, microsomal beta-keto-reductase, polygalacturonase, protein phosphatase, pyruvate kinase, Sec-independent protein translocase subunit, serine protease, thioredoxin, thioredoxin family protein, transcriptional regulator, ubiquinone biosynthesis monooxygenase, and YHR213W-protein and conferring an increased GABA content as compared to a corresponding non-transformed wild type;
c) increasing the specific activity of a protein conferring the increased expression of a protein encoded by the nucleic acid molecule of the invention or of the polypeptide of the present invention or decreasing the inhibitory regulation of the polypeptide of the invention;
d) generating or increasing the expression of an endogenous or artificial transcription factor mediating the expression of a protein conferring the increased expression of a protein encoded by the nucleic acid molecule of the invention or of the polypeptide of the invention having the herein-mentioned activity selected from the group consisting of 60S ribosomal protein, ABC transporter permease protein, acetyltransferase, acyl-carrier protein, At4g32480-protein, At5g16650-protein, ATP-binding protein, Autophagy-related protein, auxin response factor, auxin transcription factor, b1003-protein, b1522-protein, b2739-protein, b3646-protein, B4029-protein, Branched-chain amino acid permease, calcium-dependent protein kinase, cytochrome c oxidase subunit VIII, elongation factor Tu, Factor arrest protein, fumarylacetoacetate hydrolase, geranylgeranyl pyrophosphate synthase, glucose dehydrogenase, glycosyl transferase, harpin-induced family protein, homocitrate synthase, hydrolase, isochorismate synthase, MFS-type transporter protein, microsomal beta-keto-reductase, polygalacturonase, protein phosphatase, pyruvate kinase, Sec-independent protein translocase subunit, serine protease, thioredoxin, thioredoxin family protein, transcriptional regulator, ubiquinone biosynthesis monooxygenase, and YHR213W-protein and conferring an increased GABA content as compared to a corresponding non-transformed wild type;
e) stimulating activity of a protein conferring the increased expression of a protein encoded by the nucleic acid molecule of the present invention or a polypeptide of the present invention having the herein-mentioned activity selected from the group consisting of 60S ribosomal protein, ABC transporter permease protein, acetyltransferase, acyl-carrier protein, At4g32480-protein, At5g16650-protein, ATP-binding protein, Autophagy-related protein, auxin response factor, auxin transcription factor, b1003-protein, b1522-protein, b2739-protein, b3646-protein, B4029-protein, Branched-chain amino acid permease, calcium-dependent protein kinase, cytochrome c oxidase subunit VIII, elongation factor Tu, Factor arrest protein, fumarylacetoacetate hydrolase, geranylgeranyl pyrophosphate synthase, glucose dehydrogenase, glycosyl transferase, harpin-induced family protein, homocitrate synthase, hydrolase, isochorismate synthase, MFS-type transporter protein, microsomal beta-keto-reductase, polygalacturonase, protein phosphatase, pyruvate kinase, Sec-independent protein translocase subunit, serine protease, thioredoxin, thioredoxin family protein, transcriptional regulator, ubiquinone biosynthesis monooxygenase, and YHR213W-protein and conferring an increased GABA content as compared to a corresponding non-transformed wild type by adding one or more exogenous inducing factors to the organisms or parts thereof;
f) expressing a transgenic gene encoding a protein conferring the increased expression of a polypeptide encoded by the nucleic acid molecule of the present invention or a polypeptide of the present invention, having the herein-mentioned activity selected from the group consisting of 60S ribosomal protein, ABC transporter permease protein, acetyltransferase, acyl-carrier protein, At4g32480-protein, At5g16650-protein, ATP-binding protein, Autophagy-related protein, auxin response factor, auxin transcription factor, b1003-protein, b1522-protein, b2739-protein, b3646-protein, B4029-protein, Branched-chain amino acid permease, calcium-dependent protein kinase, cytochrome c oxidase subunit VIII, elongation factor Tu, Factor arrest protein, fumarylacetoacetate hydrolase, geranylgeranyl pyrophosphate synthase, glucose dehydrogenase, glycosyl transferase, harpin-induced family protein, homocitrate synthase, hydrolase, isochorismate synthase, MFS-type transporter protein, microsomal beta-keto-reductase, polygalacturonase, protein phosphatase, pyruvate kinase, Sec-independent protein translocase subunit, serine protease, thioredoxin, thioredoxin family protein, transcriptional regulator, ubiquinone biosynthesis monooxygenase, and YHR213W-protein and conferring an increased GABA content as compared to a corresponding non-transformed wild type; and/or
h) increasing the expression of the endogenous gene encoding the polypeptide of the invention or its homologs by adding positive expression or removing negative expression elements, e.g. homologous recombination can be used to either introduce positive regulatory elements like for plants the 35S enhancer into the promoter or to remove repressor elements form regulatory regions. Further gene conversion methods can be used to disrupt repressor elements or to enhance to activity of positive elements—positive elements can be randomly introduced in plants by T-DNA or transposon mutagenesis and lines can be identified in which the positive elements have been integrated near to a gene of the invention, the expression of which is thereby enhanced;
and/or
i) modulating growth conditions of the plant in such a manner, that the expression or activity of the gene encoding the protein of the invention or the protein itself is enhanced;
j) selecting of organisms with especially high activity of the proteins of the invention from natural or from mutagenized resources and breeding them into the target organisms, e.g. the elite crops.
Preferably, said mRNA is the nucleic acid molecule of the present invention and/or the protein conferring the increased expression of a protein encoded by the nucleic acid molecule of the present invention alone or linked to a transit nucleic acid sequence or transit peptide encoding nucleic acid sequence or the polypeptide having the herein mentioned activity, e.g. conferring an increased GABA content as compared to a corresponding non-transformed wild type after increasing the expression or activity of the encoded polypeptide or having the activity of a polypeptide having an activity as the protein as shown in table II column 3 or its homologs.
In general, the amount of mRNA or polypeptide in a cell or a compartment of an organism correlates with the amount of encoded protein and thus with the overall activity of the encoded protein in said volume. Said correlation is not always linear, the activity in the volume is dependent on the stability of the molecules or the presence of activating or inhibiting co-factors. Further, product and educt inhibitions of enzymes are well known and described in textbooks, e.g. Stryer, Biochemistry.
In general, the amount of mRNA, polynucleotide or nucleic acid molecule in a cell or a compartment of an organism correlates with the amount of encoded protein and thus with the overall activity of the encoded protein in said volume. Said correlation is not always linear, the activity in the volume is dependent on the stability of the molecules, the degradation of the molecules or the presence of activating or inhibiting co-factors. Further, product and educt inhibitions of enzymes are well known, e.g. Zinser et al. “Enzyminhibitoren”/Enzyme inhibitors”.
The activity of the abovementioned proteins and/or polypeptides encoded by the nucleic acid molecule of the present invention can be increased in various ways. For example, the activity in an organism or in a part thereof, like a cell, is increased via increasing the gene product number, e.g. by increasing the expression rate, like introducing a stronger promoter, or by increasing the stability of the mRNA expressed, thus increasing the translation rate, and/or increasing the stability of the gene product, thus reducing the proteins decayed. Further, the activity or turnover of enzymes can be influenced in such a way that a reduction or increase of the reaction rate or a modification (reduction or increase) of the affinity to the substrate results, is reached. A mutation in the catalytic center of an polypeptide of the invention, e.g. as enzyme, can modulate the turn over rate of the enzyme, e.g. a knock out of an essential amino acid can lead to a reduced or completely knock out activity of the enzyme, or the deletion or mutation of regulator binding sites can reduce a negative regulation like a feedback inhibition (or a substrate inhibition, if the substrate level is also increased). The specific activity of an enzyme of the present invention can be increased such that the turn over rate is increased or the binding of a co-factor is improved. Improving the stability of the encoding mRNA or the protein can also increase the activity of a gene product. The stimulation of the activity is also under the scope of the term “increased activity”.
Moreover, the regulation of the abovementioned nucleic acid sequences may be modified so that gene expression is increased. This can be achieved advantageously by means of heterologous regulatory sequences or by modifying, for example mutating, the natural regulatory sequences which are present. The advantageous methods may also be combined with each other.
In general, an activity of a gene product in an organism or part thereof, in particular in a plant cell or organelle of a plant cell, a plant, or a plant tissue or a part thereof or in a microorganism can be increased by increasing the amount of the specific encoding mRNA or the corresponding protein in said organism or part thereof. “Amount of protein or mRNA” is understood as meaning the molecule number of polypeptides or mRNA molecules in an organism, a tissue, a cell or a cell compartment. “Increase” in the amount of a protein means the quantitative increase of the molecule number of said protein in an organism, a tissue, a cell or a cell compartment such as an organelle like a plastid or mitochondria or part thereof—for example by one of the methods described herein below—in comparison to a wild type, control or reference.
The increase in molecule number amounts preferably to at least 1%, preferably to more than 10%, more preferably to 30% or more, especially preferably to 50%, 70% or more, very especially preferably to 100%, most preferably to 500% or more. However, a de novo expression is also regarded as subject of the present invention.
A modification, i.e. an increase, can be caused by endogenous or exogenous factors. For example, an increase in activity in an organism or a part thereof can be caused by adding a gene product or a precursor or an activator or an agonist to the media or nutrition or can be caused by introducing said subjects into a organism, transient or stable. Furthermore such an increase can be reached by the introduction of the inventive nucleic acid sequence or the encoded protein in the correct cell compartment for example into the nucleus, or cytoplasm respectively or into plastids either by transformation and/or targeting.
In one embodiment the increase or decrease in tolerance and/or resistance to environmental stress as compared to a corresponding non-transformed wild type plant cell in the plant or a part thereof, e.g. in a cell, a tissue, a organ, an organelle etc., is achieved by increasing the endogenous level of the polypeptide of the invention. Accordingly, in an embodiment of the present invention, the present invention relates to a process wherein the gene copy number of a gene encoding the polynucleotide or nucleic acid molecule of the invention is increased. Further, the endogenous level of the polypeptide of the invention can for example be increased by modifying the transcriptional or translational regulation of the polypeptide.
In one embodiment the increased GABA content in the cell can be altered by targeted or random mutagenesis of the endogenous genes of the invention. For example homologous recombination can be used to either introduce positive regulatory elements like for plants the 35S enhancer into the promoter or to remove repressor elements form regulatory regions. In addition gene conversion like methods described by Kochevenko and Willmitzer (Plant Physiol. 2003 May; 132(1):174-84) and citations therein can be used to disrupt repressor elements or to enhance to activity of positive regulatory elements.
Furthermore positive elements can be randomly introduced in (plant) genomes by T-DNA or transposon mutagenesis and lines can be screened for, in which the positive elements has be integrated near to a gene of the invention, the expression of which is thereby enhanced. The activation of plant genes by random integrations of enhancer elements has been described by Hayashi et al., 1992 (Science 258:1350-1353) or Weigel et al., 2000 (Plant Physiol. 122, 1003-1013) and others citated therein. Reverse genetic strategies to identify insertions (which eventually carrying the activation elements) near in genes of interest have been described for various cases e.g. Krysan et al., 1999 (Plant Cell 1999, 11, 2283-2290); Sessions et al., 2002 (Plant Cell 2002, 14, 2985-2994); Young et al., 2001, (Plant Physiol. 2001, 125, 513-518); Koprek et al., 2000 (Plant J. 2000, 24, 253-263); Jeon et al., 2000 (Plant J. 2000, 22, 561-570); Tissier et al., 1999 (Plant Cell 1999, 11, 1841-1852); Speulmann et al., 1999 (Plant Cell 1999, 11, 1853-1866). Briefly material from all plants of a large T-DNA or transposon mutagenized plant population is harvested and genomic DNA prepared. Then the genomic DNA is pooled following specific architectures as described for example in Krysan et al., 1999 (Plant Cell 1999, 11, 2283-2290). Pools of genomics DNAs are then screened by specific multiplex PCR reactions detecting the combination of the insertional mutagen (eg T-DNA or Transposon) and the gene of interest. Therefore PCR reactions are run on the DNA pools with specific combinations of T-DNA or transposon border primers and gene specific primers. General rules for primer design can again be taken from Krysan et al., 1999 (Plant Cell 1999, 11, 2283-2290) Rescreening of lower levels DNA pools lead to the identification of individual plants in which the gene of interest is activated by the insertional mutagen.
The enhancement of positive regulatory elements or the disruption or weaking of negative regulatory elements can also be achieved through common mutagenesis techniques: The production of chemically or radiation mutated populations is a common technique and known to the skilled worker. Methods for plants are described by Koorneef et al. 1982 and the citations therein and by Lightner and Caspar in “Methods in Molecular Biology” Vol 82. These techniques usually induce pointmutations that can be identified in any known gene using methods such as TILLING (Colbert et al. 2001).
Accordingly, the expression level can be increased if the endogenous genes encoding a polypeptide conferring an increased expression of the polypeptide of the present invention, in particular genes comprising the nucleic acid molecule of the present invention, are modified via homologous recombination, Tilling approaches or gene conversion. It also possible to add as mentioned herein targeting sequences to the inventive nucleic acid sequences.
Regulatory sequences preferably in addition to a target sequence or part thereof can be operatively linked to the coding region of an endogenous protein and control its transcription and translation or the stability or decay of the encoding mRNA or the expressed protein. In order to modify and control the expression, promoter, UTRs, splicing sites, processing signals, polyadenylation sites, terminators, enhancers, repressors, post transcriptional or posttranslational modification sites can be changed, added or amended. For example, the activation of plant genes by random integrations of enhancer elements has been described by Hayashi et al., 1992 (Science 258:1350-1353) or Weigel et al., 2000 (Plant Physiol. 122, 1003-1013) and others citated therein. For example, the expression level of the endogenous protein can be modulated by replacing the endogenous promoter with a stronger transgenic promoter or by replacing the endogenous 3′UTR with a 3′UTR, which provides more stability without amending the coding region. Further, the transcriptional regulation can be modulated by introduction of an artificial transcription factor as described in the examples. Alternative promoters, terminators and UTR are described below.
The activation of an endogenous polypeptide having above-mentioned activity, e.g. having the activity of a protein as shown in table II, column 3 or of the polypeptide of the invention, e.g. conferring the increased GABA content as compared to a corresponding non-transformed wild type after increase of expression or activity in the cytosol and/or in an organelle like a plastid, can also be increased by introducing a synthetic transcription factor, which binds close to the coding region of the gene encoding the protein as shown in table II, column 3 and activates its transcription. A chimeric zinc finger protein can be constructed, which comprises a specific DNA-binding domain and an activation domain as e.g. the VP16 domain of Herpes Simplex virus. The specific binding domain can bind to the regulatory region of the gene encoding the protein as shown in table II, column 3. The expression of the chimeric transcription factor in a organism, in particular in a plant, leads to a specific expression of the protein as shown in table II, column 3, see e.g. in WO01/52620, Oriz, Proc. Natl. Acad. Sci. USA, 2002, Vol. 99, 13290 or Guan, Proc. Natl. Acad. Sci. USA, 2002, Vol. 99, 13296.
In one further embodiment of the process according to the invention, organisms are used in which one of the abovementioned genes, or one of the above-mentioned nucleic acids, is mutated in a way that the activity of the encoded gene products is less influenced by cellular factors, or not at all, in comparison with the unmutated proteins. For example, well known regulation mechanism of enzymatic activity are substrate inhibition or feed back regulation mechanisms. Ways and techniques for the introduction of substitution, deletions and additions of one or more bases, nucleotides or amino acids of a corresponding sequence are described herein below in the corresponding paragraphs and the references listed there, e.g. in Sambrook et al., Molecular Cloning, Cold Spring Habour, N.Y., 1989. The person skilled in the art will be able to identify regulation domains and binding sites of regulators by comparing the sequence of the nucleic acid molecule of the present invention or the expression product thereof with the state of the art by computer software means which comprise algorithms for the identifying of binding sites and regulation domains or by introducing into a nucleic acid molecule or in a protein systematically mutations and assaying for those mutations which will lead to an increased specific activity or an increased activity per volume, in particular per cell.
It can therefore be advantageous to express in an organism a nucleic acid molecule of the invention or a polypeptide of the invention derived from a evolutionary distantly related organism, as e.g. using a prokaryotic gene in a eukaryotic host, as in these cases the regulation mechanism of the host cell may not weaken the activity (cellular or specific) of the gene or its expression product.
The mutation is introduced in such a way that the increased GABA content is not adversely affected.
Less influence on the regulation of a gene or its gene product is understood as meaning a reduced regulation of the enzymatic or biological activity leading to an increased specific or cellular activity of the gene or its product. An increase of the enzymatic or biological activity is understood as meaning an enzymatic or biological activity, which is increased by at least 10%, advantageously at least 20, 30 or 40%, especially advantageously by at least 50, 60 or 70% in comparison with the starting organism. This leads to an increased GABA content as compared to a corresponding non-transformed wild type.
The invention provides that the above methods can be performed such that the stress tolerance is increased. It is also possible to obtain a decrease in stress tolerance.
The invention is not limited to specific nucleic acids, specific polypeptides, specific cell types, specific host cells, specific conditions or specific methods etc. as such, but may vary and numerous modifications and variations therein will be apparent to those skilled in the art. It is also to be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.
The present invention also relates to isolated nucleic acids comprising a nucleic acid molecule selected from the group consisting of:
and
whereby the nucleic acid molecule according to (a) to (j) is at least in one or more nucleotides different from the sequence depicted in column 5 or 7 of Table I A and preferably which encodes a protein which differs at least in one or more amino acids from the protein sequences depicted in column 5 or 7 of Table II A.
In one embodiment the invention relates to homologs of the afore-mentioned sequences, which can be isolated advantageously from yeast, fungi, viruses, algae, bacteria, such as Acetobacter (subgen. Acetobacter) aceti; Acidithiobacillus ferrooxidans; Acinetobacter sp.; Actinobacillus sp; Aeromonas salmonicida; Agrobacterium tumefaciens; Aquifex aeolicus; Arcanobacterium pyogenes; Aster yellows phytoplasma; Bacillus sp.; Bifidobacterium sp.; Borrelia burgdorferi; Brevibacterium linens; Brucella melitensis; Buchnera sp.; Butyrivibrio fibrisolvens; Campylobacter jejuni; Caulobacter crescentus; Chlamydia sp.; Chlamydophila sp.; Chlorobium limicola; Citrobacter rodentium; Clostridium sp.; Comamonas testosteroni; Corynebacterium sp.; Coxiella burnetii; Deinococcus radiodurans; Dichelobacter nodosus; Edwardsiella ictaluri; Enterobacter sp.; Erysipelothrix rhusiopathiae; Escherichia coli; Flavobacterium sp.; Francisella tularensis; Frankia sp. Cpl1; Fusobacterium nucleatum; Geobacillus stearothermophilus; Gluconobacter oxydans; Haemophilus sp.; Helicobacter pylori; Klebsiella pneumoniae; Lactobacillus sp.; Lactococcus lactis; Listeria sp.; Mannheimia haemolytica; Mesorhizobium loti; Methylophaga thalassica; Microcystis aeruginosa; Microscilla sp. PRE1; Moraxella sp. TA144; Mycobacterium sp.; Mycoplasma sp.; Neisseria sp.; Nitrosomonas sp.; Nostoc sp. PCC 7120; Novosphingobium aromaticivorans; Oenococcus oeni; Pantoea citrea; Pasteurella multocida; Pediococcus pentosaceus; Phormidium foveolarum; Phytoplasma sp.; Plectonema boryanum; Prevotella ruminicola; Propionibacterium sp.; Proteus vulgaris; Pseudomonas sp.; Ralstonia sp.; Rhizobium sp.; Rhodococcus equi; Rhodothermus marinus; Rickettsia sp.; Riemerella anatipestifer; Ruminococcus flavefaciens; Salmonella sp.; Selenomonas ruminantium; Serratia entomophila; Shigella sp.; Sinorhizobium meliloti; Staphylococcus sp.; Streptococcus sp.; Streptomyces sp.; Synechococcus sp.; Synechocystis sp. PCC 6803; Thermotoga maritima; Treponema sp.; Ureaplasma urealyticum; Vibrio cholerae; Vibrio parahaemolyticus; Xylella fastidiosa; Yersinia sp.; Zymomonas mobilis, preferably Salmonella sp. or Escherichia coli or plants, preferably from yeasts such as from the genera Saccharomyces, Pichia, Candida, Hansenula, Torulopsis or Schizosaccharomyces or plants such as Arabidopsis thaliana, maize, wheat, rye, oat, triticale, rice, barley, soybean, peanut, cotton, borage, sunflower, linseed, primrose, rapeseed, canola and turnip rape, manihot, pepper, sunflower, tagetes, solanaceous plant such as potato, tobacco, eggplant and tomato, Vicia species, pea, alfalfa, bushy plants such as coffee, cacao, tea, Salix species, trees such as oil palm, coconut, perennial grass, such as ryegrass and fescue, and forage crops, such as alfalfa and clover and from spruce, pine or fir for example. More preferably homologs of aforementioned sequences can be isolated from Saccharomyces cerevisiae, E. coli or plants, preferably Brassica napus, Glycine max, Zea mays, cotton, or Oryza sativa.
The (GABA related) proteins of the present invention are preferably produced by recombinant DNA techniques. For example, a nucleic acid molecule encoding the protein is cloned into an expression vector, for example in to a binary vector, the expression vector is introduced into a host cell, for example the Arabidopsis thaliana wild type NASC N906 or any other plant cell as described in the examples see below, and the stress related protein is expressed in said host cell. Examples for binary vectors are pBIN19, pBI101, pBinAR, pGPTV, pCAMBIA, pBIB-HYG, pBecks, pGreen or pPZP (Hajukiewicz, P. et al., 1994, Plant Mol. Biol., 25: 989-994 and Hellens et al, Trends in Plant Science (2000) 5, 446-451.).
In one embodiment the (GABA related) protein of the present invention is preferably produced in an compartment of the cell, more preferably in the plastids. Ways of introducing nucleic acids into plastids and producing proteins in this compartment are know to the person skilled in the art have been also described in this application.
Advantageously, the nucleic acid sequences according to the invention or the gene construct together with at least one reporter gene are cloned into an expression cassette, which is introduced into the organism via a vector or directly into the genome. This reporter gene should allow easy detection via a growth, fluorescence, chemical, bioluminescence or resistance assay or via a photometric measurement. Examples of reporter genes which may be mentioned are antibiotic- or herbicide-resistance genes, hydrolase genes, fluorescence protein genes, bioluminescence genes, sugar or nucleotide metabolic genes or biosynthesis genes such as the Ura3 gene, the IIv2 gene, the luciferase gene, the β-galactosidase gene, the gfp gene, the 2-desoxyglucose-6-phosphate phosphatase gene, the □β-glucuronidase gene, β-lactamase gene, the neomycin phosphotransferase gene, the hygromycin phosphotransferase gene, a mutated acetohydroxyacid synthase (AHAS) gene, also known as acetolactate synthase (ALS) gene], a gene for a D-amino acid metabolizing enzmye or the BASTA (=gluphosinate-resistance) gene. These genes permit easy measurement and quantification of the transcription activity and hence of the expression of the genes. In this way genome positions may be identified which exhibit differing productivity.
In a preferred embodiment a nucleic acid construct, for example an expression cassette, comprises upstream, i.e. at the 5′ end of the encoding sequence, a promoter and downstream, i.e. at the 3′ end, a polyadenylation signal and optionally other regulatory elements which are operably linked to the intervening encoding sequence with one of the nucleic acids of SEQ ID NO as depicted in table I, column 5 and 7. By an operable linkage is meant the sequential arrangement of promoter, encoding sequence, terminator and optionally other regulatory elements in such a way that each of the regulatory elements can fulfill its function in the expression of the encoding sequence in due manner. The sequences preferred for operable linkage are targeting sequences for ensuring subcellular localization in plastids. However, targeting sequences for ensuring subcellular localization in the mitochondrium, in the endoplasmic reticulum (=ER), in the nucleus, in oil corpuscles or other compartments may also be employed as well as translation promoters such as the 5′ lead sequence in tobacco mosaic virus (Gallie et al., Nucl. Acids Res. 15 (1987), 8693-8711).
A nucleic acid construct, for example an expression cassette may, for example, contain a constitutive promoter or a tissue-specific promoter (preferably the USP or napin promoter) the gene to be expressed and the ER retention signal. For the ER retention signal the KDEL amino acid sequence (lysine, aspartic acid, glutamic acid, leucine) or the KKX amino acid sequence (lysine-lysine-X-stop, wherein X means every other known amino acid) is preferably employed.
For expression in a host organism, for example a plant, the expression cassette is advantageously inserted into a vector such as by way of example a plasmid, a phage or other DNA which allows optimal expression of the genes in the host organism. Examples of suitable plasmids are: in E. coli pLG338, pACYC184, pBR series such as e.g. pBR322, pUC series such as pUC18 or pUC19, M113mp series, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III113-B1, λgt11 or pBdCI; in Streptomyces pIJ101, pIJ364, pIJ702 or pIJ361; in Bacillus pUB110, pC194 or pBD214; in Corynebacterium pSA77 or pAJ667; in fungi pALS1, pIL2 or pBB116; other advantageous fungal vectors are described by Romanos, M. A. et al., [(1992) “Foreign gene expression in yeast: a review”, Yeast 8: 423-488] and by van den Hondel, C.A.M.J.J. et al. [(1991) “Heterologous gene expression in filamentous fungi” as well as in More Gene Manipulations in Fungi [J. W. Bennet & L. L. Lasure, eds., pp. 396-428: Academic Press: San Diego] and in “Gene transfer systems and vector development for filamentous fungi” [van den Hondel, C.A.M.J.J. & Punt, P.J. (1991) in: Applied Molecular Genetics of Fungi, Peberdy, J. F. et al., eds., pp. 1-28, Cambridge University Press: Cambridge]. Examples of advantageous yeast promoters are 2 μM, pAG-1, YEp6, YEp13 or pEMBLYe23. Examples of algal or plant promoters are pLGV23, pGHIac+, pBIN19, pAK2004, pVKH or pDH51 (see Schmidt, R. and Willmitzer, L., 1988). The vectors identified above or derivatives of the vectors identified above are a small selection of the possible plasmids. Further plasmids are well known to those skilled in the art and may be found, for example, in the book Cloning Vectors (Eds. Pouwels P. H. et al. Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018). Suitable plant vectors are described inter alia in “Methods in Plant Molecular Biology and Biotechnology” (CRC Press), Ch. 6/7, pp. 71-119. Advantageous vectors are known as shuttle vectors or binary vectors which replicate in E. coli and Agrobacterium.
By vectors is meant with the exception of plasmids all other vectors known to those skilled in the art such as by way of example phages, viruses such as SV40, CMV, baculovirus, adenovirus, transposons, IS elements, phasmids, phagemids, cosmids, linear or circular DNA. These vectors can be replicated autonomously in the host organism or be chromosomally replicated, chromosomal replication being preferred.
In a further embodiment of the vector the expression cassette according to the invention may also advantageously be introduced into the organisms in the form of a linear DNA and be integrated into the genome of the host organism by way of heterologous or homologous recombination. This linear DNA may be composed of a linearized plasmid or only of the expression cassette as vector or the nucleic acid sequences according to the invention.
In a further advantageous embodiment the nucleic acid sequence according to the invention can also be introduced into an organism on its own.
If in addition to the nucleic acid sequence according to the invention further genes are to be introduced into the organism, all together with a reporter gene in a single vector or each single gene with a reporter gene in a vector in each case can be introduced into the organism, whereby the different vectors can be introduced simultaneously or successively.
The vector advantageously contains at least one copy of the nucleic acid sequences according to the invention and/or the expression cassette (=gene construct) according to the invention.
The invention further provides an isolated recombinant expression vector comprising a nucleic acid encoding a polypeptide as depicted in table II, column 5 or 7, wherein expression of the vector in a host cell results in increased tolerance to environmental stress as compared to a wild type variety of the host cell. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell or a organelle upon introduction into the host cell, and thereby are replicated along with the host or organelle genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.
The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. As used herein with respect to a recombinant expression vector, “operatively linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) and Gruber and Crosby, in: Methods in Plant Molecular Biology and Biotechnology, eds. Glick and Thompson, Chapter 7, 89-108, CRC Press: Boca Raton, Fla., including the references therein. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells or under certain conditions. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce polypeptides or peptides, including fusion polypeptides or peptides, encoded by nucleic acids as described herein (e.g., GABA-related Proteins, mutant forms of GABA-related Proteins, fusion polypeptides, etc.).
The recombinant expression vectors of the invention can be designed for expression of the polypeptide of the invention in plant cells. For example, genes coding for GABA-related Proteins can be expressed in plant cells (See Schmidt, R. and Willmitzer, L., 1988, High efficiency Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana leaf and cotyledon explants, Plant Cell Rep. 583-586; Plant Molecular Biology and Biotechnology, C Press, Boca Raton, Fla., chapter 6/7, S.71-119 (1993); F. F. White, B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds. Kung and R. Wu, 128-43, Academic Press: 1993; Potrykus, 1991, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42:205-225 and references cited therein). Suitable host cells are discussed further in
Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press: San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
Expression of polypeptides in prokaryotes is most often carried out with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion polypeptides. Fusion vectors add a number of amino acids to a polypeptide encoded therein, usually to the amino terminus of the recombinant polypeptide but also to the C-terminus or fused within suitable regions in the polypeptides. Such fusion vectors typically serve three purposes: 1) to increase expression of a recombinant polypeptide; 2) to increase the solubility of a recombinant polypeptide; and 3) to aid in the purification of a recombinant polypeptide by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant polypeptide to enable separation of the recombinant polypeptide from the fusion moiety subsequent to purification of the fusion polypeptide. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin, and enterokinase.
By way of example the plant expression cassette can be installed in the pRT transformation vector ((a) Toepfer et al., 1993, Methods Enzymol., 217: 66-78; (b) Toepfer et al. 1987, Nucl. Acids. Res. 15: 5890 ff.).
Alternatively, a recombinant vector (=expression vector) can also be transcribed and translated in vitro, e.g. by using the T7 promoter and the T7 RNA polymerase.
Expression vectors employed in prokaryotes frequently make use of inducible systems with and without fusion proteins or fusion oligopeptides, wherein these fusions can ensue in both N-terminal and C-terminal manner or in other useful domains of a protein. Such fusion vectors usually have the following purposes: i.) to increase the RNA expression rate; ii.) to increase the achievable protein synthesis rate; iii.) to increase the solubility of the protein; iv.) or to simplify purification by means of a binding sequence usable for affinity chromatography. Proteolytic cleavage points are also frequently introduced via fusion proteins, which allow cleavage of a portion of the fusion protein and purification. Such recognition sequences for proteases are recognized, e.g. factor Xa, thrombin and enterokinase.
Typical advantageous fusion and expression vectors are pGEX [Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67: 31-40], pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which contains glutathione S-transferase (GST), maltose binding protein or protein A.
In one embodiment, the coding sequence of the polypeptide of the invention is cloned into a pGEX expression vector to create a vector encoding a fusion polypeptide comprising, from the N-terminus to the C-terminus, GST-thrombin cleavage site-X polypeptide. The fusion polypeptide can be purified by affinity chromatography using glutathione-agarose resin. Recombinant GABA-related Proteins unfused to GST can be recovered by cleavage of the fusion polypeptide with thrombin.
Other examples of E. coli expression vectors are pTrc [Amann et al., (1988) Gene 69:301-315] and pET vectors [Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89; Stratagene, Amsterdam, The Netherlands].
Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET 11 d vector relies on transcription from a T7 gn10-lac fusion promoter mediated by a co-expressed viral RNA polymerase (T7 gn1). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident I prophage harboring a T7 gn1 gene under the transcriptional control of the lacUV 5 promoter.
In a preferred embodiment of the present invention, the proteins of the invention which enhance the GABA content in a cell, meaning the GABA-related Proteins are expressed in plants and plants cells such as unicellular plant cells (e.g. algae) (See Falciatore et al., 1999, Marine Biotechnology 1(3):239-251 and references therein) and plant cells from higher plants (e.g., the spermatophytes, such as crop plants). A nucleic acid molecule coding for GABA-related Proteins as depicted in table II, column 5 or 7 may be “introduced” into a plant cell by any means, including transfection, transformation or transduction, electroporation, particle bombardment, agroinfection, and the like. One transformation method known to those of skill in the art is the dipping of a flowering plant into an Agrobacteria solution, wherein the Agrobacteria contains the nucleic acid of the invention, followed by breeding of the transformed gametes.
Other suitable methods for transforming or transfecting host cells including plant cells can be found in Sambrook, et al., Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, and other laboratory manuals such as Methods in Molecular Biology, 1995, Vol. 44, Agrobacterium protocols, ed: Gartland and Davey, Humana Press, Totowa, N.J. As biotic and abiotic stress tolerance is a general trait wished to be inherited into a wide variety of plants like maize, wheat, rye, oat, triticale, rice, barley, soybean, peanut, cotton, rapeseed and canola, manihot, pepper, sunflower and tagetes, solanaceous plants like potato, tobacco, eggplant, and tomato, Vicia species, pea, alfalfa, bushy plants (coffee, cacao, tea), Salix species, trees (oil palm, coconut), perennial grasses, and forage crops, these crop plants are also preferred target plants for a genetic engineering as one further embodiment of the present invention. Forage crops include, but are not limited to, Wheatgrass, Canarygrass, Bromegrass, Wildrye Grass, Bluegrass, Orchardgrass, Alfalfa, Salfoin, Birdsfoot Trefoil, Alsike Clover, Red Clover, and Sweet Clover.
In one embodiment of the present invention, transfection of a nucleic acid molecule coding for GABA-related Proteins as depicted in table II, column 5 or 7 into a plant is achieved by Agrobacterium mediated gene transfer. Agrobacterium mediated plant transformation can be performed using for example the GV3101(pMP90) (Koncz and Schell, 1986, Mol. Gen. Genet. 204:383-396) or LBA4404 (Clontech) Agrobacterium tumefaciens strain. Transformation can be performed by standard transformation and regeneration techniques (Deblaere et al., 1994, Nucl. Acids Res. 13:4777-4788; Gelvin, Stanton B. and Schilperoort, Robert A, Plant Molecular Biology Manual, 2nd Ed.—Dordrecht: Kluwer Academic Publ., 1995.—in Sect., Ringbuc Zentrale Signatur: BT11-P ISBN 0-7923-2731-4; Glick, Bernard R.; Thompson, John E., Methods in Plant Molecular Biology and Biotechnology, Boca Raton: CRC Press, 1993 360 S., ISBN 0-8493-5164-2). For example, rapeseed can be transformed via cotyledon or hypocotyl transformation (Moloney et al., 1989, Plant cell Report 8:238-242; De Block et al., 1989, Plant Physiol. 91:694-701). Use of antibiotics for Agrobacterium and plant selection depends on the binary vector and the Agrobacterium strain used for transformation. Rapeseed selection is normally performed using kanamycin as selectable plant marker. Agrobacterium mediated gene transfer to flax can be performed using, for example, a technique described by Mlynarova et al., 1994, Plant Cell Report 13:282-285. Additionally, transformation of soybean can be performed using for example a technique described in European Patent No. 0424 047, U.S. Pat. No. 5,322,783, European Patent No. 0397 687, U.S. Pat. No. 5,376,543, or U.S. Pat. No. 5,169,770. Transformation of maize can be achieved by particle bombardment, polyethylene glycol mediated DNA uptake or via the silicon carbide fiber technique. (See, for example, Freeling and Walbot “The maize handbook” Springer Verlag: New York (1993) ISBN 3-540-97826-7). A specific example of maize transformation is found in U.S. Pat. No. 5,990,387, and a specific example of wheat transformation can be found in PCT Application No. WO 93/07256.
According to the present invention, the introduced nucleic acid molecule coding for GABA-related Proteins as depicted in table II, column 5 or 7 may be maintained in the plant cell stably if it is incorporated into a non-chromosomal autonomous replicon or integrated into the plant chromosomes or organelle genome. Alternatively, the introduced gene coding or GABA-related Proteins may be present on an extrachromosomal non-replicating vector and be transiently expressed or transiently active.
In one embodiment, a homologous recombinant microorganism can be created wherein the gene coding for GABA-related Proteins is integrated into a chromosome, a vector is prepared which contains at least a portion of a nucleic acid molecule coding for GABA-related Proteins as depicted in table II, column 5 or 7 into which a deletion, addition, or substitution has been introduced to thereby alter, e.g., functionally disrupt, the GABA-related Proteins gene. Preferably, the gene encoding GABA-related Proteins is a yeast or a E. coli. or a Physcomitrella patens, or a Synechocystis or a Thermus thermophilus or a Brassica napus gene, but it can be a homolog from a related organism or plant or even from a mammalian or insect source. The vector can be designed such that, upon homologous recombination, the endogenous nucleic acid molecule coding for GABA-related Proteins as depicted in table II, column 5 or 7 is mutated or otherwise altered but still encodes a functional polypeptide (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous GABA-related Proteins). In a preferred embodiment the biological activity of the protein of the invention is increased upon homologous recombination. To create a point mutation via homologous recombination, DNA-RNA hybrids can be used in a technique known as chimeraplasty (Cole-Strauss et al., 1999, Nucleic Acids Research 27(5):1323-1330 and Kmiec, 1999 Gene therapy American Scientist. 87(3):240-247). Homologous recombination procedures in Physcomitrella patens are also well known in the art and are contemplated for use herein.
Whereas in the homologous recombination vector, the altered portion of the nucleic acid molecule coding for GABA-related Proteins as depicted in table II, column 5 or 7 is flanked at its 5′ and 3′ ends by an additional nucleic acid molecule of the gene encoding GABA-related Proteins to allow for homologous recombination to occur between the exogenous GABA-related Protein gene carried by the vector and an endogenous gene coding for GABA-related Proteins, in a microorganism or plant. The additional flanking nucleic acid molecule encoding GABA-related Proteins is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several hundreds of base pairs up to kilobases of flanking DNA (both at the 5′ and 3′ ends) are included in the vector. See, e.g., Thomas, K. R., and Capecchi, M. R., 1987, Cell 51:503 for a description of homologous recombination vectors or Strepp et al., 1998, PNAS, 95 (8):4368-4373 for cDNA based recombination in Physcomitrella patens). The vector is introduced into a microorganism or plant cell (e.g., via polyethylene glycol mediated DNA), and cells in which the introduced gene encoding GABA-related Proteins has homologously recombined with the endogenous gene coding for GABA-related Proteins are selected using art-known techniques.
Whether present in an extra-chromosomal non-replicating vector or a vector that is integrated into a chromosome, the nucleic acid molecule coding for GABA-related Proteins as depicted in table II, column 5 or 7 preferably resides in a plant expression cassette. A plant expression cassette preferably contains regulatory sequences capable of driving gene expression in plant cells that are operatively linked so that each sequence can fulfill its function, for example, termination of transcription by polyadenylation signals. Preferred polyadenylation signals are those originating from Agrobacterium tumefaciens t-DNA such as the gene 3 known as octopine synthase of the Ti-plasmid pTiACH5 (Gielen et al., 1984, EMBO J. 3:835) or functional equivalents thereof but also all other terminators functionally active in plants are suitable. As plant gene expression is very often not limited on transcriptional levels, a plant expression cassette preferably contains other operatively linked sequences like translational enhancers such as the overdrive-sequence containing the 5′-untranslated leader sequence from tobacco mosaic virus enhancing the polypeptide per RNA ratio (Gallie et al., 1987, Nucl. Acids Research 15:8693-8711). Examples of plant expression vectors include those detailed in: Becker, D. et al., 1992, New plant binary vectors with selectable markers located proximal to the left border, Plant Mol. Biol. 20: 1195-1197; and Bevan, M. W., 1984, Binary Agrobacterium vectors for plant transformation, Nucl. Acid. Res. 12:8711-8721; and Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds.: Kung and R. Wu, Academic Press, 1993, S. 15-38.
“Transformation” is defined herein as a process for introducing heterologous DNA into a plant cell, plant tissue, or plant. It may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into aprokaryotic or eukaryotic host cell. The method is selected based on the host cell being transformed and may include, but is not limited to, viral infection, electroporation, lipofection, and particle bombardment. Such “transformed” cells include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome. They also include cells which transiently express the inserted DNA or RNA for limited periods of time. Transformed plant cells, plant tissue, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof.
The terms “transformed,” “transgenic,” and “recombinant” refer to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome of the host or the nucleic acid molecule can also be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating. Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof. A “non-transformed,” “non-transgenic,” or “non-recombinant” host refers to a wild-type organism, e.g., a bacterium or plant, which does not contain the heterologous nucleic acid molecule.
A “transgenic plant”, as used herein, refers to a plant which contains a foreign nucleotide sequence inserted into either its nuclear genome or organellar genome. It encompasses further the offspring generations i.e. the T1-, T2- and consecutively generations or BC1-, BC2- and consecutively generation as well as crossbreeds thereof with non-transgenic or other transgenic plants.
The host organism (=transgenic organism) advantageously contains at least one copy of the nucleic acid according to the invention and/or of the nucleic acid construct according to the invention.
In principle all plants can be used as host organism. Preferred transgenic plants are, for example, selected from the families Aceraceae, Anacardiaceae, Apiaceae, Asteraceae, Brassicaceae, Cactaceae, Cucurbitaceae, Euphorbiaceae, Fabaceae, Malvaceae, Nymphaeaceae, Papaveraceae, Rosaceae, Salicaceae, Solanaceae, Arecaceae, Bromeliaceae, Cyperaceae, Iridaceae, Liliaceae, Orchidaceae, Gentianaceae, Labiaceae, Magnoliaceae, Ranunculaceae, Carifolaceae, Rubiaceae, Scrophulariaceae, Caryophyllaceae, Ericaceae, Polygonaceae, Violaceae, Juncaceae or Poaceae and preferably from a plant selected from the group of the families Apiaceae, Asteraceae, Brassicaceae, Cucurbitaceae, Fabaceae, Papaveraceae, Rosaceae, Solanaceae, Liliaceae or Poaceae. Preferred are crop plants such as plants advantageously selected from the group of the genus peanut, oilseed rape, canola, sunflower, safflower, olive, sesame, hazelnut, almond, avocado, bay, pumpkin/squash, linseed, soya, pistachio, borage, maize, wheat, rye, oats, sorghum and millet, triticale, rice, barley, cassava, potato, sugarbeet, egg plant, alfalfa, and perennial grasses and forage plants, oil palm, vegetables (brassicas, root vegetables, tuber vegetables, pod vegetables, fruiting vegetables, onion vegetables, leafy vegetables and stem vegetables), buckwheat, Jerusalem artichoke, broad bean, vetches, lentil, dwarf bean, lupin, clover and Lucerne for mentioning only some of them.
In one embodiment of the invention transgenic plants are selected from the group comprising corn, soy, oil seed rape (including canola and winter oil seed reap), cotton, wheat and rice.
In one preferred embodiment, the host plant is selected from the families Aceraceae, Anacardiaceae, Apiaceae, Asteraceae, Brassicaceae, Cactaceae, Cucurbitaceae, Euphorbiaceae, Fabaceae, Malvaceae, Nymphaeaceae, Papaveraceae, Rosaceae, Salicaceae, Solanaceae, Arecaceae, Bromeliaceae, Cyperaceae, Iridaceae, Liliaceae, Orchidaceae, Gentianaceae, Labiaceae, Magnoliaceae, Ranunculaceae, Carifolaceae, Rubiaceae, Scrophulariaceae, Caryophyllaceae, Ericaceae, Polygonaceae, Violaceae, Juncaceae or Poaceae and preferably from a plant selected from the group of the families Apiaceae, Asteraceae, Brassicaceae, Cucurbitaceae, Fabaceae, Papaveraceae, Rosaceae, Solanaceae, Liliaceae or Poaceae. Preferred are crop plants and in particular plants mentioned herein above as host plants such as the families and genera mentioned above for example preferred the species Anacardium occidentale, Calendula officinalis, Carthamus tinctorius, Cichorium intybus, Cynara scolymus, Helianthus annus, Tagetes lucida, Tagetes erecta, Tagetes tenuifolia; Daucus carota; Corylus avellana, Corylus columa, Borago officinalis; Brassica napus, Brassica rapa ssp., Sinapis arvensis Brassica juncea, Brassica juncea var. juncea, Brassica juncea var. crispifolia, Brassica juncea var. foliosa, Brassica nigra, Brassica sinapioides, Melanosinapis communis, Brassica oleracea, Arabidopsis thaliana, Anana comosus, Ananas ananas, Bromelia comosa, Carica papaya, Cannabis sative, Ipomoea batatus, Ipomoea pandurata, Convolvulus batatas, Convolvulus tiliaceus, Ipomoea fastigiata, Ipomoea tiliacea, Ipomoea triloba, Convolvulus panduratus, Beta vulgaris, Beta vulgaris var. altissima, Beta vulgaris var. vulgaris, Beta maritima, Beta vulgaris var. perennis, Beta vulgaris var. conditiva, Beta vulgaris var. esculenta, Cucurbita maxima, Cucurbita mixta, Cucurbita pepo, Cucurbita moschata, Olea europaea, Manihot utilissima, Janipha manihot, Jatropha manihot., Manihot aipil, Manihot dulcis, Manihot manihot, Manihot melanobasis, Manihot esculenta, Ricinus communis, Pisum sativum, Pisum arvense, Pisum humile, Medicago sativa, Medicago falcata, Medicago varia, Glycine max Dolichos soja, Glycine gracilis, Glycine hispida, Phaseolus max, Soja hispida, Soja max, Cocos nucifera, Pelargonium grossularioides, Oleum cocoas, Laurus nobilis, Persea americana, Arachis hypogaea, Linum usitatissimum, Linum humile, Linum austriacum, Linum bienne, Linum angustifolium, Linum catharticum, Linum flavum, Linum grandiflorum, Adenolinum grandiflorum, Linum lewisii, Linum narbonense, Linum perenne, Linum perenne var. lewisii, Linum pratense, Linum trigynum, Punica granatum, Gossypium hirsutum, Gossypium arboreum, Gossypium barbadense, Gossypium herbaceum, Gossypium thurberi, Musa nana, Musa acuminata, Musa paradisiaca, Musa spp., Elaeis guineensis, Papaver orientate, Papaver rhoeas, Papaver dubium, Sesamum indicum, Piper aduncum, Piper amalago, Piper angustifolium, Piper auritum, Piper betel, Piper cubeba, Piper longum, Piper nigrum, Piper retrofractum, Artanthe adunca, Artanthe elongata, Peperomia elongata, Piper elongatum, Steffensia elongata, Hordeum vulgare, Hordeum jubatum, Hordeum murinum, Hordeum secalinum, Hordeum distichon Hordeum aegiceras, Hordeum hexastichon., Hordeum hexastichum, Hordeum irregulare, Hordeum sativum, Hordeum secalinum, Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida, Sorghum bicolor, Sorghum halepense, Sorghum saccharatum, Sorghum vulgare, Andropogon drum-mondii, Holcus bicolor, Holcus sorghum, Sorghum aethiopicum, Sorghum arundinaceum, Sorghum caffrorum, Sorghum cernuum, Sorghum dochna, Sorghum drummondii, Sorghum durra, Sorghum guineense, Sorghum lanceolatum, Sorghum nervosum, Sorghum saccharatum, Sorghum subglabrescens, Sorghum verticilliflorum, Sorghum vulgare, Holcus halepensis, Sorghum miliaceum millet, Panicum militaceum, Zea mays, Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum or Triticum vulgare, Cofea spp., Coffea arabica, Coffea canephora, Coffea liberica, Capsicum annuum, Capsicum annuum var. glabriusculum, Capsicum frutescens, Capsicum annuum, Nicotiana tabacum, Solanum tuberosum, Solanum melongena, Lycopersicon esculentum, Lycopersicon lycopersicum., Lycopersicon pyriforme, Solanum integrifolium, Solanum lycopersicum Theobroma cacao or Camellia sinensis.
Anacardiaceae such as the genera Pistacia, Mangifera, Anacardium e.g. the species Pistacia vera [pistachios, Pistazie], Mangifer Indica [Mango] or Anacardium occidentale [Cashew]; Asteraceae such as the genera Calendula, Carthamus, Centaurea, Cichorium, Cynara, Helianthus, Lactuca, Locusta, Tagetes, Valeriana e.g. the species Calendula officinalis [Marigold], Carthamus tinctorius [safflower], Centaurea cyanus [cornflower], Cichorium intybus [blue daisy], Cynara scolymus [Artichoke], Helianthus annus [sunflower], Lactuca sativa, Lactuca crispa, Lactuca esculenta, Lactuca scariola L. ssp. sativa, Lactuca scariola L. var. integrata, Lactuca scariola L. var. integrifolia, Lactuca sativa subsp. romana, Locusta communis, Valeriana locusta [lettuce], Tagetes lucida, Tagetes erecta or Tagetes tenuifolia [Marigold]; Apiaceae such as the genera Daucus e.g. the species Daucus carota [carrot]; Betulaceae such as the genera Corylus e.g. the species Corylus avellana or Corylus colurna [hazelnut]; Boraginaceae such as the genera Borago e.g. the species Borago officinalis [borage]; Brassicaceae such as the genera Brassica, Melanosinapis, Sinapis, Arabadopsis e.g. the species Brassica napus, Brassica rapa ssp. [canola, oilseed rape, turnip rape], Sinapis arvensis Brassica juncea, Brassica juncea var. juncea, Brassica juncea var. crispifolia, Brassica juncea var. foliosa, Brassica nigra, Brassica sinapioides, Melanosinapis communis [mustard], Brassica oleracea [fodder beet] or Arabidopsis thaliana; Bromeliaceae such as the genera Anana, Bromelia e.g. the species Anana comosus, Ananas ananas or Bromelia comosa [pineapple]; Caricaceae such as the genera Carica e.g. the species Carica papaya [papaya]; Cannabaceae such as the genera Cannabis e.g. the species Cannabis sative [hemp], Convolvulaceae such as the genera Ipomea, Convolvulus e.g. the species Ipomoea batatus, Ipomoea pandurata, Convolvulus batatas, Convolvulus tiliaceus, Ipomoea fastigiata, Ipomoea tiliacea, Ipomoea triloba or Convolvulus panduratus [sweet potato, Man of the Earth, wild potato], Chenopodiaceae such as the genera Beta, i.e. the species Beta vulgaris, Beta vulgaris var. altissima, Beta vulgaris var. Vulgaris, Beta maritima, Beta vulgaris var. perennis, Beta vulgaris var. conditiva or Beta vulgaris var. esculenta [sugar beet]; Cucurbitaceae such as the genera Cucubita e.g. the species Cucurbita maxima, Cucurbita mixta, Cucurbita pepo or Cucurbita moschata [pumpkin, squash]; Elaeagnaceae such as the genera Elaeagnus e.g. the species Olea europaea [olive]; Ericaceae such as the genera Kalmia e.g. the species Kalmia latifolia, Kalmia angustifolia, Kalmia microphylla, Kalmia polifolia, Kalmia occidentalis, Cistus chamaerhodendros or Kalmia lucida [American laurel, broad-leafed laurel, calico bush, spoon wood, sheep laurel, alpine laurel, bog laurel, western bog-laurel, swamp-laurel]; Euphorbiaceae such as the genera Manihot, Janipha, Jatropha, Ricinus e.g. the species Manihot utilissima, Janipha manihot, Jatropha manihot., Manihot aipil, Manihot dulcis, Manihot manihot, Manihot melanobasis, Manihot esculenta [manihot, arrowroot, tapioca, cassava] or Ricinus communis [castor bean, Castor Oil Bush, Castor Oil Plant, Palma Christi, Wonder Tree]; Fabaceae such as the genera Pisum, Albizia, Cathormion, Feuillea, Inga, Pithecolobium, Acacia, Mimosa, Medicajo, Glycine, Dolichos, Phaseolus, Soja e.g. the species Pisum sativum, Pisum arvense, Pisum humile [pea], Albizia berteriana, Albizia julibrissin, Albizia lebbeck, Acacia berteriana, Acacia littoralis, Albizia berteriana, Albizzia berteriana, Cathormion berteriana, Feuillea berteriana, Inga fragrans, Pithecellobium berterianum, Pithecellobium fragrans, Pithecolobium berterianum, Pseudalbizzia berteriana, Acacia julibrissin, Acacia nemu, Albizia nemu, Feuilleea julibrissin, Mimosa julibrissin, Mimosa speciosa, Sericanrda julibrissin, Acacia lebbeck, Acacia macrophylla, Albizia lebbek, Feuilleea lebbeck, Mimosa lebbeck, Mimosa speciosa [bastard logwood, silk tree, East Indian Walnut], Medicago sativa, Medicago falcata, Medicago varia [alfalfa] Glycine max Dolichos soja, Glycine gracilis, Glycine hispida, Phaseolus max, Soja hispida or Soja max [soybean]; Geraniaceae such as the genera Pelargonium, Cocos, Oleum e.g. the species Cocos nucifera, Pelargonium grossularioides or Oleum cocois [coconut]; Gramineae such as the genera Saccharum e.g. the species Saccharum officinarum; Juglandaceae such as the genera Juglans, Wallia e.g. the species Juglans regia, Juglans ailanthifolia, Juglans sieboldiana, Juglans cinerea, Wallia cinerea, Juglans bixbyi, Juglans californica, Juglans hindsii, Juglans intermedia, Juglans jamaicensis, Juglans major, Juglans microcarpa, Juglans nigra or Wallia nigra [walnut, black walnut, common walnut, persian walnut, white walnut, butternut, black walnut]; Lauraceae such as the genera Persea, Laurus e.g. the species laurel Laurus nobilis [bay, laurel, bay laurel, sweet bay], Persea americana Persea americana, Persea gratissima or Persea persea [avocado]; Leguminosae such as the genera Arachis e.g. the species Arachis hypogaea [peanut]; Linaceae such as the genera Linum, Adenolinum e.g. the species Linum usitatissimum, Linum humile, Linum austriacum, Linum bienne, Linum angustifolium, Linum catharticum, Linum flavum, Linum grandiflorum, Adenolinum grandiflorum, Linum lewisii, Linum narbonense, Linum perenne, Linum perenne var. lewisii, Linum pratense or Linum trigynum [flax, linseed]; Lythrarieae such as the genera Punica e.g. the species Punica granatum [pomegranate]; Malvaceae such as the genera Gossypium e.g. the species Gossypium hirsutum, Gossypium arboreum, Gossypium barbadense, Gossypium herbaceum or Gossypium thurberi [cotton]; Musaceae such as the genera Musa e.g. the species Musa nana, Musa acuminata, Musa paradisiaca, Musa spp. [banana]; Onagraceae such as the genera Camissonia, Oenothera e.g. the species Oenothera biennia or Camissonia brevipes [primrose, evening primrose]; Palmae such as the genera Elacis e.g. the species Elaeis guineensis [oil plam]; Papaveraceae such as the genera Papaver e.g. the species Papaver orientale, Papaver rhoeas, Papaver dubium [poppy, oriental poppy, corn poppy, field poppy, shirley poppies, field poppy, long-headed poppy, long-pod poppy]; Pedaliaceae such as the genera Sesamum e.g. the species Sesamum indicum [sesame]; Piperaceae such as the genera Piper, Artanthe, Peperomia, Steffensia e.g. the species Piper aduncum, Piper amalago, Piper angustifolium, Piper auritum, Piper betel, Piper cubeba, Piper longum, Piper nigrum, Piper retrofracturn, Artanthe adunca, Artanthe elongata, Peperomia elongata, Piper elongatum, Steffensia elongata. [Cayenne pepper, wild pepper]; Poaceae such as the genera Hordeum, Secale, Avena, Sorghum, Andropogon, Holcus, Panicum, Oryza, Zea, Triticum e.g. the species Hordeum vulgare, Hordeum jubatum, Hordeum murinum, Hordeum secalinum, Hordeum distichon Hordeum aegiceras, Hordeum hexastichon., Hordeum hexastichum, Hordeum irregulare, Hordeum sativum, Hordeum secalinum [barley, pearl barley, foxtail barley, wall barley, meadow barley], Secale cereale [rye], Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida [oat], Sorghum bicolor, Sorghum halepense, Sorghum saccharatum, Sorghum vulgare, Andropogon drummondii, Holcus bicolor, Holcus sorghum, Sorghum aethiopicum, Sorghum arundinaceum, Sorghum caffrorum, Sorghum cemuum, Sorghum dochna, Sorghum drummondii, Sorghum durra, Sorghum guineense, Sorghum lanceolatum, Sorghum nervosum, Sorghum saccharatum, Sorghum subglabrescens, Sorghum verticilliflorum, Sorghum vulgare, Holcus halepensis, Sorghum miliaceum millet, Panicum militaceum [Sorghum, millet], Oryza sativa, Oryza latifolia [rice], Zea mays [corn, maize] Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybemum, Triticum macha, Triticum sativum or Triticum vulgare [wheat, bread wheat, common wheat], Proteaceae such as the genera Macadamia e.g. the species Macadamia intergrifolia [macadamia]; Rubiaceae such as the genera Coffea e.g. the species Cofea spp., Coffea arabica, Coffea canephora or Coffea liberica [coffee]; Scrophulariaceae such as the genera Verbascum e.g. the species Verbascum blattaria, Verbascum chaixii, Verbascum densiflorum, Verbascum lagurus, Verbascum longifolium, Verbascum lychnitis, Verbascum nigrum, Verbascum olympicum, Verbascum phlomoides, Verbascum phoenicum, Verbascum pulverulentum or Verbascum thapsus [mullein, white moth mullein, nettle-leaved mullein, dense-flowered mullein, silver mullein, long-leaved mullein, white mullein, dark mullein, greek mullein, orange mullein, purple mullein, hoary mullein, great mullein]; Solanaceae such as the genera Capsicum, Nicotiana, Solanum, Lycopersicon e.g. the species Capsicum annuum, Capsicum annuum var. glabriusculum, Capsicum frutescens [pepper], Capsicum annuum [paprika], Nicotiana tabacum, Nicotiana alata, Nicotiana attenuata, Nicotiana glauca, Nicotiana langsdorffii, Nicotiana obtusifolia, Nicotiana quadrivalvis, Nicotiana repanda, Nicotiana rustica, Nicotiana sylvestris [tobacco], Solanum tuberosum [potato], Solanum melongena [egg-plant] (Lycopersicon esculentum, Lycopersicon lycopersicum., Lycopersicon pyriforme, Solanum integrifolium or Solanum lycopersicum [tomato]; Sterculiaceae such as the genera Theobroma e.g. the species Theobroma cacao [cacao]; Theaceae such as the genera Camellia e.g. the species Camellia sinensis) [tea].
The introduction of the nucleic acids according to the invention, the expression cassette or the vector into organisms, plants for example, can in principle be done by all of the methods known to those skilled in the art. The introduction of the nucleic acid sequences gives rise to recombinant or transgenic organisms.
Unless otherwise specified, the terms “polynucleotides”, “nucleic acid” and “nucleic acid molecule” as used herein are interchangeably. Unless otherwise specified, the terms “peptide”, “polypeptide” and “protein” are interchangeably in the present context. The term “sequence” may relate to polynucleotides, nucleic acids, nucleic acid molecules, peptides, polypeptides and proteins, depending on the context in which the term “sequence” is used. The terms “gene(s)”, “polynucleotide”, “nucleic acid sequence”, “nucleotide sequence”, or “nucleic acid molecule(s)” as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. The terms refer only to the primary structure of the molecule.
Thus, the terms “gene(s)”, “polynucleotide”, “nucleic acid sequence”, “nucleotide sequence”, or “nucleic acid molecule(s)” as used herein include double- and single-stranded DNA and RNA. They also include known types of modifications, for example, methylation, “caps”, substitutions of one or more of the naturally occurring nucleotides with an analog. Preferably, the DNA or RNA sequence of the invention comprises a coding sequence encoding the herein defined polypeptide.
A “coding sequence” is a nucleotide sequence, which is transcribed into mRNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5′-terminus and a translation stop codon at the 3′-terminus. A coding sequence can include, but is not limited to mRNA, cDNA, recombinant nucleotide sequences or genomic DNA, while introns may be present as well under certain circumstances.
The transfer of foreign genes into the genome of a plant is called transformation. In doing this the methods described for the transformation and regeneration of plants from plant tissues or plant cells are utilized for transient or stable transformation. Suitable methods are protoplast transformation by poly(ethylene glycol)-induced DNA uptake, the “biolistic” method using the gene cannon—referred to as the particle bombardment method, electroporation, the incubation of dry embryos in DNA solution, microinjection and gene transfer mediated by Agrobacterium. Said methods are described by way of example in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S. D. Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225). The nucleic acids or the construct to be expressed is preferably cloned into a vector which is suitable for transforming Agrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711). Agrobacteria transformed by such a vector can then be used in known manner for the transformation of plants, in particular of crop plants such as by way of example tobacco plants, for example by bathing bruised leaves or chopped leaves in an agrobacterial solution and then culturing them in suitable media. The transformation of plants by means of Agrobacterium tumefaciens is described, for example, by Höfgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or is known inter alia from F. F. White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S. D. Kung and R. Wu, Academic Press, 1993, pp. 15-38.
Agrobacteria transformed by an expression vector according to the invention may likewise be used in known manner for the transformation of plants such as test plants like Arabidopsis or crop plants such as cereal crops, corn, oats, rye, barley, wheat, soybean, rice, cotton, sugar beet, canola, sunflower, flax, hemp, potatoes, tobacco, tomatoes, carrots, paprika, oilseed rape, tapioca, cassava, arrowroot, tagetes, alfalfa, lettuce and the various tree, nut and vine species, in particular of oil-containing crop plants such as soybean, peanut, castor oil plant, sunflower, corn, cotton, flax, oilseed rape, coconut, oil palm, safflower (Carthamus tinctorius) or cocoa bean, e.g. by bathing bruised leaves or chopped leaves in an agrobacterial solution and then culturing them in suitable media.
The genetically modified plant cells may be regenerated by all of the methods known to those skilled in the art. Appropriate methods can be found in the publications referred to above by S. D. Kung and R. Wu, Potrykus or Höfgen and Willmitzer.
Accordingly, a further aspect of the invention relates to transgenic organisms transformed by at least one nucleic acid sequence, expression cassette or vector according to the invention as well as cells, cell cultures, tissue, parts—such as, for example, leaves, roots, etc. in the case of plant organisms—or reproductive material derived from such organisms. The terms “host organism”, “host cell”, “recombinant (host) organism” and “transgenic (host) cell” are used here interchangeably. Of course these terms relate not only to the particular host organism or the particular target cell but also to the descendants or potential descendants of these organisms or cells. Since, due to mutation or environmental effects certain modifications may arise in successive generations, these descendants need not necessarily be identical with the parental cell but nevertheless are still encompassed by the term as used here.
For the purposes of the invention “transgenic” or “recombinant” means with regard for example to a nucleic acid sequence, an expression cassette (=gene construct, nucleic acid construct) or a vector containing the nucleic acid sequence according to the invention or an organism transformed by the nucleic acid sequences, expression cassette or vector according to the invention all those constructions produced by genetic engineering methods in which either
a) the nucleic acid sequence depicted in table I, column 5 or 7 or its derivatives or parts thereof or b) a genetic control sequence functionally linked to the nucleic acid sequence described under (a), for example a 3′- and/or 5′-genetic control sequence such as a promoter or terminator, or
c) (a) and (b)
are not found in their natural, genetic environment or have been modified by genetic engineering methods, wherein the modification may by way of example be a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. Natural genetic environment means the natural genomic or chromosomal locus in the organism of origin or inside the host organism or presence in a genomic library. In the case of a genomic library the natural genetic environment of the nucleic acid sequence is preferably retained at least in part. The environment borders the nucleic acid sequence at least on one side and has a sequence length of at least 50 bp, preferably at least 500 bp, particularly preferably at least 1,000 bp, most particularly preferably at least 5,000 bp. A naturally occurring expression cassette—for example the naturally occurring combination of the natural promoter of the nucleic acid sequence according to the invention with the corresponding delta-8-desaturase, delta-9-elongase and/or delta-5-desaturase gene—turns into a transgenic expression cassette when the latter is modified by unnatural, synthetic (“artificial”) methods such as by way of example a mutagenation. Appropriate methods are described by way of example in U.S. Pat. No. 5,565,350 or WO 00/15815.
Suitable organisms or host organisms for the nucleic acid, expression cassette or vector according to the invention are advantageously in principle all organisms, which are suitable for the expression of recombinant genes as described above. Further examples which may be mentioned are plants such as Arabidopsis, Asteraceae such as Calendula or crop plants such as soybean, peanut, castor oil plant, sunflower, flax, corn, cotton, flax, oilseed rape, coconut, oil palm, safflower (Carthamus tinctorius) or cocoa bean.
In one embodiment of the invention host plants for the nucleic acid, expression cassette or vector according to the invention are selected from the group comprising corn, soy, oil seed rape (including canola and winter oil seed reap), cotton, wheat and rice.
A further object of the invention relates to the use of a nucleic acid construct, e.g. an expression cassette, containing DNA sequences encoding polypeptides shown in table II or DNA sequences hybridizing therewith for the transformation of plant cells, tissues or parts of plants.
In doing so, depending on the choice of promoter, the sequences shown in table I can be expressed specifically in the leaves, in the seeds, the nodules, in roots, in the stem or other parts of the plant. Those transgenic plants overproducing sequences as depicted in table I, the reproductive material thereof, together with the plant cells, tissues or parts thereof are a further object of the present invention.
The expression cassette or the nucleic acid sequences or construct according to the invention containing sequences according to table I can, moreover, also be employed for the transformation of the organisms identified by way of example above such as bacteria, yeasts, filamentous fungi and plants.
Within the framework of the present invention, increased GABA content means, for example, the artificially acquired trait of increased GABA content, concentration, activity due to functional over expression of polypeptide sequences of table II encoded by the corresponding nucleic acid molecules as depicted in table I, column 5 or 7 and/or homologs in the organisms according to the invention, advantageously in the transgenic plants according to the invention, by comparison with the nongenetically modified initial plants at least for the duration of at least one plant generation.
A constitutive expression of the polypeptide sequences of the of table II encoded by the corresponding nucleic acid molecule as depicted in table I, column 5 or 7 and/or homologs is, moreover, advantageous. On the other hand, however, an inducible expression may also appear desirable. Expression of the polypeptide sequences of the invention can be either direct to the cytsoplasm or the organelles preferably the plastids of the host cells, preferably the plant cells.
The efficiency of the expression of the sequences of the of table II encoded by the corresponding nucleic acid molecule as depicted in table I, column 5 or 7 and/or homologs can be determined, for example, in vitro by shoot meristem propagation. In addition, an expression of the sequences of of table II encoded by the corresponding nucleic acid molecule as depicted in table I, column 5 or 7 and/or homologs modified in nature and level and its effect on the metabolic pathways performance can be tested on test plants in greenhouse trials.
An additional object of the invention comprises transgenic organisms such as transgenic plants transformed by an expression cassette containing sequences of as depicted in table I, column 5 or 7 according to the invention or DNA sequences hybridizing therewith, as well as transgenic cells, tissue, parts and reproduction material of such plants. Particular preference is given in this case to transgenic crop plants such as by way of example barley, wheat, rye, oats, corn, soybean, rice, cotton, sugar beet, oilseed rape and canola, sunflower, flax, hemp, thistle, potatoes, tobacco, tomatoes, tapioca, cassava, arrowroot, alfalfa, lettuce and the various tree, nut and vine species.
In one embodiment of the invention transgenic plants transformed by an expression cassette containing sequences of as depicted in table I, column 5 or 7 according to the invention or DNA sequences hybridizing therewith are selected from the group comprising corn, soy, oil seed rape (including canola and winter oil seed rape), cotton, wheat and rice.
For the purposes of the invention plants are mono- and dicotyledonous plants, mosses or algae.
A further refinement according to the invention are transgenic plants as described above which contain a nucleic acid sequence or construct according to the invention or a expression cassette according to the invention.
However, transgenic also means that the nucleic acids according to the invention are located at their natural position in the genome of an organism, but that the sequence has been modified in comparison with the natural sequence and/or that the regulatory sequences of the natural sequences have been modified. Preferably, transgenic/recombinant is to be understood as meaning the transcription of the nucleic acids of the invention and shown in table I, occurs at a non-natural position in the genome, that is to say the expression of the nucleic acids is homologous or, preferably, heterologous. This expression can be transiently or of a sequence integrated stably into the genome.
The term “transgenic plants” used in accordance with the invention also refers to the progeny of a transgenic plant, for example the T1, T2, T3 and subsequent plant generations or the BC1, BC2, BC3 and subsequent plant generations. Thus, the transgenic plants according to the invention can be raised and selfed or crossed with other individuals in order to obtain further transgenic plants according to the invention. Transgenic plants may also be obtained by propagating transgenic plant cells vegetatively.
The present invention also relates to transgenic plant material, which can be derived from a transgenic plant population according to the invention. Such material includes plant cells and certain tissues, organs and parts of plants in all their manifestations, such as seeds, leaves, anthers, fibers, tubers, roots, root hairs, stems, embryo, calli, cotelydons, petioles, harvested material, plant tissue, reproductive tissue and cell cultures, which are derived from the actual transgenic plant and/or can be used for bringing about the transgenic plant.
Any transformed plant obtained according to the invention can be used in a conventional breeding scheme or in in vitro plant propagation to produce more transformed plants with the same characteristics and/or can be used to introduce the same characteristic in other varieties of the same or related species. Such plants are also part of the invention. Seeds obtained from the transformed plants genetically also contain the same characteristic and are part of the invention. As mentioned before, the present invention is in principle applicable to any plant and crop that can be transformed with any of the transformation method known to those skilled in the art.
Advantageous inducible plant promoters are by way of example the PRP1 promoter [Ward et al., Plant. Mol. Biol. 22(1993), 361-366], a promoter inducible by benzenesulfonamide (EP 388 186), a promoter inducible by tetracycline [Gatz et al., (1992) Plant J. 2,397-404], a promoter inducible by salicylic acid (WO 95/19443), a promoter inducible by abscisic acid (EP 335 528) and a promoter inducible by ethanol or cyclohexanone (WO93/21334). Other examples of plant promoters which can advantageously be used are the promoter of cytosolic FBPase from potato, the ST-LSI promoter from potato (Stockhaus et al., EMBO J. 8 (1989) 2445-245), the promoter of phosphoribosyl pyrophosphate amidotransferase from Glycine max (see also gene bank accession number U87999) or a nodiene-specific promoter as described in EP 249 676. Particular advantageous are those promoters which ensure expression expression upon the early onset of environmental stress like for example drought or cold.
In one embodiment seed-specific promoters may be used for monocotylodonous or dicotylodonous plants.
In principle all natural promoters with their regulation sequences can be used like those named above for the expression cassette according to the invention and the method according to the invention. Over and above this, synthetic promoters may also advantageously be used.
In the preparation of an expression cassette various DNA fragments can be manipulated in order to obtain a nucleotide sequence, which usefully reads in the correct direction and is equipped with a correct reading frame. To connect the DNA fragments (=nucleic acids according to the invention) to one another regulatory element or adaptors or linkers may be attached to the fragments.
The promoter and the terminator regions can usefully be provided in the transcription direction with a linker or polylinker containing one or more restriction points for the insertion of this sequence. Generally, the linker has 1 to 10, mostly 1 to 8, preferably 2 to 6, restriction points. In general the size of the linker inside the regulatory region is less than 100 bp, frequently less than 60 bp, but at least 5 bp. The promoter may be both native or homologous as well as foreign or heterologous to the host organism, for example to the host plant. In the 5′-3′ transcription direction the expression cassette contains the promoter, a DNA sequence which shown in table I and a region for transcription termination. Different termination regions can be exchanged for one another in any desired fashion.
As also used herein, the terms “nucleic acid” and “nucleic acid molecule” are intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. This term also encompasses untranslated sequence located at both the 3′ and 5′ ends of the coding region of the gene: at least about 1000 nucleotides of sequence upstream from the 5′ end of the coding region and at least about 200 nucleotides of sequence downstream from the 3′ end of the coding region of the gene. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
An “isolated” nucleic acid molecule is one that is substantially separated from other nucleic acid molecules, which are present in the natural source of the nucleic acid. That means other nucleic acid molecules are present in an amount less than 5% based on weight of the amount of the desired nucleic acid, preferably less than 2% by weight, more preferably less than 1% by weight, most preferably less than 0.5% by weight. Preferably, an “isolated” nucleic acid is free of some of the sequences that naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated stress related protein encoding nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be free from some of the other cellular material with which it is naturally associated, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.
A nucleic acid molecule of the present invention, e.g., a nucleic acid molecule encoding an GABA-related Proteins or a portion thereof which confers tolerance and/or resistance to environmental stress and increased biomass production in plants, can be isolated using standard molecular biological techniques and the sequence information provided herein. For example, an Arabidopsis thaliana stress related protein encoding cDNA can be isolated from a A. thaliana c-DNA library or a Synechocystis sp., Brassica napus, Glycine max, Zea mays or Oryza sativa stress related protein encoding cDNA can be isolated from a Synechocystis sp., Brassica napus, Glycine max, Zea mays or Oryza sativa c-DNA library respectively using all or portion of one of the sequences shown in table I. Moreover, a nucleic acid molecule encompassing all or a portion of one of the sequences of table I can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon this sequence. For example, mRNA can be isolated from plant cells (e.g., by the guanidinium-thiocyanate extraction procedure of Chirgwin et al., 1979 Biochemistry 18:5294-5299) and c-DNA can be prepared using reverse transcriptase (e.g., Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, Md.; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Petersburg, Fla.). Synthetic oligonucleotide primers for polymerase chain reaction amplification can be designed based upon one of the nucleotide sequences shown in table I. A nucleic acid molecule of the invention can be amplified using cDNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid molecule so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to GABA-related Proteins encoding nucleotide sequence can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
In a preferred embodiment, an isolated nucleic acid molecule of the invention comprises one of the nucleotide sequences shown in table I encoding the GABA-related Proteins (i.e., the “coding region”), as well as 5′ untranslated sequences and 3′ untranslated sequences.
Moreover, the nucleic acid molecule of the invention can comprise only a portion of the coding region of one of the sequences Of the nucleic acid of table I, for example, a fragment which can be used as a probe or primer or a fragment encoding a biologically active portion of a GABA-related Proteins.
Portions of proteins encoded by the GABA-related Proteins encoding nucleic acid molecules of the invention are preferably biologically active portions described herein. As used herein, the term “biologically active portion of” a GABA-related Proteins is intended to include a portion, e.g., a domain/motif, of GABA-related protein that participates in GABA increase and preferably in enhanced nutrient efficiency use or stress tolerance and/or resistance response in a plant. To determine whether a GABA-related Proteins, or a biologically active portion thereof, results in GABA increase and preferably in increased stress tolerance or nutrient efficiency use in a plant, a metabolite analysis of a plant comprising the GABA-related Proteins may be performed. Such analysis methods are well known to those skilled in the art, as detailed in the Examples. More specifically, nucleic acid fragments encoding biologically active portions of a GABA-related Proteins can be prepared by isolating a portion of one of the sequences of the nucleic acid of table I expressing the encoded portion of the GABA-related Proteins or peptide (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the GABA-related Proteins or peptide.
Biologically active portions of a GABA-related Protein are encompassed by the present invention and include peptides comprising amino acid sequences derived from the amino acid sequence of a GABA-related Protein encoding gene, or the amino acid sequence of a protein homologous to a GABA-related Protein, which include fewer amino acids than a full length GABA-related Proteinsor the full length protein which is homologous to a GABA-related Protein, and exhibits at least some enzymatic or biological activity of a GABA-related Protein. Typically, biologically active portions (e.g., peptides which are, for example, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids in length) comprise a domain or motif with at least one activity of a GABA-related Protein. Moreover, other biologically active portions in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the activities described herein. Preferably, the biologically active portions of a GABA-related Protein include one or more selected domains/motifs or portions thereof having biological activity.
The term “biological active portion” or “biological activity” means a polypeptide as depicted in table II, column 3 or a portion of said polypeptide which still has at least 10% or 20%, preferably 20%, 30%, 40% or 50%, especially preferably 60%, 70% or 80% of the enzymatic or biological activity of the natural or starting enzyme or protein.
In the process according to the invention nucleic acid sequences can be used, which, if appropriate, contain synthetic, non-natural or modified nucleotide bases, which can be incorporated into DNA or RNA. Said synthetic, non-natural or modified bases can for example increase the stability of the nucleic acid molecule outside or inside a cell. The nucleic acid molecules of the invention can contain the same modifications as afore-mentioned.
As used in the present context the term “nucleic acid molecule” may also encompass the untranslated sequence located at the 3′ and at the 5′ end of the coding gene region, for example at least 500, preferably 200, especially preferably 100, nucleotides of the sequence upstream of the 5′ end of the coding region and at least 100, preferably 50, especially preferably 20, nucleotides of the sequence downstream of the 3′ end of the coding gene region. It is often advantageous only to choose the coding region for cloning and expression purposes.
Preferably, the nucleic acid molecule used in the process according to the invention or the nucleic acid molecule of the invention is an isolated nucleic acid molecule.
An “isolated” polynucleotide or nucleic acid molecule is separated from other polynucleotides or nucleic acid molecules, which are present in the natural source of the nucleic acid molecule. An isolated nucleic acid molecule may be a chromosomal fragment of several kb, or preferably, a molecule only comprising the coding region of the gene. Accordingly, an isolated nucleic acid molecule of the invention may comprise chromosomal regions, which are adjacent 5′ and 3′ or further adjacent chromosomal regions, but preferably comprises no such sequences which naturally flank the nucleic acid molecule sequence in the genomic or chromosomal context in the organism from which the nucleic acid molecule originates (for example sequences which are adjacent to the regions encoding the 5′- and 3′-UTRs of the nucleic acid molecule). In various embodiments, the isolated nucleic acid molecule used in the process according to the invention may, for example comprise less than approximately 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb nucleotide sequences which naturally flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid molecule originates.
The nucleic acid molecules used in the process, for example the polynucleotide of the invention or of a part thereof can be isolated using molecular-biological standard techniques and the sequence information provided herein. Also, for example a homologous sequence or homologous, conserved sequence regions at the DNA or amino acid level can be identified with the aid of comparison algorithms. The former can be used as hybridization probes under standard hybridization techniques (for example those described in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) for isolating further nucleic acid sequences useful in this process.
A nucleic acid molecule encompassing a complete sequence of the nucleic acid molecules used in the process, for example the polynucleotide of the invention, or a part thereof may additionally be isolated by polymerase chain reaction, oligonucleotide primers based on this sequence or on parts thereof being used. For example, a nucleic acid molecule comprising the complete sequence or part thereof can be isolated by polymerase chain reaction using oligonucleotide primers which have been generated on the basis of this very sequence. For example, mRNA can be isolated from cells (for example by means of the guanidinium thiocyanate extraction method of Chirgwin et al. (1979) Biochemistry 18:5294-5299) and cDNA can be generated by means of reverse transcriptase (for example Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, Md., or AMV reverse transcriptase, obtainable from Seikagaku America, Inc., St. Petersburg, Fla.).
Synthetic oligonucleotide primers for the amplification, e.g. as shown in table III, column 7, by means of polymerase chain reaction can be generated on the basis of a sequence shown herein, for example the sequence shown in table I, columns 5 and 7 or the sequences derived from table II, columns 5 and 7.
Moreover, it is possible to identify conserved protein motif or domain by carrying out protein sequence alignments with the polypeptide encoded by the nucleic acid molecules of the present invention, in particular with the sequences encoded by the nucleic acid molecule shown in, column 5 or 7 of Table I, from which conserved regions, and in turn, degenerate primers can be derived.
Conserved regions are those, which show a very little variation in the amino acid in one particular position of several homologs from different origin. The consensus sequence and polypeptide motifs shown in column 7 of Table IV are derived from said alignments. Moreover, it is possible to identify conserved regions from various organisms by carrying out protein sequence alignments with the polypeptide encoded by the nucleic acid of the present invention, in particular with the sequences encoding the polypeptide molecule shown in column 5 or 7 of Table II, from which conserved regions, and in turn, degenerate primers can be derived.
In one advantageous embodiment, in the method of the present invention the activity of a polypeptide is increased comprising or consisting of a consensus sequence or a polypeptide motif shown in table IV column 7 and in one another embodiment, the present invention relates to a polypeptide comprising or consisting of a consensus sequence or a polypeptide motif shown in table IV, column 7 whereby 20 or less, preferably 15 or 10, preferably 9, 8, 7, or 6, more preferred 5 or 4, even more preferred 3, even more preferred 2, even more preferred 1, most preferred 0 of the amino acids positions indicated can be replaced by any amino acid. In one embodiment not more than 15%, preferably 10%, even more preferred 5%, 4%, 3%, or 2%, most preferred 1% or 0% of the amino acid position indicated by a letter are/is replaced another amino acid. In one embodiment 20 or less, preferably 15 or 10, preferably 9, 8, 7, or 6, more preferred 5 or 4, even more preferred 3, even more preferred 2, even more preferred 1, most preferred 0 amino acids are inserted into a consensus sequence or protein motif.
The consensus sequence was derived from a multiple alignment of the sequences as listed in table II. The letters represent the one letter amino acid code and indicate that the amino acids are conserved in at least 80% of the aligned proteins, whereas the letter X stands for amino acids, which are not conserved in at least 80% of the aligned sequences. The consensus sequence starts with the first conserved amino acid in the alignment, and ends with the last conserved amino acid in the alignment of the investigated sequences. The number of given X indicates the distances between conserved amino acid residues, e.g. Y-x(21,23)-F means that conserved tyrosine and phenylalanine residues in the alignment are separated from each other by minimum 21 and maximum 23 amino acid residues in the alignment of all investigated sequences.
Conserved domains were identified from all sequences and are described using a subset of the standard Prosite notation, e.g the pattern Y-x(21,23)-[FW] means that a conserved tyrosine is separated by minimum 21 and maximum 23 amino acid residues from either a phenylalanine or tryptophane. Patterns had to match at least 80% of the investigated proteins.
Conserved patterns were identified with the software tool MEME version 3.5.1 or manually. MEME was developed by Timothy L. Bailey and Charles Elkan, Dept. of Computer Science and Engineering, University of California, San Diego, USA and is described by Timothy L. Bailey and Charles Elkan [Fitting a mixture model by expectation maximization to discover motifs in biopolymers, Proceedings of the Second International Conference on Intelligent Systems for Molecular Biology, pp. 28-36, AAAI Press, Menlo Park, Calif., 1994]. The source code for the stand-alone program is public available from the San Diego Supercomputer center (meme.sdsc.edu).
For identifying common motifs in all sequences with the software tool MEME, the following settings were used: -maxsize 500000, -nmotifs 15, -evt 0.001, -maxw 60, -distance 1e-3, -minsites number of sequences used for the analysis. Input sequences for MEME were non-aligned sequences in Fasta format. Other parameters were used in the default settings in this software version.
Prosite patterns for conserved domains were generated with the software tool Pratt version 2.1 or manually. Pratt was developed by Inge Jonassen, Dept. of Informatics, University of Bergen, Norway and is described by Jonassen et al. [I. Jonassen, J. F. Collins and D. G. Higgins, Finding flexible patterns in unaligned protein sequences, Protein Science 4 (1995), pp. 1587-1595; I. Jonassen, Efficient discovery of conserved patterns using a pattern graph, Submitted to CABIOS Febr. 1997]. The source code (ANSI C) for the stand-alone program is public available, e.g. at established Bioinformatic centers like EBI (European Bioinformatics Institute).
For generating patterns with the software tool Pratt, following settings were used: PL (max Pattern Length): 100, PN (max Nr of Pattern Symbols): 100, PX (max Nr of consecutive x's): 30, FN (max Nr of flexible spacers): 5, FL (max Flexibility): 30, FP (max Flex.Product): 10, ON (max number patterns): 50. Input sequences for Pratt were distinct regions of the protein sequences exhibiting high similarity as identified from software tool MEME. The minimum number of sequences, which have to match the generated patterns (CM, min Nr of Seqs to Match) was set to at least 80% of the provided sequences. Parameters not mentioned here were used in their default settings.
The Prosite patterns of the conserved domains can be used to search for protein sequences matching this pattern. Various established Bioinformatic centers provide public internet portals for using those patterns in database searches (e.g. PIR [Protein Information Resource, located at Georgetown University Medical Center] or ExPASy [Expert Protein Analysis System]). Alternatively, stand-alone software is available, like the program Fuzzpro, which is part of the EMBOSS software package. For example, the program Fuzzpro not only allows to search for an exact pattern-protein match but also allows to set various ambiguities in the performed search.
The alignment was performed with the software ClustalW (version 1.83) and is described by Thompson et al. [Thompson, J. D., Higgins, D. G. and Gibson, T.J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22:4673-4680]. The source code for the stand-alone program is public available from the European Molecular Biology Laboratory; Heidelberg, Germany. The analysis was performed using the default parameters of ClustalW v1.83 (gap open penalty: 10.0; gap extension penalty: 0.2; protein matrix: Gonnet; pprotein/DNA endgap: −1; protein/DNA gapdist: 4).
Degenerated primers can then be utilized by PCR for the amplification of fragments of novel proteins having above-mentioned activity, e.g. conferring the increased GABA content as compared to a corresponding non-transformed wild type after increasing the expression or activity or having the activity of a protein as shown in table II, column 3 or further functional homologs of the polypeptide of the invention from other organisms.
These fragments can then be utilized as hybridization probe for isolating the complete gene sequence. As an alternative, the missing 5′ and 3′ sequences can be isolated by means of RACE-PCR. A nucleic acid molecule according to the invention can be amplified using cDNA or, as an alternative, genomic DNA as template and suitable oligonucleotide primers, following standard PCR amplification techniques. The nucleic acid molecule amplified thus can be cloned into a suitable vector and characterized by means of DNA sequence analysis. Oligonucleotides, which correspond to one of the nucleic acid molecules used in the process can be generated by standard synthesis methods, for example using an automatic DNA synthesizer.
Nucleic acid molecules which are advantageously for the process according to the invention can be isolated based on their homology to the nucleic acid molecules disclosed herein using the sequences or part thereof as hybridization probe and following standard hybridization techniques under stringent hybridization conditions. In this context, it is possible to use, for example, isolated nucleic acid molecules of at least 15, 20, 25, 30, 35, 40, 50, 60 or more nucleotides, preferably of at least 15, 20 or 25 nucleotides in length which hybridize under stringent conditions with the above-described nucleic acid molecules, in particular with those which encompass a nucleotide sequence of the nucleic acid molecule used in the process of the invention or encoding a protein used in the invention or of the nucleic acid molecule of the invention. Nucleic acid molecules with 30, 50, 100, 250 or more nucleotides may also be used.
The term “homology” means that the respective nucleic acid molecules or encoded proteins are functionally and/or structurally equivalent. The nucleic acid molecules that are homologous to the nucleic acid molecules described above and that are derivatives of said nucleic acid molecules are, for example, variations of said nucleic acid molecules which represent modifications having the same biological function, in particular encoding proteins with the same or substantially the same biological function. They may be naturally occurring variations, such as sequences from other plant varieties or species, or mutations. These mutations may occur naturally or may be obtained by mutagenesis techniques. The allelic variations may be naturally occurring allelic variants as well as synthetically produced or genetically engineered variants. Structurally equivalents can, for example, be identified by testing the binding of said polypeptide to antibodies or computer based predictions. Structurally equivalent have the similar immunological characteristic, e.g. comprise similar epitopes.
By “hybridizing” it is meant that such nucleic acid molecules hybridize under conventional hybridization conditions, preferably under stringent conditions such as described by, e.g., Sambrook (Molecular Cloning; A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)) or in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989), 6.3.1-6.3.6.
According to the invention, DNA as well as RNA molecules of the nucleic acid of the invention can be used as probes. Further, as template for the identification of functional homologues Northern blot assays as well as Southern blot assays can be performed. The Northern blot assay advantageously provides further informations about the expressed gene product: e.g. expression pattern, occurance of processing steps, like splicing and capping, etc. The Southern blot assay provides additional information about the chromosomal localization and organization of the gene encoding the nucleic acid molecule of the invention.
A preferred, nonlimiting example of stringent hydridization conditions are hybridizations in 6×sodium chloride/sodium citrate (=SSC) at approximately 45° C., followed by one or more wash steps in 0.2×SSC, 0.1% SDS at 50 to 65° C., for example at 50° C., 55° C. or 60° C. The skilled worker knows that these hybridization conditions differ as a function of the type of the nucleic acid and, for example when organic solvents are present, with regard to the temperature and concentration of the buffer. The temperature under “standard hybridization conditions” differs for example as a function of the type of the nucleic acid between 42° C. and 58° C., preferably between 45° C. and 50° C. in an aqueous buffer with a concentration of 0.1×0.5×, 1×, 2×, 3×, 4× or 5×SSC (pH 7.2). If organic solvent(s) is/are present in the abovementioned buffer, for example 50% formamide, the temperature under standard conditions is approximately 40° C., 42° C. or 45° C. The hybridization conditions for DNA:DNA hybrids are preferably for example 0.1×SSC and 20° C., 25° C., 30° C., 35° C., 40° C. or 45° C., preferably between 30° C. and 45° C. The hybridization conditions for DNA:RNA hybrids are preferably for example 0.1×SSC and 30° C., 35° C., 40° C., 45° C., 50° C. or 55° C., preferably between 45° C. and 55° C. The abovementioned hybridization temperatures are determined for example for a nucleic acid approximately 100 bp (=base pairs) in length and a G+C content of 50% in the absence of formamide. The skilled worker knows to determine the hybridization conditions required with the aid of textbooks, for example the ones mentioned above, or from the following textbooks: Sambrook et al., “Molecular Cloning”, Cold Spring Harbor Laboratory, 1989; Hames and Higgins (Ed.) 1985, “Nucleic Acids Hybridization: A Practical Approach”, IRL Press at Oxford University Press, Oxford; Brown (Ed.) 1991, “Essential Molecular Biology: A Practical Approach”, IRL Press at Oxford University Press, Oxford.
A further example of one such stringent hybridization condition is hybridization at 4×SSC at 65° C., followed by a washing in 0.1×SSC at 65° C. for one hour. Alternatively, an exemplary stringent hybridization condition is in 50% formamide, 4×SSC at 42° C. Further, the conditions during the wash step can be selected from the range of conditions delimited by low-stringency conditions (approximately 2×SSC at 50° C.) and high-stringency conditions (approximately 0.2×SSC at 50° C., preferably at 65° C.) (20×SSC: 0.3M sodium citrate, 3M NaCl, pH 7.0). In addition, the temperature during the wash step can be raised from low-stringency conditions at room temperature, approximately 22° C., to higher-stringency conditions at approximately 65° C. Both of the parameters salt concentration and temperature can be varied simultaneously, or else one of the two parameters can be kept constant while only the other is varied. Denaturants, for example formamide or SDS, may also be employed during the hybridization. In the presence of 50% formamide, hybridization is preferably effected at 42° C. Relevant factors like i) length of treatment, ii) salt conditions, iii) detergent conditions, iv) competitor DNAs, v) temperature and vi) probe selection can be combined case by case so that not all possibilities can be mentioned herein.
Thus, in a preferred embodiment, Northern blots are prehybridized with Rothi-HybriQuick buffer (Roth, Karlsruhe) at 68° C. for 2 h. Hybridzation with radioactive labelled probe is done overnight at 68° C. Subsequent washing steps are performed at 68° C. with 1×SSC.
For Southern blot assays the membrane is prehybridized with Rothi-Hybri-Quick buffer (Roth, Karlsruhe) at 68° C. for 2 h. The hybridzation with radioactive labelled probe is conducted over night at 68° C. Subsequently the hybridization buffer is discarded and the filter shortly washed using 2×SSC; 0.1% SDS. After discarding the washing buffer new 2×SSC; 0.1% SDS buffer is added and incubated at 68° C. for 15 minutes. This washing step is performed twice followed by an additional washing step using 1×SSC; 0.1% SDS at 68° C. for 10 min.
Some examples of conditions for DNA hybridization (Southern blot assays) and wash step are shown hereinbelow:
Polypeptides having above-mentioned activity, i.e. conferring the increased GABA content as compared to a corresponding non-transformed wild type, derived from other organisms, can be encoded by other DNA sequences which hybridize to the sequences shown in table I, columns 5 and 7 under relaxed hybridization conditions and which code on expression for peptides conferring the increased GABA content as compared to a corresponding non-transformed wild type.
Further, some applications have to be performed at low stringency hybridisation conditions, without any consequences for the specificity of the hybridisation. For example, a Southern blot analysis of total DNA could be probed with a nucleic acid molecule of the present invention and washed at low stringency (55° C. in 2×SSPE, 0.1% SDS). The hybridisation analysis could reveal a simple pattern of only genes encoding polypeptides of the present invention or used in the process of the invention, e.g. having herein-mentioned activity of increasing the tolerance and/or resistance to environmental stress and the biomass production as compared to a corresponding non-transformed wild type plant cell, plant or part thereof. A further example of such low-stringent hybridization conditions is 4×SSC at 50° C. or hybridization with 30 to 40% formamide at 42° C. Such molecules comprise those which are fragments, analogues or derivatives of the polypeptide of the invention or used in the process of the invention and differ, for example, by way of amino acid and/or nucleotide deletion(s), insertion(s), substitution (s), addition(s) and/or recombination (s) or any other modification(s) known in the art either alone or in combination from the above-described amino acid sequences or their underlying nucleotide sequence(s). However, it is preferred to use high stringency hybridisation conditions.
Hybridization should advantageously be carried out with fragments of at least 5, 10, 15, 20, 25, 30, 35 or 40 bp, advantageously at least 50, 60, 70 or 80 bp, preferably at least 90, 100 or 110 bp. Most preferably are fragments of at least 15, 20, 25 or 30 bp. Preferably are also hybridizations with at least 100 bp or 200, very especially preferably at least 400 bp in length. In an especially preferred embodiment, the hybridization should be carried out with the entire nucleic acid sequence with conditions described above.
The terms “fragment”, “fragment of a sequence” or “part of a sequence” mean a truncated sequence of the original sequence referred to. The truncated sequence (nucleic acid or protein sequence) can vary widely in length; the minimum size being a sequence of sufficient size to provide a sequence with at least a comparable function and/or activity of the original sequence referred to or hybidizing with the nucleic acid molecule of the invention or used in the process of the invention under stringend conditions, while the maximum size is not critical. In some applications, the maximum size usually is not substantially greater than that required to provide the desired activity and/or function(s) of the original sequence.
Typically, the truncated amino acid sequence will range from about 5 to about 310 amino acids in length. More typically, however, the sequence will be a maximum of about 250 amino acids in length, preferably a maximum of about 200 or 100 amino acids. It is usually desirable to select sequences of at least about 10, 12 or 15 amino acids, up to a maximum of about 20 or 25 amino acids.
The term “epitope” relates to specific immunoreactive sites within an antigen, also known as antigenic determinates. These epitopes can be a linear array of monomers in a polymeric composition—such as amino acids in a protein—or consist of or comprise a more complex secondary or tertiary structure. Those of skill will recognize that immunogens (i.e., substances capable of eliciting an immune response) are antigens; however, some antigen, such as haptens, are not immunogens but may be made immunogenic by coupling to a carrier molecule. The term “antigen” includes references to a substance to which an antibody can be generated and/or to which the antibody is specifically immunoreactive.
In one embodiment the present invention relates to a epitope of the polypeptide of the present invention or used in the process of the present invention and confers an increased GABA content as compared to a corresponding non-transformed wild type.
The term “one or several amino acids” relates to at least one amino acid but not more than that number of amino acids, which would result in a homology of below 50% identity. Preferably, the identity is more than 70% or 80%, more preferred are 85%, 90%, 91%, 92%, 93%, 94% or 95%, even more preferred are 96%, 97%, 98%, or 99% identity.
Further, the nucleic acid molecule of the invention comprises a nucleic acid molecule, which is a complement of one of the nucleotide sequences of above mentioned nucleic acid molecules or a portion thereof. A nucleic acid molecule which is complementary to one of the nucleotide sequences shown in table I, columns 5 and 7 is one which is sufficiently complementary to one of the nucleotide sequences shown in table I, columns 5 and 7 such that it can hybridize to one of the nucleotide sequences shown in table I, columns 5 and 7, thereby forming a stable duplex. Preferably, the hybridisation is performed under stringent hybrization conditions. However, a complement of one of the herein disclosed sequences is preferably a sequence complement thereto according to the base pairing of nucleic acid molecules well known to the skilled person. For example, the bases A and G undergo base pairing with the bases T and U or C, resp. and visa versa. Modifications of the bases can influence the base-pairing partner.
The nucleic acid molecule of the invention comprises a nucleotide sequence which is at least about 30%, 35%, 40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more preferably at least about 70%, 80%, or 90%, and even more preferably at least about 95%, 97%, 98%, 99% or more homologous to a nucleotide sequence shown in table I, columns 5 and 7, or a portion thereof and preferably has above mentioned activity, in particular having a tolerance and/or resistance to environmental stress and biomass production increasing activity after increasing the activity or an activity of a gene product as shown in table II, column 3 by for example expression either in the cytsol or in an organelle such as a plastid or mitochondria or both, preferably in plastids.
The nucleic acid molecule of the invention comprises a nucleotide sequence which hybridizes, preferably hybridizes under stringent conditions as defined herein, to one of the nucleotide sequences shown in table I, columns 5 and 7, or a portion thereof and encodes a protein having above-mentioned activity, e.g. conferring an increased GABA content as compared to a corresponding non-transformed wild type by for example expression either in the cytsol or in an organelle such as a plastid or mitochondria or both, preferably in plastids, and optionally, the activity selected from the group consisting of: 60S ribosomal protein, ABC transporter permease protein, acetyltransferase, acyl-carrier protein, At4g32480-protein, At5g16650-protein, ATP-binding protein, Autophagy-related protein, auxin response factor, auxin transcription factor, b1003-protein, b1522-protein, b2739-protein, b3646-protein, B4029-protein, Branched-chain amino acid permease, calcium-dependent protein kinase, cytochrome c oxidase subunit VIII, elongation factor Tu, Factor arrest protein, fumarylacetoacetate hydrolase, geranylgeranyl pyrophosphate synthase, glucose dehydrogenase, glycosyl transferase, harpin-induced family protein, homocitrate synthase, hydrolase, isochorismate synthase, MFS-type transporter protein, microsomal beta-keto-reductase, polygalacturonase, protein phosphatase, pyruvate kinase, Sec-independent protein translocase subunit, serine protease, thioredoxin, thioredoxin family protein, transcriptional regulator, ubiquinone biosynthesis monooxygenase, and YHR213W-protein.
Throughout the context of this application the expression of the nucleotide sequences comprising the nucleotide sequences shown in table I, columns 5 and 7 or of the nucleotide sequences which encode a protein comprising the polypeptide sequences as shown in table II columns 5 or 7 in plastids is especially preferred if these sequences are shown in table I or II in the same line as an ORF (column 3), for which table I, II, III or IV shown “plastidic” in the column “target”.
Moreover, the nucleic acid molecule of the invention can comprise only a portion of the coding region of one of the sequences shown in table I, columns 5 and 7, for example a fragment which can be used as a probe or primer or a fragment encoding a biologically active portion of the polypeptide of the present invention or of a polypeptide used in the process of the present invention, i.e. having above-mentioned activity, e.g. conferring an increase of the tolerance and/or resistance to environmental stress and biomass production as compared to a corresponding non-transformed wild type plant cell, plant or part thereof if its activity is increased by for example expression either in the cytsol or in an organelle such as a plastid or mitochondria or both, preferably in plastids. The nucleotide sequences determined from the cloning of the present protein-according-to-the-invention-encoding gene allows for the generation of probes and primers designed for use in identifying and/or cloning its homologues in other cell types and organisms. The probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 15 preferably about 20 or 25, more preferably about 40, 50 or 75 consecutive nucleotides of a sense strand of one of the sequences set forth, e.g., in table I, columns 5 and 7, an anti-sense sequence of one of the sequences, e.g., set forth in table I, columns 5 and 7, or naturally occurring mutants thereof. Primers based on a nucleotide of invention can be used in PCR reactions to clone homologues of the polypeptide of the invention or of the polypeptide used in the process of the invention, e.g. as the primers described in the examples of the present invention, e.g. as shown in the examples. A PCR with the primers shown in table III, column 7 will result in a fragment of the gene product as shown in table II, column 3.
Primer sets are interchangable. The person skilled in the art knows to combine said primers to result in the desired product, e.g. in a full length clone or a partial sequence. Probes based on the sequences of the nucleic acid molecule of the invention or used in the process of the present invention can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. The probe can further comprise a label group attached thereto, e.g. the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a genomic marker test kit for identifying cells which express an polypeptide of the invention or used in the process of the present invention, such as by measuring a level of an encoding nucleic acid molecule in a sample of cells, e.g., detecting mRNA levels or determining, whether a genomic gene comprising the sequence of the polynucleotide of the invention or used in the process of the present invention has been mutated or deleted.
The nucleic acid molecule of the invention encodes a polypeptide or portion thereof which includes an amino acid sequence which is sufficiently homologous to the amino acid sequence shown in table II, columns 5 and 7 such that the protein or portion thereof maintains the ability to participate in the increase of the GABA content and preferably increase of further yield related trait as compared to a corresponding non-transformed wild type plant cell, plant or part thereof, in particular increasing the activity as mentioned above or as described in the examples in plants is comprised.
As used herein, the language “sufficiently homologous” refers to proteins or portions thereof which have amino acid sequences which include a minimum number of identical or equivalent amino acid residues (e.g., an amino acid residue which has a similar side chain as an amino acid residue in one of the sequences of the polypeptide of the present invention) to an amino acid sequence shown in table II, columns 5 and 7 such that the protein or portion thereof is able to participate in the increase of GABA content as compared to a corresponding non-transformed wild type. For examples having the activity of a protein as shown in table II, column 3 and as described herein.
In one embodiment, the nucleic acid molecule of the present invention comprises a nucleic acid that encodes a portion of the protein of the present invention. The protein is at least about 30%, 35%, 40%, 45% or 50%, preferably at least about 55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about 95%, 97%, 98%, 99% or more homologous to an entire amino acid sequence of table II, columns 5 and 7 and having above-mentioned activity, e.g. conferring an increased GABA content as compared to a corresponding non-transformed wild type by for example expression either in the cytsol or in an organelle such as a plastid or mitochondria or both, preferably in plastids.
Portions of proteins encoded by the nucleic acid molecule of the invention are preferably biologically active, preferably having above-mentioned annotated activity, e.g. conferring an increased GABA content as compared to a corresponding non-transformed wild type cell after increase of activity.
As mentioned herein, the term “biologically active portion” is intended to include a portion, e.g., a domain/motif, that confers increased GABA content as compared to a corresponding non-transformed wild type or has an immunological activity such that it is binds to an antibody binding specifically to the polypeptide of the present invention or a polypeptide used in the process of the present invention for increased GABA content as compared to a corresponding non-transformed wild type.
The invention further relates to nucleic acid molecules that differ from one of the nucleotide sequences shown in table I A, columns 5 and 7 (and portions thereof) due to degeneracy of the genetic code and thus encode a polypeptide of the present invention, in particular a polypeptide having above mentioned activity, e.g. as that polypeptides depicted by the sequence shown in table II, columns 5 and 7 or the functional homologues. Advantageously, the nucleic acid molecule of the invention comprises, or in an other embodiment has, a nucleotide sequence encoding a protein comprising, or in an other embodiment having, an amino acid sequence shown in table II, columns 5 and 7 or the functional homologues. In a still further embodiment, the nucleic acid molecule of the invention encodes a full length protein which is substantially homologous to an amino acid sequence shown in table II, columns 5 and 7 or the functional homologues. However, in a preferred embodiment, the nucleic acid molecule of the present invention does not consist of the sequence shown in table I, preferably table IA, columns 5 and 7.
In addition, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences may exist within a population. Such genetic polymorphism in the gene encoding the polypeptide of the invention or comprising the nucleic acid molecule of the invention may exist among individuals within a population due to natural variation.
As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding the polypeptide of the invention or comprising the nucleic acid molecule of the invention or encoding the polypeptide used in the process of the present invention, preferably from a crop plant or from a microorgansim useful for the method of the invention. Such natural variations can typically result in 1-5% variance in the nucleotide sequence of the gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in genes encoding a polypeptide of the invention or comprising a the nucleic acid molecule of the invention that are the result of natural variation and that do not alter the functional activity as described are intended to be within the scope of the invention.
Nucleic acid molecules corresponding to natural variants homologues of a nucleic acid molecule of the invention, which can also be a cDNA, can be isolated based on their homology to the nucleic acid molecules disclosed herein using the nucleic acid molecule of the invention, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
Accordingly, in another embodiment, a nucleic acid molecule of the invention is at least 15, 20, 25 or 30 nucleotides in length. Preferably, it hybridizes under stringent conditions to a nucleic acid molecule comprising a nucleotide sequence of the nucleic acid molecule of the present invention or used in the process of the present invention, e.g. comprising the sequence shown in table I, columns 5 and 7. The nucleic acid molecule is preferably at least 20, 30, 50, 100, 250 or more nucleotides in length.
The term “hybridizes under stringent conditions” is defined above. In one embodiment, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 30%, 40%, 50% or 65% identical to each other typically remain hybridized to each other. Preferably, the conditions are such that sequences at least about 70%, more preferably at least about 75% or 80%, and even more preferably at least about 85%, 90% or 95% or more identical to each other typically remain hybridized to each other.
Preferably, nucleic acid molecule of the invention that hybridizes under stringent conditions to a sequence shown in table I, columns 5 and 7 corresponds to a naturally-occurring nucleic acid molecule of the invention. As used herein, a “naturally-occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein). Preferably, the nucleic acid molecule encodes a natural protein having above-mentioned activity, e.g. conferring the tolerance and/or resistance to environmental stress and biomass production increase after increasing the expression or activity thereof or the activity of a protein of the invention or used in the process of the invention by for example expression the nucleic acid sequence of the gene product in the cytsol and/or in an organelle such as a plastid or mitochondria, preferably in plastids.
In addition to naturally-occurring variants of the sequences of the polypeptide or nucleic acid molecule of the invention as well as of the polypeptide or nucleic acid molecule used in the process of the invention that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into a nucleotide sequence of the nucleic acid molecule encoding the polypeptide of the invention or used in the process of the present invention, thereby leading to changes in the amino acid sequence of the encoded said polypeptide, without altering the functional ability of the polypeptide, preferably not decreasing said activity.
For example, nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues can be made in a sequence of the nucleic acid molecule of the invention or used in the process of the invention, e.g. shown in table I, columns 5 and 7.
A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of one without altering the activity of said polypeptide, whereas an “essential” amino acid residue is required for an activity as mentioned above, e.g. leading to an increase in the tolerance and/or resistance to environmental stress and biomass production as compared to a corresponding non-transformed wild type plant cell, plant or part thereof in an organism after an increase of activity of the polypeptide. Other amino acid residues, however, (e.g., those that are not conserved or only semi-conserved in the domain having said activity) may not be essential for activity and thus are likely to be amenable to alteration without altering said activity.
Further, a person skilled in the art knows that the codon usage between organisms can differ. Therefore, he may adapt the codon usage in the nucleic acid molecule of the present invention to the usage of the organism or the cell compartment for example of the plastid or mitochondria in which the polynuclestide or polypeptide is expressed.
Accordingly, the invention relates to nucleic acid molecules encoding a polypeptide having above-mentioned activity, in an organisms or parts thereof by for example expression either in the cytsol or in an organelle such as a plastid or mitochondria or both, preferably in plastids that contain changes in amino acid residues that are not essential for said activity. Such polypeptides differ in amino acid sequence from a sequence contained in the sequences shown in table II, columns 5 and 7 yet retain said activity described herein. The nucleic acid molecule can comprise a nucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence at least about 50% identical to an amino acid sequence shown in table II, columns 5 and 7 and is capable of participation in the increased GABA content production as compared to a corresponding non-transformed wild type plant cell, plant or part thereof after increasing its activity, e.g. its expression by for example expression either in the cytsol or in an organelle such as a plastid or mitochondria or both, preferably in plastids. Preferably, the protein encoded by the nucleic acid molecule is at least about 60% identical to the sequence shown in table II, columns 5 and 7, more preferably at least about 70% identical to one of the sequences shown in table II, columns 5 and 7, even more preferably at least about 80%, 90%, 95% homologous to the sequence shown in table II, columns 5 and 7, and most preferably at least about 96%, 97%, 98%, or 99% identical to the sequence shown in table II, columns 5 and 7.
To determine the percentage homology (=identity, herein used interchangeably) of two amino acid sequences or of two nucleic acid molecules, the sequences are written one underneath the other for an optimal comparison (for example gaps may be inserted into the sequence of a protein or of a nucleic acid in order to generate an optimal alignment with the other protein or the other nucleic acid).
The amino acid residues or nucleic acid molecules at the corresponding amino acid positions or nucleotide positions are then compared. If a position in one sequence is occupied by the same amino acid residue or the same nucleic acid molecule as the corresponding position in the other sequence, the molecules are homologous at this position (i.e. amino acid or nucleic acid “homology” as used in the present context corresponds to amino acid or nucleic acid “identity”. The percentage homology between the two sequences is a function of the number of identical positions shared by the sequences (i.e. % homology=number of identical positions/total number of positions ×100). The terms “homology” and “identity” are thus to be considered as synonyms.
For the determination of the percentage homology (=identity) of two or more amino acids or of two or more nucleotide sequences several computer software programs have been developed. The homology of two or more sequences can be calculated with for example the software fasta, which presently has been used in the version fasta 3 (W. R. Pearson and D. J. Lipman (1988), Improved Tools for Biological Sequence Comparison.PNAS 85:2444-2448; W. R. Pearson (1990) Rapid and Sensitive Sequence Comparison with FASTP and FASTA, Methods in Enzymology 183:63 98; W. R. Pearson and D. J. Lipman (1988) Improved Tools for Biological Sequence Comparison.PNAS 85:2444-2448; W. R. Pearson (1990); Rapid and Sensitive Sequence Comparison with FASTP and FASTAMethods in Enzymology 183:63-98).
Another useful program for the calculation of homologies of different sequences is the standard blast program, which is included in the Biomax pedant software (Biomax, Munich, Federal Republic of Germany). This leads unfortunately sometimes to sub-optimal results since blast does not always include complete sequences of the subject and the querry. Nevertheless as this program is very efficient it can be used for the comparison of a huge number of sequences. The following settings are typically used for such a comparisons of sequences:
-p Program Name [String]; -d Database [String]; default=nr; -i Query File [File In]; default=stdin; -e Expectation value (E) [Real]; default=10.0; -m alignment view options: 0=pairwise; 1=query-anchored showing identities; 2=query-anchored no identities; 3=flat query-anchored, show identities; 4=flat query-anchored, no identities; 5=query-anchored no identities and blunt ends; 6=flat query-anchored, no identities and blunt ends; 7=XML Blast output; 8=tabular; 9 tabular with comment lines [Integer]; default=0; -o BLAST report Output File [File Out] Optional; default=stdout; -F Filter query sequence (DUST with blastn, SEG with others) [String]; default=T; -G Cost to open a gap (zero invokes default behavior) [Integer]; default=0; -E Cost to extend a gap (zero invokes default behavior) [Integer]; default=0; -X X dropoff value for gapped alignment (in bits) (zero invokes default behavior); blastn 30, megablast 20, tblastx 0, all others 15 [Integer]; default=0; -I Show GI's in deflines [T/F]; default=F; -q Penalty for a nucleotide mismatch (blastn only) [Integer]; default=−3; -r Reward for a nucleotide match (blastn only) [Integer]; default=1; -v Number of database sequences to show one-line descriptions for (V) [Integer]; default=500; -b Number of database sequence to show alignments for (B) [Integer]; default=250; -f Threshold for extending hits, default if zero; blastp 11, blastn 0, blastx 12, tblastn 13; tblastx 13, megablast 0 [Integer]; default=0; -g Perfom gapped alignment (not available with tblastx) [T/F]; default=T; -Q Query Genetic code to use [Integer]; default=1; -D DB Genetic code (for tblast[nx] only) [Integer]; default=1; -a Number of processors to use [Integer]; default=1; -O SeqAlign file [File Out] Optional; -J Believe the query defline [T/F]; default=F; -M Matrix [String]; default=BLOSUM62; -W Word size, default if zero (blastn 11, megablast 28, all others 3) [Integer]; default=0; -z Effective length of the database (use zero for the real size) [Real]; default=0; -K Number of best hits from a region to keep (off by default, if used a value of 100 is recommended) [Integer]; default=0; -P 0 for multiple hit, 1 for single hit [Integer]; default=0; -Y Effective length of the search space (use zero for the real size) [Real]; default=0; -S Query strands to search against database (for blast[nx], and tblastx); 3 is both, 1 is top, 2 is bottom [Integer]; default=3; -T Produce HTML output [T/F]; default=F; -I Restrict search of database to list of GI's [String] Optional; -U Use lower case filtering of FASTA sequence [T/F] Optional; default=F; -y X dropoff value for ungapped extensions in bits (0.0 invokes default behavior); blastn 20, megablast 10, all others 7 [Real]; default=0.0; -Z X dropoff value for final gapped alignment in bits (0.0 invokes default behavior); blastn/megablast 50, tblastx 0, all others 25 [Integer]; default=0; -R PSITBLASTN checkpoint file [File In] Optional; -n MegaBlast search [T/F]; default=F; -L Location on query sequence [String] Optional; -A Multiple Hits window size, default if zero (blastn/megablast 0, all others 40 [Integer]; default=0; -w Frame shift penalty (00F algorithm for blastx) [Integer]; default=0; -t Length of the largest intron allowed in tblastn for linking HSPs (0 disables linking) [Integer]; default=0.
Results of high quality are reached by using the algorithm of Needleman and Wunsch or Smith and Waterman. Therefore programs based on said algorithms are preferred. Advantageously the comparisons of sequences can be done with the program PileUp (J. Mol. Evolution., 25, 351 (1987), Higgins et al., CABIOS 5, 151 (1989)) or preferably with the programs “Gap” and “Needle”, which are both based on the algorithms of Needleman and Wunsch (J. Mol. Biol. 48; 443 (1970)), and “BestFit”, which is based on the algorithm of Smith and Waterman (Adv. Appl. Math. 2; 482 (1981)). “Gap” and “BestFit” are part of the GCG software-package (Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711 (1991); Altschul et al., (Nucleic Acids Res. 25, 3389 (1997)), “Needle” is part of the The European Molecular Biology Open Software Suite (EMBOSS) (Trends in Genetics 16 (6), 276 (2000)). Therefore preferably the calculations to determine the percentages of sequence homology are done with the programs “Gap” or “Needle” over the whole range of the sequences. The following standard adjustments for the comparison of nucleic acid sequences were used for “Needle”: matrix: EDNAFULL, Gap_penalty: 10.0, Extend_penalty: 0.5. The following standard adjustments for the comparison of nucleic acid sequences were used for “Gap”: gap weight: 50, length weight: 3, average match: 10.000, average mismatch: 0.000.
For example a sequence, which has 80% homology with sequence SEQ ID NO: 42 at the nucleic acid level is understood as meaning a sequence which, upon comparison with the sequence SEQ ID NO: 42 by the above program “Needle” with the above parameter set, has a 80% identity.
Homology between two polypeptides is understood as meaning the identity of the amino acid sequence over in each case the entire sequence length which is calculated by comparison with the aid of the above program “Needle” using Matrix: EBLOSUM62, Gap_penalty: 8.0, Extend_penalty: 2.0.
For example a sequence which has a 80% homology with sequence SEQ ID NO: 43 at the protein level is understood as meaning a sequence which, upon comparison with the sequence SEQ ID NO: 43 by the above program “Needle” with the above parameter set, has a 80% identity.
Functional equivalents derived from one of the polypeptides as shown in table II, columns 5 and 7 according to the invention by substitution, insertion or deletion have at least 30%, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70% by preference at least 80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or 94%, very especially preferably at least 95%, 97%, 98% or 99% homology with one of the polypeptides as shown in table II, columns 5 and 7 according to the invention and are distinguished by essentially the same properties as the polypeptide as shown in table II, columns 5 and 7.
Functional equivalents derived from the nucleic acid sequence as shown in table I, columns 5 and 7 according to the invention by substitution, insertion or deletion have at least 30%, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70% by preference at least 80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or 94%, very especially preferably at least 95%, 97%, 98% or 99% homology with one of the polypeptides as shown in table II, columns 5 and 7 according to the invention and encode polypeptides having essentially the same properties as the polypeptide as shown in table II, columns 5 and 7.
“Essentially the same properties” of a functional equivalent is above all understood as meaning that the functional equivalent has above mentioned activity, by for example expression either in the cytsol or in an organelle such as a plastid or mitochondria or both, preferably in plastids while increasing the amount of protein, activity or function of said functional equivalent in an organism, e.g. a microorgansim, a plant or plant or animal tissue, plant or animal cells or a part of the same.
A nucleic acid molecule encoding an homologous sequence to a protein sequence of table II, columns 5 and 7 can be created by introducing one or more nucleotide substitutions, additions or deletions into a nucleotide sequence of the nucleic acid molecule of the present invention, in particular of table I, columns 5 and 7 such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into the encoding sequences of table I, columns 5 and 7 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophane), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophane, histidine).
Thus, a predicted nonessential amino acid residue in a polypeptide of the invention or a polypeptide used in the process of the invention is preferably replaced with another amino acid residue from the same family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a coding sequence of a nucleic acid molecule of the invention or used in the process of the invention, such as by saturation mutagenesis, and the resultant mutants can be screened for activity described herein to identify mutants that retain or even have increased above mentioned activity, e.g. conferring an increased GABA content as compared to a corresponding non-transformed wild type.
Following mutagenesis of one of the sequences of shown herein, the encoded protein can be expressed recombinantly and the activity of the protein can be determined using, for example, assays described herein (see Examples).
A hight homology of the nucleic acid molecule used in the process according to the invention was found for the following database entries by Gap search.
Homologues of the nucleic acid sequences used, with the sequence shown in table I, columns 5 and 7, comprise also allelic variants with at least approximately 30%, 35%, 40% or 45% homology, by preference at least approximately 50%, 60% or 70%, more preferably at least approximately 90%, 91%, 92%, 93%, 94% or 95% and even more preferably at least approximately 96%, 97%, 98%, 99% or more homology with one of the nucleotide sequences shown or the abovementioned derived nucleic acid sequences or their homologues, derivatives or analogues or parts of these. Allelic variants encompass in particular functional variants which can be obtained by deletion, insertion or substitution of nucleotides from the sequences shown, preferably from table I, columns 5 and 7, or from the derived nucleic acid sequences, the intention being, however, that the enzyme activity or the biological activity of the resulting proteins synthesized is advantageously retained or increased.
In another embodiment the nucleic acid molecule of the invention or used in the process of the invention comprises the sequences shown in table I column 5 or 7 and in addition the natural 5′ and/or 3′ untranslated sequences or parts thereof.
In one embodiment of the present invention, the nucleic acid molecule of the invention or used in the process of the invention comprises the sequences shown in any of the table I, columns 5 and 7. It is preferred that the nucleic acid molecule comprises as little as possible other nucleotides not shown in any one of table I, columns 5 and 7. In one embodiment, the nucleic acid molecule comprises less than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50 or 40 further nucleotides. In a further embodiment, the nucleic acid molecule comprises less than 30, 20 or 10 further nucleotides. In one embodiment, the nucleic acid molecule use in the process of the invention is identical to the sequences shown in table I, columns 5 and 7.
Also preferred is that the nucleic acid molecule used in the process of the invention encodes a polypeptide comprising the sequence shown in table II, columns 5 and 7. In one embodiment, the nucleic acid molecule encodes less than 150, 130, 100, 80, 60, 50, 40 or 30 further amino acids. In a further embodiment, the encoded polypeptide comprises less than 20, 15, 10, 9, 8, 7, 6 or 5 further amino acids. In one embodiment used in the inventive process, the encoded polypeptide is identical to the sequences shown in table II, columns 5 and 7.
In one embodiment, the nucleic acid molecule of the invention or used in the process encodes a polypeptide comprising the sequence shown in table II, columns 5 and 7 comprises less than 100 further nucleotides. In a further embodiment, said nucleic acid molecule comprises less than 30 further nucleotides. In one embodiment, the nucleic acid molecule used in the process is identical to a coding sequence of the sequences shown in table I, columns 5 and 7.
Polypeptides (=proteins), which still have the essential biological or enzymatic activity of the polypeptide of the present invention conferring an increased GABA content production as compared to a corresponding non-transformed wild type plant cell, plant or part thereof i.e. whose activity is essentially not reduced, are polypeptides with at least 10% or 20%, by preference 30% or 40%, especially preferably 50% or 60%, very especially preferably 80% or 90 or more of the wild type biological activity or enzyme activity, advantageously, the activity is essentially not reduced in comparison with the activity of a polypeptide shown in table II, columns 5 and 7 expressed under identical conditions.
Homologues of table I, columns 5 and 7 or of the derived sequences of table II, columns 5 and 7 also mean truncated sequences, cDNA, single-stranded DNA or RNA of the coding and noncoding DNA sequence. Homologues of said sequences are also understood as meaning derivatives, which comprise noncoding regions such as, for example, UTRs, terminators, enhancers or promoter variants. The promoters upstream of the nucleotide sequences stated can be modified by one or more nucleotide substitution(s), insertion(s) and/or deletion(s) without, however, interfering with the functionality or activity either of the promoters, the open reading frame (=ORF) or with the 3′-regulatory region such as terminators or other 3′ regulatory regions, which are far away from the ORF. It is furthermore possible that the activity of the promoters is increased by modification of their sequence, or that they are replaced completely by more active promoters, even promoters from heterologous organisms. Appropriate promoters are known to the person skilled in the art and are mentioned herein below.
In addition to the nucleic acid molecules encoding the GABA-related Proteins described above, another aspect of the invention pertains to negative regulators of the activity of a nucleic acid molecules selected from the group according to table I, column 5 and/or 7, preferably column 7. Antisense polynucleotides thereto are thought to inhibit the downregulating activity of those negative regulators by specifically binding the target polynucleotide and interfering with transcription, splicing, transport, translation, and/or stability of the target polynucleotide. Methods are described in the prior art for targeting the antisense polynucleotide to the chromosomal DNA, to a primary RNA transcript, or to a processed mRNA. Preferably, the target regions include splice sites, translation initiation codons, translation termination codons, and other sequences within the open reading frame.
The term “antisense,” for the purposes of the invention, refers to a nucleic acid comprising a polynucleotide that is sufficiently complementary to all or a portion of a gene, primary transcript, or processed mRNA, so as to interfere with expression of the endogenous gene. “Complementary” polynucleotides are those that are capable of base pairing according to the standard Watson-Crick complementarity rules. Specifically, purines will base pair with pyrimidines to form a combination of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. It is understood that two polynucleotides may hybridize to each other even if they are not completely complementary to each other, provided that each has at least one region that is substantially complementary to the other. The term “antisense nucleic acid” includes single stranded RNA as well as double-stranded DNA expression cassettes that can be transcribed to produce an antisense RNA. “Active” antisense nucleic acids are antisense RNA molecules that are capable of selectively hybridizing with a negative regulator of the activity of a nucleic acid molecules encoding a polypeptide having at least 80% sequence identity with the polypeptide selected from the group according to table II, column 5 and/or 7, preferably column 7.
The antisense nucleic acid can be complementary to an entire negative regulator strand, or to only a portion thereof. In an embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence encoding a GABA-related Proteins. The term “noncoding region” refers to 5′ and 3′ sequences that flank the coding region that are not translated into amino acids (i.e., also referred to as 5′ and 3′ untranslated regions). The antisense nucleic acid molecule can be complementary to only a portion of the noncoding region of GABA-related Proteins mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of GABA-related Proteins mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. Typically, the antisense molecules of the present invention comprise an RNA having 60-100% sequence identity with at least 14 consecutive nucleotides of a noncoding region of one of the nucleic acid of table I. Preferably, the sequence identity will be at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 98% and most preferably 99%.
An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
In yet another embodiment, the antisense nucleic acid molecule of the invention is an alpha-anomeric nucleic acid molecule. An alpha-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual b-units, the strands run parallel to each other (Gaultier et al., 1987, Nucleic Acids. Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al., 1987, Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBS Lett. 215:327-330).
The antisense nucleic acid molecules of the invention are typically administered to a cell or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an anti-sense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. The antisense molecule can be modified such that it specifically binds to a receptor or an antigen expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecule to a peptide or an antibody which binds to a cell surface receptor or antigen. The antisense nucleic acid molecule can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the anti-sense nucleic acid molecule is placed under the control of a strong prokaryotic, viral, or eukaryotic (including plant) promoter are preferred.
As an alternative to antisense polynucleotides, ribozymes, sense polynucleotides, or double stranded RNA (dsRNA) can be used to reduce expression of a GABA increasing polypeptide of the invention. By “ribozyme” is meant a catalytic RNA-based enzyme with ribonuclease activity which is capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which it has a complementary region. Ribozymes (e.g., hammerhead ribozymes described in Haselhoff and Gerlach, 1988, Nature 334:585-591) can be used to catalytically cleave GABA increasing polypeptide of the invention mRNA transcripts to thereby inhibit translation of GABA increasing polypeptide of the invention mRNA. A ribozyme having specificity for a nucleic acid encoding a GABA increasing polypeptide of the invention can be designed based upon the nucleotide sequence of a GABA increasing polypeptide of the invention cDNA, as disclosed herein or on the basis of a heterologous sequence to be isolated according to methods taught in this invention. For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a GABA-related Proteins—encoding mRNA. See, e.g., U.S. Pat. Nos. 4,987,071 and 5,116,742 to Cech et al. Alternatively,
GABA increasing polypeptide of the invention mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J. W., 1993, Science 261:1411-1418. In preferred embodiments, the ribozyme will contain a portion having at least 7, 8, 9, 10, 12, 14, 16, 18 or 20 nucleotides, and more preferably 7 or 8 nucleotides, that have 100% complementarity to a portion of the target RNA. Methods for making ribozymes are known to those skilled in the art. See, e.g., U.S. Pat. Nos. 6,025,167; 5,773,260; and 5,496,698.
The term “dsRNA,” as used herein, refers to RNA hybrids comprising two strands of RNA. The dsRNAs can be linear or circular in structure. In a preferred embodiment, dsRNA is specific for a polynucleotide encoding either the polypeptide according to table II or a polypeptide having at least 70% sequence identity with a polypeptide according to table II. The hybridizing RNAs may be substantially or completely complementary. By “substantially complementary,” is meant that when the two hybridizing RNAs are optimally aligned using the BLAST program as described above, the hybridizing portions are at least 95% complementary. Preferably, the dsRNA will be at least 100 base pairs in length. Typically, the hybridizing RNAs will be of identical length with no over hanging 5′ or 3′ ends and no gaps. However, dsRNAs having 5′ or 3′ overhangs of up to 100 nucleotides may be used in the methods of the invention.
The dsRNA may comprise ribonucleotides or ribonucleotide analogs, such as 2′-O-methyl ribosyl residues, or combinations thereof. See, e.g., U.S. Pat. Nos. 4,130,641 and 4,024,222. A dsRNA polyriboinosinic acid:polyribocytidylic acid is described in U.S. Pat. No. 4,283,393. Methods for making and using dsRNA are known in the art. One method comprises the simultaneous transcription of two complementary DNA strands, either in vivo, or in a single in vitro reaction mixture. See, e.g., U.S. Pat. No. 5,795,715. In one embodiment, dsRNA can be introduced into a plant or plant cell directly by standard transformation procedures. Alternatively, dsRNA can be expressed in a plant cell by transcribing two complementary RNAs.
Other methods for the inhibition of endogenous gene expression, such as triple helix formation (Moser et al., 1987, Science 238:645-650 and Cooney et al., 1988, Science 241:456-459) and cosuppression (Napoli et al., 1990, The Plant Cell 2:279-289) are known in the art. Partial and full-length cDNAs have been used for the cosuppression of endogenous plant genes. See, e.g., U.S. Pat. Nos. 4,801,340, 5,034,323, 5,231,020, and 5,283,184; Van der Kroll et al., 1990, The Plant Cell 2:291-299; Smith et al., 1990, Mol. Gen. Genetics 224:477-481 and Napoli et al., 1990, The Plant Cell 2:279-289.
For sense suppression, it is believed that introduction of a sense polynucleotide blocks transcription of the corresponding target gene. The sense polynucleotide will have at least 65% sequence identity with the target plant gene or RNA. Preferably, the percent identity is at least 80%, 90%, 95% or more. The introduced sense polynucleotide need not be full length relative to the target gene or transcript. Preferably, the sense polynucleotide will have at least 65% sequence identity with at least 100 consecutive nucleotides of one of the nucleic acids as depicted in Table I. The regions of identity can comprise introns and and/or exons and untranslated regions. The introduced sense polynucleotide may be present in the plant cell transiently, or may be stably integrated into a plant chromosome or extrachromosomal replicon.
Further, object of the invention is an expression vector comprising a nucleic acid molecule comprising a nucleic acid molecule selected from the group consisting of:
The invention further provides an isolated recombinant expression vector comprising a stress related protein encoding nucleic acid as described above, wherein expression of the vector or stress related protein encoding nucleic acid, respectively in a host cell results in increased GABA content as compared to the corresponding non-transformed wild type of the host cell. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Further types of vectors can be linearized nucleic acid sequences, such as transposons, which are pieces of DNA which can copy and insert themselves. There have been 2 types of transposons found: simple transposons, known as Insertion Sequences and composite transposons, which can have several genes as well as the genes that are required for transposition.
Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors”. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
A plant expression cassette preferably contains regulatory sequences capable of driving gene expression in plant cells and operably linked so that each sequence can fulfill its function, for example, termination of transcription by polyadenylation signals. Preferred polyadenylation signals are those originating from Agrobacterium tumefaciens T-DNA such as the gene 3 known as octopine synthase of the Ti-plasmid pTiACH5 (Gielen et al., 1984 EMBO J. 3:835) or functional equivalents thereof but also all other terminators functionally active in plants are suitable.
As plant gene expression is very often not limited on transcriptional levels, a plant expression cassette preferably contains other operably linked sequences like translational enhancers such as the overdrive-sequence containing the 5′-untranslated leader sequence from tobacco mosaic virus enhancing the protein per RNA ratio (Gallie et al., 1987 Nucl. Acids Research 15:8693-8711).
Plant gene expression has to be operably linked to an appropriate promoter conferring gene expression in a timely, cell or tissue specific manner. Preferred are promoters driving constitutive expression (Benfey et al., 1989 EMBO J. 8:2195-2202) like those derived from plant viruses like the 35S CaMV (Franck et al., 1980 Cell 21:285-294), the 19S CaMV (see also U.S. Pat. No. 5,352,605 and PCT Application No. WO 8402913) or plant promoters like those from Rubisco small subunit described in U.S. Pat. No. 4,962,028.
Additional advantageous regulatory sequences are, for example, included in the plant promoters such as CaMV/35S [Franck et al., Cell 21 (1980) 285-294], PRP1 [Ward et al., Plant. Mol. Biol. 22 (1993)], SSU, OCS, lib4, usp, STLS1, B33, LEB4, nos or in the ubiquitin, napin or phaseolin promoter. Also advantageous in this connection are inducible promoters such as the promoters described in EP-A-0 388 186 (benzyl sulfonamide inducible), Plant J. 2, 1992: 397-404 (Gatz et al., Tetracyclin inducible), EP-A-0 335 528 (abscisic acid inducible) or WO 93/21334 (ethanol or cyclohexenol inducible). Additional useful plant promoters are the cytosolic FBPase promoter or ST-LSI promoter of the potato (Stockhaus et al., EMBO J. 8, 1989, 2445), the phosphorybosyl phyrophoshate amido transferase promoter of Glycine max (gene bank accession No. U87999) or the noden specific promoter described in EP-A-0 249 676. Additional particularly advantageous promoters are seed specific promoters which can be used for monokotyledones or dikotyledones and are described in U.S. Pat. No. 5,608,152 (napin promoter from rapeseed), WO 98/45461 (phaseolin promoter from Arobidopsis), U.S. Pat. No. 5,504,200 (phaseolin promoter from Phaseolus vulgaris), WO 91/13980 (Bce4 promoter from Brassica) and Baeumlein et al., Plant J., 2, 2, 1992: 233-239 (LEB4 promoter from leguminosa). Said promoters are useful in dikotyledones. The following promoters are useful for example in monokotyledones Ipt-2- or Ipt-1-promoter from barley (WO 95/15389 and WO 95/23230) or hordein promoter from barley. Other useful promoters are described in WO 99/16890.
It is possible in principle to use all natural promoters with their regulatory sequences like those mentioned above for the novel process. It is also possible and advantageous in addition to use synthetic promoters.
The gene construct may also comprise further genes which are to be inserted into the organisms and which are for example involved in stress resistance and biomass production increase. It is possible and advantageous to insert and express in host organisms regulatory genes such as genes for inducers, repressors or enzymes which intervene by their enzymatic activity in the regulation, or one or more or all genes of a biosynthetic pathway. These genes can be heterologous or homologous in origin. The inserted genes may have their own promoter or else be under the control of same promoter as the sequences of the nucleic acid of table I or their homologs.
The gene construct advantageously comprises, for expression of the other genes present, additionally 3′ and/or 5′ terminal regulatory sequences to enhance expression, which are selected for optimal expression depending on the selected host organism and gene or genes.
These regulatory sequences are intended to make specific expression of the genes and protein expression possible as mentioned above. This may mean, depending on the host organism, for example that the gene is expressed or overexpressed only after induction, or that it is immediately expressed and/or overexpressed.
The regulatory sequences or factors may moreover preferably have a beneficial effect on expression of the introduced genes, and thus increase it. It is possible in this way for the regulatory elements to be enhanced advantageously at the transcription level by using strong transcription signals such as promoters and/or enhancers. However, in addition, it is also possible to enhance translation by, for example, improving the stability of the mRNA.
Other preferred sequences for use in plant gene expression cassettes are targeting-sequences necessary to direct the gene product in its appropriate cell compartment (for review see Kermode, 1996 Crit. Rev. Plant Sci. 15(4):285-423 and references cited therein) such as the vacuole, the nucleus, all types of plastids like amyloplasts, chloroplasts, chromoplasts, the extracellular space, mitochondria, the endoplasmic reticulum, oil bodies, peroxisomes and other compartments of plant cells.
Plant gene expression can also be facilitated via an inducible promoter (for review see Gatz, 1997 Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:89-108). Chemically inducible promoters are especially suitable if gene expression is wanted to occur in a time specific manner.
Table V lists several examples of promoters that may be used to regulate transcription of the stress related protein nucleic acid coding sequences.
Other promoters, e.g. Super promotor (Ni et al., Plant Journal 7, 1995: 661-676), Ubiquitin promoter (Callis et al., J. Biol. Chem., 1990, 265: 12486-12493; U.S. Pat. No. 5,510,474; U.S. Pat. No. 6,020,190; Kawalleck et al., Plant. Molecular Biology, 1993, 21: 673-684) or 34S promoter (GenBank Accession numbers M59930 and X16673) were similar useful for the present invention and are known to a person skilled in the art.
Developmental stage-preferred promoters are preferentially expressed at certain stages of development. Tissue and organ preferred promoters include those that are preferentially expressed in certain tissues or organs, such as leaves, roots, seeds, or xylem. Examples of tissue preferred and organ preferred promoters include, but are not limited to fruit-preferred, ovule-preferred, male tissue-preferred, seed-preferred, integument-preferred, tuber-preferred, stalk-preferred, pericarp-preferred, and leafpreferred, stigma-preferred, pollen-preferred, anther-preferred, a petal-preferred, sepalpreferred, pedicel-preferred, silique-preferred, stem-preferred, root-preferred promoters, and the like. Seed preferred promoters are preferentially expressed during seed development and/or germination. For example, seed preferred promoters can be embryo-preferred, endosperm preferred, and seed coat-preferred. See Thompson et al., 1989, BioEssays 10:108. Examples of seed preferred promoters include, but are not limited to, cellulose synthase (celA), Cim1, gamma-zein, globulin-1, maize 19 kD zein (cZ19B1), and the like.
Other promoters useful in the expression cassettes of the invention include, but are not limited to, the major chlorophyll a/b binding protein promoter, histone promoters, the Ap3 promoter, the β-conglycin promoter, the napin promoter, the soybean lectin promoter, the maize 15 kD zein promoter, the 22 kD zein promoter, the 27 kD zein promoter, the g-zein promoter, the waxy, shrunken 1, shrunken 2 and bronze promoters, the Zm13 promoter (U.S. Pat. No. 5,086,169), the maize polygalacturonase promoters (PG) (U.S. Pat. Nos. 5,412,085 and 5,545,546), and the SGB6 promoter (U.S. Pat. No. 5,470,359), as well as synthetic or other natural promoters.
Additional flexibility in controlling heterologous gene expression in plants may be obtained by using DNA binding domains and response elements from heterologous sources (i.e., DNA binding domains from non-plant sources). An example of such a heterologous DNA binding domain is the LexA DNA binding domain (Brent and Ptashne, 1985, Cell 43:729-736).
The invention further provides a recombinant expression vector comprising a GABA increasing polypeptide of the invention DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to a GABA increasing polypeptide of the invention mRNA. Regulatory sequences operatively linked to a nucleic acid molecule cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types. For instance, viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific, or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid, or attenuated virus wherein antisense nucleic acids are produced under the control of a high efficiency regulatory region. The activity of the regulatory region can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes, see Weintraub, H. et al., 1986, Antisense RNA as a molecular tool for genetic analysis, Reviews—Trends in Genetics, Vol. 1(1), and Mol et al., 1990, FEBS Letters 268:427-430.
Another aspect of the invention pertains to isolated GABA increasing polypeptide of the invention, and biologically active portions thereof. An “isolated” or “purified” polypeptide or biologically active portion thereof is free of some of the cellular material when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. The language “substantially free of cellular material” includes preparations of GABA increasing polypeptide of the invention in which the polypeptide is separated from some of the cellular components of the cells in which it is naturally or recombinantly produced. In one embodiment, the language “substantially free of cellular material” includes preparations of a GABA increasing polypeptide of the invention having less than about 30% (by dry weight) of non-GABA increasing polypeptide of the invention material (also referred to herein as a “contaminating polypeptide”), more preferably less than about 20% of non-GABA increasing polypeptide of the invention material, still more preferably less than about 10% of non-GABA increasing polypeptide of the invention material, and most preferably less than about 5% non-GABA increasing polypeptide of the invention material.
When the GABA increasing polypeptide of the invention or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the polypeptide preparation. The language “substantially free of chemical precursors or other chemicals” includes preparations of GABA increasing polypeptide of the invention in which the polypeptide is separated from chemical precursors or other chemicals that are involved in the synthesis of the polypeptide. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of a GABA increasing polypeptide of the invention having less than about 30% (by dry weight) of chemical precursors or non-GABA increasing polypeptide of the invention chemicals, more preferably less than about 20% chemical precursors or non-GABA increasing polypeptide of the invention chemicals, still more preferably less than about 10% chemical precursors or non-GABA increasing polypeptide of the invention chemicals, and most preferably less than about 5% chemical precursors or non-GABA increasing polypeptide of the invention chemicals. In preferred embodiments, isolated polypeptides, or biologically active portions thereof, lack contaminating polypeptides from the same organism from which the GABA increasing polypeptide of the invention is derived. Typically, such polypeptides are produced by recombinant expression of, for example, a Saccharomyces cerevisiae, E. coli or Brassica napus, Glycine max, Zea mays or Oryza sativa GABA increasing polypeptide of the invention in plants other than Saccharomyces cerevisiae, E. coli, or microorganisms such as C. glutamicum, ciliates, algae or fungi.
The nucleic acid molecules, polypeptides, polypeptide homologs, fusion polypeptides, primers, vectors, and host cells described herein can be used in one or more of the following methods: identification of Saccharomyces cerevisiae, E. coli or Brassica napus, Glycine max, Zea mays or Oryza sativa and related organisms; mapping of genomes of organisms related to Saccharomyces cerevisiae, E. coli; identification and localization of Saccharomyces cerevisiae, E. coli or Brassica napus, Glycine max, Zea mays or Oryza sativa sequences of interest; evolutionary studies; determination of regions required for function in the GABA increasing polypeptide of the invention; modulation of a GABA increasing polypeptide activity; modulation of the metabolism of one or more cell functions; modulation of the transmembrane transport of one or more compounds; modulation of stress resistance; and modulation of expression of GABA increasing polypeptide nucleic acids.
The nucleic acid molecules of the invention are also useful for evolutionary and polypeptide structural studies. The metabolic and transport processes in which the molecules of the invention participate are utilized by a wide variety of prokaryotic and eukaryotic cells; by comparing the sequences of the nucleic acid molecules of the present invention to those encoding similar enzymes from other organisms, the evolutionary relatedness of the organisms can be assessed. Similarly, such a comparison permits an assessment of which regions of the sequence are conserved and which are not, which may aid in determining those regions of the polypeptide that are essential for the functioning of the enzyme. This type of determination is of value for polypeptide engineering studies and may give an indication of what the polypeptide can tolerate in terms of mutagenesis without losing function.
Manipulation of the nucleic acid molecules of the invention may result in the production of having functional differences from the wild-type. These polypeptides may be improved in efficiency or activity, may be present in greater numbers in the cell than is usual, or may be decreased in efficiency or activity.
There are a number of mechanisms by which the alteration of a GABA increasing polypeptide of the invention of the invention may directly affect stress response and/or stress tolerance. In the case of plants expressing GABA increasing polypeptide of the invention, increased transport can lead to improved salt and/or solute partitioning within the plant tissue and organs. By either increasing the number or the activity of transporter molecules which export ionic molecules from the cell, it may be possible to affect the salt and cold tolerance of the cell.
The effect of the genetic modification in plants, on stress tolerance can be assessed by growing the modified plant under less than suitable conditions and then analyzing the growth characteristics and/or metabolism of the plant. Such analysis techniques are well known to one skilled in the art, and include dry weight, wet weight, polypeptide synthesis, carbohydrate synthesis, lipid synthesis, evapotranspiration rates, general plant and/or crop yield, flowering, reproduction, seed setting, root growth, respiration rates, photosynthesis rates, etc. (Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, vol. 17; Rehm et al., 1993 Biotechnology, vol. 3, Chapter III: Product recovery and purification, page 469-714, VCH: Weinheim; Belter, P. A. et al., 1988, Bioseparations: downstream processing for biotechnology, John Wiley and Sons; Kennedy, J. F. and Cabral, J. M. S., 1992, Recovery processes for biological materials, John Wiley and Sons; Shaeiwitz, J. A. and Henry, J. D., 1988, Biochemical separations, in: Ulmann's Encyclopedia of Industrial Chemistry, vol. B3, Chapter 11, page 1-27, VCH: Weinheim; and Dechow, F.J., 1989, Separation and purification techniques in biotechnology, Noyes Publications).
For example, yeast expression vectors comprising the nucleic acids disclosed herein, or fragments thereof, can be constructed and transformed into Saccharomyces cerevisiae using standard protocols. The resulting transgenic cells can then be assayed for fail or alteration of their tolerance to drought, salt, and cold stress. Similarly, plant expression vectors comprising the nucleic acids disclosed herein, or fragments thereof, can be constructed and transformed into an appropriate plant cell such as Arabidopsis, soy, rape, maize, cotton, rice, wheat, Medicago truncatula, etc., using standard protocols. The resulting transgenic cells and/or plants derived therefrom can then be assayed for fail or alteration of their tolerance to drought, salt, cold stress.
The engineering of one or more genes according to table I and coding for the GABA increasing polypeptide of the invention of table II of the invention may also result in GABA increasing polypeptide of the invention having altered activities which indirectly impact the stress response and/or stress tolerance of algae, plants, ciliates, or fungi, or other microorganisms like C. glutamicum.
Additionally, the sequences disclosed herein, or fragments thereof, can be used to generate knockout mutations in the genomes of various organisms, such as bacteria, mammalian cells, yeast cells, and plant cells (Girke, T., 1998, The Plant Journal 15:39-48). The resultant knockout cells can then be evaluated for their ability or capacity to tolerate various stress conditions, their response to various stress conditions, and the effect on the phenotype and/or genotype of the mutation. For other methods of gene inactivation, see U.S. Pat. No. 6,004,804 “Non-Chimeric Mutational Vectors” and Puttaraju et al., 1999, Spliceosome-mediated RNA trans-splicing as a tool for gene therapy, Nature Biotechnology 17:246-252.
The aforementioned mutagenesis strategies for GABA-related Proteins resulting in increased stress resistance are not meant to be limiting; variations on these strategies will be readily apparent to one skilled in the art. Using such strategies, and incorporating the mechanisms disclosed herein, the nucleic acid and polypeptide molecules of the invention may be utilized to generate algae, ciliates, plants, fungi, or other microorganisms like C. glutamicum expressing mutated GABA-related Proteins nucleic acid and polypeptide molecules such that the stress tolerance is improved.
The present invention also provides antibodies that specifically bind to a GABA increasing polypeptide of the invention, or a portion thereof, as encoded by a nucleic acid described herein. Antibodies can be made by many well-known methods (See, e.g. Harlow and Lane, “Antibodies; A Laboratory Manual,” Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1988)). Briefly, purified antigen can be injected into an animal in an amount and in intervals sufficient to elicit an immune response. Antibodies can either be purified directly, or spleen cells can be obtained from the animal. The cells can then fused with an immortal cell line and screened for antibody secretion. The antibodies can be used to screen nucleic acid clone libraries for cells secreting the antigen. Those positive clones can then be sequenced. See, for example, Kelly et al., 1992, Bio/Technology 10:163-167; Bebbington et al., 1992, Bio/Technology 10:169-175.
The phrases “selectively binds” and “specifically binds” with the polypeptide refer to a binding reaction that is determinative of the presence of the polypeptide in a heterogeneous population of polypeptides and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bound to a particular polypeptide do not bind in a significant amount to other polypeptides present in the sample. Selective binding of an antibody under such conditions may require an antibody that is selected for its specificity for a particular polypeptide. A variety of immunoassay formats may be used to select antibodies that selectively bind with a particular polypeptide. For example, solid-phase ELISA immunoassays are routinely used to select antibodies selectively immunoreactive with a polypeptide. See Harlow and Lane, “Antibodies, A Laboratory Manual,” Cold Spring Harbor Publications, New York, (1988), for a description of immunoassay formats and conditions that could be used to determine selective binding.
In some instances, it is desirable to prepare monoclonal antibodies from various hosts. A description of techniques for preparing such monoclonal antibodies may be found in Stites et al., eds., “Basic and Clinical Immunology,” (Lange Medical Publications, Los Altos, Calif., Fourth Edition) and references cited therein, and in Harlow and Lane, “Antibodies, A Laboratory Manual,” Cold Spring Harbor Publications, New York, (1988).
Gene expression in plants is regulated by the interaction of protein transcription factors with specific nucleotide sequences within the regulatory region of a gene. One example of transcription factors are polypeptides that contain zinc finger (ZF) motifs. Each ZF module is approximately 30 amino acids long folded around a zinc ion. The DNA recognition domain of a ZF protein is a α-helical structure that inserts into the major grove of the DNA double helix. The module contains three amino acids that bind to the DNA with each amino acid contacting a single base pair in the target DNA sequence. ZF motifs are arranged in a modular repeating fashion to form a set of fingers that recognize a contiguous DNA sequence. For example, a three-fingered ZF motif will recognize 9 bp of DNA. Hundreds of proteins have been shown to contain ZF motifs with between 2 and 37 ZF modules in each protein (Isalan M, et al., 1998 Biochemistry 37(35):12026-33; Moore M, et al., 2001 Proc. Natl. Acad. Sci. USA 98(4):1432-1436 and 1437-1441; US patents U.S. Pat. No. 6,007,988 and U.S. Pat. No. 6,013,453).
The regulatory region of a plant gene contains many short DNA sequences (cis-acting elements) that serve as recognition domains for transcription factors, including ZF proteins. Similar recognition domains in different genes allow the coordinate expression of several genes encoding enzymes in a metabolic pathway by common transcription factors. Variation in the recognition domains among members of a gene family facilitates differences in gene expression within the same gene family, for example, among tissues and stages of development and in response to environmental conditions.
Typical ZF proteins contain not only a DNA recognition domain but also a functional domain that enables the ZF protein to activate or repress transcription of a specific gene. Experimentally, an activation domain has been used to activate transcription of the target gene (U.S. Pat. No. 5,789,538 and patent application WO9519431), but it is also possible to link a transcription repressor domain to the ZF and thereby inhibit transcription (patent applications WO00/47754 and WO2001002019). It has been reported that an enzymatic function such as nucleic acid cleavage can be linked to the ZF (patent application WO00/20622)
The invention provides a method that allows one skilled in the art to isolate the regulatory region of one or more stress related protein encoding genes from the genome of a plant cell and to design zinc finger transcription factors linked to a functional domain that will interact with the regulatory region of the gene. The interaction of the zinc finger protein with the plant gene can be designed in such a manner as to alter expression of the gene and preferably thereby to confer increased GABA content.
In particular, the invention provides a method of producing a transgenic plant with a stress related protein coding nucleic acid, wherein expression of the nucleic acid(s) in the plant results in increased tolerance to environmental stress as compared to a wild type plant comprising: (a) transforming a plant cell with an expression vector comprising a stress related protein encoding nucleic acid, and (b) generating from the plant cell a transgenic plant with an increased GABA content as compared to a wild type plant. For such plant transformation, binary vectors such as pBinAR can be used (Höfgen and Willmitzer, 1990 Plant Science 66:221-230). Moreover suitable binary vectors are for example pBIN19, pBI101, pGPTV or pPZP (Hajukiewicz, P. et al., 1994, Plant Mol. Biol., 25: 989-994).
Construction of the binary vectors can be performed by ligation of the cDNA into the T-DNA. 5′ to the cDNA a plant promoter activates transcription of the cDNA. A polyadenylation sequence is located 3′ to the cDNA. Tissue-specific expression can be achieved by using a tissue specific promoter as listed above. Also, any other promoter element can be used. For constitutive expression within the whole plant, the CaMV 35S promoter can be used. The expressed protein can be targeted to a cellular compartment using a signal peptide, for example for plastids, mitochondria or endoplasmic reticulum (Kermode, 1996 Crit. Rev. Plant Sci. 4(15):285-423). The signal peptide is cloned 5′ in frame to the cDNA to archive subcellular localization of the fusion protein. Additionally, promoters that are responsive to abiotic stresses can be used with, such as the Arabidopsis promoter RD29A. One skilled in the art will recognize that the promoter used should be operatively linked to the nucleic acid such that the promoter causes transcription of the nucleic acid which results in the synthesis of a mRNA which encodes a polypeptide.
Alternate methods of transfection include the direct transfer of DNA into developing flowers via electroporation or Agrobacterium mediated gene transfer. Agrobacterium mediated plant transformation can be performed using for example the GV3101(pMP90) (Koncz and Schell, 1986 Mol. Gen. Genet. 204:383-396) or LBA4404 (Ooms et al., Plasmid, 1982, 7: 15-29; Hoekema et al., Nature, 1983, 303: 179-180) Agrobacterium tumefaciens strain. Transformation can be performed by standard transformation and regeneration techniques (Deblaere et al., 1994 Nucl. Acids. Res. 13:4777-4788; Gelvin and Schilperoort, Plant Molecular Biology Manual, 2nd Ed. -Dordrecht: Kluwer Academic Publ., 1995.-in Sect., Ringbuc Zentrale Signatur: BT11-P ISBN 0-7923-2731-4; Glick, B R and Thompson, J E, Methods in Plant Molecular Biology and Biotechnology, Boca Raton: CRC Press, 1993.-360 S., ISBN 0-8493-5164-2). For example, rapeseed can be transformed via cotyledon or hypocotyl transformation (Moloney et al., 1989 Plant Cell Reports 8:238-242; De Block et al., 1989 Plant Physiol. 91:694-701). Use of antibiotics for Agrobacterium and plant selection depends on the binary vector and the Agrobacterium strain used for transformation. Rapeseed selection is normally performed using kanamycin as selectable plant marker. Agrobacterium mediated gene transfer to flax can be performed using, for example, a technique described by Mlynarova et al., 1994 Plant Cell Report 13:282-285. Additionally, transformation of soybean can be performed using for example a technique described in European Patent No. 0424 047, U.S. Pat. No. 5,322,783, European Patent No. 0397 687, U.S. Pat. No. 5,376,543 or U.S. Pat. No. 5,169,770. Transformation of maize can be achieved by particle bombardment, polyethylene glycol mediated DNA uptake or via the silicon carbide fiber technique (see, for example, Freeling and Walbot “The maize handbook” Springer Verlag: New York (1993) ISBN 3-540-97826-7). A specific example of maize transformation is found in U.S. Pat. No. 5,990,387 and a specific example of wheat transformation can be found in PCT Application No. WO 93/07256.
Growing the modified plants under stress conditions and then screening and analyzing the growth characteristics and/or metabolic activity assess the effect of the genetic modification in plants on increased GABA content. Such analysis techniques are well known to one skilled in the art. They include next to screening (Römpp Lexikon Biotechnologie, Stuttgart/New York: Georg Thieme Verlag 1992, “screening” p. 701) dry weight, wet weight, protein synthesis, carbohydrate synthesis, lipid synthesis, evapotranspiration rates, general plant and/or crop yield, flowering, reproduction, seed setting, root growth, respiration rates, photosynthesis rates, etc. (Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, vol. 17; Rehm et al., 1993 Biotechnology, vol. 3, Chapter III: Product recovery and purification, page 469-714, VCH: Weinheim; Belter, P. A. et al., 1988 Bioseparations: downstream processing for biotechnology, John Wiley and Sons; Kennedy, J. F. and Cabral, J. M. S., 1992 Recovery processes for biological materials, John Wiley and Sons; Shaeiwitz, J. A. and Henry, J. D., 1988 Biochemical separations, in: Ulmann's Encyclopedia of Industrial Chemistry, vol. B3, Chapter 11, page 1-27, VCH: Weinheim; and Dechow, F.J. (1989) Separation and purification techniques in biotechnology, Noyes Publications).
In one embodiment, the present invention relates to a method for the identification of a gene product conferring increased GABA content as compared to a corresponding non-transformed wild type in a cell of an organism for example plant, comprising the following steps:
a) contacting, e.g. hybridising, some or all nucleic acid molecules of a sample, e.g. cells, tissues, plants or microorganisms or a nucleic acid library, which can contain a candidate gene encoding a gene product conferring increased GABA content, with a nucleic acid molecule as shown in column 5 or 7 of Table I A or B or a functional homologue thereof;
b) identifying the nucleic acid molecules, which hybridize under relaxed stringent conditions with said nucleic acid molecule, in particular to the nucleic acid molecule sequence shown in column 5 or 7 of Table I and, optionally, isolating the full length cDNA clone or complete genomic clone;
c) identifying the candidate nucleic acid molecules or a fragment thereof in host cells, preferably in a plant cell
d) increasing the expressing of the identified nucleic acid molecules in the host cells for which increased GABA content as desired
e) assaying the level of increased GABA content of the host cells; and
f) identifying the nucleic acid molecule and its gene product which increased expression confers increased GABA content in the host cell compared to the wild type.
Relaxed hybridisation conditions are: After standard hybridisation procedures washing steps can be performed at low to medium stringency conditions usually with washing conditions of 40°-55° C. and salt conditions between 2×SSC and 0.2×SSC with 0.1% SDS in comparison to stringent washing conditions as e.g. 60° to 68° C. with 0.1% SDS. Further examples can be found in the references listed above for the stringend hybridization conditions. Usually washing steps are repeated with increasing stringency and length until a useful signal to noise ratio is detected and depend on many factors as the target, e.g. its purity, GC-content, size etc, the probe, e.g. its length, is it a RNA or a DNA probe, salt conditions, washing or hybridisation temperature, washing or hybridisation time etc.
In another embodiment, the present invention relates to a method for the identification of a gene product the expression of which confers an increased GABA content in a cell, comprising the following steps:
a) identifying a nucleic acid molecule in an organism, which is at least 20%, preferably 25%, more preferably 30%, even more preferred are 35%. 40% or 50%, even more preferred are 60%, 70% or 80%, most preferred are 90% or 95% or more homolog to the nucleic acid molecule encoding a protein comprising the polypeptide molecule as shown in column 5 or 7 of Table II or comprising a consensus sequence or a polypeptide motif as shown in column 7 of Table IV or being encoded by a nucleic acid molecule comprising a polynucleotide as shown in column 5 or 7 of Table I or a homologue thereof as described herein, for example via homology search in a data bank;
b) enhancing the expression of the identified nucleic acid molecules in the host cells;
c) assaying the level of increased GABA content in the host cells; and
d) identifying the host cell, in which the enhanced expression confers increased GABA content in the host cell compared to a wild type.
Further, the nucleic acid molecule disclosed herein, in particular the nucleic acid molecule shown column 5 or 7 of Table I A or B, may be sufficiently homologous to the sequences of related species such that these nucleic acid molecules may serve as markers for the construction of a genomic map in related organism or for association mapping. Furthermore natural variation in the genomic regions corresponding to nucleic acids disclosed herein, in particular the nucleic acid molecule shown column 5 or 7 of Table I A or B, or homologous thereof may lead to variation in the activity of the proteins disclosed herein, in particular the proteins comprising polypeptides as shown in column 5 or 7 of Table II A or B or comprising the consensus sequence or the polypeptide motif as shown in column 7 of Table IV, and their homolgous and in consequence in natural variation in GABA content.
In consequence natural variation eventually also exists in form of more active allelic variants leading already to a relative increase in the GABA content. Different variants of the nucleic acids molecule disclosed herein, in particular the nucleic acid comprising the nucleic acid molecule as shown column 5 or 7 of Table I A or B, which corresponds to different GABA concentration levels can be identified and used for marker assisted breeding for increased GABA content.
Accordingly, the present invention relates to a method for breeding plants for increased GABA content, comprising
a) selecting a first plant variety with increased GABA content based on increased expression of a nucleic acid of the invention as disclosed herein, in particular of a nucleic acid molecule comprising a nucleic acid molecule as shown in column 5 or 7 of Table I A or B or a polypeptide comprising a polypeptide as shown in column 5 or 7 of Table II A or B or comprising a consensus sequence or a polypeptide motif as shown in column 7 of Table IV, or a homologue thereof as described herein;
b) associating the level of GABA concentration with the expression level or the genomic structure of a gene encoding said polypeptide or said nucleic acid molecule;
c) crossing the first plant variety with a second plant variety, which significantly differs in its level of GABA concentration and
e) identifying, which of the offspring varieties has got increased levels of GABA concentration by the expression level of said polypeptide or nucleic acid molecule or the genomic structure of the genes encoding said polypeptide or nucleic acid molecule of the invention.
In one embodiment, the expression level of the gene according to step (b) is increased.
Yet another embodiment of the invention relates to a process for the identification of a compound conferring increased GABA content as compared to a corresponding non-transformed wild type in a plant cell, a plant or a part thereof, a plant or a part thereof, comprising the steps:
a) culturing a plant cell; a plant or a part thereof maintaining a plant expressing the polypeptide as shown in column 5 or 7 of Table II or being encoded by a nucleic acid molecule comprising a polynucleotide as shown in column 5 or 7 of Table I or a homologue thereof as described herein or a polynucleotide encoding said polypeptide and conferring an increased GABA content as compared to a corresponding non-transformed wild type and providing a readout system capable of interacting with the polypeptide under suitable conditions which permit the interaction of the polypeptide with this readout system in the presence of a chemical compound or a sample comprising a plurality of chemical compounds and capable of providing a detectable signal in response to the binding of a chemical compound to said polypeptide under conditions which permit the expression of said readout system and of the protein as shown in column 5 or 7 of Table II or being encoded by a nucleic acid molecule comprising a polynucleotide as shown in column 5 or 7 of Table I or a homologue thereof as described herein; and
b) identifying if the chemical compound is an effective agonist by detecting the presence or absence or decrease or increase of a signal produced by said readout system.
Said compound may be chemically synthesized or microbiologically produced and/or comprised in, for example, samples, e.g., cell extracts from, e.g., plants, animals or microorganisms, e.g. pathogens. Furthermore, said compound(s) may be known in the art but hitherto not known to be capable of suppressing the polypeptide of the present invention. The reaction mixture may be a cell free extract or may comprise a cell or tissue culture. Suitable set ups for the process for identification of a compound of the invention are known to the person skilled in the art and are, for example, generally described in Alberts et al., Molecular Biology of the Cell, third edition (1994), in particular Chapter 17. The compounds may be, e.g., added to the reaction mixture, culture medium, injected into the cell or sprayed onto the plant.
If a sample containing a compound is identified in the process, then it is either possible to isolate the compound from the original sample identified as containing the compound capable of activating or increasing yield production under condition of transient and repetitive abiotic stress as compared to a corresponding non-transformed wild type, or one can further subdivide the original sample, for example, if it consists of a plurality of different compounds, so as to reduce the number of different substances per sample and repeat the method with the subdivisions of the original sample. Depending on the complexity of the samples, the steps described above can be performed several times, preferably until the sample identified according to the said process only comprises a limited number of or only one substance(s). Preferably said sample comprises substances of similar chemical and/or physical properties, and most preferably said substances are identical. Preferably, the compound identified according to the described method above or its derivative is further formulated in a form suitable for the application in plant breeding or plant cell and tissue culture.
The compounds which can be tested and identified according to said process may be expression libraries, e.g., cDNA expression libraries, peptides, proteins, nucleic acids, antibodies, small organic compounds, hormones, peptidomimetics, PNAs or the like (Milner, Nature Medicine 1 (1995), 879-880; Hupp, Cell 83 (1995), 237-245; Gibbs, Cell 79 (1994), 193-198 and references cited supra). Said compounds can also be functional derivatives or analogues of known inhibitors or activators. Methods for the preparation of chemical derivatives and analogues are well known to those skilled in the art and are described in, for example, Beilstein, Handbook of Organic Chemistry, Springer edition New York Inc., 175 Fifth Avenue, New York, N.Y. 10010 U.S.A. and Organic Synthesis, Wiley, New York, USA. Furthermore, said derivatives and analogues can be tested for their effects according to methods known in the art. Furthermore, peptidomimetics and/or computer aided design of appropriate derivatives and analogues can be used, for example, according to the methods described above. The cell or tissue that may be employed in the process preferably is a host cell, plant cell or plant tissue of the invention described in the embodiments hereinbefore.
Thus, in a further embodiment the invention relates to a compound obtained or identified according to the method for identifying an agonist of the invention said compound being an antagonist of the polypeptide of the present invention.
Accordingly, in one embodiment, the present invention further relates to a compound identified by the method for identifying a compound of the present invention.
In one embodiment, the invention relates to an antibody specifically recognizing the compound or agonist of the present invention.
The invention also relates to a diagnostic composition comprising at least one of the aforementioned nucleic acid molecules, antisense nucleic acid molecule, RNAi, snRNA, dsRNA, siRNA, miRNA, ta-siRNA, cosuppression molecule, ribozyme, vectors, proteins, antibodies or compounds of the invention and optionally suitable means for detection.
The diagnostic composition of the present invention is suitable for the isolation of mRNA from a cell and contacting the mRNA so obtained with a probe comprising a nucleic acid probe as described above under hybridizing conditions, detecting the presence of mRNA hybridized to the probe, and thereby detecting the expression of the protein in the cell. Further methods of detecting the presence of a protein according to the present invention comprise immunotechniques well known in the art, for example enzyme linked immunoadsorbent assay. Furthermore, it is possible to use the nucleic acid molecules according to the invention as molecular markers or primers in plant breeding. Suitable means for detection are well known to a person skilled in the art, e.g. buffers and solutions for hydridization assays, e.g. the afore-mentioned solutions and buffers, further and means for Southern-, Western-, Northern-etc. -blots, as e.g. described in Sambrook et al. are known. In one embodiment diagnostic composition contain PCR primers designed to specifically detect the presense or the expression level of the nucleic acid molecule to be reduced in the process of the invention, e.g. of the nucleic acid molecule of the invention, or to descriminate between different variants or alleles of the nucleic acid molecule of the invention or which activity is to be reduced in the process of the invention.
In another embodiment, the present invention relates to a kit comprising the nucleic acid molecule, the vector, the host cell, the polypeptide, or the anti-sense, RNAi, snRNA, dsRNA, siRNA, miRNA, ta-siRNA, cosuppression molecule, or ribozyme molecule, or the viral nucleic acid molecule, the antibody, plant cell, the plant or plant tissue, the harvestable part, the propagation material and/or the compound and/or agonist identified according to the method of the invention.
The compounds of the kit of the present invention may be packaged in containers such as vials, optionally with/in buffers and/or solution. If appropriate, one or more of said components might be packaged in one and the same container. Additionally or alternatively, one or more of said components might be adsorbed to a solid support as, e.g. a nitrocellulose filter, a glass plate, a chip, or a nylon membrane or to the well of a micro titerplate. The kit can be used for any of the herein described methods and embodiments, e.g. for the production of the host cells, transgenic plants, pharmaceutical compositions, detection of homologous sequences, identification of antagonists or agonists, as food or feed or as a supplement thereof or as supplement for the treating of plants, etc.
Further, the kit can comprise instructions for the use of the kit for any of said embodiments.
In one embodiment said kit comprises further a nucleic acid molecule encoding one or more of the aforementioned protein, and/or an antibody, a vector, a host cell, an anti-sense nucleic acid, a plant cell or plant tissue or a plant. In another embodiment said kit comprises PCR primers to detect and discriminate the nucleic acid molecule to be reduced in the process of the invention, e.g. of the nucleic acid molecule of the invention.
In a further embodiment, the present invention relates to a method for the production of an agricultural composition providing the nucleic acid molecule for the use according to the process of the invention, the nucleic acid molecule of the invention, the vector of the invention, the antisense, RNAi, snRNA, dsRNA, siRNA, miRNA, ta-siRNA, cosuppression molecule, ribozyme, or antibody of the invention, the viral nucleic acid molecule of the invention, or the polypeptide of the invention or comprising the steps of the method according to the invention for the identification of said compound or agonist; and formulating the nucleic acid molecule, the vector or the polypeptide of the invention or the agonist, or compound identified according to the methods or processes of the present invention or with use of the subject matters of the present invention in a form applicable as plant agricultural composition.
In another embodiment, the present invention relates to a method for the production of the plant culture composition comprising the steps of the method of the present invention; and formulating the compound identified in a form acceptable as agri-cultural composition.
Under “acceptable as agricultural composition” is understood, that such a composition is in agreement with the laws regulating the content of fungicides, plant nutrients, herbicides, etc. Preferably such a composition is without any harm for the protected plants and the animals (humans included) fed therewith.
The effect of the genetic modification in the host cell on the production of gamma-aminobutyric acid can be determined by growing the modified microorganisms or the modified plant under suitable conditions (such as those described above) and analyzing the medium and/or the cellular components for the elevated production of gamma-aminobutyric acid. These analytical techniques are known to the skilled worker and comprise spectroscopy, thin-layer chromatography, various types of staining methods, enzymatic and microbiological methods and analytical chromatography such as high-performance liquid chromatography (see, for example, Ullman, Encyclopedia of Industrial Chemistry, Vol. A2, p. 89-90 and p. 443-613, VCH: Weinheim (1985); Fallon, A., et al., (1987) “Applications of HPLC in Biochemistry” in: Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 17; Rehm et al. (1993) Biotechnology, Vol. 3, Chapter III: “Product recovery and purification”, p. 469-714, VCH: Weinheim; Belter, P. A., et al. (1988) Bioseparations: downstream processing for Biotechnology, John Wiley and Sons; Kennedy, J. F., and Cabral, J. M. S. (1992) Recovery processes for biological Materials, John Wiley and Sons; Shaeiwitz, J. A., and Henry, J. D. (1988) Biochemical Separations, in: Ullmann's Encyclopedia of Industrial Chemistry, Vol. B3; Chapter 11, p. 1-27, VCH: Weinheim; and Dechow, F.J. (1989) Separation and purification techniques in biotechnology, Noyes Publications).
Gamma-aminobutyric acid can for example be detected advantageously via HPLC, LC or GC separation methods. The unambiguous detection for the presence of gamma-aminobutyric acid containing products can be obtained by analyzing recombinant organisms using analytical standard methods: LC, LC-MS, MS or TLC). The material to be analyzed can be disrupted by sonication, grinding in a glass mill, liquid nitrogen and grinding, cooking, or via other applicable methods.
The GABA can be isolated and purified.
The unambiguous detection for the presence of gamma-aminobutyric acid can be obtained by analyzing recombinant organisms using analytical standard methods: LC, LCMSMS or TLC, as described. The total amount produced in the organism for example in yeasts used in the inventive process can be analysed for example according to the following procedure:
The material such as yeasts, E. coli or plants to be analyzed can be disrupted by sonication, grinding in a glass mill, liquid nitrogen and grinding or via other applicable methods.
Plant material is initially homogenized mechanically by comminuting in a pestle and mortar to make it more amenable to extraction.
A typical sample pretreatment consists of a total lipid extraction using such polar organic solvents as acetone or alcohols as methanol, or ethers, saponification, partition between phases, seperation of non-polar epiphase from more polar hypophasic derivatives and chromatography.
For analysis, solvent delivery and aliquot removal can be accomplished with a robotic system comprising a single injector valve Gilson 232XL and a 402 2S1V diluter [Gilson, Inc. USA, 3000 W. Beltline Highway, Middleton, Wis.]. For saponification, 3 ml of 50% potassium hydroxide hydro-ethanolic solution (4 water−1 ethanol) can be added to each vial, followed by the addition of 3 ml of octanol. The saponification treatment can be conducted at room temperature with vials maintained on an IKA HS 501 horizontal shaker [Labworld-online, Inc., Wilmington, N.C.] for fifteen hours at 250 movements/minute, followed by a stationary phase of approximately one hour.
Following saponification, the supernatant can be diluted with 0.17 ml of methanol. The addition of methanol can be conducted under pressure to ensure sample homogeneity. Using a 0.25 ml syringe, a 0.1 ml aliquot can be removed and transferred to HPLC vials for analysis.
For HPLC analysis, a Hewlett Packard 1100 HPLC, complete with a quaternary pump, vacuum degassing system, six-way injection valve, temperature regulated autosampler, column oven and Photodiode Array detector can be used [Agilent Technologies available through Ultra Scientific Inc., 250 Smith Street, North Kingstown, R.I.]. The column can be a Waters YMC30, 5-micron, 4.6×250 mm with a guard column of the same material [Waters, 34 Maple Street, Milford, Mass.]. The solvents for the mobile phase can be 81 methanol: 4 water: 15 tetrahydrofuran (THF) stabilized with 0.2% BHT (2,6-di-tert-butyl-4-methylphenol). Injections were 20 I. Separation can be isocratic at 30° C. with a flow rate of 1.7 ml/minute. The peak responses can be measured by absorbance at 447 nm.
If required and desired, further chromatography steps with a suitable resin may follow. Advantageously, the gamma-aminobutyric acid can be further purified with a so-called RTHPLC. As eluent acetonitrile/water or chloroform/acetonitrile mixtures can be used. If necessary, these chromatography steps may be repeated, using identical or other chromatography resins. The skilled worker is familiar with the selection of suitable chromatography resin and the most effective use for a particular molecule to be purified.
Abbreviations; GC-MS, gas liquid chromatography/mass spectrometry; TLC, thin-layer chromatography.
The identity and purity of the compound(s) isolated can be determined by prior-art techniques. They encompass high-performance liquid chromatography (HPLC), gas chromatography (GC), spectroscopic methods, mass spectrometry (MS), staining methods, thin-layer chromatography, NIRS, enzyme assays or microbiological assays. These analytical methods are compiled in: Patek et al. (1994) Appl. Environ. Microbiol. 60:133-140; Malakhova et al. (1996) Biotekhnologiya 11 27-32; and Schmidt et al. (1998) Bioprocess Engineer. 19:67-70. Ulmann's Encyclopedia of Industrial Chemistry (1996) Bd. A27, VCH Weinheim, pp. 89-90, pp. 521-540, pp. 540-547, pp. 559-566, 575-581 and pp. 581-587; Michal, G (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, vol. 17.
The gamma-aminobutyric acid obtained in the process are suitable as starting material for the synthesis of further products of value. For example, they can be used in combination with each other or alone for the production of pharmaceuticals, foodstuffs, animal feeds or cosmetics. Accordingly, the present invention relates a method for the production of pharmaceuticals, food stuff, animal feeds, nutrients or cosmetics comprising the steps of the process according to the invention, including the isolation of the gamma-aminobutyric acid composition produced or the GABA produced if desired and formulating the product with a pharmaceutical acceptable carrier or formulating the product in a form acceptable for an application in agriculture. A further embodiment according to the invention is the use of the gamma-aminobutyric acid produced in the process or of the transgenic organisms in animal feeds, foodstuffs, medicines, food supplements, cosmetics or pharmaceuticals or for the production of gamma-aminobutyric acid e.g. after isolation of the GABA or without, e.g. in situ, e.g in the organism used for the process for the production of the GABA.
The plants of the invention, e.g. having an increase GABA content, have an increase nitrogen uptake. Additionally these plants have an increase nitrogen assimilation and utilization, preferably at low nitrogen disposal and/or nitrogen deprivation.
In one embodiment of the present invention, an enhanced nitrogen uptake leads into an increased nitrogen use efficiency. An increased nitrogen use efficiency is further in one embodiment an enhanced nitrogen uptake, assimilation and utilization.
In one embodiment of the present invention, an enhanced nitrogen uptake leads into an increased plant yield. So an increased yield is mediated by increasing the “nitrogen use efficiency of a plant”.
In one embodiment the plants of the invention show an increase GABA content and an increase nitrogen uptake. In one embodiment these plants have additionally an increase nitrogen assimilation and utilization, preferably at low nitrogen disposal and/or nitrogen deprivation.
In one embodiment of the plants of the invention show an increased GABA content and increased nitrogen use efficiency.
In one embodiment of the plants of the present invention have an increased GABA content and increased plant yield.
In a further embodiment, an increased tolerance to abiotic environmental stress and/or increased yield related trait, in particular increased nutrient use efficiency, preferably nitrogen use efficiency, compared to a corresponding non-modified, e.g. a non-transformed, wild type plant is conferred if the activity of a polypeptide comprising the polypeptide shown in SEQ ID NO. 43, or encoded by a nucleic acid molecule comprising the nucleic acid molecule shown in SEQ ID NO. 42, or a homolog of said nucleic acid molecule or polypeptide, is increased or generated. For example, the activity of a corresponding nucleic acid molecule or a polypeptide derived from Saccharomyces cerevisiae is increased or generated, preferably comprising the nucleic acid molecule shown in SEQ ID NO. 42 or polypeptide shown in SEQ ID NO. 43, respectively, or a homolog thereof. E.g. an increased tolerance to abiotic environmental stress and/or increased yield related trait, in particular increased nutrient use efficiency, preferably nitrogen use efficiency, compared to a corresponding non-modified, e.g. a non-transformed, wild type plant is conferred if the activity “Factor arrest protein” or if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, depicted in table I, II or IV, column 7, respective same line as SEQ ID NO.: 42 or SEQ ID NO.: 43, respectively, is increased or generated in a plant or part thereof. Preferably, the increase occurs cytoplasmic. Particularly, an increase of yield from 1.05-fold to 1.28-fold, for example plus at least 100% thereof, is conferred compared to a corresponding control, e.g. an non-modified, e.g. non-transformed, wild type plant.
In a further embodiment, an increased tolerance to abiotic environmental stress and/or increased yield related trait, in particular increased nutrient use efficiency, preferably nitrogen use efficiency, compared to a corresponding non-modified, e.g. a non-transformed, wild type plant is conferred if the activity of a polypeptide comprising the polypeptide shown in SEQ ID NO. 7138, or encoded by a nucleic acid molecule comprising the nucleic acid molecule shown in SEQ ID NO. 7137, or a homolog of said nucleic acid molecule or polypeptide, is increased or generated. For example, the activity of a corresponding nucleic acid molecule or a polypeptide derived from Saccharomyces cerevisiae is increased or generated, preferably comprising the nucleic acid molecule shown in SEQ ID NO. 7137 or polypeptide shown in SEQ ID NO. 7138, respectively, or a homolog thereof. E.g. an increased tolerance to abiotic environmental stress and/or increased yield related trait, in particular increased nutrient use efficiency, preferably nitrogen use efficiency, compared to a corresponding non-modified, e.g. a non-transformed, wild type plant is conferred if the activity “microsomal beta-keto-reductase” or if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, depicted in table I, II or IV, column 7, respective same line as SEQ ID NO.: 7137 or SEQ ID NO.: 7138, respectively, is increased or generated in a plant or part thereof. Preferably, the increase occurs cytoplasmic. Particularly, an increase of yield from 1.05-fold to 1.38-fold, for example plus at least 100% thereof, is conferred compared to a corresponding control, e.g. an non-modified, e.g. non-transformed, wild type plant. In a further embodiment, an increased tolerance to abiotic environmental stress and/or increased yield related trait, in particular increased nutrient use efficiency, preferably nitrogen use efficiency, compared to a corresponding non-modified, e.g. a non-transformed, wild type plant is conferred if the activity of a polypeptide comprising the polypeptide shown in SEQ ID NO. 8240, or encoded by a nucleic acid molecule comprising the nucleic acid molecule shown in SEQ ID NO. 8239, or a homolog of said nucleic acid molecule or polypeptide, is increased or generated. For example, the activity of a corresponding nucleic acid molecule or a polypeptide derived from Saccharomyces cerevisiae is increased or generated, preferably comprising the nucleic acid molecule shown in SEQ ID NO. 8239 or polypeptide shown in SEQ ID NO. 8240, respectively, or a homolog thereof. E.g. an increased tolerance to abiotic environmental stress and/or increased yield related trait, in particular increased nutrient use efficiency, preferably nitrogen use efficiency, compared to a corresponding non-modified, e.g. a non-transformed, wild type plant is conferred if the activity “60S ribosomal protein” or if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, depicted in table I, II or IV, column 7, respective same line as SEQ ID NO.: 8239 or SEQ ID NO.: 8240, respectively, is increased or generated in a plant or part thereof. Preferably, the increase occurs cytoplasmic. Particularly, an increase of yield from 1.05-fold to 1.223-fold, for example plus at least 100% thereof, is conferred compared to a corresponding control, e.g. an non-modified, e.g. non-transformed, wild type plant.
In a further embodiment, an increased tolerance to abiotic environmental stress and/or increased yield related trait, in particular increased nutrient use efficiency, preferably nitrogen use efficiency, compared to a corresponding non-modified, e.g. a non-transformed, wild type plant is conferred if the activity of a polypeptide comprising the polypeptide shown in SEQ ID NO. 8228, or encoded by a nucleic acid molecule comprising the nucleic acid molecule shown in SEQ ID NO. 8227, or a homolog of said nucleic acid molecule or polypeptide, is increased or generated. For example, the activity of a corresponding nucleic acid molecule or a polypeptide derived from Saccharomyces cerevisiae is increased or generated, preferably comprising the nucleic acid molecule shown in SEQ ID NO. 8227 or polypeptide shown in SEQ ID NO. 8228, respectively, or a homolog thereof. E.g. an increased tolerance to abiotic environmental stress and/or increased yield related trait, in particular increased nutrient use efficiency, preferably nitrogen use efficiency, compared to a corresponding non-modified, e.g. a non-transformed, wild type plant is conferred if the activity “cytochrome c oxidase subunit VIII” or if the activity of a nucleic acid molecule or a polypeptide comprising the nucleic acid or polypeptide or the consensus sequence or the polypeptide motif, depicted in table I, II or IV, column 7, respective same line as SEQ ID NO.: 8227 or SEQ ID NO.: 8228, respectively, is increased or generated in a plant or part thereof. Preferably, the increase occurs cytoplasmic. Particularly, an increase of yield from 1.05-fold to 1.56-fold, for example plus at least 100% thereof, is conferred compared to a corresponding control, e.g. an non-modified, e.g. non-transformed, wild type plant.
It was further observed that increasing or generating the activity of a gene shown in Table IX, e.g. a nucleic acid molecule derived from the nucleic acid molecule shown in Table IX in A. thaliana conferred increased nutrient use efficiency, preferably nitrogen use efficiency, compared to the wild type control. Thus, in one embodiment, a nucleic acid molecule indicated in Table IX or its homolog as indicated in Table I or the expression product is used in the method of the present invention to increased nutrient use efficiency, preferably nitrogen use efficiency, of the plant compared to the wild type control.
In one embodiment of the invention the enhanced NUE is determinated and quantified according to the following methods:
Procedure 1:
Biomass Production on Agar Plates:
For screening of transgenic plants a specific culture facility is used. For high-throughput purposes plants are screened for biomass production on agar plates with limited supply of nitrogen (adapted from Estelle and Somerville, 1987). This screening pipeline consists of two levels. Transgenic lines are subjected to subsequent level if biomass production was significantly improved in comparison to wild type plants. With each level number of replicates and statistical stringency was increased.
For the sowing, the seeds, which can be stored in the refrigerator (at −20° C.), can be removed from the Eppendorf tubes with the aid of a toothpick and transferred onto the above-mentioned agar plates, with limited supply of nitrogen (0.05 mM KNO3). In total, approximately 15-30 seeds can be distributed horizontally on each plate (12×12 cm). After the seeds are sown, plates are subjected to stratification for 2-4 days in the dark at 4° C. After the stratification, the test plants are grown for 22 to 25 days at a 16-h-light, 8-h-dark rhythm at 20° C., an atmospheric humidity of 60% and a CO2 concentration of approximately 400 ppm. The light sources to be used generate a light resembling the solar color spectrum with a light intensity of approximately 100 μE/m2s. After 10 to 11 days the plants are individualized. Improved growth under nitrogen limited conditions is assessed by biomass production of shoots and roots of transgenic plants in comparison to wild type control plants after 20-25 days growth.
Transgenic lines showing a significant improved biomass production in comparison to wild type plants are subjected to following experiment of the subsequent level:
Biomass Production on Soil:
Arabidopsis thaliana seeds are sown in pots containing a 1:1 (v/v) mixture of nutrient depleted soil (“Einheitserde Typ 0”, 30% clay, Tantau, Wansdorf Germany) and sand. Germination is induced by a four day period at 4° C., in the dark. Subsequently the plants are grown under standard growth conditions (photoperiod of 16 h light and 8 h dark, 20° C., 60% relative humidity, and a photon flux density of 200 μE/m2s). The plants are grown and cultured, inter alia they are watered every second day with a N-depleted nutrient solution. The N-depleted nutrient solution e.g. contains beneath water
After 9 to 10 days the plants are individualized. After a total time of 29 to 31 days the plants are harvested and rated by the fresh weight of the aerial parts of the plants. The biomass increase can be measured as ratio of the fresh weight of the aerial parts of the respective transgene plant and the non-transgenic wild type plant.
Procedure 2:
Procedure 2 can be performed like procedure 1, however, the screening on agar plates is omitted and a one-level screen on soil is performed.
Throughout this application, various publications are referenced. The disclosures of all of these publications and those references cited within those publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
It should also be understood that the foregoing relates to preferred embodiments of the present invention and that numerous changes and variations may be made therein without departing from the scope of the invention. The invention is further illustrated by the following examples, which are not to be construed in any way as limiting. On the contrary, it is to be clearly understood that various other embodiments, modifications and equivalents thereof, which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention and/or the scope of the claims.
Unless otherwise specified, standard methods as described in Sambrook et al., Molecular Cloning: A laboratory manual, Cold Spring Harbor 1989, Cold Spring Harbor Laboratory Press are used.
The inventive sequences as shown in table I, column 5, were amplified by PCR as described in the protocol of the Pfu Ultra, Pfu Turbo or Herculase DNA polymerase (Stratagene).
The composition for the protocol of the Pfu Ultra, Pfu Turbo or Herculase DNA polymerase was as follows: 1×PCR buffer (Stratagene), 0.2 mM of each dNTP, 100 ng genomic DNA of Saccharomyces cerevisiae (strain S288C; Research Genetics, Inc., now Invitrogen), Escherichia coli (strain MG1655; E. coli Genetic Stock Center), Synechocystis sp. (strain PCC6803), Azotobacter vinelandii (strain N. R. Smith, 16), Thermus thermophilus (HB8) or 50 ng cDNA from various tissues and development stages of Arabidopsis thaliana (ecotype Columbia), Physcomitrella patens, Glycine max (variety Resnick), or Zea mays (variety B73, Mo17, A188), 50 μmol forward primer, 50 μmol reverse primer, with or without 1 M Betaine, 2.5u Pfu Ultra, Pfu Turbo or Herculase DNA polymerase.
The Amplification cycles were as follows:
1 cycle of 2-3 minutes at 94-95° C., then 25-36 cycles with 30-60 seconds at 94-95° C., 30-45 seconds at 50-60° C. and 210-480 seconds at 72° C., followed by 1 cycle of 5-10 minutes at 72° C., then 4-16° C.—preferably for Saccharomyces cerevisiae; Escherichia coli, Synechocystis sp., Azotobacter vinelandii, Thermus thermophilus.
In case of Arabidopsis thaliana, Brassica napus, Glycine max, Oryza sativa, Physcomitrella patens, Zea mays the amplification cycles were as follows:
1 cycle with 30 seconds at 94° C., 30 seconds at 61° C., 15 minutes at 72° C., then 2 cycles with 30 seconds at 94° C., 30 seconds at 60° C., 15 minutes at 72° C., then 3 cycles with 30 seconds at 94° C., 30 seconds at 59° C., 15 minutes at 72° C., then 4 cycles with 30 seconds at 94° C., 30 seconds at 58° C., 15 minutes at 72° C., then 25 cycles with 30 seconds at 94° C., 30 seconds at 57° C., 15 minutes at 72° C., then 1 cycle with 10 minutes at 72° C.,
then finally 4-16° C.
RNA were generated with the RNeasy Plant Kit according to the standard protocol (Qiagen) and Supersript II Reverse Transkriptase was used to produce double stranded cDNA according to the standard protocol (Invitrogen).
ORF specific primer pairs for the genes to be expressed are shown in table III, column 7. The following adapter sequences were added to Saccharomyces cerevisiae ORF specific primers for cloning purposes:
These adaptor sequences allow cloning of the ORF into the various vectors containing the Resgen adaptors, see table column E of table VI.
The following adapter sequences were added to Saccharomyces cerevisiae, Escherichia coli, Synechocystis sp., Azotobacter vinelandii, Thermus thermophilus, Arabidopsis thaliana, Brassica napus or Physcomitrella patens ORF specific primers for cloning purposes:
The adaptor sequences allow cloning of the ORF into the various vectors containing the Colic adaptors, see column E of table VI.
Therefore for amplification and cloning of Saccharomyces cerevisiae SEQ ID NO: 42, a primer consisting of the adaptor sequence i) and the ORF specific sequence SEQ ID NO: 48 and a second primer consisting of the adaptor sequence ii) and the ORF specific sequence SEQ ID NO: 49 or a primer consisting of the adaptor sequence iii) and the ORF specific sequence SEQ ID NO: 48 and a second primer consisting of the adaptor sequence iiii) and the ORF specific sequence SEQ ID NO: 49 were used.
For amplification and cloning of Echerichia coli SEQ ID NO: 4068, a primer consisting of the adaptor sequence iii) and the ORF specific sequence SEQ ID NO: 4160 and a second primer consisting of the adaptor sequence iiii) and the ORF specific sequence SEQ ID NO: 4161 were used.
For amplification and cloning of Synechocystis sp. SEQ ID NO: 6041, a primer consisting of the adaptor sequence iii) and the ORF specific sequence SEQ ID NO: 6461 and a second primer consisting of the adaptor sequence iiii) and the ORF specific sequence SEQ ID NO: 6462 were used.
For amplification and cloning of Azotobacter vinelandii SEQ ID NO: 2553, a primer consisting of the adaptor sequence iii) and the ORF specific sequence SEQ ID NO: 3397 and a second primer consisting of the adaptor sequence iiii) and the ORF specific sequence SEQ ID NO: 3398 were used.
For amplification and cloning of Thermus thermophilus SEQ ID NO: 6469, a primer consisting of the adaptor sequence iii) and the ORF specific sequence SEQ ID NO: 6735 and a second primer consisting of the adaptor sequence iiii) and the ORF specific sequence SEQ ID NO: 6736 were used.
For amplification and cloning of Arabidopsis thaliana SEQ ID NO: 654, a primer consisting of the adaptor sequence iii) and the ORF specific sequence SEQ ID NO: 694 and a second primer consisting of the adaptor sequence iiii) and the ORF specific sequence SEQ ID NO: 695 were used.
For amplification and cloning of Brassica napus SEQ ID NO: 53, a primer consisting of the adaptor sequence iii) and the ORF specific sequence SEQ ID NO: 649 and a second primer consisting of the adaptor sequence iiii) and the ORF specific sequence SEQ ID NO: 650 were used.
For amplification and cloning of Physcomitrella patens SEQ ID NO: 5458, a primer consisting of the adaptor sequence iii) and the ORF specific sequence SEQ ID NO:
6038 and a second primer consisting of the adaptor sequence iiii) and the ORF specific sequence SEQ ID NO: 6039 were used.
Following these examples every sequence disclosed in table I, preferably column 5, can be cloned by fusing the adaptor sequences to the respective specific primer sequences as disclosed in table III, column 7 using the respective vectors shown in table VI.
“Non-targeted” expression in this context means, that no additional targeting sequence was added to the ORF to be expressed.
For non targeted expression in preferentially green tissues the following binary vectors were used for cloning: pMTX155, VC-MME220-1qcz and VC-MME221-1qcz.
For constitutive expression of ORFs from Saccharomyces cerevisiae in preferentially green tissues the enhanced 35S (Big35 S) promoter (Comai et al., Plant Mol Biol 15, 373-383 (1990)) in context of the vector pMTX155 was used.
For constitutive expression of ORFs from Echerichia coli in preferentially green tissues an artifical promoter A(ocs)3AmasPmas promoter (Super promoter) (Ni et al., Plant Journal 7, 661 (1995), WO 95/14098) in context of the vector VC-MME220-1qcz was used.
For constitutive expression in preferentially green tissues and seeds the PcUbi promoter from parsley (Kawalleck et al., Plant. Molecular Biology, 21, 673 (1993), WO 2003/102198) was used in context of the vector VC-MME221-1qcz for ORFs from Saccharomyces cerevisiae, Echerichia coli, Synechocystis sp., Azotobacter vinelandii, Thermus thermophilus, Arabidopsis thaliana, Brassica napus, Glycine max, Oryza sativa, Physcomitrella patens, or Zea mays.
Amplification of the Plastid Targeting Sequence of the Gene FNR from Spinacia oleracea and Construction of Vector for Plastid-Targeted Expression in Preferential Green Tissues or Preferential in Seeds.
In order to amplify the targeting sequence of the FNR gene from S. oleracea, genomic DNA was extracted from leaves of 4 weeks old S. oleracea plants (DNeasy Plant Mini Kit, Qiagen, Hilden). The gDNA was used as the template for a PCR.
To enable cloning of the transit sequence into the vector VC-MME489-1 QCZ, an EcoRI restriction enzyme recognition sequence was added to both the forward and reverse primers, whereas for cloning in the vector VC-MME220-1qcz and VC-MME221-1qcz a Pmel restriction enzyme recognition sequence was added to the forward primer and a Ncol site was added to the reverse primer.
The resulting sequence SEC) ID NO: 28 amplified from genomic spinach DNA, comprised a 5′UTR (bp 1-165), and the coding region (bp 166-273 and 351-419). The coding sequence is interrupted by an intronic sequence from by 274 to by 350:
The PCR fragment derived with the primers FNR5EcoResgen and FNR3EcoResgen was digested with EcoRI and ligated in the vector VC-MME489-1 QCZ that had been digested with EcoRI. The correct orientation of the FNR targeting sequence was tested by sequencing. The vector generated in this ligation step was VC-MME354-1 QCZ.
The PCR fragment derived with the primers FNR5PmeColic and FNR3NcoColic was digested with Pmel and Ncol and ligated in the vector VC-MME220-1qcz and VC-MME221-1qcz that had been digested with Smal and Ncol. The vector generated in this ligation step was VC-MME432-1qcz and pMTX447korrp.
For plastidic-targeted constitutive expression in preferentially green tissues an artifical promoter A(ocs)3AmasPmas promoter (Super promotor)) (Ni et al., Plant Journal 7, 661 (1995), WO 95/14098) was used in context of the vector VC-MME354-1QCZ for ORFs from Saccharomyces cerevisiae and in context of the vector VC-MME432-1qcz for ORFs from Escherichia coli, resulting in “in-frame” fusion of the FNR targeting sequence with the ORFs.
For plastidic-targeted constitutive expression in preferentially green tissues and seeds the PcUbi promoter was use in context of the vector pMTX447korrp for ORFs from Saccharomyces cerevisiae, Echerichia coli, Synechocystis sp., Azotobacter vinelandii, Thermus thermophilus, Arabidopsis thaliana, Brassica napus, Glycine max, Oryza sativa, Physcomitrella patens, or Zea mays, resulting in “in-frame” fusion of the FNR targeting sequence with the ORFs.
For cloning the ORFs from S. cerevisiae into vectors containing the Resgen adaptor sequence the respective vector DNA was treated with the restriction enzyme Ncol. For cloning of ORFs from Saccharomyces cerevisiae into vectors containing the Colic adaptor sequence, the respective vector DNA was treated with the restriction enzymes Pacl and Ncol following the standard protocol (MBI Fermentas). For cloning of ORFs from Escherichia coli, Synechocystis sp., Azotobacter vinelandii, Thermus thermophilus, Arabidopsis thaliana, Brassica napus, Glycine max, Oryza sativa, Physcomitrella patens, or Zea mays the vector DNA was treated with the restriction enzymes Pacl and Ncol following the standard protocol (MBI Fermentas). In all cases the reaction was stopped by inactivation at 70° C. for 20 minutes and purified over QIAquick or Nucleo-Spin Extract II columns following the standard protocol (Qiagen or Macherey-Nagel).
Then the PCR-product representing the amplified ORF with the respective adapter sequences and the vector DNA were treated with T4 DNA polymerase according to the standard protocol (MBI Fermentas) to produce single stranded overhangs with the parameters 1 unit T4 DNA polymerase at 37° C. for 2-10 minutes for the vector and 1-2u T4 DNA polymerase at 15-17° C. for 10-60 minutes for the PCR product representing.
The reaction was stopped by addition of high-salt buffer and purified over QIAquick or Nucleo-Spin Extract II columns following the standard protocol (Qiagen or Macherey-Nagel).
According to this example the skilled person is able to clone all sequences disclosed in table I, preferably column 5.
Approximately 30-60 ng of prepared vector and a defined amount of prepared amplificate were mixed and hybridized at 65° C. for 15 minutes followed by 37° C. 0.1° C./1 seconds, followed by 37° C. 10 minutes, followed by 0.1° C./1 seconds, then 4-10° C.
The ligated constructs were transformed in the same reaction vessel by addition of competent E. coli cells (strain DH5alpha) and incubation for 20 minutes at 1° C. followed by a heat shock for 90 seconds at 42° C. and cooling to 1-4° C. Then, complete medium (SOC) was added and the mixture was incubated for 45 minutes at 37° C. The entire mixture was subsequently plated onto an agar plate with 0.05 mg/ml kanamycine and incubated overnight at 37° C.
The outcome of the cloning step was verified by amplification with the aid of primers which bind upstream and downstream of the integration site, thus allowing the amplification of the insertion. The amplifications were carried out as described in the protocol of Taq DNA polymerase (Gibco-BRL).
The amplification cycles were as follows:
1 cycle of 1-5 minutes at 94° C., followed by 35 cycles of in each case 15-60 seconds at 94° C., 15-60 seconds at 50-66° C. and 5-15 minutes at 72° C., followed by 1 cycle of 10 minutes at 72° C., then 4-16° C.
Several colonies were checked, but only one colony for which a PCR product of the expected size was detected was used in the following steps.
A portion of this positive colony was transferred into a reaction vessel filled with complete medium (LB) supplemented with kanamycin and incubated overnight at 37° C.
The plasmid preparation was carried out as specified in the Qiaprep or Nucleo-Spin Multi-96 Plus standard protocol (Qiagen or Macherey-Nagel).
Generation of transgenic plants which express SEQ ID NO: 42 or any other sequence disclosed in table I, preferably column 5
1-5 ng of the plasmid DNA isolated was transformed by electroporation or transformation into competent cells of Agrobacterium tumefaciens, of strain GV 3101 μMP90 (Koncz and Schell, Mol. Gen. Gent. 204, 383 (1986)). Thereafter, complete medium (YEP) was added and the mixture was transferred into a fresh reaction vessel for 3 hours at 28° C. Thereafter, all of the reaction mixture was plated onto YEP agar plates supplemented with the respective antibiotics, e.g. rifampicine (0.1 mg/ml), gentamycine (0.025 mg/ml and kanamycine (0.05 mg/ml) and incubated for 48 hours at 28° C.
The agrobacteria that contains the plasmid construct were then used for the transformation of plants.
A colony was picked from the agar plate with the aid of a pipette tip and taken up in 3 ml of liquid TB medium, which also contained suitable antibiotics as described above. The preculture was grown for 48 hours at 28° C. and 120 rpm.
400 ml of LB medium containing the same antibiotics as above were used for the main culture. The preculture was transferred into the main culture. It was grown for 18 hours at 28° C. and 120 rpm. After centrifugation at 4 000 rpm, the pellet was resuspended in infiltration medium (MS medium, 10% sucrose).
In order to grow the plants for the transformation, dishes (Piki Saat 80, green, provided with a screen bottom, 30×20×4.5 cm, from Wiesauplast, Kunststofftechnik, Germany) were half-filled with a GS 90 substrate (standard soil, Werkverband E.V., Germany). The dishes were watered overnight with 0.05% Proplant solution (Chimac-Apriphar, Belgium). Arabidopsis thaliana C24 seeds (Nottingham Arabidopsis Stock Centre, UK; NASC Stock N906) were scattered over the dish, approximately 1 000 seeds per dish. The dishes were covered with a hood and placed in the stratification facility (8 h, 110 μmol/m2s1, 22° C.; 16 h, dark, 6° C.). After 5 days, the dishes were placed into the short-day controlled environment chamber (8 h, 130 μmol/m2s1, 22° C.; 16 h, dark, 20° C.), where they remained for approximately 10 days until the first true leaves had formed.
The seedlings were transferred into pots containing the same substrate (Teku pots, 7 cm, LC series, manufactured by Pöppelmann GmbH & Co, Germany). Five plants were pricked out into each pot. The pots were then returned into the short-day controlled environment chamber for the plant to continue growing.
After 10 days, the plants were transferred into the greenhouse cabinet (supplementary illumination, 16 h, 340 μE/m2s, 22° C.; 8 h, dark, 20° C.), where they were allowed to grow for further 17 days.
For the transformation, 6-week-old Arabidopsis plants, which had just started flowering were immersed for 10 seconds into the above-described agrobacterial suspension which had previously been treated with 10 μl Silwett L77 (Crompton S. A., Osi Specialties, Switzerland). The method in question is described by Clough J. C. and Bent A. F. (Plant J. 16, 735 (1998)).
The plants were subsequently placed for 18 hours into a humid chamber. Thereafter, the pots were returned to the greenhouse for the plants to continue growing. The plants remained in the greenhouse for another 10 weeks until the seeds were ready for harvesting.
Depending on the resistance marker used for the selection of the transformed plants the harvested seeds were planted in the greenhouse and subjected to a spray selection or else first sterilized and then grown on agar plates supplemented with the respective selection agent. Since the vector contained the bar gene as the resistance marker, plantlets were sprayed four times at an interval of 2 to 3 days with 0.02% BASTA® and transformed plants were allowed to set seeds.
The seeds of the transgenic A. thaliana plants were stored in the freezer (at −20° C.).
For the bioanalytical analyses of the transgenic plants, the latter were grown uniformly in a specific culture facility. To this end the GS-90 substrate was introduced into the potting machine (Laible System GmbH, Singen, Germany) and filled into the pots. Thereafter, 35 pots were combined in one dish and treated with Proplant. For the treatment, 15 ml of Proplant were taken up in 10 I of tap water (0.15% solution). This amount was sufficient for the treatment of approximately 280 pots. The pots were placed into the Proplant solution and additionally irrigated overhead. 3 I Proplant solution (0.15%) for 210 pots. They were used within five days.
For sowing, the seeds, which had been stored in the refrigerator (at −20° C.) were dispersed from the Eppendorf tubes into the pots. In total, approximately 5 to 10 seeds were distributed in the middle of the pot.
After the seeds had been sown, the dishes with the pots were covered with matching plastic hoods and placed into the stratification chamber for 4 days in the dark at 4° C. The humidity was approximately 90%. After the stratification, the test plants were grown for 22 to 23 days at a 16-h-light, 8-h-dark rhythm at 20° C., an atmospheric humidity of 60% and a CO2 concentration of approximately 400 ppm. The light sources used were Powerstar HQI-T 250 W/D Daylight lamps from Osram, which generate a light resembling the solar color spectrum with a light intensity of approximately 220 E/m2/s−1.
Selection of transgenic plants was depending on the used resistance marker. In case of the bar gene as the resistance marker plantlets were sprayed three times at days 8-10 after sowing with 0.02% BASTA®, Bayer CropScience, Germany, Leverkusen. The resistance plants were thinned when they had reached the age of 14 days. The plants, which had grown best in the center of the pot were considered the target plants. All the remaining plants were removed carefully with the aid of metal tweezers and discarded.
During their growth, the plants received overhead irrigation with distilled water and bottom irrigation into the placement grooves. Once the grown plants had reached the age of 23 or 24 days, they were harvested.
Metabolic Analysis of Transformed Plants
The modifications identified in accordance with the invention, in the content of above-described metabolites, were identified by the following procedure.
a) Sampling and Storage of the Samples
Sampling was performed directly in the controlled-environment chamber. The plants were cut using small laboratory scissors, rapidly weighed on laboratory scales, transferred into a pre-cooled extraction thimble and placed into an aluminum rack cooled by liquid nitrogen. If required, the extraction thimble can be stored in the freezer at −80° C. The time elapsing between cutting the plant to freezing it in liquid nitrogen amounted to not more than 10 to 20 seconds.
b) Lyophilization
During the experiment, care was taken that the plants either remained in the deep-frozen state (temperatures <−40° C.) or were freed from water by lyophilization until the first contact with solvents.
The aluminum rack with the plant samples in the extraction thimbles was placed into the pre-cooled (−40° C.) lyophilization facility. The initial temperature during the main drying phase was −35° C. and the pressure was 0.120 mbar. During the drying phase, the parameters were altered following a pressure and temperature program. The final temperature after 12 hours was +30° C. and the final pressure was 0.001 to 0.004 mbar. After the vacuum pump and the refrigerating machine had been switched off, the system was flushed with air (dried via a drying tube) or argon.
c) Extraction
Immediately after the lyophilization apparatus had been flushed, the extraction thimbles with the lyophilized plant material were transferred into the 5 ml extraction cartridges of the ASE device (Accelerated Solvent Extractor ASE 200 with Solvent Controller and AutoASE software (DIONEX)).
The 24 sample positions of an ASE device (Accelerated Solvent Extractor ASE 200 with Solvent Controller and AutoASE software (DIONEX)) were filled with plant samples, including some samples for testing quality control.
The polar substances were extracted with approximately 10 ml of methanol/water (80/20, v/v) at T=70° C. and p=140 bar, 5 minutes heating-up phase, 1 minute static extraction. The more lipophilic substances were extracted with approximately 10 ml of methanol/dichloromethane (40/60, v/v) at T=70° C. and p=140 bar, 5 minute heating-up phase, 1 minute static extraction. The two solvent mixtures were extracted into the same glass tubes (centrifuge tubes, 50 ml, equipped with screw cap and pierceable septum for the ASE (DIONEX)).
The solution was treated with commercial available internal standards, such as ribitol, L-glycine-2,2-d2, L-alanine-2,3,3,3-d4, methionine-d3, Arginine_(13C), Tryptophan-d5, and α-methylglucopyranoside and methyl nonadecanoate, methyl undecanoate, methyl tridecanoate, methyl pentadecanoate, methyl nonacosanoate.
The total extract was treated with 8 ml of water. The solid residue of the plant sample and the extraction sleeve were discarded.
The extract was shaken and then centrifuged for 5 to 10 minutes at least 1400 g in order to accelerate phase separation. 1 ml of the supernatant methanol/water phase (“polar phase”, colorless) was removed for the further GC analysis, and 1 ml was removed for the LC analysis. The remainder of the methanol/water phase was discarded. 0.5 ml of the organic phase (“lipid phase”, dark green) was removed for the further GC analysis and 0.5 ml was removed for the LC analysis. All the portions removed were evaporated to dryness using the IR Dancer infrared vacuum evaporator (Hettich). The maximum temperature during the evaporation process did not exceed 40° C. Pressure in the apparatus was not less than 10 mbar.
d) Processing the Lipid and Polar Phase for the LC/MS or LC/MS/MS Analysis
The lipid extract, which had been evaporated to dryness was taken up in mobile phase. The polar extract, which had been evaporated to dryness was taken up in mobile phase.
e) LC-MS Analyisis
The LC part was carried out on a commercially available LCMS system from Agilent Technologies, USA. For polar extracts 10 μl are injected into the system at a flow rate of 200 μl/min. The separation column (Reversed Phase C18) was maintained at 15° C. during chromatography. For lipid extracts 5 μl are injected into the system at a flow rate of 200 μl/min. The separation column (Reversed Phase C18) was maintained at 30° C. HPLC was performed with gradient elution.
The mass spectrometric analysis was performed on an Applied Biosystems API 4000 triple quadrupole instrument with turbo ion spray source. For polar extracts the instrument measures in negative ion mode in MRM-mode and fullscan mode from 100-1000 amu. For lipid extracts the instrument measures in positive ion mode in MRM-mode fullscan mode from 100-1000 amu. MS analysis is described in more detail in patent publication number WO 03/073464 (Walk and Dostler).
f) Derivatization of the Lipid Phase for the GC/MS Analysis
For the transmethanolysis, a mixture of 140 μl of chloroform, 37 μl of hydrochloric acid (37% by weight HCL in water), 320 μl of methanol and 20 μl of toluene was added to the evaporated extract. The vessel was sealed tightly and heated for 2 hours at 100° C., with shaking. The solution was subsequently evaporated to dryness. The residue was dried completely.
The methoximation of the carbonyl groups was carried out by reaction with methoxyamine hydrochloride (5 mg/ml in pyridine, 100 μl for 1.5 hours at 60° C.) in a tightly sealed vessel. 20 μl of a solution of odd-numbered, straight-chain fatty acids (solution of each 0.3 mg/mL of fatty acids from 7 to 25 carbon atoms and each 0.6 mg/mL of fatty acids with 27, 29 and 31 carbon atoms in 3/7 (v/v) pyridine/toluene) were added as time standards. Finally, the derivatization with 100 μl of N-methyl-N-(trimethylsilyl)-2,2,2-trifluoroacetamide (MSTFA) was carried out for 30 minutes at 60° C., again in the tightly sealed vessel. The final volume before injection into the GC was 220 μl.
g) Derivatization of the Polar Phase for the GC/MS Analysis
The methoximation of the carbonyl groups was carried out by reaction with methoxyamine hydrochloride (5 mg/ml in pyridine, 50 μl for 1.5 hours at 60° C.) in a tightly sealed vessel. 10 μl of a solution of odd-numbered, straight-chain fatty acids (solution of each 0.3 mg/mL of fatty acids from 7 to 25 carbon atoms and each 0.6 mg/ml of fatty acids with 27, 29 and 31 carbon atoms in 3/7 (v/v) pyridine/toluene were added as time standards. Finally, the derivatization with 50 μl of N-methyl-N-(trimethylsilyl)-2,2,2-trifluoroacetamide (MSTFA) was carried out for 30 minutes at 60° C., again in the tightly sealed vessel. The final volume before injection into the GC was 110 μl.
h) GC-MS Analysis
The GC-MS systems consist of an Agilent 6890 GC coupled to an Agilent 5973 MSD. The autosamplers are CompiPaI or GCPaI from CTC. For the analysis usual commercial capillary separation columns (30 m×0.25 mm×0.25 μm) with different polymethyl-siloxane stationary phases containing 0% up to 35% of aromatic moieties, depending on the analysed sample materials and fractions from the phase separation step, are used (for example: DB-1 ms, HP-5 ms, DB-XLB, DB-35 ms, Agilent Technologies). Up to 1 μl of the final volume is injected splitless and the oven temperature program is started at 70° C. and ended at 340° C. with different heating rates depending on the sample material and fraction from the phase separation step in order to achieve a sufficient chromatographic separation and number of scans within each analyte peak. Usual GC-MS standard conditions, for example constant flow with nominal 1 to 1.7 ml/min and helium as the mobile phase gas are used. Ionisation is done by electron impact with 70 eV, scanning within a m/z range from 15 to 600 with scan rates from 2.5 to 3 scans/sec and standard tune conditions.
i) The samples were measured in individual series of 20 to 21 plant or seed samples each (also referred to as sequences), each sequence containing at least 5 wild-type plants or seed samples as controls. Seed samples were from individual plants. The peak area of each analyte was divided by the peak area of the respective internal standard. The data were standardized for the fresh weight established for the plant or seed sample, respectively. The values calculated thus were related to the wild-type control group by being divided by the mean of the corresponding data of the wild-type control group of the same sequence. The values obtained were referred to as ratio_by_WT, they are comparable between sequences and indicate how much the analyte concentration in the mutant differs in relation to the wild-type control. Appropriate controls were done before to proof that the vector and transformation procedure itself has no significant influence on the metabolic composition of the plants. Therefore the described changes in comparison with wildtypes were caused by the introduced gene constructs. At least 3-5 independent lines were analyzed in two independent experiments for each construct.
A regenerating clone of alfalfa (Medicago sativa) is transformed using the method of (McKersie et al., Plant Physiol 119, 839(1999)). Regeneration and transformation of alfalfa is genotype dependent and therefore a regenerating plant is required. Methods to obtain regenerating plants have been described. For example, these can be selected from the cultivar Rangelander (Agriculture Canada) or any other commercial alfalfa variety as described by Brown D. C. W. and Atanassov A. (Plant Cell Tissue Organ Culture 4, 111(1985)). Alternatively, the RA3 variety (University of Wisconsin) is selected for use in tissue culture (Walker et al., Am. J. Bot. 65, 654 (1978)).
Petiole explants are cocultivated with an overnight culture of Agrobacterium tumefaciens C58C1 μMP90 (McKersie et al., Plant Physiol 119, 839(1999)) or LBA4404 containing a binary vector. Many different binary vector systems have been described for plant transformation (e.g. An G., in Agrobacterium Protocols, Methods in Molecular Biology, Vol 44, pp 47-62, Gartland K. M. A. and Davey M. R. eds. Humana Press, Totowa, N.J.). Many are based on the vector pBIN19 described by Bevan (Nucleic Acid Research. 12, 8711 (1984)) that includes a plant gene expression cassette flanked by the left and right border sequences from the Ti plasmid of Agrobacterium tumefaciens. A plant gene expression cassette consists of at least two genes—a selection marker gene and a plant promoter regulating the transcription of the cDNA or genomic DNA of the trait gene. Various selection marker genes can be used including the Arabidopsis gene encoding a mutated acetohydroxy acid synthase (AHAS) enzyme (U.S. Pat. Nos. 5,7673,666 and 6,225,105). Similarly, various promoters can be used to regulate the trait gene that provides constitutive, developmental, tissue or environmental regulation of gene transcription. In this example, the 34S promoter (GenBank Accession numbers M59930 and X16673) is used to provide constitutive expression of the trait gene.
The explants are cocultivated for 3 days in the dark on SH induction medium containing 288 mg/L Pro, 53 mg/L thioproline, 4.35 g/L K2SO4, and 100 μm acetosyringinone.
The explants are washed in half-strength Murashige-Skoog medium (Murashige and Skoog, 1962) and plated on the same SH induction medium without acetosyringinone but with a suitable selection agent and suitable antibiotic to inhibit Agrobacterium growth. After several weeks, somatic embryos are transferred to BOi2Y development medium containing no growth regulators, no antibiotics, and 50 g/L sucrose. Somatic embryos are subsequently germinated on half-strength Murashige-Skoog medium. Rooted seedlings are transplanted into pots and grown in a greenhouse.
T1 or T2 generation seeds plants are produced and subjected to experiments similar as described above to determine their fine chemical content in comparison to respective control material
Seeds of several different ryegrass varieties may be used as explant sources for transformation, including the commercial variety Gunne available from Svalöf Weibull seed company or the variety Affinity. Seeds are surface-sterilized sequentially with 1% Tween-20 for 1 minute, 100% bleach for 60 minutes, 3 rinses with 5 minutes each with deionized and distilled H2O, and then germinated for 3-4 days on moist, sterile filter paper in the dark. Seedlings are further sterilized for 1 minute with 1% Tween-20, 5 minutes with 75% bleach, and rinsed 3 times with dd H2O, 5 min each.
Surface-sterilized seeds are placed on the callus induction medium containing Murashige and Skoog basal salts and vitamins, 20 g/L sucrose, 150 mg/L asparagine, 500 mg/L casein hydrolysate, 3 g/L Phytagel, 10 mg/L BAP, and 5 mg/L dicamba. Plates are incubated in the dark at 25° C. for 4 weeks for seed germination and embryogenic callus induction.
After 4 weeks on the callus induction medium, the shoots and roots of the seedlings are trimmed away, the callus is transferred to fresh media, maintained in culture for another 4 weeks, and then transferred to MSO medium in light for 2 weeks. Several pieces of callus (11-17 weeks old) are either strained through a 10 mesh sieve and put onto callus induction medium, or cultured in 100 ml of liquid ryegrass callus induction media (same medium as for callus induction with agar) in a 250 ml flask. The flask is wrapped in foil and shaken at 175 rpm in the dark at 23° C. for 1 week. Sieving the liquid culture with a 40-mesh sieve collected the cells. The fraction collected on the sieve is plated and cultured on solid ryegrass callus induction medium for 1 week in the dark at 25° C. The callus is then transferred to and cultured on MS medium containing 1% sucrose for 2 weeks.
Transformation can be accomplished with either Agrobacterium or with particle bombardment methods. An expression vector is created containing a constitutive plant promoter and the cDNA of the gene in a pUC vector. The plasmid DNA is prepared from E. coli cells using Qiagen kit according to manufacturer's instruction. Approximately 2 g of embryogenic callus is spread in the center of a sterile filter paper in a Petri dish. An aliquot of liquid MSO with 10 g/L sucrose is added to the filter paper. Gold particles (1.0 μm in size) are coated with plasmid DNA according to method of Sanford et al., 1993 and delivered to the embryogenic callus with the following parameters: 500 μg particles and 2 μg DNA per shot, 1300 psi and a target distance of 8.5 cm from stopping plate to plate of callus and 1 shot per plate of callus.
After the bombardment, calli are transferred back to the fresh callus development medium and maintained in the dark at room temperature for a 1-week period. The callus is then transferred to growth conditions in the light at 25° C. to initiate embryo differentiation with the appropriate selection agent, e.g. 250 nM Arsenal, 5 mg/L PPT or 50 mg/L kanamycin. Shoots resistant to the selection agent are appearing and once rotted are transferred to soil.
Samples of the primary transgenic plants (T0) are analyzed by PCR to confirm the presence of T-DNA. These results are confirmed by Southern hybridization in which DNA is electrophoresed on a 1% agarose gel and transferred to a positively charged nylon membrane (Roche Diagnostics). The PCR DIG Probe Synthesis Kit (Roche Diagnostics) is used to prepare a digoxigenin-labelled probe by PCR, and used as recommended by the manufacturer.
Transgenic T0 ryegrass plants are propagated vegetatively by excising tillers. The transplanted tillers are maintained in the greenhouse for 2 months until well established. The shoots are defoliated and allowed to grow for 2 weeks.
T1 or T2 generation seeds plants are produced and subjected to experiments similar as described above to determine their fine chemical content in comparison to respective control material.
Soybean is transformed according to the following modification of the method described in the Texas A&M patent U.S. Pat. No. 5,164,310. Several commercial soybean varieties are amenable to transformation by this method. The cultivar Jack (available from the Illinois Seed Foundation) is commonly used for transformation. Seeds are sterilized by immersion in 70% (v/v) ethanol for 6 min and in 25% commercial bleach (NaOCI) supplemented with 0.1% (v/v) Tween for 20 min, followed by rinsing 4 times with sterile double distilled water. Seven-day seedlings are propagated by removing the radicle, hypocotyl and one cotyledon from each seedling. Then, the epicotyl with one cotyledon is transferred to fresh germination media in petri dishes and incubated at 25° C. under a 16-h photoperiod (approx. 100 μE/m2s) for three weeks. Axillary nodes (approx. 4 mm in length) were cut from 3-4 week-old plants. Axillary nodes are excised and incubated in Agrobacterium LBA4404 culture.
Many different binary vector systems have been described for plant transformation (e.g. An G., in Agrobacterium Protocols. Methods in Molecular Biology Vol. 44, p. 47-62, Gartland K. M. A. and Davey M. R. eds. Humana Press, Totowa, N.J.). Many are based on the vector pBIN19 described by Bevan (Nucleic Acid Research. 12, 8711 (1984)) that includes a plant gene expression cassette flanked by the left and right border sequences from the Ti plasmid of Agrobacterium tumefaciens. A plant gene expression cassette consists of at least two genes—a selection marker gene and a plant promoter regulating the transcription of the cDNA or genomic DNA of the trait gene. Various selection marker genes can be used including the Arabidopsis gene encoding a mutated acetohydroxy acid synthase (AHAS) enzyme (US Pat. Nos. 5,7673,666 and 6,225,105). Similarly, various promoters can be used to regulate the trait gene to provide constitutive, developmental, tissue or environmental regulation of gene transcription. In this example, the 34S promoter (GenBank Accession numbers M59930 and X16673) can be used to provide constitutive expression of the trait gene.
After the co-cultivation treatment, the explants are washed and transferred to selection media supplemented with 500 mg/L timentin. Shoots are excised and placed on a shoot elongation medium. Shoots longer than 1 cm are placed on rooting medium for two to four weeks prior to transplanting to soil.
The primary transgenic plants (T0) are analyzed by PCR to confirm the presence of T-DNA. These results are confirmed by Southern hybridization in which DNA is electrophoresed on a 1 agarose gel and transferred to a positively charged nylon membrane (Roche Diagnostics). The PCR DIG Probe Synthesis Kit (Roche Diagnostics) is used to prepare a digoxigenin-labelled probe by PCR, and used as recommended by the manufacturer.
T1 or T2 generation seeds plants are produced and subjected to experiments similar as described above to determine their fine chemical content in comparison to respective control material.
Cotyledonary petioles and hypocotyls of 5-6 day-old young seedlings are used as explants for tissue culture and transformed according to Babic et al. (Plant Cell Rep 17, 183 (1998)). The commercial cultivar Westar (Agriculture Canada) is the standard variety used for transformation, but other varieties can be used.
Agrobacterium tumefaciens LBA4404 containing a binary vector can be used for canola transformation. Many different binary vector systems have been described for plant transformation (e.g. An G., in Agrobacterium Protocols. Methods in Molecular Biology Vol. 44, p. 47-62, Gartland K. M. A. and Davey M. R. eds. Humana Press, Totowa, N.J.) Many are based on the vector pBIN19 described by Bevan (Nucleic Acid Research. 12, 8711(1984)) that includes a plant gene expression cassette flanked by the left and right border sequences from the Ti plasmid of Agrobacterium tumefaciens. A plant gene expression cassette consists of at least two genes—a selection marker gene and a plant promoter regulating the transcription of the cDNA or genomic DNA of the trait gene. Various selection marker genes can be used including the Arabidopsis gene encoding a mutated acetohydroxy acid synthase (AHAS) enzyme (U.S. Pat. Nos. 5,7673,666 and 6,225,105). Similarly, various promoters can be used to regulate the trait gene to provide constitutive, developmental, tissue or environmental regulation of gene transcription. In this example, the 34S promoter (GenBank Accession numbers M59930 and X16673) can be used to provide constitutive expression of the trait gene.
Canola seeds are surface-sterilized in 70% ethanol for 2 min., and then in 30% Clorox with a drop of Tween-20 for 10 min, followed by three rinses with sterilized distilled water. Seeds are then germinated in vitro 5 days on half strength MS medium without hormones, 1% sucrose, 0.7% Phytagar at 23° C., 16 h light. The cotyledon petiole explants with the cotyledon attached are excised from the in vitro seedlings, and inoculated with Agrobacterium by dipping the cut end of the petiole explant into the bacterial suspension. The explants are then cultured for 2 days on MSBAP-3 medium containing 3 mg/L BAP, 3% sucrose, 0.7% Phytagar at 23° C., 16 h light. After two days of co-cultivation with Agrobacterium, the petiole explants are transferred to MSBAP-3 medium containing 3 mg/L BAP, cefotaxime, carbenicillin, or timentin (300 mg/L) for 7 days, and then cultured on MSBAP-3 medium with cefotaxime, carbenicillin, or timentin and selection agent until shoot regeneration. When the shoots were 5-10 mm in length, they are cut and transferred to shoot elongation medium (MSBAP-0.5, containing 0.5 mg/L BAP). Shoots of about 2 cm in length are transferred to the rooting medium (MSO) for root induction.
Samples of the primary transgenic plants (T0) are analyzed by PCR to confirm the presence of T-DNA. These results are confirmed by Southern hybridization in which DNA is electrophoresed on a 1 agarose gel and transferred to a positively charged nylon membrane (Roche Diagnostics). The PCR DIG Probe Synthesis Kit (Roche Diagnostics) is used to prepare a digoxigenin-labelled probe by PCR, and used as recommended by the manufacturer.
T1 or T2 generation seeds plants are produced and subjected to experiments similar as described above to determine their fine chemical content in comparison to respective control material.
Transformation of maize (Zea Mays L.) is performed with a modification of the method described by Ishida et al. (Nature Biotech 14745 (1996)). Transformation is genotype-dependent in corn and only specific genotypes are amenable to transformation and regeneration. The inbred line A188 (University of Minnesota) or hybrids with A188 as a parent are good sources of donor material for transformation (From et al. Biotech 8, 833 (1990)), but other genotypes can be used successfully as well. Ears are harvested from corn plants at approximately 11 days after pollination (DAP) when the length of immature embryos is about 1 to 1.2 mm. Immature embryos are co-cultivated with Agrobacterium tumefaciens that carry “super binary” vectors and transgenic plants are recovered through organogenesis. The super binary vector system of Japan Tobacco is described in WO patents WO 94/00977 and WO 95/06722. Vectors were constructed as described. Various selection marker genes can be used including the maize gene encoding a mutated acetohydroxy acid synthase (AHAS) enzyme (U.S. Pat. No. 6,025,541). Similarly, various promoters can be used to regulate the trait gene to provide constitutive, developmental, tissue or environmental regulation of gene transcription. In this example, the 34S promoter (GenBank Accession numbers M59930 and X16673) was used to provide constitutive expression of the trait gene.
Excised embryos are grown on callus induction medium, then maize regeneration medium, containing imidazolinone as a selection agent. The Petri plates are incubated in the light at 25° C. for 2-3 weeks, or until shoots develop. The green shoots are transferred from each embryo to maize rooting medium and incubated at 25° C. for 2-3 weeks, until roots develop. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the imidazolinone herbicides and which are PCR positive for the transgenes.
The T1 transgenic plants are then evaluated for their enhanced NUE and/or increased biomass production according to the method described in Example 3. The T1 generation of single locus insertions of the T-DNA will segregate for the transgene in a 3:1 ratio. Those progeny containing one or two copies of the transgene are tolerant regarding the imidazolinone herbicide, and exhibit an enhancement of NUE and/or increased biomass production than those progeny lacking the transgenes.
T1 or T2 generation plants are produced and subjected to experiments similar as described in WO 2006092449, Example 15c to determine their fine chemical content in comparison to respective control material.
Transformation of wheat is performed with the method described by Ishida et al. (Nature Biotech. 14745 (1996)). The cultivar Bobwhite (available from CYMMIT, Mexico) is commonly used in transformation. Immature embryos are co-cultivated with Agrobacterium tumefaciens that carry “super binary” vectors, and transgenic plants are recovered through organogenesis. The super binary vector system of Japan Tobacco is described in WO patents WO 94/00977 and WO 95/06722. Vectors were constructed as described. Various selection marker genes can be used including the maize gene encoding a mutated acetohydroxy acid synthase (AHAS) enzyme (U.S. Pat. No. 6,025,541). Similarly, various promoters can be used to regulate the trait gene to provide constitutive, developmental, tissue or environmental regulation of gene transcription. In this example, the 34S promoter (GenBank Accession numbers M59930 and X16673) was used to provide constitutive expression of the trait gene.
After incubation with Agrobacterium, the embryos are grown on callus induction medium, then regeneration medium, containing imidazolinone as a selection agent. The Petri plates are incubated in the light at 25° C. for 2-3 weeks, or until shoots develop. The green shoots are transferred from each embryo to rooting medium and incubated at 25° C. for 2-3 weeks, until roots develop. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the imidazolinone herbicides and which are PCR positive for the transgenes.
T1 or T2 generation seeds plants are produced and subjected to experiments similar as described above to determine their fine chemical content in comparison to respective control material.
Rice Transformation:
The two Agrobacterium strains each containing an expression vector, are used independently to transform Oryza sativa plants. Mature dry seeds of the rice japonica cultivar Nipponbare are dehusked. Sterilization is carried out by incubating for one minute in 70% ethanol, followed by 30 minutes in 0.2% HgCl2, followed by a 6 times 15 min wash with sterile distilled water. The sterile seeds are then germinated on a medium containing 2,4-D (callus inducing medium). After incubation in the dark for four weeks, embryonic, scutellum derived calli are excised and propagated on the same medium. After two weeks, the calli are multiplied or propagated by subculture on the same medium for another 2 weeks. Embryonic callus pieces are sub-cultured on fresh medium 3 days before co-cultivation.
Agrobacterium strain LB4404 or other useful Agrobacterium strain, dependent on the expression vector of choice, containing the expression vector is used for co-cultivation. Agrobacterium is inoculated on AB medium (EXPLAIN) with the appropriate antibiotics and cultured for 3 days at 28° C. The bacteria are then collected and suspended in liquid co-cultivation medium at a density OD600) of about 1. The suspension is then transferred to a Petri dish and the calli immersed in the suspension for 15 minutes. The callus tissue were then bolotted dry on a filter paper and transferred to solified, co-cultivation medium and incubated for 3 days in the dark at 25° C. Co-cultivation calli are grown on 2,4-D-containing medium for 4 weeks in the dark at 28° C. in the presence of a selection agent, which dependent of the resistance marker of the used vector. During this period, rapidly growing resistant callus develop. After the transfer of this material to a regeneration medium and incubation in the light, the embryonic potential is released and shoots develop in the next four to five weeks. Shoots are excised from the calli and incubated for 2 to 3 weeks on an auxin-containing medium from which they are transferred to soil. Hardened shoots are grown under high humidity and short days in a greenhouse.
After a quantitative PCR analysis to verify copy number and the T-DNA insert, only single copy transgenic plants that exhibit tolerance to the selection agent are kept to harvest of T1 seed. Seeds are then harvested three to five months after transplanting. Seeds or plants from various independent lines are then used for analysis of the fine chemical content.
Gene sequences can be used to identify identical or heterologous genes from cDNA or genomic libraries. Identical genes (e. g. full-length cDNA clones) can be isolated via nucleic acid hybridization using for example cDNA libraries. Depending on the abundance of the gene of interest, 100,000 up to 1,000,000 recombinant bacteriophages are plated and transferred to nylon membranes. After denaturation with alkali, DNA is immobilized on the membrane by e. g. UV cross linking. Hybridization is carried out at high stringency conditions. In aqueous solution, hybridization and washing is performed at an ionic strength of 1 M NaCl and a temperature of 68° C. Hybridization probes are generated by e.g. radioactive (32P) nick transcription labeling (High Prime, Roche, Mannheim, Germany). Signals are detected by autoradiography.
Partially identical or heterologous genes that are related but not identical can be identified in a manner analogous to the above-described procedure using low stringency hybridization and washing conditions. For aqueous hybridization, the ionic strength is normally kept at 1 M NaCl while the temperature is progressively lowered from 68° C. to 42° C.
Isolation of gene sequences with homology (or sequence identity/similarity) only in a distinct domain of (for example 10-20 amino acids) can be carried out by using synthetic radio labeled oligonucleotide probes. Radiolabeled oligonucleotides are prepared by phosphorylation of the 5-prime end of two complementary oligonucleotides with T4 polynucleotide kinase. The complementary oligonucleotides are annealed and ligated to form concatemers. The double stranded concatemers are than radiolabeled by, for example, nick transcription. Hybridization is normally performed at low stringency conditions using high oligonucleotide concentrations.
Oligonucleotide hybridization solution:
6×SSC
0.01 M sodium phosphate
1 mM EDTA (pH 8)
0.5% SDS
100 μg/ml denatured salmon sperm DNA
0.1% nonfat dried milk
During hybridization, temperature is lowered stepwise to 5-10° C. below the estimated oligonucleotide Tm or down to room temperature followed by washing steps and autoradiography. Washing is performed with low stringency such as 3 washing steps using 4×SSC. Further details are described by Sambrook J. et al., 1989, “Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory Press or Ausubel F.M. et al., 1994, “Current Protocols in Molecular Biology,” John Wiley & Sons.
cDNA clones can be used to produce recombinant polypeptide for example in E. coli (e.g. Qiagen QIAexpress pQE system). Recombinant polypeptides are then normally affinity purified via Ni-NTA affinity chromatography (Qiagen). Recombinant polypeptides are then used to produce specific antibodies for example by using standard techniques for rabbit immunization. Antibodies are affinity purified using a Ni-NTA column saturated with the recombinant antigen as described by Gu et al., BioTechniques 17, 257 (1994). The antibody can than be used to screen expression cDNA libraries to identify identical or heterologous genes via an immunological screening (Sambrook, J. et al., 1989, “Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory Press or Ausubel, F. M. et al., 1994, “Current Protocols in Molecular Biology”, John Wiley & Sons).
In vivo mutagenesis of microorganisms can be performed by passage of plasmid (or other vector) DNA through E. coli or other microorganisms (e.g. Bacillus spp. or yeasts such as Saccharomyces cerevisiae) which are impaired in their capabilities to maintain the integrity of their genetic information. Typical mutator strains have mutations in the genes for the DNA repair system (e.g., mutHLS, mutD, mutT, etc.; for reference, see Rupp W. D., DNA repair mechanisms, in: Escherichia coli and Salmonella, p. 2277-2294, ASM, 1996, Washington.) Such strains are well known to those skilled in the art. The use of such strains is illustrated, for example, in Greener A. and Callahan M., Strategies 7, 32 (1994). Transfer of mutated DNA molecules into plants is preferably done after selection and testing in microorganisms. Transgenic plants are generated according to various examples within the exemplification of this document.
Plants with an increased activity of a polypeptide mentioned in Table VIII and IX under the column SEQ ID NO: or locus were used.
Two different procedures were used for screening:
Procedure 1:
Biomass Production on Agar Plates:
For screening of transgenic plants a specific culture facility was used. For high-throughput purposes plants were screened for biomass production on agar plates with limited supply of nitrogen (adapted from Estelle and Somerville, 1987). This screening pipeline consists of two levels. Transgenic lines were subjected to subsequent level if biomass production was significantly improved in comparison to wild type plants. With each level number of replicates and statistical stringency was increased.
For the sowing, the seeds were removed from the Eppendorf tubes with the aid of a toothpick and transferred onto the above-mentioned agar plates, with limited supply of nitrogen (0.05 mM KNO3). In total, approximately 15-30 seeds were distributed horizontally on each plate (12×12 cm).
After the seeds had been sown, plates were subjected to stratification for 2-4 days in the dark at 4° C. After the stratification, the test plants were grown for 22 to 25 days at a 16-h-light, 8-h-dark rhythm at 20° C., an atmospheric humidity of 60% and a CO2 concentration of approximately 400 ppm. The light sources used generate a light resembling the solar color spectrum with a light intensity of approximately 100 μE/m2s. After 10 to 11 days the plants are individualized. Improved growth under nitrogen limited conditions was assessed by biomass production of shoots and roots of transgenic plants in comparison to wild type control plants after 20-25 days growth.
Transgenic lines showing a significant improved biomass production in comparison to wild type plants were subjected to following experiment of the subsequent level:
Arabidopsis thaliana seeds were sown in pots containing a 1:1 (v/v) mixture of nutrient depleted soil (“Einheitserde Typ 0”, 30% clay, Tantau, Wansdorf Germany) and sand. Germination was induced by a four day period at 4° C., in the dark. Subsequently the plants were grown under standard growth conditions (photoperiod of 16 h light and 8 h dark, 20° C., 60% relative humidity, and a photon flux density of 200 μE/m2s). The plants were grown and cultured, inter alia they were watered every second day with a N-depleted nutrient solution. The N-depleted nutrient solution e.g. contains beneath water
After 9 to 10 days the plants were individualized. After a total time of 29 to 31 days the plants were harvested and rated by the fresh weight of the aerial parts of the plants. The results thereof are summarized in table VIII. The biomass increase has been measured as ratio of the fresh weight of the aerial parts of the respective transgene plant and the non-transgenic wild type plant.
Procedure 2:
Procedure 2 was performed like procedure 1, however, the screening on agar plates was omitted and a one-level screen on soil was performed. Per transgenic construct 4 independent transgenic lines (=events) were tested (16 plants per construct). The results thereof are summarized in table IX.
In this experiment, a plant screening for yield increase (in this case: biomass yield increase) under standardised growth conditions in the absence of substantial abiotic stress has been performed. In a standard experiment soil is prepared as 3.5:1 (v/v) mixture of nutrient rich soil (GS90, Tantau, Wansdorf, Germany) and quarz sand. Alternatively, plants were sown on nutrient rich soil (GS90, Tantau, Germany). Pots were filled with soil mixture and placed into trays. Water was added to the trays to let the soil mixture take up appropriate amount of water for the sowing procedure. The seeds for transgenic A. thaliana plants and their non-trangenic wild-type controls were sown in pots (6 cm diameter). Then the filled tray was covered with a transparent lid and transferred into a precooled (4° C.-5° C.) and darkened growth chamber. Stratification was established for a period of 3-4 days in the dark at 4° C.-5° C. Germination of seeds and growth was initiated at a growth condition of 20° C., approximately 60% relative humidity, 16 h photoperiod and illumination at approximately 200 μmol/m2s. Covers were removed 7-8 days after sowing. BASTA selection was done at day 10 or day 11 (9 or 10 days after sowing) by spraying pots with plantlets from the top. In the standard experiment, a 0.07% (v/v) solution of BASTA concentrate (183 g/I glufosinate-ammonium) in tap water was sprayed once or, alternatively, a 0.02% (v/v) solution of BASTA was sprayed three times. The wild-type control plants were sprayed with tap water only (instead of spraying with BASTA dissolved in tap water) but were otherwise treated identically. Plants were individualized 13-15 days after sowing by removing the surplus of seedlings and leaving one seedling in soil.
Watering was carried out every two days after removing the covers in a standard experiment or, alternatively, every day. For measuring biomass performance, plant fresh weight was determined at harvest time (24-29 days after sowing; 20-26 days after stratification) by cutting shoots and weighing them. Usually, plants were in the stage prior to flowering and prior to growth of inflorescence when harvested. Transgenic plants were compared to the non-transgenic wild-type control plants of the same age, grown in the same culture facility and harvested at the same day.
In the cycling drought assay repetitive stress is applied to plants without leading to desiccation. In a standard experiment soil is prepared as 1:1 (v/v) mixture of nutrient rich soil (GS90, Tantau, Wansdorf, Germany) and quarz sand. Pots (6 cm diameter) were filled with this mixture and placed into trays. Water was added to the trays to let the soil mixture take up appropriate amount of water for the sowing procedure (day 1) and subsequently seeds of transgenic A. thaliana plants and their wild-type controls were sown in pots. Then the filled tray was covered with a transparent lid and transferred into a precooled (4° C.-5° C.) and darkened growth chamber. Stratification was established for a period of 3 days in the dark at 4° C.-5° C. or, alternatively, for 4 days in the dark at 4° C. Germination of seeds and growth was initiated at a growth condition of 20° C., 60% relative humidity, 16 h photoperiod and illumination with fluorescent light at approximately 200 μmol/m2s. Covers were removed 7-8 days after sowing. BASTA selection was done at day 10 or day 11 (9 or 10 days after sowing) by spraying pots with plantlets from the top. In the standard experiment, a 0.07% (v/v) solution of BASTA concentrate (183 g/I glufosinate-ammonium) in tap water was sprayed once or, alternatively, a 0.02% (v/v) solution of BASTA was sprayed three times. The wild-type control plants were sprayed with tap water only (instead of spraying with BASTA dissolved in tap water) but were otherwise treated identically. Plants were individualized 13-14 days after sowing by removing the surplus of seedlings and leaving one seedling in soil. Transgenic events and wild-type control plants were evenly distributed over the chamber.
The water supply throughout the experiment was limited and plants were subjected to cycles of drought and re-watering. Watering was carried out at day 1 (before sowing), day 14 or day 15, day 21 or day 22, and, finally, day 27 or day 28. For measuring biomass production, plant fresh weight was determined one day after the final watering (day 28 or day 29) by cutting shoots and weighing them. Plants were in the stage prior to flowering and prior to growth of inflorescence when harvested. Significance values for the statistical significance of the biomass changes were calculated by applying the ‘student's’ t test (parameters: two-sided, unequal variance).
Up to five lines (events) per transgenic construct were tested in successive experimental levels. Transgenic lines showing increased biomass production compared to wild-type plants were subjected to the next experimental level. Usually in the first level five plants per construct were tested and in the subsequent levels 14-40 plants were tested. Biomass performance was evaluated as described above. Data from level 3 are shown in table XI.
Biomass production was measured by weighing plant rosettes. Biomass increase was calculated as ratio of average weight for transgenic plants compared to average weight of wild type control plants from the same experiment. The mean biomass increase of transgenic constructs is given (significance value <0.05).
In a standard experiment soil was prepared as 3.5:1 (v/v) mixture of nutrient rich soil (GS90, Tantau, Wansdorf, Germany) and sand. Pots were filled with soil mixture and placed into trays. Water was added to the trays to let the soil mixture take up appropriate amount of water for the sowing procedure. The seeds for transgenic A. thaliana plants were sown in pots (6 cm diameter). Stratification was established for a period of 3 days in the dark at 4° C.-5° C. Germination of seeds and growth was initiated at a growth condition of 20° C., approx. 60% relative humidity, 16 h photoperiod and illumination with fluorescent light at 150-200 μmol/m2s. BASTA selection was done at day 9 after sowing by spraying pots with plantlets from the top. Therefore, a 0.07% (v/v) solution of BASTA concentrate (183 g/I glufosinate-ammonium) in tap water was sprayed. The wild-type control plants were sprayed with tap water only (instead of spraying with BASTA dissolved in tap water) but were otherwise treated identically. Watering was carried out every two days after covers were removed from the trays. Plants were individualized 12-13 days after sowing by removing the surplus of seedlings leaving one seedling in a pot. Cold (chilling to 11° C.-12° C.) was applied 14-16 days after sowing until the end of the experiment. For measuring biomass performance, plant fresh weight was determined at harvest time (35-37 days after sowing) by cutting shoots and weighing them. Plants were in the stage prior to flowering and prior to growth of inflorescence when harvested. Transgenic plants were compared to the non-transgenic wild-type control plants harvested at the same day. Significance values for the statistical significance of the biomass changes were calculated by applying the ‘student's’ t test (parameters: two-sided, unequal variance).
Up to five lines per transgenic construct were tested in 2 to 3 successive experimental levels. Only constructs that displayed positive performance were subjected to the next experimental level. In the final experimental level 20-28 plants were tested. Biomass performance was evaluated as described above. Data are shown for constructs that displayed increased biomass performance in at least two successive experimental levels.
S. cerevisiae
A. th.
A. th.
A. th.
A. th.
A. th.
A. th.
A. th.
A. th.
A. th.
A. th.
A. vinelandii
A. vinelandii
A. vinelandii
A. vinelandii
E. coli
E. coli
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E. coli
E. coli
E. coli
E. coli
E. coli
P. patens
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T.
thermophilus
T.
thermophilus
S. cerevisiae
S. cerevisiae
B. napus
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
Oryza
sativa
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A. th.
A. th.
A. th.
A. th.
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A. th.
A. th.
A. th.
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A. th.
A. vinelandii
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thermophilus
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sativa
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cerevisiae
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vinelandii
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patens
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T.
thermophilus
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thermophilus
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cerevisiae
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cerevisiae
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napus
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cerevisiae
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cerevisiae
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cerevisiae
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cerevisiae
S.
cerevisiae
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cerevisiae
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cerevisiae
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cerevisiae
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sativa
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cerevisiae
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A. th.
A. th.
A. th.
A. th.
A.
vinelandii
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vinelandii
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vinelandii
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vinelandii
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P.
patens
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T.
thermophilus
T.
thermophilus
S.
cerevisiae
S.
cerevisiae
B.
napus
S.
cerevisiae
S.
cerevisiae
S.
cerevisiae
S.
cerevisiae
S.
cerevisiae
S.
cerevisiae
S.
cerevisiae
S.
cerevisiae
Oryza
sativa
S. cerevisiae
A. th.
A. th.
A. th.
A. th.
A. th.
A. th.
A. th.
A. th.
A. th.
A. th.
A. vinelandii
A. vinelandii
A. vinelandii
A. vinelandii
E. coli
E. coli
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E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
P. patens
Synechocystis sp.
T. thermophilus
T. thermophilus
S. cerevisiae
S. cerevisiae
B. napus
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
Oryza sativa
S. cerevisiae
A. th.
A. th.
A. th.
A. th.
A. th.
A. th.
A. th.
A. th.
A. th.
A. th.
A. vinelandii
A. vinelandii
A. vinelandii
A. vinelandii
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
P. patens
Synechocystis sp.
T. thermophilus
T. thermophilus
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
Oryza sativa
| Number | Date | Country | Kind |
|---|---|---|---|
| 08167450.9 | Oct 2008 | EP | regional |
This application is a divisional of patent application Ser. No. 13/125,338 filed on Apr. 21, 2011, which is a national stage application (under 35 U.S.C. §371) of PCT/EP2009/063979, filed Oct. 23, 2009, which claims benefit of European application 08167450.9, filed Oct. 23, 2008. The entire content of each afore-mentioned application is hereby incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13125338 | Apr 2011 | US |
| Child | 14753128 | US |
