Patent Application
20120199144
The invention relates to genetic constructs used in the preparation of transgenic plants. The constructs can have the ability to cause plants to accumulate threonine in their leaves, particularly during leaf senescence. The invention extends to plant cells transformed with such constructs, and to the transgenic plants themselves. The invention also relates to methods of producing transgenic plants, and to methods of increasing the concentration of threonine in senescent plants. The invention also relates to harvested plant leaves, for example tobacco leaves, that have been transformed with the genetic constructs, and to smoking articles comprising such harvested plant leaves.
A primary target in increasing the flavour of flue-cured tobacco is the production of the amino acid threonine. Accumulation of high levels of threonine in the leaves of mutant tobacco plants gives significant flavour and aroma benefits. Normally, however, threonine production is tightly regulated, in conjunction with the production of other amino acids of the aspartate family, namely methionine, lysine and isoleucine. Therefore, modification of the biosynthetic pathway which produces threonine is required to achieve the flavour and aroma benefits.
As shown in
It has been previously shown that transgenic plants containing feedback-insensitive aspartate kinase, and which are able to overproduce threonine in comparison with wild-type plants, grew poorly and demonstrated a catastrophic fitness cost if such plants were homozygous for that mutation. Poor growth and a fitness cost is any non-beneficial change in the growth of the plants which the farmer notices. Clearly, any such change is not desirable.
The inventors of the present invention therefore set out to provide a transgenic plant which is able to accumulate threonine in leaves by overcoming the feedback inhibition loop discussed above, but ideally without any cost to its fitness. With this in mind, the inventors developed a number of genetic constructs, in which a gene encoding a threonine insensitive aspartate kinase (AK) enzyme was placed under the control of a promoter, to determine what effect, if any, over-expression of this gene had on threonine levels in senescent leaves.
Leaf senescence is a phase of plant development during which the cells undergo distinct metabolic and structural changes prior to cell death. Physiological and genetic studies indicate that senescence is a highly regulated process. The progression of a leaf through senescence is visibly marked by the loss of chlorophyll and consequent yellowing, which results from the disassembly of the chloroplasts. The decreasing levels of leaf chlorophyll, characteristic of this developmental stage, can be measured, e.g. by solvent extraction and spectrophotometric measurement, or by a chlorophyll content meter. A decreased leaf chlorophyll level in comparison with an earlier leaf chlorophyll level recorded for the same plant, preferably grown under constant conditions, indicates senescence.
Molecular studies indicate that senescence is associated with changes in gene expression. The levels of mRNAs encoding proteins involved in photosynthesis decrease during senescence, whilst mRNA levels of genes encoding proteins thought to be involved in the senescence increase. Senescence is a highly organised process regulated by genes known as Senescence Associates Genes (SAGs). Leaf senescence involves the degradation of proteins, nucleic acids and membranes, and the subsequent transport of the nutrients resulting from this degradation to other regions of the plant, such as the developing seeds, leaves or storage organs. One problem of plant senescence is that many useful minerals and nutrients that are present in senescent leaves will remain in the leaves, and will therefore be effectively lost as the leaves die. For example, threonine, as well as many other amino acids, that are present in the senescent leaves, will go to waste, if they are not removed from the dying leaves.
As described in Example 2, the inventors carried out experiments which involved working with genetic constructs expressing threonine insensitive AK at specific locations within the plant, i.e. by use of the leaf-specific pea-plastocyanin promoter. However, they found that such transgenic plants had leaves which were pale, thickened, brittle and strap-like. The internodes were shortened and showed browning as maturity increased, and the buds either did not develop or were misshapen. Thus, such transgenic plants were unable to overcome the fitness cost.
Consequently, the inventors have developed a series of genetic constructs in which the feedback insensitive aspartate kinase (AK) activity is expressed only after the plant has entered senescence (under the control of the SAG12 promoter), and thus been allowed to develop normally. The inventors have observed that the constructs that they have developed, which allow transgenic plants transformed with these constructs to grow normally to maturity (i.e. to senescence) before the feedback insensitive aspartate kinase (AK) is switch on, are surprisingly able to overcome the fitness cost.
Therefore, according to a first aspect of the invention, there is provided a genetic construct comprising a senescence-specific promoter operably linked to a coding sequence encoding a polypeptide having threonine insensitive aspartate kinase activity.
The inventors have used a senescence-specific promoter linked to a coding sequence encoding a polypeptide having a threonine insensitve aspartate kinase activity to form the construct of the first aspect, which was then used to transform a plant. As a result of their studies, the inventors surprisingly found that the construct according to the invention, resulted in increased levels of threonine in senescent leaves. Furthermore, this temporal limitation on the expression of the transgene controlled by the senescence-specific promoter overcomes the negative effect of the fitness cost that was previously seen in earlier attempts to produce a threonine-accumulating plant. As shown in the Examples, the resulting transgenic plant produces threonine at a greater than wild-type level during leaf senescence. Threonine accumulation was demonstrated to occur in the leaves, and these increased levels are thought to positively contribute to the flavour of tobacco leaves containing the construct, and thus smoking articles made from such leaves.
The promoter in the genetic construct of the first aspect may be capable of inducing RNA polymerase to bind to, and start transcribing, the coding sequence encoding the polypeptide having threonine insensitive aspartate kinase activity.
A “senescence-specific promoter” (SAG) can be any promoter, which is associated with controlling the expression of a senescence-associated gene. Hence, the promoter can restrict expression of a coding sequence (i.e. a gene) to which it is operably linked substantially exclusively in senescing tissue. Therefore, a senescence-specific promoter can be a promoter capable of preferentially promoting gene expression in a plant tissue in a developmentally-regulated manner such that expression of a 3′ protein-coding region occurs substantially only when the plant tissue is undergoing senescence. It will be appreciated that senescence tends to occur in the older parts of the plant, such as the older leaves, and not in the younger parts of the plants, such as the seeds.
One example of a plant which is known to express numerous senescence-associated genes is Arabidopsis. Hence, the promoter present in the construct according to the first aspect may be isolated from a senescence-associated gene in Arabidopsis, Gepstein et al. (The Plant Journal, 2003, 36, 629-642) conducted a detailed study of SAGs and their promoters using Arabidopsis as a model. Thus, the genetic construct may comprise a promoter from any of the SAGs disclosed in this paper. For example, a suitable promoter may be selected from a group consisting of SAG12, SAG13, SAG101, SAG21, and SAG18, or a functional variant or a functional fragment thereof.
Preferred promoters are SAG12 and SAG13 promoters. In one embodiment, the promoter is a SAG12 promoter, which will be known to the skilled technician, or a functional variant or a fragment thereof (Gan & Amasino, 1997, Plant physiology, 113: 313-319). The DNA sequence encoding the SAG12 promoter is shown in
Therefore, the promoter in the construct of the invention may comprise a nucleotide sequence substantially as set out in SEQ ID No.1, or a functional variant or functional fragment thereof. The SAG12 promoter sequence may be obtained from Arabidopsis thaliana, as described in U.S. Pat. No. 5,689,042. In embodiments where the promoter is SAG12, it will be appreciated that the promoter may comprise each of the bases 1-2093 of SEQ ID No.1. However, functional variants or functional fragments of the promoter may also be used in the genetic constructs of the invention.
A “functional variant or functional fragment of a promoter” can be a derivative or a portion of the promoter that is functionally sufficient to initiate expression of any coding region that is operably linked thereto. For example, in embodiments where the promoter is based on SAG12, the skilled technician will appreciate that SEQ ID No:1 may be modified, or that only portions of the SAG12 promoter may be required, such that it would still initiate gene expression, in the construct, of the polypeptide having threonine insensitive aspartate kinase activity. Similar modifications may be made to the nucleotide sequences of any of the other known SAG promoters, such as SAG13, SAG101, SAG21 and SAG18.
Functional variants and functional fragments of the promoter may be readily identified by assessing whether or not transcriptase will bind to a putative promoter region, and then lead to the transcription of the coding sequence into the polypeptide having threonine insensitive aspartate kinase activity. Alternatively, such functional variants and fragments may be examined by conducting mutagenesis on the promoter, when associated with a coding region, and assessing whether or not gene expression may occur.
The genetic construct of the first aspect may be capable of causing, during senescence, expression of a polypeptide having threonine insensitive aspartate kinase activity. The promoter may induce expression of a coding sequence encoding a polypeptide exhibiting threonine insensitive aspartate kinase activity. Therefore, the genetic construct may comprise at least one coding sequence, which encodes threonine insensitive aspartate kinase (AK), or a functional variant or fragment thereof. Hence, in the first embodiment, the genetic construct may comprise the senescence-specific promoter and a coding sequence encoding threonine insensitive aspartate kinase (AK), or a functional variant or fragment thereof.
As described in Example 3-4, the inventors have found that expression of threonine insensitive aspartate kinase in a host cell, by transforming a plant with the construct of the invention, caused a significant increase in threonine levels. Furthermore, advantageously they found that the construct did not have any detrimental effect the fitness of the transformed plant.
As illustrated in
As shown in
Elevating leaf threonine (Thr) content depends on overcoming the negative feedback control on the synthetic pathway. For example, transgenic tobacco expressing a lysine-insensitive AK from E. coli has been produced, which exhibited a threonine accumulating phenotype. In these transformants, the bacterial AK was expressed under the control. of 35S in tobacco, either targeted to the cytoplasm or the chloroplast. The endogenous activity of AK was still entirely susceptible to inhibition by both lysine and threonine. The chloroplast-directed transgene gave higher AK activity. Higher threonine levels were found in the plants expressing the chloroplastic form. However, poor plant growth was noted and the homozygous plants demonstrated a fitness loss including wrinkled upper leaves, delayed flowering and partial sterility.
Work carried out in Arabidopsis (Paris et al, 2003, The Journal of Biological Chemistry, Vol 278, no. 7, pp 5361-5366) has demonstrated that the regulatory domain of the AK:HSDH enzyme contains two homologous sub-domains defined by a common loop-α helix-loop-β strand-loop-β strand motif. Site-directed mutagenesis was used to elucidate the threonine binding sites. It was found that each regulatory domain of the monomers of aspartate kinase-homoserine dehydrogenase possessess two non-equivalent threonine binding sites constituted in part by Gln443 and Gln524. The binding of threonine to Gln443 inhibits AK activity and also facilitates the binding of a second threonine to Gln524 which leads to inhibition of HSDH.
Conformational modification of the second sub-domain would induce the binding of a second threonine leading to conformational modifications of HSDH catalytic domain and HSDH inhibition.
The mutation of these glutamine residues to alanine rendered the threonine inhibition of the enzyme ineffective. The mutations do not affect the kinetics of the HSDH activity, only its sensitivity to threonine; the AK kinetics are only slightly modified. However, unfortunately, transgenic plants in which feedback insensitive AK:HSDH from Arabidopsis has been introduced and expressed also leads to a fitness cost.
In contrast, the genetic construct of the present invention causes expression of a polypeptide having threonine insensitive aspartate activity during senescence, and does not show any detrimental effect on the fitness of the transgenic plant. Thus, the genetic construct of the first aspect may encode a threonine insensitive aspartate kinase (AK), or a threonine insensitive bifunctional aspartate kinase-homoserine dehydrogenase enzyme (AK-HSDH), or functional variants or functional fragments thereof.
The threonine insensitive AK or bifunctional AK-HSDH, or functional variant or fragment thereof, may be derived from any suitable source, such as a plant. The coding sequence, which encodes the polypeptide having threonine insensitive aspartate kinase activity may be derived from Arabidopsis spp., Zea spp., Flaveria spp., or Cleome spp. The coding sequence, which encodes the polypeptide having threonine insensitive aspartate kinase activity may be derived from Arabidopsis thaliana, Zea mays, Flaveria trinervia, Flaveria bidentis, Flaveria brownie or Cleome gynandra. Preferably, the coding sequence of the enzyme may be derived from Arabidopsis spp., such as Arabidopsis thaliana.
A particularly preferred threonine insensitive enzyme is a mutated AK-HSDH in which at least one of the threonine binding sites has been altered. Preferably, one or both of the threonine binding sites constititued in part by Gln443 and Gln524 have been mutated. For example, the Arabidopsis AK-HSDH may be mutated at Gln443 and/or Gln524. Preferably, the Arabidopsis AK-HSDH is mutated at Gln443 and Gln524. The mutated AK:HSDH gene used in the present invention is shown in
Accordingly, the DNA sequence encoding one embodiment (i.e. Gln443Ala single mutant) of Arabidopsis threonine insensitive aspartate kinase is provided herein as SEQ ID No:2, as follows:
In SEQ ID No:2 (i.e. Q443A), GCT, as highlighted, corresponds to mutated Gln443 encoding alanine, and CAR, as highlighted, corresponds to wild-type Gln524, where R may be either G or A.
The DNA sequence encoding another embodiment (i.e. Gln524Ala single mutant) of Arabidopsis threonine insensitive aspartate kinase is provided herein as SEQ ID No:3, as follows:
In SEQ ID No:3 (i.e. Q524A), GCT, as highlighted, corresponds to mutated Gln524 encoding alanine, and CAR, as highlighted, corresponds to wild-type Gln443, where R may be either G or A.
The DNA sequence encoding another embodiment (i.e. Gln443Ala; Gln524Ala double mutant) of Arabidopsis threonine insensitive aspartate kinase is provided herein as SEQ ID No:4, as follows:
In SEQ ID No:4 (i.e. Q443A; Q524A), GCT, as highlighted, correspond to mutated Gln443 and Gln524 both encoding alanine.
Accordingly, the coding sequence, which encodes the polypeptide having threonine insensitive aspartate kinase activity, may comprise a nucleic acid sequence substantially as set out in any one of SEQ ID No:2, 3 or 4, or a functional variant or a fragment thereof.
The polypeptide sequence of one embodiment (i.e. the Gln443Ala single mutant) of the threonine insensitive aspartate kinase is provided herein as SEQ ID No:5, as follows:
In SEQ ID No:5, the mutant Alanine (A) at position 443, and wild-type glutamine (Q) at position 524, are highlighted.
The polypeptide sequence of another embodiment (i.e. the Gln524Ala single mutant) of the threonine insensitive aspartate kinase is provided herein as SEQ ID No:6, as follows:
In SEQ ID No:6, the mutant Alanine (A) at position 524, and wild-type glutamine (Q) at position 443, are highlighted.
The polypeptide sequence of another embodiment (i.e. Gln443Ala; Gln524Ala double mutant) of the threonine insensitive aspartate kinase is provided herein as SEQ ID No:7, as follows:
In SEQ ID No:7, the mutant Alanine (A) at positions 443 and 524, are highlighted.
Accordingly, the polypeptide sequence having threonine insensitive asparate kinase activity may comprise an amino acid sequence substantially as set out in any one of SEQ ID No:5, 6 or 7, or a functional variant or a fragment thereof.
Genetic constructs of the invention may be in the form of an expression cassett, which may be suitable for expression of the coding sequence in a host cell. The genetic construct of the invention may be introduced in to a host cell without it being incorporated in a vector. For instance, the genetic construct, which may be a nucleic acid molecule, may be incorporated within a liposome or a virus particle. Alternatively, a purified nucleic acid molecule (e.g. histone-free DNA, or naked DNA) may be inserted directly into a host cell by suitable means, e.g. direct endocytotic uptake. The genetic construct may be introduced directly in to cells of a host subject (e.g. a plant) by transfection, infection, microinjection, cell fusion, protoplast fusion or ballistic bombardment. Alternatively, genetic constructs of the invention may be introducted directly into a host cell using a particle gun. Alternatively, the genetic construct may be harboured within a recombinant vector, for expression in a suitable host cell.
Hence, in a second aspect, there is provided a recombinant vector comprising the genetic construct according to the first aspect.
The recombinant vector may be a plasmid, cosmid or phage. Such recombinant vectors are highly useful for transforming host cells with the genetic construct of the first aspect, and for replicating the expression cassette therein. The skilled technician will appreciate that genetic constructs of the invention may be combined with many types of backbone vector for expression purposes. The backbone vector may be a binary vector, for example one which can replicate in both E. coli and Agrobacterium tumefaciens. For example, a suitable vector may be a pBIN plasmid, such as pBIN19. However, a preferred backbone vector is BNP1380000001, which is based on pBINPLUS (F. A. van Engelen et al. Transgenic Research (1995) 4, 288-290), and which harbours the SAG12 promoter. An embodiment of this vector is shown in
Recombinant vectors may include a variety of other functional elements in addition to the promoter (e.g. a senescence-associated promoter), and the at least one coding sequence (encoding mutated AK-HSDH). For instance, the recombinant vector may be designed such that it autonomously replicates in the cytosol of the host cell. In this case, elements which induce or regulate DNA replication may be required in the recombinant vector. Alternatively, the recombinant vector may be designed such that it integrates into the genome of a host cell. In this case, DNA sequences which favour targeted integration (e.g. by homologous recombination) are envisaged.
The recombinant vector may also comprise DNA coding for a gene that may be used as a selectable marker in the cloning process, i.e. to enable selection of cells that have been transfected or transformed, and to enable the selection of cells harbouring vectors incorporating heterologous DNA. Alternatively, the selectable marker gene may be in a different vector to be used simultaneously with vector containing the gene of interest. The vector may also comprise DNA involved with regulating expression of the coding sequence, or for targeting the expressed polypeptide to a certain part of the host cell, e.g. the chloroplast. Hence, the vector of the second aspect may comprise at least one additional element selected from a group consisting of: a selectable marker gene (e.g. an antibiotic resistance gene); a polypeptide termination signal; and a protein targeting sequence (e.g. a chloroplast transit peptide).
Examples of suitable marker genes include antibiotic resistance genes such as those conferring resistance to Kanamycin, Geneticin (G418) and Hygromycin (npt-II, byg-B); herbicide resistance genes, such as those conferring resistance to phosphinothricin and sulphonamide based herbicides (bar and suI respectively; EP-A-242246, EP-A-0249637); and screenable markers such as beta-glucuronidase (GB2197653), luciferase and green fluorescent protein (GFP).
The marker gene may be controlled by a second promoter (which may or may not be a senescence-associated promoter), which allows expression in cells, which may or may not be in the seed, thereby allowing the selection of cells or tissue containing the marker at any stage of development of the plant. Suitable second promoters are the promoter of nopaline synthase gene of Agrobacterium and the promoter derived from the gene which encodes the 35S cauliflower mosaic virus (CaMV) transcript. However, any other suitable second promoter may be used.
Various embodiments of genetic constructs of the invention may be prepared using a suitable cloning procedure, which is described in Example 2, and which may be summarised as follows. The gene encoding wild-type AK-HSDH may be amplified from either the genomic or cDNA templates by PCR using suitable primers. Suitable primers for amplification of the wild-type AK-HSDH gene may be SEQ ID No:8 and/or SEQ ID No:9. PCR products may be examined using agarose gel electrophoresis. Site-directed mutagenesis using suitable pairs of primers may then be carried out in order to mutate the wild-type codons at positions 443 and/or 524 to produce the Gln443Ala and Gln524Ala single mutants or the double mutant. For example, suitable primers for changing the codon for Gln443 may be SEQ ID No:10 and/or SEQ ID No:11. Suitable primers for changing the codon for Gln524 may be SEQ ID No:12 and/or SEQ ID No:13.
The PCR products encoding either of the two single mutants or the double mutant may then be ligated into a suitable vector for cloning purposes, for example that which is available under the trade name the pCR4 Blunt-TOPO vector, which may be obtained from Invitrogen. Vectors harbouring the PCR products may then be grown up in a suitable host, such as E. coli. E. coli colonies may then be screened by PCR using suitable primers, and inserts in plasmids showing the correct restriction enzyme digest Pattern may be sequenced using suitable primers. E. coli colonies carrying TOPO-cDNA (AK-HSDH) may be cultured to produce a suitable amount of plasmid, which may then be purified. The plasmid may then be digested to release a DNA fragment encoding mutant AK-HSDH, which may then be cloned into a vector harbouring a suitable promoter, for example a SAG promoter (preferably, SAG12), such as a pBNP plasmid. The resultant plasmid was named pBNP138-0453-001. An embodiment of the vector according to the second aspect may be substantially as set out in
In a third aspect, there is provided a method of producing a transgenic plant which accumulates a higher level of threonine in the leaves than a corresponding wild type plant cultured under the same conditions, the method comprising:
(i) transforming a plant cell with a genetic construct of the first aspect, or the vector of the second aspect; and
(ii) regenerating a plant from the transformed cell.
Methods for determining the level of threonine in plant leaves, and plant growth rates, are set out in Example 1. The method of the third aspect may comprise transforming a test plant cell with a genetic construct according to the first aspect, or a vector according to the second aspect. The genetic construct or the vector may be introduced into a host cell by any suitable means.
In a fourth aspect, there is provided a cell comprising the genetic construct according to first aspect, or the recombinant vector according to the second aspect.
The cell may be a plant cell. As the inventors have observed that expressing the threonine-insensitive aspartate kinase under the control of the senescence-specific promoter in a host cell is surprisingly effective at increasing threonine concentrations in senescent leaves without compromising fitness, the cell of the fourth aspect may comprise one or more constructs of the first aspect, or one or more vectors of the second aspect.
The cell may be transformed with the genetic construct or the vector according to the invention, using known techniques. Suitable means for introducing the genetic construct into the host cell may include use of a disarmed Ti-plasmid vector carried by Agrobacterium by procedures known in the art, for example as described in EP-A-0116718 and EP-A-0270822. A further method may be to transform a plant protoplast, which involves first removing the cell wall and introducing the nucleic acid, and then reforming the cell wall. The transformed cell may then be gown into a plant.
In a fifth aspect, there is provided a transgenic plant comprising the genetic construct according to first aspect, or the vector according to the second aspect.
The transgenic plant according to the fifth aspect may include the Brassicaceae family, such as Brassica spp. The plant may be Brassica napus (oilseed rape).
Further examples of transgenic plants according to the fifth aspect include the family Poales, such as Triticeae spp. The plant mey be Triticum spp. (wheat). Increasing the grain protein content in wheat may result in increased volume of food products comprising such wheat, such as bread.
Further examples of suitable transgenic plants according to the fifth aspect include the Solanaceae family of plants which include, for example jiimson weed, eggplant, mandrake, deadly nightshade (belladonna), capsicum (paprika, chilli pepper), potato and tobacco. One example of a suitable genus of Solanaceae is Nicotiana. A suitable species of Nicotiana may be referred to as tobacco plant, or simply tobacco. Various methods for transforming plants with the genetic construct of the first aspect, or vector of the second aspect, are known and can be used in the present invention.
For example, tobacco may be transformed as follows. Nicotiana tabacum is transformed using the method of leaf disk co-cultivation essentially as described by Horsch et al. (Science 227: 1229-1231, 1985). The youngest two expanded leaves may be taken from 7 week old tobacco plants and may be surface sterilised in 8% Domestos™ for 10 minutes and washed 6 times with sterile distilled water. Leaf disks may be cut using a number 6 cork borer and placed in the Agrobacterium suspension, containing the appropriate binary vectors (according to the invention), for approximately two minutes. The discs may be gently blotted between two sheets of sterile filter paper. Ten disks may be placed on LS 3% sucrose+2 μM BAP+0.2 μM NAA plates, which may then be incubated for 2 days in the growth room.
Discs may be transferred to plates of LS+3% sucrose+2 μM BAP+0.2 μM NAA supplemented with 500 g/l claforan and 100 g/l kanamycin. The discs may be transferred onto fresh plates of above medium after 2 weeks. After a further two weeks, the leaf disks may be transferred onto plates containing LS+3% sucrose+0.5 μM BAP supplemented with 500 mg/l claforan and 100 mg/l kanamycin. The leaf disks may be transferred onto fresh medium every two weeks. As shoots appear, they may be excised and transferred to jars of LS+3% sucrose supplemented with 500 mg/l claforan. The shoots in jars may be transferred to LS+3% sucrose+250 mg/l claforan after approximately 4 weeks. After a further 3-4 weeks the plants may be transferred to LS+3% sucrose (no antibiotics) and rooted. Once the plants are rooted they may be transferred to soil in the greenhouse.
In a sixth aspect, there is provided a plant propagation product obtainable from the transgenic plant according to the fifth aspect.
A “plant propagation product” can be any plant matter taken from a plant from which further plants may be produced. Suitably, the plant propagation product may be a seed.
The present invention also embraces harvested leaves from a transgenic plant of the present invention in which the harvested leaves contain a higher level of threonine than harvested leaves from a corresponding wild type plant cultured under the same conditions.
Therefore, in a seventh aspect, there is provided a harvested leaf containing a higher level of threonine than harvested leaves than the corresponding level of threonine in a harvested leaf taken from a wild-type plant cultured under the same conditions, wherein the leaf is harvested from the transgenic plant according to the fifth aspect, or produced by the method according to the third aspect.
An eighth aspect of the invention provides a smoking article comprising threonine-enriched tobacco obtained from a mutant tobacco plant, which mutant is capable of over-producing threonine in senescent leaves.
Advantageously, and preferably, the mutant tobacco plant may have been transformed with a genetic construct or vector of the invention. The smoking article may be a cigarette, cigar, cigarillo, or rolling tobacco, or the like.
Threonine-reduced tobacco can include tobacco in which the threonine concentration is more than the corresponding concentration in a wild-type plant cultured under the same conditions. Such a smoking article may comprise tobacco obtained from a mutant tobacco plant, which may have been transformed with a genetic construct according to the first aspect of the invention, or a vector according to the second aspect. The flavour and aroma of the threonine-enriched tobacco is improved.
It will be appreciated that the present invention provides a method of increasing the level of threonine in plant leaves above the corresponding wild type level without compromising plant fitness, this comprises altering plant metabolism to achieve increased production of threonine after initiation of leaf senescence.
Hence, in a ninth aspect of the invention, there is provided a method of increasing the level of threonine in plant leaves above the corresponding wild type level without compromising plant fitness, the method comprising altering plant metabolism to achieve increased production of threonine after the initiation of leaf senescence.
Preferably, and advantageously, the methods according to the invention do not compromise the health of fitness of the plant that is generated. Preferably, the methods comprise transforming the test plant, and preferably its leaves, with the genetic construct of the first aspect, or the vector of the second aspect.
As described in Example 4, in addition to measuring threonine levels in the transformed plants of the invention, and showing that threonine concentrations increase in senescent leaves, the inventors have also measured the concentrations of other amino acids in the transgenic plant, including glutamine, glutamic acid, aspartic acid and histidine. As shown in
It will be appreciated that the invention extends to any nucleic acid or peptide or variant, derivative or analogue thereof, which comprises substantially the amino acid or nucleic acid sequences of any of the sequences referred to herein, including functional variants or functional fragments thereof. The terms “substantially the amino acid/polynucleotide/polypeptide sequence”, “functional variant” and “functional fragment”, can be a sequence that has at least 40% sequence identity with the amino acid/polynucleotide/polypeptide sequence of any one of the sequences referred to herein, for example 40% identity with the promoter identified as SEQ ID No:1 (i.e. SAG12 promoter) or the gene identified as SEQ ID No.2, 3 or 4 (which encode various embodiments of the AK-HSDH enzyme), or 40% identity with the polypeptide identified as SEQ ID No.5, 6 or 7 (i.e. various embodiments of the mutant AK-HSDH enzyme), and so on.
Amino acid/polynucleotide/polypeptide sequences with a sequence identity which is greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, and still more preferably greater than 80% sequence identity to any of the sequences referred to are also envisaged. Preferably, the amino acid/polynucleotide/polypeptide sequence has at least 85% identity with any of the sequences referred to, more preferably at least 90% identity, even more preferably at least 92% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 98% identity and, most preferably at least 99% identity with any of the sequences referred to herein.
The skilled technician will appreciate how to calculate the percentage identity between two amino acd/polynucleotide/polypeptide sequences. In order to calculate the percentage identity between two amino acid/polynucleotide/polypeptide sequences, an alignment of the two sequences must first be prepared, followed by calculation of the sequence identity value. The percentage identity for two sequences may take different values depending on: (i) the method used to align the sequences, for example, ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, for example, local vs global alignment, the pair-score matrix used (e.g. BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.g. functional form and constants.
Having made the alignment, there are many different ways of calculating percentage identity between the two sequences. For example, one may divide the number of identities by: (i) the length of shortest sequence; (ii) the length of alignment, (iii) the mean length of sequence; (iv) the number of non-gap positions; or (iv) the number of equivalenced positions excluding overhangs. Furthermore, it will be appreciated that percentage identity is also strongly length dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance.
Hence, it will be appreciated that the accurate alignment of protein or DNA sequences is a complex process. The popular multiple alignment program ClustalW (Thompson et al., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al., 1997, Nucleic Acids Research, 24, 4876-4882) is a preferred way for generating multiple alignments of proteins or DNA in accordance with the invention. Suitable parameters for ClustalW may be as follows: For DNA alignments: Gap Open Penalty=15.0, Gap Extension Penalty=6.66, and Matrix=Identity. For protein alignments: Gap Open Penalty=10.0, Gap Extension Penalty=0.2, and Matrix=Gonnet. For DNA and Protein alignments: ENDGAP=−1, and GAPDIST=4. Those skilled in the art will be aware that it may be necessary to vary these and other parameters for optimal sequence alignment.
Preferably, calculation of percentage identities between two amino acid/polynucleotide/polypeptide sequences may then be calculated from such an alignment as (N/T)*100, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps but excluding overhangs. Hence, a most preferred method for calculating percentage identity between two sequences comprises (i) preparing a sequence alignment using the ClustalW program using a suitable set of parameters, for example, as set out above; and (ii) inserting the values of N and T into the following formula: Sequence Identity=(N/T)*100.
Alternative methods for identifying similar sequences will be known to those skilled in the art. For example, a substantially similar nucleotide sequence will be encoded by a sequence which hybridizes to the sequences shown in SEQ ID No's: 1, 2, 3 or 4, or their complements under stringent conditions. By stringent conditions, we mean the nucleotide hybridises to filter-bound DNA or RNA in 3×sodium chloride/sodium citrate (SSC) at approximately 45° C. followed by at least one wash in 0.2×SSC/0.1% SDS at approximately 20-65° C. Alternatively, a substantially similar polypeptide may differ by at least 1, but less than 5, 10, 20, 50 or 100 amino acids from the sequences shown in SEQ ID No: 5, 6 or 7.
Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof. suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent change. Other suitable variants are those having homologous nucleotide sequences but comprising all, or portions of, sequence, which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change. For example small non-polar, hydrophobic amino acids include glycine, alanine, Leucine, isoleucine, valine, proline, and methionine. Large non-polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine. The polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine. The positively charged (basic) amino acids include lysine, arginine and histidine. The positively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore be appreciated which amino acids may be replaced with an amino acid having similar biophysical properties, and the skill technician will known the nucleotide sequences encoding these amino acids.
All of the features described herein (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.
For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which:
As described in Example 2, the inventors have developed transgenic plants in which modified feedback insensitive aspartate kinase (AK:HSDH) was expressed under the control of a leaf-specific promoter. However, they found that localising expression of modified AK:HSDH to leaves resulted in a fitness cost to the transgenic plant. Therefore, as described in Examples 3-4, the inventors then used a senescence-specific promoter (SAG12) linked to a coding sequence encoding a polypeptide having a threonine insensitive aspartate kinase activity (AK:HSDH). The resulting transgenic plant produced threonine at a greater than wild type level during leaf senescence without compromising the plant's fitness.
The test for threonine levels was carried out on green or yellowing leaf. Leaf discs were taken and used for analysis so the measurements were based on the amounts of threonine per leaf disc (i.e. amount of thr/leaf area) or were related to the amount of protein in the supernatant (i.e. amount of thr/mg protein). The leaf disc was mashed with a set volume of water and centrifuged to sediment the insoluble leaf debris. Supernatant from this process was then processed using the Phenomenex EZfaast Kit. This is a proprietary kit which is used to derivatise the amino acids in the extract such that they can be quantified using a standard lc/ms set-up.
Calibration is by means of external standard for each amino acid to be quantified and the efficiency of the derivatisation steps is normalised between samples by the inclusion in the process of internal standards. The chromatograms are assessed by peak area and related to concentration using the integration algorithms in the lc/ms software. If no peak can be confidently identified by the software or operator, this is stated as “below limits of detection”. This was found to be the case for some of the empty-vector controls and some of the segregating null plants in the next generations.
The inventors performed site-directed mutagenesis on the bifunctional AK:HSDH wild-type sequence from Arabidopsis thaliana (At4g19710). Hence, the wild-type sequence was first isolated from a leaf specific cDNA library from Arabidopsis thaliana by PCR using the following primers:
The wild-type sequence was modified in one of three ways: (i) the AK domain only was mutated, (ii) the HSDH domain was mutated, or (iii) both domains were mutated to disallow regulation by Thr binding. The specific mutations were on Gln443 & Gln524, both in the enzyme regulatory domains, both of which were mutated by site-directed mutagensis to Alanine (Paris et al (2003), “Mechanism of control of Arabidopsis thaliana aspartate kinase-homoserine dehydrogenase by threonine”, J.Biol.Chem 278:5361-5366, and Frankard et al (1992), “Two feedback-insensitive enzymes of the aspartate pathway in Nicotiana sylvestris” Plant Physiol 99:1285-1293.
The Stratagene ® QuikChange® Site-Directed Mutagenesis Kit (Catalog #200518) was used for this procedure. To change the codon coding for Glu443, the following primers were used in the site-directed mutagenesis reactions:
For Gln524, the following primer pair was used:
These three mutant sequences were used individually to transform Nicotiana tabacum plants, as was the wild-type Arabidopsis AK:HSDH. In all cases, the gene of interest was expressed under the control of the leaf-specific pea-plastocyanin promoter. Plants of all populations were generated through Agrobacterium-mediated transformation and were grown under glasshouse conditions in Cambridge, UK. EZfaast amino acid kit (Phenomenex®) was used to extract and derivatise the free amino acids in each sample. Quantification was then carried out by LC/MS.
The results are shown in
The inventors achieved elevated leaf threonine levels in all populations transformed with the Arabidopsis sequence—including that transformed with the non-mutated Arabidopsis sequence. The populations transformed with sequences mutated at the AK domain gave the highest leaf Thr levels. These correlated with the highest proportion of the populations showning severely compromised fitness. Although the inventors had used a leaf-specific promoter with the intention of reducing the impact of the modification on fertility, the effect that they had was sufficient that the entire plant was still affected by the metabolic consequences of de-regulating the feedback control on the enzyme.
However, in all plants showing elevated threonine, there was a correlation with altered growth habit. Leaves were pale, thickened, brittle and strap-like. Internodes were shortened and shoed browning as maturity increased. Buds either did not develop or were misshapen. In conclusion, the site-directed mutagenesis was successful in providing elevated leaf threonine. However, the fitness costs of releasing the feedback control on the aspartate kinase still needed to be overcome if an agronomically viable plant with high leaf threonine was to result from this approach.
bld = below limit of detection
aNegligible phenotype: slightly pale, slightly short
bMild phenotype: stunted by c. ⅓, leaves appear normal shape but
cStrong phenotype: very stunted, deformed leaves, pale &/or mottled
Nicotiana tabacum plants, cultivar K326, were used to provide leaf discs which were co-cultivated with Agrobacterium tumefasciens which had been previously transformed (via electroporation) with a binary vector carrying the gene of interest (i.e. mutated AK:HSDH, the sequence of which is shown in
The results are shown in
Plant cell lines selected from the experimental population of SAG12:Aspartate Kinase (pBNP 138-0253-001) were field grown during 2008 in North Carolina. The leaf was flue-cured and analysed for the presence of selected free amino acids. The analysis was by LC/MS, validated through internal and external standard calibration. Where the analyte falls above the range used for calibration it is indicated in the table as “>S” in Table 2, and, in the Figures, by a star over the relevant bar.
The sample list is divided into “AK” numbers, which are the K326 background lines modified with the genetic construct of the invention, and three representative controls; unmodified K326, unmodified KV1, unmodified NC71, which were all grown and cured at the same time under the same conditions.
Referring to
The inventors also assessed the concentration of several other amino acids (GLN: glutamine; GLU: glutamic acid; ASN: asparagine; ASP: aspartic acid; HIS: histidine) in the cured leaves produced from the field trial plants, and the data are shown in
As can be seen in
Referring to
In summary,
| Number | Date | Country | Kind |
|---|---|---|---|
| 0814927.0 | Aug 2008 | GB | national |
This application is a National Stage Entry entitled to and hereby claims priority under 35 U.S.C. §§365 and 371 to corresponding PCT Application No. PCT/EP2009/060582, filed Aug. 14, 2009, which in turn claims priority to British Patent Application Serial No. GB 0814927.0, filed Aug. 15, 2008. The entire contents of the aforementioned applications are herein expressly incorporated by reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2009/060582 | 8/14/2009 | WO | 5/16/2011 |
| Number | Date | Country | |
|---|---|---|---|
| 20110226267 A1 | Sep 2011 | US |
