Patent Application
20160138039
The ASCII file, entitled 65113SequenceListing.txt, created on Jan. 25, 2016, comprising 425,492 bytes, submitted concurrently with the filing of this application is incorporated herein by reference.
The present invention relates to methods of increasing abiotic stress tolerance and/or biomass in plants and, more particularly, to plants expressing exogenous abiotic stress-tolerance genes.
Abiotic stress (also referred to as “environmental stress”) conditions such as salinity, drought, flood, suboptimal temperature and toxic chemical pollution, cause substantial damage to agricultural plants. Most plants have evolved strategies to protect themselves against these conditions. However, if the severity and duration of the stress conditions are too great, the effects on plant development, growth and yield of most crop plants are profound. Furthermore, most crop plants are very susceptible to abiotic stress (ABS) and thus necessitate optimal growth conditions for commercial crop yields. Continuous exposure to stress causes major alterations in plant metabolism which ultimately lead to cell death and consequently yields losses. Thus, despite extensive research and the use of sophisticated and intensive crop-protection measures, losses due to abiotic stress conditions remain in the billions of dollars annually (1,2).
Developing stress-tolerant plants is a strategy that has the potential to solve or mediate at least some of these problems. However, traditional plant breeding strategies used to develop new lines of plants that exhibit tolerance to ABS are relatively inefficient since they are tedious, time consuming and of unpredictable outcome. Furthermore, limited germplasm resources for stress tolerance and incompatibility in crosses between distantly related plant species represent significant problems encountered in conventional breeding. Additionally, the cellular processes leading to ABS tolerance are complex in nature and involve multiple mechanisms of cellular adaptation and numerous metabolic pathways (4-7).
Genetic engineering efforts, aimed at conferring abiotic stress tolerance to transgenic crops, have been described in the prior art. Studies by Apse and Blumwald (Curr Opin Biotechnol. 13:146-150, 2002), Quesada et al. (Plant Physiol. 130:951-963, 2002), Holmström et al. (Nature 379: 683-684, 1996), Xu et al. (Plant Physiol 110: 249-257, 1996), Pilon-Smits and Ebskamp (Plant Physiol 107: 125-130, 1995) and Tarczynski et al. (Science 259: 508-510, 1993) have all attempted at generating stress tolerant plants.
In addition, several U.S. patents and patent applications also describe polynucleotides associated with stress tolerance and their use in generating stress tolerant plants. U.S. Pat. Nos. 5,296,462 and 5,356,816 describe transforming plants with polynucleotides encoding proteins involved in cold adaptation in Arabidopsis thaliana, to thereby promote cold tolerance in the transformed plants.
U.S. Pat. No. 6,670,528 describes transforming plants with polynucleotides encoding polypeptides binding to stress responsive elements, to thereby promote tolerance of the transformed plants to abiotic stress.
U.S. Pat. No. 6,720,477 describes transforming plants with a polynucleotide encoding a signal transduction stress-related protein, capable of increasing tolerance of the transformed plants to abiotic stress.
U.S. application Ser. Nos. 09/938,842 and 10/342,224 describe abiotic stress-related genes and their use to confer upon plants tolerance to abiotic stress.
U.S. application Ser. No. 10/231,035 describes overexpressing a molybdenum cofactor sulfurase in plants to thereby increase their tolerance to abiotic stress.
Although the above described studies were at least partially successful in generating stress tolerant plants, there remains a need for stress tolerant genes which can be utilized to generate plants tolerant of a wide range of abiotic stress conditions.
While reducing the present invention to practice, the present inventors have identified through bioinformatic and laboratory studies several novel abiotic stress-tolerance genes, which can be utilized to increase tolerance to abiotic stress and/or biomass in plants.
According to one aspect of the present invention there is provided a method of increasing tolerance of a plant to an abiotic stress. The method includes expressing within the plant an exogenous polynucleotide at least 90% homologous to a polynucleotide selected from the group consisting of SEQ ID NOs: 1-18, 93-98 and 247-252.
According to an additional aspect of the present invention there is provided a method of increasing tolerance of a plant to an abiotic stress. The method includes expressing within the plant an exogenous polynpeptide including an amino acid sequence selected from the group consisting of SEQ ID NOs: 39-92 and 105-230.
According to another aspect of the present invention there is provided a method of increasing biomass and/or yield of a plant. The method includes expressing within the plant an exogenous polynucleotide at least 90% homologous to a polynucleotide selected from the group consisting of SEQ ID NOs: 1-18, 93-98 and 247-252.
According to still additional aspect of the present invention there is provided a method of increasing biomass and/or yield of a plant. The method includes expressing within the plant an exogenous polypeptide including an amino acid sequence selected from the group consisting of SEQ ID NOs: 39-92 and 105-230.
According to yet another aspect of the present invention there is provided a plant cell comprising an exogenous polynucleotide at least 90% homologous to a polynucleotide selected from the group consisting of SEQ ID NOs: 1-18, 93-98 and 247-252.
According to yet another aspect of the present invention there is provided a plant cell comprising an exogenous polynucleotide encoding a polypeptide including an amino acid sequence selected from the group consisting of SEQ ID NOs: 39-92 and 105-230.
According to still another aspect of the present invention there is provided a nucleic acid construct, including a polynucleotide at least 90% homologous to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-18, 93-98 and 247-252 and a promoter capable of directing transcription of the polynucleotide in a host cell.
According to another aspect of the present invention there is provided a nucleic acid construct, including a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 39-92 and 105-230 and a promoter capable of directing transcription of the polynucleotide in a host cell.
According to further yet another aspect of the present invention there is provided an isolated polypeptide, including an amino acid sequence at least 90% homologous to the amino acid sequence encoded by a polynucleotide selected from the group consisting of SEQ ID NOs: 1-18, 93-98 and 247-252.
According to an additional aspect of the present invention there is provided an isolated polypeptide including an amino acid sequence selected from the group consisting of SEQ ID NOs: 39-92 and 105-230.
According to further features in preferred embodiments of the invention described below, the expressing is effected by (i) transforming a cell of the plant with the exogenous polynucleotide; (ii) generating a mature plant from the cell; and (iii) cultivating the mature plant under conditions suitable for expressing the exogenous polynucleotide within the mature plant.
According to still further features in the described preferred embodiments the transforming is effected by introducing to the plant cell a nucleic acid construct including the exogenous polynucleotide and at least one promoter capable of directing transcription of the exogenous polynucleotide in the plant cell.
According to still further features in the described preferred embodiments the at least one promoter is a constitutive promoter.
According to still further features in the described preferred embodiments the constitutive promoter is CaMV 35S promoter.
According to still further features in the described preferred embodiments the constitutive promoter is At6669 promoter.
According to still further features in the described preferred embodiments the at least one promoter is an inducible promoter.
According to still further features in the described preferred embodiments the inducible promoter is an abiotic stress inducible promoter.
According to still further features in the described preferred embodiments the at least one promoter is a tissue-specific promoter.
According to still further features in the described preferred embodiments the expressing is effected by infecting the plant with a virus including the exogenous polynucleotide.
According to still further features in the described preferred embodiments the virus is an avirulent virus.
According to still further features in the described preferred embodiments the abiotic stress is selected from the group consisting of salinity, water deprivation, low temperature, high temperature, heavy metal toxicity, anaerobiosis, nutrient deficiency, nutrient excess, atmospheric pollution and UV irradiation.
According to still further features in the described preferred embodiments the plant is a dicotyledonous plant.
According to still further features in the described preferred embodiments the plant is a monocotyledonous plant.
According to still further features in the described preferred embodiments the plant cell forms a part of a plant.
The present invention successfully addresses the shortcomings of the presently known configurations by providing methods of utilizing novel abiotic stress-tolerance genes to increase plants tolerance to abiotic stress and/or biomass and/or commercial yield.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.
The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.
The present invention is of methods of increasing plants tolerance to abiotic stress and/or biomass by utilizing novel abiotic stress tolerance genes and of plants exhibiting increased tolerance to stress conditions and/or increased capacity to accumulate biomass.
The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions.
Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of the components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.
While reducing the present invention to practice, the present inventors while employing bioinformatic techniques, identified polynucleotide sequences which encode putative abiotic-stress tolerance (ABST) proteins (Examples 1 and 11). Selected sequences were isolated (Examples 3 and 12), cloned into expression vectors (Example 4 and 13) and introduced into Arabidopsis thaliana plants (Example 8) and tomato plants (Example 10). These plants, which were grown under salinity stress conditions, or under normal conditions, exhibited significantly higher biomass as compared with similar plants not carrying the exogenous ABST genes (Examples 9 and 10).
Thus, according to one aspect of the present invention, there is provided a method of increasing tolerance of a plant to an abiotic stress and/or plant biomass. The method includes expressing within the plant an exogenous polynucleotide at least 70% homologous, preferably at least 80% homologous, more preferably at least 85% homologous, most preferably at least 90% homologous to a polynucleotide selected from the group consisting of SEQ ID NOs: 1-18, 93-98 and 247-252. Alternatively, the exogenous polynucleotide of the present invention encodes a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 39-92 and 105-230.
As demonstrated in Example 8 herein below, introduction of SEQ ID NOs: 1, 4, 9, 13 and 14 into Arabidopsis thaliana plants increased plant tolerance to abiotic stress, such as a salinity stress as measured by an increase in fresh weight, dry weight, and/or seed weight. Transgenic tomato plants showed an increase in fresh canopy weight, dry weight, root weight, seed weight and increase in total fruit yield during abiotic stress, such as salinity or drought stress following introduction of these polynucleotide sequences as demonstrated in Example 10.
The nucleic acid sequences of the present invention may be altered, to further improve expression levels for example, by optimizing the nucleic acid sequence in accordance with the preferred codon usage for a particular plant cell type which is selected for the expression of the polypeptides of the present invention.
Construction of synthetic genes by altering the codon usage is described in for example PCT Patent Application WO 93/07278.
Alternatively, ortholog sequences in a particular plant may be identified (e.g. by bioinformatics techniques) as described in Example 10. Following qualification, these may be used to direct the expression of the polypeptides of the present invention in a particular plant species. Since this may increase the probability of gene silencing, it may be preferable to optimize the nucleic acid sequence in accordance with the preferred codon usage as described above.
The phrase “abiotic stress” used herein refers to any adverse effect on metabolism, growth, reproduction and/or viability of a plant. Accordingly, abiotic stress can be induced by suboptimal environmental growth conditions such as, for example, salinity, water deprivation, flooding, low or high temperature, heavy metal toxicity, anaerobiosis, nutrient deficiency, atmospheric pollution or UV irradiation.
The phrase “abiotic stress tolerance” as used herein refers to the ability of a plant to endure an abiotic stress without suffering a substantial alteration in metabolism, growth, productivity and/or viability.
The polynucleotides of the present invention may enhance abiotic stress tolerance by any mechanism. The polynucleotides may enhance abiotic stress tolerance by encoding for polypeptides which increase the amount of water available to the plant. For example, SEQ ID NO: 13 encodes a polypeptide that enhances symplastic water transport. Alternatively the polynucleotides may enhance abiotic stress tolerance by encoding for polypeptides which are involved in enhancing the expression of other proteins involved in abiotic stress tolerance. For example, SEQ ID NO: 1 encodes a cytoplasmic ribosomal protein and SEQ ID NO: 14 is a transcription factor.
A suitable plant for use with the method of the present invention can be any monocotyledonous or dicotyledonous plant including, but not limited to, maize, wheat, barely, rye, oat, rice, soybean, peanut, pea, lentil and alfalfa, cotton, rapeseed, canola, pepper, sunflower, potato, tobacco, tomato, eggplant, eucalyptus, a tree, an ornamental plant, a perennial grass and a forage crop.
As used herein, the term “exogenous polynucleotide” refers to a nucleic acid sequence which is not naturally expressed within the plant but which, when introduced into the plant either in a stable or transient manner, produces at least one polypeptide product.
Expressing the exogenous polynucleotide of the present invention within the plant can be effected by transforming one or more cells of the plant with the exogenous polynucleotide, followed by generating a mature plant from the transformed cells and cultivating the mature plant under conditions suitable for expressing the exogenous polynucleotide within the mature plant.
Preferably, the transformation is effected by introducing to the plant cell a nucleic acid construct which includes the exogenous polynucleotide of the present invention and at least one promoter capable of directing transcription of the exogenous polynucleotide in the plant cell. Further details of suitable transformation approaches are provided hereinbelow.
As used herein, the term “promoter” refers to a region of DNA which lies upstream of the transcriptional initiation site of a gene to which RNA polymerase binds to initiate transcription of RNA. The promoter controls where (e.g., which portion of a plant, which organ within an animal, etc.) and/or when (e.g., which stage or condition in the lifetime of an organism) the gene is expressed.
Any suitable promoter sequence can be used by the nucleic acid construct of the present invention. Preferably the promoter is a constitutive promoter, a tissue-specific, or an abiotic stress-inducible promoter.
Suitable constitutive promoters include, for example, CaMV 35S promoter (SEQ ID NO: 19; Odell et al., Nature 313:810-812, 1985); Arabidopsis At6669 promoter (SEQ ID NO: 20); maize Ubi 1 (Christensen et al., Plant Sol. Biol. 18:675-689, 1992); rice actin (McElroy et al., Plant Cell 2:163-171, 1990); pEMU (Last et al., Theor. Appl. Genet. 81:581-588, 1991); and Synthetic Super MAS (Ni et al., The Plant Journal 7: 661-76, 1995). Other constitutive promoters include those in U.S. Pat. Nos. 5,659,026, 5,608,149; 5.608,144; 5,604,121; 5,569,597: 5,466,785; 5,399,680; 5,268,463; and 5,608,142.
Suitable tissue-specific promoters include, but not limited to, leaf-specific promoters such as described, for example, by Yamamoto et al., Plant J. 12:255-265, 1997; Kwon et al., Plant Physiol. 105:357-67, 1994; Yamamoto et al., Plant Cell Physiol. 35:773-778, 1994; Gotor et al., Plant J. 3:509-18, 1993; Orozco et al., Plant Mol. Biol. 23:1129-1138, 1993; and Matsuoka et al., Proc. Natl. Acad. Sci. USA 90:9586-9590, 1993.
Suitable abiotic stress-inducible promoters include, but not limited to, salt-inducible promoters such as RD29A (Yamaguchi-Shinozalei et al., Mol. Gen. Genet. 236:331-340, 1993); drought-inducible promoters such as maize rab17 gene promoter (Pla et. al., Plant Mol. Biol. 21:259-266, 1993), maize rab28 gene promoter (Busk et. al., Plant J. 11:1285-1295, 1997) and maize Ivr2 gene promoter (Pelleschi et. al., Plant Mol. Biol. 39:373-380, 1999); and heat-inducible promoters such as heat tomato hsp80-promoter from tomato (U.S. Pat. No. 5,187,267).
The nucleic acid construct of the present invention preferably further includes an appropriate selectable marker and/or an origin of replication. Preferably, the nucleic acid construct utilized is a shuttle vector, which can propagate both in E. coli (wherein the construct comprises an appropriate selectable marker and origin of replication) and be compatible for propagation in cells. The construct according to the present invention can be, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus or an artificial chromosome.
The nucleic acid construct of the present invention can be utilized to stably or transiently transform plant cells. In stable transformation, the exogenous polynucleotide of the present invention is integrated into the plant genome and as such it represents a stable and inherited trait. In transient transformation, the exogenous polynucleotide is expressed by the cell transformed but it is not integrated into the genome and as such it represents a transient trait.
There are various methods of introducing foreign genes into both monocotyledonous and dicotyledonous plants (Potrykus, I., Annu. Rev. Plant. Physiol., Plant. Mol. Biol. (1991) 42:205-225; Shimamoto et al., Nature (1989) 338:274-276).
The principle methods of causing stable integration of exogenous DNA into plant genomic DNA include two main approaches:
(i) Agrobacterium-mediated gene transfer: Klee et al. (1987) Annu. Rev. Plant Physiol. 38:467-486; Klee and Rogers in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego, Calif. (1989) p. 2-25; Gatenby, in Plant Biotechnology, eds. Kung, S. and Arntzen, C. J., Butterworth Publishers, Boston, Mass. (1989) p. 93-112.
(ii) Direct DNA uptake: Paszkowski et al., in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego, Calif. (1989) p. 52-68; including methods for direct uptake of DNA into protoplasts, Toriyama, K. et al. (1988) Bio/Technology 6:1072-1074. DNA uptake induced by brief electric shock of plant cells: Zhang et al. Plant Cell Rep. (1988) 7:379-384. Fromm et al. Nature (1986) 319:791-793. DNA injection into plant cells or tissues by particle bombardment, Klein et al. Bio/Technology (1988) 6:559-563; McCabe et al. Bio/Technology (1988) 6:923-926; Sanford, Physiol. Plant. (1990) 79:206-209; by the use of micropipette systems: Neuhaus et al., Theor. Appl. Genet. (1987) 75:30-36; Neuhaus and Spangenberg, Physiol. Plant. (1990) 79:213-217; glass fibers or silicon carbide whisker transformation of cell cultures, embryos or callus tissue, U.S. Pat. No. 5,464,765 or by the direct incubation of DNA with germinating pollen, DeWet et al. in Experimental Manipulation of Ovule Tissue, eds. Chapman, G. P. and Mantell, S. H. and Daniels, W. Longman, London, (1985) p. 197-209; and Ohta, Proc. Natl. Acad. Sci. USA (1986) 83:715-719.
The Agrobacterium system includes the use of plasmid vectors that contain defined DNA segments that integrate into the plant genomic DNA. Methods of inoculation of the plant tissue vary depending upon the plant species and the Agrobacterium delivery system. A widely used approach is the leaf disc procedure which can be performed with any tissue explant that provides a good source for initiation of whole plant differentiation. Horsch et al. in Plant Molecular Biology Manual A5, Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. A supplementary approach employs the Agrobacterium delivery system in combination with vacuum infiltration. The Agrobacterium system is especially viable in the creation of transgenic dicotyledonous plants.
There are various methods of direct DNA transfer into plant cells. In electroporation, the protoplasts are briefly exposed to a strong electric field. In microinjection, the DNA is mechanically injected directly into the cells using very small micropipettes. In microparticle bombardment, the DNA is adsorbed on microprojectiles such as magnesium sulfate crystals or tungsten particles, and the microprojectiles are physically accelerated into cells or plant tissues.
Following stable transformation plant propagation is exercised. The most common method of plant propagation is by seed. Regeneration by seed propagation, however, has the deficiency that due to heterozygosity there is a lack of uniformity in the crop, since seeds are produced by plants according to the genetic variances governed by Mendelian rules. Basically, each seed is genetically different and each will grow with its own specific traits. Therefore, it is preferred that the transformed plant be produced such that the regenerated plant has the identical traits and characteristics of the parent transgenic plant. Therefore, it is preferred that the transformed plant be regenerated by micropropagation which provides a rapid, consistent reproduction of the transformed plants.
Micropropagation is a process of growing new generation plants from a single piece of tissue that has been excised from a selected parent plant or cultivar. This process permits the mass reproduction of plants having the preferred tissue expressing the fusion protein. The new generation plants which are produced are genetically identical to, and have all of the characteristics of, the original plant. Micropropagation allows mass production of quality plant material in a short period of time and offers a rapid multiplication of selected cultivars in the preservation of the characteristics of the original transgenic or transformed plant. The advantages of cloning plants are the speed of plant multiplication and the quality and uniformity of plants produced.
Micropropagation is a multi-stage procedure that requires alteration of culture medium or growth conditions between stages. Thus, the micropropagation process involves four basic stages: Stage one, initial tissue culturing; stage two, tissue culture multiplication; stage three, differentiation and plant formation; and stage four, greenhouse culturing and hardening. During stage one, initial tissue culturing, the tissue culture is established and certified contaminant-free. During stage two, the initial tissue culture is multiplied until a sufficient number of tissue samples are produced to meet production goals. During stage three, the tissue samples grown in stage two are divided and grown into individual plantlets. At stage four, the transformed plantlets are transferred to a greenhouse for hardening where the plants' tolerance to light is gradually increased so that it can be grown in the natural environment.
Preferably, mature transformed plants generated as described above are further selected for abiotic stress tolerance. Accordingly, transformed and non-transformed (wild type) plants are exposed to an abiotic stress condition, such as water depravation, suboptimal temperature, nutrient deficiency, or preferably a salt stress condition. Salt stress can be effected in many ways such as, for example, by irrigating the plants with a hyperosmotic solution, by cultivating the plants hydroponically in a hyperosmotic growth solution (e.g., Hoagland solution), or by culturing the plants in a hyperosmotic growth medium (e.g., MS medium). Since different plants vary considerably in their tolerance to salinity, the salt concentration in the irrigation water, growth solution, or growth medium is preferably adjusted according to the specific characteristics of the specific plant cultivar or variety, so as to inflict a mild or moderate effect on the physiology and/or morphology of the plants (for guidelines as to appropriate concentration see, Bernstein and Kafkafi, Root Growth Under Salinity Stress In: Plant Roots, The Hidden Half 3rd ed. Waisel Y, Eshel A and Kafkafi U. (editors) Marcel Dekker Inc., New York, 2002, and reference therein). Following exposure to the stress condition the plants are frequently monitored until substantial physiological and/or morphological effects appear in wild type plants. Subsequently, transformed plants not exhibiting substantial physiological and/or morphological effects, or exhibiting higher biomass than wild-type plants, are identified as abiotic stress tolerant plants.
Although stable transformation is presently preferred, transient transformation of leaf cells, meristematic cells or the whole plant is also envisaged by the present invention.
Transient transformation can be effected by any of the direct DNA transfer methods described above or by viral infection using modified plant viruses.
Viruses that have been shown to be useful for the transformation of plant hosts include CaMV, TMV and BV. Transformation of plants using plant viruses is described in U.S. Pat. No. 4,855,237 (BGV), EP-A 67,553 (TMV), Japanese Published Application No. 63-14693 (TMV), EPA 194,809 (BV), EPA 278,667 (BV); and Gluzman, Y. et al., Communications in Molecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, New York, pp. 172-189 (1988). Pseudovirus particles for use in expressing foreign DNA in many hosts, including plants, is described in WO 87/06261.
Preferably, the virus of the present invention is avirulent and thus is incapable of causing severe symptoms such as reduced growth rate, mosaic, ring spots, leaf roll, yellowing, streaking, pox formation, tumor formation and pitting. A suitable avirulent virus may be a naturally occurring avirulent virus or an artificially attenuated virus. Virus attenuation may be effected by using methods well known in the art including, but not limited to, sub-lethal heating, chemical treatment or by directed mutagenesis techniques such as described, for example, by Kurihara and Watanabe (Molecular Plant Pathology 4:259-269, 2003), Gal-on et al. (1992), Atreya et al. (1992) and Huet et al. (1994).
Suitable virus strains can be obtained from available sources such as, for example, the American Type culture Collection (ATCC) or by isolation from infected plants. Isolation of viruses from infected plant tissues can be effected by techniques well known in the art such as described, for example by Foster and Tatlor, Eds. “Plant Virology Protocols: From Virus Isolation to Transgenic Resistance (Methods in Molecular Biology (Humana Pr), Vol 81)”, Humana Press, 1998. Briefly, tissues of an infected plant believed to contain a high concentration of a suitable virus, preferably young leaves and flower petals, are ground in a buffer solution (e.g., phosphate buffer solution) to produce a virus infected sap which can be used in subsequent inoculations.
Construction of plant RNA viruses for the introduction and expression of non-viral exogenous polynucleotide sequences in plants is demonstrated by the above references as well as by Dawson, W. O. et al., Virology (1989) 172:285-292; Takamatsu et al. EMBO J. (1987) 6:307-311; French et al. Science (1986) 231:1294-1297; and Takamatsu et al. FEBS Letters (1990) 269:73-76.
When the virus is a DNA virus, suitable modifications can be made to the virus itself. Alternatively, the virus can first be cloned into a bacterial plasmid for ease of constructing the desired viral vector with the foreign DNA. The virus can then be excised from the plasmid. If the virus is a DNA virus, a bacterial origin of replication can be attached to the viral DNA, which is then replicated by the bacteria. Transcription and translation of this DNA will produce the coat protein which will encapsidate the viral DNA. If the virus is an RNA virus, the virus is generally cloned as a cDNA and inserted into a plasmid. The plasmid is then used to make all of the constructions. The RNA virus is then produced by transcribing the viral sequence of the plasmid and translation of the viral genes to produce the coat protein(s) which encapsidate the viral RNA.
Construction of plant RNA viruses for the introduction and expression in plants of non-viral exogenous polynucleotide sequences such as those included in the construct of the present invention is demonstrated by the above references as well as in U.S. Pat. No. 5,316,931.
In one embodiment, a plant viral polynucleotide is provided in which the native coat protein coding sequence has been deleted from a viral polynucleotide, a non-native plant viral coat protein coding sequence and a non-native promoter, preferably the subgenomic promoter of the non-native coat protein coding sequence, capable of expression in the plant host, packaging of the recombinant plant viral polynucleotide, and ensuring a systemic infection of the host by the recombinant plant viral polynucleotide, has been inserted. Alternatively, the coat protein gene may be inactivated by insertion of the non-native polynucleotide sequence within it, such that a protein is produced. The recombinant plant viral polynucleotide may contain one or more additional non-native subgenomic promoters. Each non-native subgenomic promoter is capable of transcribing or expressing adjacent genes or polynucleotide sequences in the plant host and incapable of recombination with each other and with native subgenomic promoters. Non-native (foreign) polynucleotide sequences may be inserted adjacent the native plant viral subgenomic promoter or the native and a non-native plant viral subgenomic promoters if more than one polynucleotide sequence is included. The non-native polynucleotide sequences are transcribed or expressed in the host plant under control of the subgenomic promoter to produce the desired products.
In a second embodiment, a recombinant plant viral polynucleotide is provided as in the first embodiment except that the native coat protein coding sequence is placed adjacent one of the non-native coat protein subgenomic promoters instead of a non-native coat protein coding sequence.
In a third embodiment, a recombinant plant viral polynucleotide is provided in which the native coat protein gene is adjacent its subgenomic promoter and one or more non-native subgenomic promoters have been inserted into the viral polynucleotide. The inserted non-native subgenomic promoters are capable of transcribing or expressing adjacent genes in a plant host and are incapable of recombination with each other and with native subgenomic promoters. Non-native polynucleotide sequences may be inserted adjacent the non-native subgenomic plant viral promoters such that the sequences are transcribed or expressed in the host plant under control of the subgenomic promoters to produce the desired product.
In a fourth embodiment, a recombinant plant viral polynucleotide is provided as in the third embodiment except that the native coat protein coding sequence is replaced by a non-native coat protein coding sequence.
The viral vectors are encapsidated by the coat proteins encoded by the recombinant plant viral polynucleotide to produce a recombinant plant virus. The recombinant plant viral polynucleotide or recombinant plant virus is used to infect appropriate host plants. The recombinant plant viral polynucleotide is capable of replication in the host, systemic spread in the host, and transcription or expression of foreign gene(s) (exogenous polynucleotide) in the host to produce the desired protein.
Techniques for inoculation of viruses to plants may be found in Foster and Taylor, eds. “Plant Virology Protocols: From Virus Isolation to Transgenic Resistance (Methods in Molecular Biology (Humana Pr), Vol 81)”, Humana Press, 1998; Maramorosh and Koprowski, eds. “Methods in Virology” 7 vols, Academic Press, New York 1967-1984; Hill, S. A. “Methods in Plant Virology”, Blackwell, Oxford, 1984; Walkey, D. G. A. “Applied Plant Virology”, Wiley, New York, 1985; and Kado and Agrawa, eds. “Principles and Techniques in Plant Virology”, Van Nostrand-Reinhold, New York.
In addition to the above, the polynucleotide of the present invention can also be introduced into a chloroplast genome thereby enabling chloroplast expression.
A technique for introducing exogenous polynucleotide sequences to the genome of the chloroplasts is known. This technique involves the following procedures. First, plant cells are chemically treated so as to reduce the number of chloroplasts per cell to about one. Then, the exogenous polynucleotide is introduced via particle bombardment into the cells with the aim of introducing at least one exogenous polynucleotide molecule into the chloroplasts. The exogenous polynucleotides selected such that it is integratable into the chloroplast's genome via homologous recombination which is readily effected by enzymes inherent to the chloroplast. To this end, the exogenous polynucleotide includes, in addition to a gene of interest, at least one polynucleotide stretch which is derived from the chloroplast's genome. In addition, the exogenous polynucleotide includes a selectable marker, which serves by sequential selection procedures to ascertain that all or substantially all of the copies of the chloroplast genomes following such selection will include the exogenous polynucleotide. Further details relating to this technique are found in U.S. Pat. Nos. 4,945,050; and 5,693,507 which are incorporated herein by reference. A polypeptide can thus be produced by the protein expression system of the chloroplast and become integrated into the chloroplast's inner membrane.
Since abiotic stress tolerance in plants can involve multiple genes acting additively or in synergy (see, for example, in Quesda et al., Plant Physiol. 130:951-063, 2002), the present invention also envisages expressing a plurality of exogenous polynucleotides in a single host plant to thereby achieve superior abiotic stress tolerance.
Expressing a plurality of exogenous polynucleotides in a single host plant can be effected by co-introducing multiple nucleic acid constructs, each including a different exogenous polynucleotide, into a single plant cell. The transformed cell can than be regenerated into a mature plant using the methods described hereinabove.
Alternatively, expressing a plurality of exogenous polynucleotides in a single host plant can be effected by co-introducing into a single plant-cell a single nucleic-acid construct including a plurality of different exogenous polynucleotides. Such a construct can be designed with a single promoter sequence which can transcribe a polycistronic message including all the different exogenous polynucleotide sequences. To enable co-translation of the different polypeptides encoded by the polycistronic message, the polynucleotide sequences can be inter-linked via an internal ribosome entry site (IRES) sequence which facilitates translation of polynucleotide sequences positioned downstream of the IRES sequence. In this case, a transcribed polycistronic RNA molecule encoding the different polypeptides described above will be translated from both the capped 5′ end and the two internal IRES sequences of the polycistronic RNA molecule to thereby produce in the cell all different polypeptides. Alternatively, the construct can include several promoter sequences each linked to a different exogenous polynucleotide sequence.
The plant cell transformed with the construct including a plurality of different exogenous polynucleotides, can be regenerated into a mature plant, using the methods described hereinabove.
Alternatively, expressing a plurality of exogenous polynucleotides in a single host plant can be effected by introducing different nucleic acid constructs, including different exogenous polynucleotides, into a plurality of plants. The regenerated transformed plants can then be cross-bred and resultant progeny selected for superior abiotic stress tolerance and/or biomass traits, using conventional plant breeding techniques.
Hence, the present application provides methods of utilizing novel abiotic stress-tolerance genes to increase tolerance to abiotic stress and/or biomass and/or yield in a wide range of economical plants, safely and cost effectively.
Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.
Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.
Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J., eds. (1985); “Transcription and Translation” Hames, B. D., and Higgins S. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide to Molecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317, Academic Press; “PCR Protocols: A Guide To Methods And Applications”, Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategies for Protein Purification and Characterization—A Laboratory Course Manual” CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below.
Putative abiotic stress-tolerance (ABST) genes were selected from NCBI databases of tomato expressed sequence tags (ESTs) and cDNAs. The database sequences were clustered and assembled using the LEADS™ software (Compugen). Clustering resulted in more than 20,000 clusters, each representing a different gene. An expression profile summary was compiled for each cluster by pooling all keywords included in the sequence records comprising the cluster. The clusters were then screened to include polynucleotides originating from libraries identified by keywords relating to ABST. The selected clusters were further filtered to exclude any cluster which included more than 100 ESTs per cluster and/or any cluster in which less than 50% of the sequences were annotated by ABST-related keywords.
Prior art ABST plant genes were identified from the publications of Quesada et al. (Plant Physiol. 130:951-963, 2002); Apse and Blumwald (Curr Opin Biotechnol. 13:146-150, 2002); Rontein et al. (Metab Eng 4:49-56, 2002); and references therein. Known plant ABST genes were aligned with the clustered tomato nucleic-acid sequences using the BLAST program. The tomato sequences having an e-score value lower than 5 were identified as ABST orthologes. Additional prior art tomato ABST genes were identified by searching the clustered tomato sequence records using the keywords “root”, “crown gall”, “nutrient”, “callus”, “disease”, “pathogen”, “elicitor” and “pseudomonas”.
Finally, all identified prior art ABST genes were matched (by sequence alignment using the BLAST software) with the output set of tomato gene clusters, selected as described above. Consequently, about 40% of the genes selected in the output set of clusters which matched with prior art ABST genes proved to be known ABST genes, indicating that the remaining genes of the selected clusters are potentially capable of increasing abiotic stress tolerance in plants.
The selected polynucleotide sequences (Table 1A), were analyzed for presence of ORFs using Vector NTI suite (InforMax, U.K.) version 6 (Hasting Software, Inc: World Wide Web (dot) Generunner (dot) com/). ORFs identified in each of these polynucleotide sequences were compared to Genbank database sequences, using Blast (World Wide Web (dot) ncbi (dot) nlm (dot) nih (dot) gov/BLAST/); the ORF displaying the highest homology to a GenBank sequence or sequences, was mapped in order to identify an ATG start codon. The position of the ATG start codon of this ORF was then compared with that of the identified polynucleotide sequence in order to verify that each of the eighteen sequences described herein includes a full length ORF and an ATG start codon (thus qualifying as a “putative ABST gene”).
ABST polypeptide homologues were identified from the NCBI databases using BLAST software (Table 1B).
Five of these genes were selected as having the most potential of being putative ABS tolerance genes on the basis of digital expression profiles (i.e. known to be up-regulated under different stress conditions) as listed in Tables 2-6, and homologies to public protein sequences and domains through multi sequence alignment searches. Also, only genes with low and medium expression levels were included.
The five genes are listed together with their functions in Table 1C below.
Digital expression, also known as electronic northern blot, is a tool for virtually displaying the expression profile of query genes based on the EST sequences forming the cluster. The tool can provide the expression profile of a cluster in terms of plant anatomy (in what tissues/organs is the gene expressed), developmental stage (the developmental stages at which a gene can be found) and profile of treatment (provides the physiological conditions under which a gene is expressed such as drought, cold, pathogen infection, etc). Given a random distribution of ESTs in the different clusters, the digital expression provides a probability value that describes the probability of a cluster having a total of N ESTs to contain X ESTs from a certain collection of libraries. For the probability calculations, various considerations are taken: a) the number of ESTs in the cluster, b) the number of ESTs of the implicated and related libraries, c) the overall number of ESTs available representing the species. Thereby clusters with low probability values are highly enriched with ESTs from the group of libraries of interest indicating a specialized expression.
The digital expression profile for ABST_1 (SEQ ID NO:1) is summarized below in Table 2.
Agrobacterium
tumefaciens
The digital expression profile for ABST_6 (SEQ ID NO:4) is summarized below in Table 3.
pseudomonas
The digital expression profile for ABST_22 (SEQ ID NO:9) is summarized below in Table 4.
Agrobacterium
tumefaciens
The digital expression profile for ABST_36 (SEQ ID NO:13) is summarized below in Table 5.
Agrobacterium
tumefaciens
pseudomonas
The digital expression profile for ABST_37 (SEQ ID NO:14) is summarized below in Table 6.
The five genes listed in Table 1C were validated in situ as putative ABST genes by analyzing their expression profile in tomato plants under favorable, salt and drought stress conditions.
The expression studies were carried out on three tomato lines—sensitive tomato variety (Evoline 1), processing tomato line with moderate tolerance to salt (Evoline 3, also referred to herein as M82) and drought and high salt tolerant tomato line (Evoline 2). All lines were tested for several seasons for their levels of tolerance to salt and other soil stresses such as drought. All lines are commercially available from Evogene, Rehovot, Israel.
Methods
Salt Induction:
Salt stress induction was performed by introducing the roots of 14 days old tomato seedlings, of different tomato homozygote varieties, into a water bath which contained a solution of Hogland (comprising KNO3—8 mM, MgSO4—1 mM, KH2PO4—1 mM, and microelements, all dissolved in water, at pH 6.5), and 300 mM NaCl. Plants were placed on a floating tray, such that only the roots were dipped in the solution. Plants were grown in salt solution for 5 weeks during which the degree of tolerance to the salt stress was measured, by comparing plant development and biomass. The experiment was performed in 3 sequential seasons and with 5 repeats for each line in each experiment. The experiments identified 3 tomato lines showing consistent level of either weak (Evoline1), moderate (Evoline3), or high (Evoline2) level of tolerance. During the last experiment RNA samples from leaves and roots from Evoline1 and Evoline3 were taken 5, 9, 72, 240 hours after introducing the plants to salt solution.
Drought Induction:
Levels of tolerance to drought induction were tested on Evoline 1, Evoline2, and Evoline3 tomato plants. The plants were grown in CYG Germination Pouches, (Mega International, MN, USA), from germination until 4 true leaf stage in a regular nutrient solution. Drought conditions were applied by adding polyethylene glycol-PEG to the growth solution to a final concentration of 15%. RNA samples from leaves and roots were taken at time 0, 0.5 h, 3 h, 6 h and 48 h following drought induction. RNA expression level was measured by using quantitative RT PCR.
Results
As illustrated in
Tables 7-9 below summarise the up-regulation change in gene expression in response to various stress conditions (e.g. drought and salt) of polynucleotides of the present invention in tomato plants as calculated by quantitative RT-PCR.
As seen from tables 7-9, ABST_1, 6, 22, 27, 36 and 37 all showed induction of expression under different stress conditions. The changes in gene expression as a response to various stresses could be classified into two main categories—immediate up-regulation e.g. ABST_36 and ABST_1 (from 30 minutes to 6 hours following induction) and delayed up-regulation e.g. ABST_37 and ABST_6 (24 to 240 hours following induction).
The expression profile of most the genes was also affected by the genotype of the plants (highly tolerant line vs. sensitive line) and the specific stress the plants were exposed to (i.e. salt or PEG). Three genes showed changes in expression in leaves only (ABST_1, 22, 36). Only ABST_6 and 37 showed up-regulation of expression in both leaves and roots.
Significant changes were not detected in the expression of the ABST_19 as a response to salt or drought stress.
Table 10 below summarises the main expression modes of the ABST genes of the present invention under salt and osmotic stress.
RNA was extracted from 4 week-old tomato root and leaf tissues using Tri Reagent (Molecular Research Center, Inc), following the protocol provided by the manufacturer (World Wide Web (dot) Mrcgene (dot) com/tri (dot) htm). Complementary DNA molecules were produced from the extracted mRNA using M-MuLV reverse-transcriptase (RT) enzyme (Roche) and T16NN DNA primer, according to the manufacturer's instructions. The cDNA sequences set forth in SEQ ID NOs: 1, 4, 8-9 and 12-14, were amplified by PCR using the primers described in Table 11 below, with PFU proof reading DNA polymerase enzyme (Promega-World Wide Web (dot) Promega (dot) com/pnotes/68/7381_07/7381_07 (dot) html), following the protocol provided by the manufacturer. Additional restriction endonuclease sites were added to the 5′ prime end of each primer to facilitate cloning of the ABS tolerance genes of the present invention in binary vectors.
The resulting PCR blunt ended products were purified using PCR Purification Kit (Qiagen, Germany), digested with the appropriate restriction enzymes (Roche) and then inserted into the binary plasmid vector pPI. The plasmid pPI was constructed by inserting a synthetic poly-(A) signal sequence, originating from pGL3 basic plasmid vector (Promega, Acc No U47295; by 4658-4811) into the HindIII restriction site of the binary vector pBI101.3 (Clontech, Acc. No. U12640).
The resulting pPI plasmid was digested with restriction enzymes (BamHI and SacI; MBI Fermentas) and purified using PCR Purification Kit (Qiagen, Germany). The open pPI construct was then ligated with each of the seven PCR products described hereinabove. The ligation was effected using a ligation mixture containing T4 DNA ligase enzyme (Roche) and was performed according to the manufacturer's instructions.
The pPI constructs harboring ABST genes of the present invention were introduced to E. coli DH5 competent cells by electroporation, using a MicroPulser electroporator (Biorad), 0.2 cm cuvettes (Biorad) and EC-2 electroporation program (Biorad). The treated cells were cultured in LB liquid medium at 37° C. for 1 hr, then plated over LB agar supplemented with kanamycin (50 mg/L; Sigma) and incubated at 37° C. for 16 hrs. Colonies which developed on the selective medium were analyzed by PCR using the primers set forth in SEQ ID NOs: 35-36, which were designed to span the inserted sequence in the pPI plasmid. The resulting PCR products were separated on 1.5% agarose gels and the DNA fragment having the predicted size were isolated and sequenced using the ABI 377 sequencer (Amersham Biosciences Inc) in order to verify that the correct DNA sequences were properly introduced to the E. coli cells.
Generating Binary Vectors Comprising the Cauliflower Mosaic Virus 35S Promoter:
The Cauliflower Mosaic Virus 35S promoter sequence (set forth in SEQ ID NO: 19) was inserted upstream of the ABST genes of the present invention in each of the pPI constructs described above. The promoter was isolated from the pBI121 plasmid (Clontech, Accession No. AF485783) using the restriction endonucleases HindIII and BamHI (Roche). The isolated promoter was ligated into the pPI constructs digested with the same enzymes. Altogether, seven pPI constructs were generated, each comprising the CaMV 35S promoter positioned upstream of the ABST genes of the present invention having a sequence set forth in SEQ ID NO: 1, 4, 8, 9, 12, 13 or 14.
Generating Binary Vectors Comprising the At6669 Promoter:
The At6669 promoter sequence (set forth in SEQ ID NO: 20) was inserted upstream of the ABST genes of the present invention in each of the pPI binary constructs described above. The promoter was isolated from Arabidopsis thaliana var Col0 genomic DNA by PCR amplification using the primers set forth in SEQ ID NOs: 37-38. The PCR product was purified (Qiagen, Germany) and digested with the restriction endonucleases HindIII and BamHI (Roche). The resulting promoter sequence was introduced into the open binary constructs digested with the same enzymes. Altogether, seven pPI constructs were generated, each comprising the At6669 promoter positioned upstream of the ABST genes of the present invention having a sequence set forth in SEQ ID NO: 1, 4, 8, 9, 12, 13 or 14.
The capacity of At-6669 promoter to regulate transcription of genes carried by the pPI vector in plants was tested. Accordingly, the promoter At6669 was inserted into the pPI binary vector upstream of a Luciferase reporter gene. The binary vector was introduced to Arabidopsis thaliana plants using the procedure as described in Example 6 below. Mature transformed T2 Arabidopsis plants were assayed for bio-illumination in a darkroom using an ultra-low light detection camera (Princeton Instruments Inc., USA) using the procedure described by Meissner et al. (Plant J. 22:265, 2000). Illumination indicating positive Luciferase activity was observed in the flower and root meristem tissues of transformed plants (
To study the regulation mode of the promoter under stress conditions, the 6669 promoter and 35S promoter were both fused to the Arabidopsis Rab7 gene. Rab7 expression under the 6669 promoter gave significantly higher salt and osmotic tolerance performance compared to 35S in Arabidopsis, as determined by vegetative growth.
The promoter was further validated by comparing tolerance level of Arabidopsis plants containing the ABST genes of the present invention under the regulation of 6669 and under the regulation of 35S promoter (
Each of the binary vectors described in Example 5 above were used to transform Agrobacterium cells. Two additional binary constructs, having the Luciferase reporter gene replacing an ABST gene (positioned downstream of the 35S or At6669 promoter), were used as negative controls.
The binary vectors were introduced to Agrobacterium tumefaciens GV301, or LB4404 competent cells (about 109 cells/mL) by electroporation. The electroporation was effected by using a MicroPulser electroporator (Biorad), 0.2 cm cuvettes (Biorad) and EC-2 electroporation program (Biorad). The treated cells were cultured in LB liquid medium at 28° C. for 3 hr, then plated over LB agar supplemented with gentamycin (50 mg/L; for Agrobacterium strains GV301) or streptomycin (300 mg/L; for Agrobacterium strain LB4404) and kanamycin (50 mg/L) at 28° C. for 48 hrs. Agrobacterium colonies which developed on the selective media were analyzed by PCR using the primers set forth in SEQ ID NOs: 35-36, which were designed to span the inserted sequence in the pPI plasmid. The resulting PCR products were isolated and sequenced as described in Example 5 above, to verify that the correct ABST sequences were properly introduced to the Agrobacterium cells.
Arabidopsis thaliana Columbia plants (T0 plants) were transformed using the Floral Dip procedure described by Clough and Bent (10) and by Desfeux et al. (11), with minor modifications. Briefly, T0 Plants were sown in 250 ml pots filled with wet peat-based growth mix. The pots were covered with aluminum foil and a plastic dome, kept at 4° C. for 3-4 days, then uncovered and incubated in a growth chamber at 18-24° C. under 16/8 hr light/dark cycles. The T0 plants were ready for transformation six days before anthesis.
Single colonies of Agrobacterium carrying the binary constructs, generated as described in Example 6 above, were cultured in LB medium supplemented with kanamycin (50 mg/L) and gentamycin (50 mg/L). The cultures were incubated at 28° C. for 48 hrs under vigorous shaking and then centrifuged at 4000 rpm for 5 minutes. The pellets comprising Agrobacterium cells were re-suspended in a transformation medium containing half-strength (2.15 g/L) Murashig-Skoog (Duchefa); 0.044 μM benzylamino purine (Sigma); 112 μg/L B5 Gambourg vitamins (Sigma); 5% sucrose; and 0.2 ml/L Silwet L-77 (OSI Specialists, CT) in double-distilled water, at a pH of 5.7.
Transformation of T0 plants was effected by inverting each plant into an Agrobacterium suspension, such that the above ground plant tissue was submerged for 3-5 seconds. Each inoculated T0 plant was immediately placed in a plastic tray, then covered with a clear plastic dome to maintain humidity and was kept in the dark at room temperature for 18 hrs, to facilitate infection and transformation. Transformed (transgenic) plants were then uncovered and transferred to a greenhouse for recovery and maturation. The transgenic T0 plants were grown in the greenhouse for 3-5 weeks until siliques were brown and dry. Seeds were harvested from plants and kept at room temperature until sowing.
For generating T1 and T2 transgenic plants harboring the genes, seeds collected from transgenic T0 plants were surface-sterilized by soaking in 70% ethanol for 1 minute, followed by soaking in 5% sodium hypochloride and 0.05% triton for 5 minutes. The surface-sterilized seeds were thoroughly washed in sterile distilled water then placed on culture plates containing half-strength Murashig-Skoog (Duchefa); 2% sucrose; 0.8% plant agar; 50 mM kanamycin; and 200 mM carbenicylin (Duchefa). The culture plates were incubated at 4° C. for 48 hours then transferred to a growth room at 25° C. for an additional week of incubation. Vital T1 Arabidopsis plants were transferred to a fresh culture plates for another week of incubation. Following incubation, the T1 plants were removed from culture plates and planted in growth mix contained in 250 ml pots. The transgenic plants were allowed to grow in a greenhouse to maturity. Seeds harvested from T1 plants were cultured and grown to maturity as T2 plants under the same conditions as used for culturing and growing the T1 plants.
Methods:
T1 or T2 transgenic plants generated as described above were individually transplanted into pots containing a growth mixture of peat and vermiculite (volume ratio 3:2, respectively). The pots were covered for a 24 hr period for hardening, then placed in the greenhouse in random order and irrigated with tap water (provided from the pots' bottom every 3-5 days) for seven days. Thereafter, half of the plants were irrigated with a salt solution (100 mM NaCl and 5 mM CaCl2) to induce salinity stress (stress conditions). The other half was irrigated with tap water throughout (normal conditions). All plants were grown in the greenhouse at 100% RH for 28 days and then harvested (the above ground tissue).
Vigor Measurement:
Fresh and dry mass were measured as a function of plant vigor. Dry mass was measured immediately following drying in an oven at 50° C. for seven days.
Results:
Fresh Weight:
No significant differences in plant fresh weights were observed between the T1 plants transformed with 3 different ABST genes and plants transformed with the Luciferase reporter gene, grown either under normal or stress conditions (
1N Rows represent number of independent transformation event plants measured. For each transgene, 3-5 independent transformation events with 1-3 plants per a single transformation event were used.
T2 plants transformed with SEQ ID NOs: 7 or 14 positioned under the regulatory control of the 35S promoter accumulated significantly higher biomass than control plants, regardless of growth conditions. As shown in
Similar phenomenon was observed with T2 plants transformed with SEQ ID NO: 4 positioned under the regulatory control of the 35S promoter. Accordingly, as shown in
1N Rows represent number of independent transformation event plants measured. For each transgene, 3-5 independent transformation events with 1-3 plants per a single transformation event were used.
T2 plants transformed with SEQ ID NOs: 1 and 13 positioned under the regulatory control of the At6669 promoter and grown under stress conditions, exhibited significantly higher biomass than control plants grown under similar stress conditions. The mean fresh weight of T2 plants transformed with SEQ ID NOs: 1 and 13 positioned under the regulatory control of the At6669 promoter, and grown under stress conditions, were 37% and 21%, respectively, higher than the mean fresh weight control plants grown under similar stress conditions (
1N Rows represent number of independent transformation event plants measured. For each transgene, 3-5 independent transformation events with 1-3 plants per a single transformation event were used.
The results illustrate that the isolated ABST genes of the present invention, set forth in SEQ ID NOs: 1 and 13, are capable of increasing plant tolerance to abiotic stress, such as a salinity stress. In addition, the isolated ABST genes of the present invention as set forth in SEQ ID NOs: 7, 14 (and possibly also 4), are capable of substantially promoting biomass in plants grown under stress, as well as under normal conditions.
Dry Mass:
Table 15 summarises the results the dry mass of T1 plants grown under 100 mM NaCl.
Table 16 summarises the results of the absolute and relative dry mass of the of the T2 transgenic lines overexpressing the ABST genes under regular growth conditions.
As summarized in Table 16 above, transgenic T2 Arabidopsis plants over-expressing ABST genes of the present invention as set forth in SEQ ID NOs: 1 (ABST_1) and 4 (ABST_6) showed significant elevation of dry mass both under the 35S promoter and the 6669 promoter (ABST_1: 18%, 49% respectively, ABST_6: 10%, 44% respectively). SEQ ID NO: 13 (ABST_36) over-expression caused elevation in dry mass only under the regulation of 6669 promoter (24%). ABST_22 and _37 over-expression (SEQ ID NOs: 9 and 14 respectively), under the regulation of the 35S promoter, caused more than a 40% increase in the dry mass of the transgenic lines.
The results showed that all five tested genes improve vegetative growth development under favorable conditions. ABST_1 and 6 improve plant vigor in both regulation modes (6669 and 35S promoters).
The specific gene-promoter combination has a significant effect on dry mass elevation.
To further examine if elevation in plant vigor has a direct effect on total seed weight, T2 plant seeds (grown under regular conditions) over-expressing ABST_1, (SEQ ID NO: 1) 6 (SEQ ID NO: 4), 36 (SEQ ID NO: 13) and 37 (SEQ ID NO: 14) under the 6669 promoter were weighed. The seeds from plants over-expressing ABST_1 and 36 weighed 50% more compared to control lines as illustrated in
The effect of over-expression of ABST genes in plants subjected to salt stress on dry mass was tested using the same plant populations grown under continuous irrigation of saline water.
As summarized in Table 18 below, ABST_6 and 36 (SEQ ID NOs: 4 and 13 respectively) increased the plant dry mass under both the 35S and 6669 promoters (ABST_6: 25%, 16% respectively; ABST_36: 15%, 33% respectively). Plants over-expressing ABST_1 (SEQ ID NO: 1) showed higher dry mass only under the 6669 promoter (66%). ABST_22 and 37 (SEQ ID NOs: 9 and 14 respectively) were tested only under the 35S promoter and showed a significant increase (>43%) of plant dry mass compared to control line.
Table 19 below summarizes the results of all the dry mass and seed measurements that were performed on the transgenic arabidopsis over-expressing the ABST genes. (calculated as percentage compare negative control).
The M82 tomato variety strain (Evoline 3) was used to determine whether improvement of plant vigor under stress may be translated into improvement of commercial yield. ABST genes 1, 6 and 36 (SEQ I.D. NOs. 1, 4 and 13 respectively) were transformed under the regulation of the 6669 promoter. As described in Example 9, all three gene combinations showed significant improvement of stress tolerance in arabidopsis plants. The genes were introduced into M82 variety by crossing transgenic miniature tomato lines with M82 plants. To represent the variation of the position effect, a pool of pollen from transgenic lines representing 4 different insertion events from each one of the genes were used as the male parent. The F1 hybrids were used for further evaluation.
The segregating F1 populations were divided into two isogenic populations, i.e. plants over-expressing the ABST genes of the present invention and plants that were not transformed to over-express ABST genes (NT) from the same populations used as negative controls. During the first three weeks, all the plants were grown in a nursery under regular conditions. Following this period the plants were transplanted into a commercial greenhouse under two different treatments. The first group of plants was grown under favorable conditions while the second group was grown under continuous irrigation of saline water (180 mM NaCl). Each transgenic line was compared to a NT plant derived from the same F1 population. The plants were evaluated for their plant and fruit performance at a stage where the percentage of red fruit was on average 80%.
Results:
The plants over-expressing ABST genes showed improved vigor and larger root systems than the NT plants. In addition, the plants over-expressing ABST genes showed improved yield under salt stress conditions.
Specifically, plants over-expressing ABST genes showed an elevation in the fresh weight of both canopies and roots. The populations expressing ABST_1, 6 and 36 showed elevations of 165%, 162%, 206% respectively in canopy fresh weight compared to NT populations (Table 20) and higher root weight of 162%, 168% and 121% respectively compared to NT plants.
To further prove that this elevation in weight was due to accumulation of biomass and not only due to water accumulation, the dry mass of both the canopies and roots was measured. The dry mass of the canopies increased by 156%, 145% and 161% in the lines expressing ABST_1, 6, 36 respectively compared to the corresponding NT lines. Similar effects were observed in the roots of these lines. Roots of the plants over-expressing ABST genes contained 40% (as summarized in Table 21 below) more dry matter than roots of the NT control plants (ABST_1—168%, 6−159% and 36—140%).
Only transgenic plants over expressing ABST_36 had a lower root/shoot mass ratio than control plants (Table 22 below). This lower ratio (0.067 compared to more than 0.08 in control plants) is the result of a nearly 50% increase in shoot fresh weight rather than a major decrease in root fresh weight. This finding suggests that under stress growth conditions, the ABST_36 over-expressing plants require a relatively lower root mass to support shoot growth and development.
Fruit yield was analyzed by measuring the number of fruit clusters, the number of green and red fruits and weight of the green and red fruits in each one of the lines.
Plants over-expressing ABST_36 comprise (37%) significantly more fruit clusters compared to control line suggesting a link between vigor and yield potential as summarized in Table 23 below.
All three tested ABST genes of the present invention improved total fruit yield as illustrated in Table 24. Specifically, ABST_1 increased fruit yield by 137%. ABST_6 increased fruit yield by 151%. ABST_36 increased fruit yield by 191%. The relative large internal variation within populations reflects the variation that exists between different insertion events.
A more detailed analysis of the yield determinants showed that most of the additional yield (50% to 90%) was due to elevation in the weight of green fruit (Table 25). In addition most of the elevation in yield was due to an increase in the number of fruits rather than enlargement of fruit size (Table 26).
The significant difference in the number of green fruits between the lines largely depends on the physiological status of the plants. The control NT plants wilted much earlier while the plants over-expression ABST genes continued to develop for a longer period.
Comparison between lines expressing ABST_1, 6, 36 under favorable conditions to control lines under favorable conditions did not show any significant changes in the vigor of the vegetative parts or in the fruit yield. The following parameters were tested: fresh and dry weight of the roots and canopies, green fruit weight, red fruit weight and the average green and red fruit diameter. In all these parameters no differences were detected.
Table 27 below summarizes the results on crop yield following over-expression of ABST in tomato plants.
Tomato plants that were crossed between transgenic miniature tomatoes and the M82 line (line 3) were also grown under both salt stress and favorable conditions. The salt stress was carried out by continuous irrigation of saline water containing 180 mM NaCl.
The miniature populations expressing ABST_1 and 22 showed the highest improvement in salt tolerance compare to the control line based on plant and root mass and yield performance. The dry mass of the transgenic lines expressing ABST_1, 22 was increased by 300% and 257% respectively. ABST_36 improved both shoot mass and yield by about 30% as compared to control plants. The lines expressing ABST_1 and 22 showed the highest yield performance (165% and 140% respectively) compared to control population.
ABST_36 and 37 were also over-expressed in a line 2 tolerant tomato plant (Y361) and its expression was compared to that in a line 1 sensitive tomato plant (Shirly). As illustrated in
Hence, the results from Examples 1-10, clearly indicate that the abiotic stress tolerance genes of the present invention described herein can be readily isolated and utilized to substantially increase tolerance to abiotic stress and/or biomass in plants.
Monocot ortholog sequences for the 5 putative ABST tomato genes (SEQ I.D. NOs. 1,4, 9, 13 and 14) were sought. Monocot genomic databases namely NCBI (Hypertext Transfer Protocol://World Wide Web (dot) ncbi (dot) nlm (dot) nih (dot) gov/) and TIGR (Hypertext Transfer Protocol://World Wide Web (dot) tigr (dot) org/) databases of Maize, Sorghum and Barley were initially screened. The expressed sequence tags (ESTs) and cDNA sequences were clustered and assembled using the LEADS™ software (Compugen) and compared to the TIGR (Hypertext Transfer Protocol://World Wide Web (dot) tigr (dot) org/) databases of the above monocots. Overall, clustering of 372,000 maize ESTs resulted in 41,990 clusters among them 19,870 singletons. In Sorghum, about 190,000 ESTs were clustered into 39,000 clusters while in barley 370,500 ESTs generated 50,000 different clusters each representing a different gene. A digital expression profile summary was compiled for each cluster according to all keywords included in the sequence records comprising the cluster.
Yet, while comparing the monocot sequences to the tomato ABST genes, sequence homology levels differed dramatically, ranging from 45% to 88%. Moreover, the in-silico expression profile of the monocot genes did not always fit the profile of a gene involved in ABS tolerance.
In an attempt to identify the best orthologues for the tomato ABST genes various additional factors were analyzed. First, the sequences of the 5 tomato ABST genes (SEQ ID NO: 1, 4, 9, 13 and 14) and their deduced polypeptide sequences (SEQ ID NOs: 236-240) were compared to all monocot putative proteins, encoded by DNA sequences of gene clusters mentioned above. The comparison was performed on the protein level looking for identity higher than 45% along the entire protein sequences. Table 28 shows the best homologous genes and their identity level to the tomato ABST proteins. Next, these monocot proteins originating from different monocot species (barley, sorghum and maize) were screened based on their expression pattern during the development of several monocot species. This screening was based on digital expression of the genes, as described above. The genes were selected based on three criteria: genes with higher expression in roots, roots and leaves and or induce expression by treatments representing soil stress conditions (drought, salinity, soil deficiencies). The increase of expression was only counted in cases where the increase was greater than 2 fold (relative to the random EST distribution) with significance probability lower than 0.05. Table 29 summarizes the expression profile of the genes in different organ or tissues and the treatments that set off significant elevation in their expression level.
Sorghum
Sorghum
Sorghum
Sorghum
Sorghum
Sorghum
Sorghum
Sorghum
Sorghum
Sorghum
Sorghum
Sorghum
Sorghum
Sorghum
Sorghum
Sorghum
A combination of the above screening as described in Table 28 and in Table 29 revealed the final list of six monocot genes that are predicted to be the most related to the tomato ABST genes (SEQ ID NOs. 93, 94, 95, 96, 97 and 98).
Another type of sequence alignment for finding putative orthologous sequences from barley, rice, maize and sorghum, using the tomato ABST genes as involved the use of an evology system. Digital expression analysis was performed on these genes allowing for the identification of other putative monocot orthologs. The results were corroborated by phylogenetic analysis which studies the relationships between the tomato ABST genes and the putative monocot orthologs.
The Evology system is a method for constructing ortholog groups across multiple eukaryotic taxa, using the Markov cluster algorithm to group putative orthologs and paralogs. The method coherent with the groups identified by EGO (Hypertext Transfer Protocol://World Wide Web (dot) tigr (dot) org/tdb/tgi/ego/index (dot) shtml) but improved the identification of “recent paralogs” since EGO is easily misled by the functional redundancy of multiple paralogs and by the absence of true orthologs within incomplete genome data set as in most of the plant species.
The Evologs is a tool for large-scale automated eukaryotic ortholog group identification. To resolve the many-to-many orthologous relationships inherent in comparisons across multiple genomes, Evologs applied the Markov Cluster algorithm (Hypertext Transfer Protocol://micans (dot) org/mcl/), which is based on probability and graph flow theory and allows simultaneous classification of global relationships in a similarity space. MCL simulates random walks on a graph using Markov matrices to determine the transition probabilities among nodes of the graph. The MCL algorithm has previously been exploited for clustering a large set of protein sequences, where it was found to be very fast and reliable in dealing with complicated domain structures. Evologs generates clusters of at least two proteins, where each cluster consists of orthologs or paralogs from at least one species.
The putative orthologs were obtained using three levels of stringency. The first group with the lowest level (p value<=1e-20 and identity>=50%), the second group with moderate level of stringency (p value<=1e-42 and identity>=50%) and the third group with the highest stringency include p value<=1e-70 and identity>=70%.
These genes were subjected to digital expression analysis. Genes that were identified as being up-regulated under stress conditions underwent phylogenetic analysis. The phylogenetic trees showed similar distances between tomato, Arabidopsis and monocots supporting the claim that conservation in function in Arabidopsis and tomato strongly indicates conservation in function in monocot (data not shown).
A final list of ten candidate monocot ortholog genes was drawn up, as detailed in Table 30 below.
Sorghum
Sorghum
Sorghum
Sorghum
Sorghum
The digital expression profile for TC104838 (SEQ ID NO:93) is listed in Tables 31-33 herein below.
The digital expression profile for TC94284 (SEQ ID NO:94) is listed in Tables 34-36 herein below.
The digital expression profile for TC93449 (SEQ ID NO:95) is listed in Tables 37-39 herein below.
Colletotrichum
graminicola
Colletotrichum
graminicola
The digital expression profile for TC102291 (SEQ ID NO: 241 and 247) is listed in Tables 40-42 herein below.
Colletotrichum
graminicola
The digital expression profile for TC131030 (SEQ ID NO:242 and 248) is listed in Tables 43-45 herein below.
The digital expression profile for TC249366 (SEQ ID NO:243 and 251) is listed in Tables 46-48 herein below.
The digital expression profile for TC249365 (SEQ ID NO:244 and 250) is listed in Tables 49-51 herein below.
Fusarium, 6 h
The digital expression profile for AFO57183 (SEQ ID NO:245 and 249) is listed in Tables 52-54 herein below.
Fusarium,
Fusarium,
The digital expression profile for TC263205 (SEQ ID NO:246 and 252) is listed in Tables 55-57 herein below.
The digital expression profile for TC103772 (SEQ ID NO:98) is listed in Tables 58-60 herein below.
Colletotrichum
graminicola
Selected polynucleotide sequences (SEQ ID NOs: 93-98) were analyzed for presence of ORFs using Vector NTI suite (InforMax, U.K.) version 6 (Hasting Software, Inc: World Wide Web (dot) generunner (dot) com/). ORFs identified in each of these polynucleotide sequences were compared to Genbank database sequences, using Blast (World Wide Web (dot) ncbi (dot) nlm (dot) nih (dot) gov/BLAST/); the ORF displaying the highest homology to a GenBank sequence or sequences, was mapped in order to identify an ATG start codon. The position of the ATG start codon of this ORF was then compared with that of the identified polynucleotide sequence in order to verify that each of the five sequences described herein includes a full length ORF and an ATG start codon (thus qualifies as a “putative monocot ABST gene”).
Polypeptides with significant homology to monocot ABST genes (SEQ ID NOs: 93-98) have been identified from the NCBI databases using BLAST software (Table 61).
Zea mays
Saccharum officinarum
sorghum
Saccharum officinarum
sorghum
Oryza sativa
Zea mays
Hordeum vulgare
Oryza sativa
Triticum aestivum
Triticum aestivum
Hordeum vulgare
Triticum aestivum
Zea mays
Saccharum officinarum
Triticum aestivum
Zea mays
Triticum aestivum
sorghum
Saccharum officinarum
sorghum
Saccharum officinarum
Saccharum officinarum
sorghum
Saccharum officinarum
Zea mays
Zea mays
sorghum
Zea mays
Saccharum officinarum
Solanum tuberosum
Gossypium
Gossypium
Lycopersicon
esculentum
Lycopersicon
esculentum
Lycopersicon
esculentum
Lycopersicon
esculentum
Solanum tuberosum
Solanum tuberosum
Solanum tuberosum
Solanum tuberosum
Capsicum annum
arabidopsis
Gossypium
Gossypium
Gossypium
arabidopsis
Glycine max
Capsicum annum
populus
medicago
populus
medicago
populus
medicago
Hordeum vulgare
Triticum aestivum
Oryza sativa
Oryza sativa
Triticum aestivum
Hordeum vulgare
arabidopsis
Hordeum vulgare
Saccharum officinarum
Hordeum vulgare
populus
Hordeum vulgare
Oryza sativa
Glycine max
populus
Triticum aestivum
Triticum aestivum
populus
Oryza sativa
Triticum aestivum
Oryza sativa
Hordeum vulgare
Triticum aestivum
Zea mays
Triticum aestivum
Zea mays
Triticum aestivum
Zea mays
Triticum aestivum
Hordeum vulgare
Oryza sativa
Zea mays
Triticum aestivum
Oryza sativa
Oryza sativa
Gossypium
Solanum tuberosum
Triticum aestivum
Triticum aestivum
Hordeum vulgare
Triticum aestivum
Oryza sativa
Saccharum officinarum
Oryza sativa
Glycine max
Triticum aestivum
Triticum aestivum
Solanum tuberosum
Saccharum officinarum
Populus
Solanum tuberosum
Glycine max
Solanum tuberosum
Zea mays
Zea mays
Oryza sativa
Lycopersicon
esculentum
Capsicum annuum
Hordeum vulgare
Gossypium
Oryza sativa
Triticum aestivum
Hordeum vulgare
Oryza sativa
DNA sequences of six putative Monocot ABST genes were synthesized by GeneArt (Hypertext Transfer Protocol://World Wide Web (dot) geneart (dot) com/). Synthetic DNA was designed in silico, based on the encoded amino-acid sequences of the Monocot ABST genes (SEQ ID NOs: 99, 100, 101, 102, 103 and 104), and by using plant-based codon-usage. The synthetic sequences and the plant native orthologues were compared. At least 1 mutation per 20 nucleotide base pairs was added to avoid possible silencing, when over-expressing the gene in favorable monocot species, such as maize. The planned sequences were bordered with the following restriction enzymes sites polylinker—SalI, XbaI, BamHI, SmaI at the 5′ end and SacI at the 3′ end. The sequences were cloned in double strand, PCR Script plasmid (GeneArt).
The PCR Script plasmids harboring the synthetic, monocot-based ABST genes were digested with the restriction endonucleases XbaI and SacI (Roche), purified using PCR Purification Kit (Qiagen, Germany), and inserted via DNA ligation using T4 DNA ligase enzyme (Roche) and according to the manufacturer's instructions, into pKG(NOSter), (SEQ ID NO: 233) and pKG(35S+NOSter), (SEQ ID NO: 234), plant expression vector plasmids, also digested with XbaI and SacI (Roche) and purified. pKG plasmid is based on the PCR Script backbone (GeneArt), with several changes in the polylinker site to facilitate cloning a gene of interest downstream to a promoter and upstream to a terminator, suitable for expression in plant cells. Moreover, the inserted gene, together with the promoter and the terminator could be easily moved to a binary vector.
The resulting pKG(NOSter) and pKG(35S+NOSter) harboring putative Monocot ABST genes were introduced into E. coli DH5 competent cells by electroporation, using a MicroPulser electroporator (Biorad), 0.2 cm cuvettes (Biorad) and EC-2 electroporation program (Biorad). The treated cells were cultured in LB liquid medium at 37° C. for 1 hour, plated over LB agar supplemented with ampicillin (100 mg/L; Duchefa) and incubated at 37° C. for 16 hrs. Colonies that developed on the selective medium were analyzed by PCR using the primers of SEQ ID NO: 231 and SEQ ID NO: 232, which were designed to span the inserted sequence in the pKG plasmids. The resulting PCR products were separated on 1% agarose gels and from the colonies having the DNA fragment of the predicted size, a plasmid was isolated using miniprep Plasmid Kit (Qiagen) and sequenced using the ABI 377 sequencer (Amersham Biosciences Inc) in order to verify that the correct DNA sequences were properly introduced to the E. coli cells.
Positive pKG(NOSter) plasmids harboring putative Monocot ABST genes were digested with the restriction enzymes HindIII and SalI (Roche), purified using PCR Purification Kit (Qiagen, Germany), and then ligated (as described above) with At6669 promoter sequence (set forth in SEQ ID NO: 20) digested from pPI+At6669 plasmid with the same enzymes and purified. The resulting plasmids were introduced into E. coli DH5 competent cells by electroporation, the treated cells were cultured in LB liquid medium at 37° C. for 1 hr, subsequently plated over LB agar supplemented with ampicillin (100 mg/L; Duchefa) and incubated at 37° C. for 16 hours. Colonies grown on the selective medium were analyzed by PCR using the primers SEQ ID NO: 235 and SEQ ID NO: 232. Positive plasmids were identified isolated and sequenced as described above.
The plasmid pPI was constructed by inserting a synthetic poly-(A) signal sequence, originating from pGL3 basic plasmid vector (Promega, Acc No U47295; by 4658-4811) into the HindIII restriction site of the binary vector pBI101.3 (Clontech, Acc. No. U12640).
The At6669 promoter was isolated from Arabidopsis thaliana var Col0 genomic DNA by PCR amplification using the primers set forth in SEQ ID NOs: 37 and 38. The PCR product is purified (Qiagen, Germany) and digested with the restriction endonucleases HindIII and SalI (Roche). The resulting promoter sequence was introduced into the open binary pPI vector digested with the same enzymes, to produce pPI+At6669 plasmid.
Generating Binary Vectors Comprising the Cauliflower Mosaic Virus 35S Promoter:
The five pKG(35S+NOSter) constructs harboring putative Monocot ABST genes (SEQ ID Nos: 93, 94, 95, 96, 97 and 98) were digested with HindIII and EcoRI (Roche) restriction endonucleases in order to excise expression cassettes and ligated to pPI plasmid digested with the same endonucleases and purified (as described above). Altogether, five pPI constructs were generated, each comprising putative Monocot ABST gene having a sequence set forth in SEQ ID NOs: 93, 94, 95, 96, 97 and 98 positioned downstream to the Cauliflower Mosaic Virus 35S promoter and upstream to the Nopaline Synthase (NOS) terminator, which was originated from the digestion of pBI101.3 (Clontech, Acc. No. U12640), using the restriction sites SacI and EcoRI.
Generating Binary Vectors Comprising the At6669 Promoter:
The five pKG(At6669+NOSter) constructs harboring putative Monocot ABST genes downstream to At6669 promoter sequence (set forth in SEQ ID NO: 20), and upstream to the Nopaline Synthase (NOS) terminator, were digested with HindIII and EcoRI (Roche) in order to excise expression cassettes and ligated into pPI plasmid which was digested with the same restriction endonucleases and purified (as described above). Altogether, five pPI constructs were generated, each comprising the At6669 promoter positioned upstream of a putative Monocot ABST gene having a sequence set forth in SEQ ID NOs: 93, 94, 95, 96, 97 and 98.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.
Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents, patent applications and sequences identified by their accession numbers mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application or sequence identified by their accession number was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.
This application is a division of U.S. patent application Ser. No. 13/874,500 filed on May 1, 2013, which is a continuation of U.S. patent application Ser. No. 12/457,199 filed on Jun. 3, 2009, now U.S. Pat. No. 8,481,812, which is a continuation of U.S. patent application Ser. No. 11/284,236 filed on Nov. 22, 2005, now U.S. Pat. No. 7,554,007, which is a Continuation-In-Part (CIP) of PCT Patent Application No. PCT/IL2004/000431 filed on May 20, 2004, which claims the benefit of priority of U.S. Provisional Patent Application No. 60/472,433 filed on May 22, 2003. U.S. patent application Ser. No. 11/284,236 also claims the benefit of priority of U.S. Provisional Patent Application No. 60/707,957 filed on Aug. 15, 2005. The contents of the above applications are all incorporated by reference as if fully set forth herein in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 60472433 | May 2003 | US | |
| 60707957 | Aug 2005 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13874500 | May 2013 | US |
| Child | 15005043 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12457199 | Jun 2009 | US |
| Child | 13874500 | US | |
| Parent | 11284236 | Nov 2005 | US |
| Child | 12457199 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/IL2004/000431 | May 2004 | US |
| Child | 11284236 | US |
