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WO2005047472A2 - Increasing seed threonine content through alteration of threonine aldolase activity - Google Patents

Increasing seed threonine content through alteration of threonine aldolase activity Download PDF

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WO2005047472A2
WO2005047472A2 PCT/US2004/037369 US2004037369W WO2005047472A2 WO 2005047472 A2 WO2005047472 A2 WO 2005047472A2 US 2004037369 W US2004037369 W US 2004037369W WO 2005047472 A2 WO2005047472 A2 WO 2005047472A2
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seq
nucleic acid
plant
threonine
acid molecule
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French (fr)
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WO2005047472A3 (en
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Georg Jander
Vijay Joshi
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Boyce Thompson Institute For Plant Research
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1014—Hydroxymethyl-, formyl-transferases (2.1.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)

Definitions

  • the present invention relates to a nucleic acid constructs configured for enhancement of threonine and isoleucine content in plants, and methods for using these nucleic acid constructs.
  • the present invention also relates to methods of increasing threonine and/or isoleucine content in plants.
  • Glu, Gin, Asp and Asn are sources of nitrogen and precursors for a wide variety of metabolic pathways. These are also major nitrogen transport molecules that are regulated in response to environmental signals such as light, and may in fact act as signaling molecules in the plant (Brenner et al., "Arabidopsis Mutants Resistant to S(+)-Beta-Methyl- Alpha, Beta-Diaminopropionic Acid, a Cycad-Derived Glutamate Receptor Agonist," Plant Physiol 124: 1615-1624 (2000); Lam et al., "Reciprocal Regulation of Distinct Asparagine Synthetase Genes by Light and Metabolites in Arabidopsis Thaliana," Plant J 16:345-353 (1998)).
  • the role of amino acids in plant morphogenesis and stress adaptation is widespread, with a variety of amino acids acting as precursors for the synthesis of hormones and secondary metabolites
  • Lys ketoglutarate reductase was also effectively employed to increase Lys levels in Arabidopsis seeds (Zhu et al, "A T-DNA Insertion Knockout of the Bifunctional Lysine-Ketoglutarate Reductase/Saccharopine Dehydrogenase Gene
  • Aspartate kinase as the committing enzyme in the pathways is subject to feedback inhibition by the downstream metabolites threonine, s-adenosylmethionine, and lysine (Ben-Tzvi Tzchori et al., "Lysine and Threonine Metabolism Are Subject to Complex Patterns of Regulation in Arabidopsis," Plant Mol Biol 32:727-734 (1996); Rognes et al., "S-Adenosylmethionfne--a Novel Regulator of Aspartate Kinase," Nature 287:357-359 (1980)).
  • Elevated threonine levels also inhibit the homoserine dehydrogenase portion of the bi- functional aspartate kinase-homoserine dehydrogenase enzyme (Paris et al., "Mechanism of Control of Arabidopsis Thaliana Aspartate Kinase-Homoserine Dehydrogenase by Threonine," JBiol Chem 278:5361- 5366 (2003)).
  • CGS cystathionine gamma-synthase
  • CGS cystathionine gamma-synthase
  • S-adenosylmethionfne allosterically activates tl reonine synthase, which competes with cystathionine gamma-synthase for the common substrate homeserine 4-phosphate (Curien et al., "Allosteric Activation of Arabidopsis Threonine Synthase by S-Adenosylmethionine," Biochemisti ⁇ 37:13212- 13221 (1998)).
  • the present invention relates to a nucleic acid construct having a nucleic acid molecule configured to silence tlireonine aldolase expression.
  • the construct also includes a 5' DNA promoter sequence and a 3' terminator sequence.
  • the nucleic acid molecule, the promoter, and the terminator are operatively coupled to permit transcription of the nucleic acid molecule.
  • Another aspect of the present invention is a method of increasing tlireonine content in a plant. This method involves providing a transgenic plant or plant seed transformed with a nucleic acid construct having a nucleic acid molecule configured to silence threonine aldolase expression, a 5' DNA promoter sequence, and a 3' terminator sequence. The method involves growing the transgenic plant or a transgenic plant grown from the transgenic plant seed under conditions effective to increase the threonine content of the transgenic plant or the plant grown from the transgenic plant seed.
  • the present invention also relates to expression vectors, host cells, plant cells, plants, and plant seeds having a nucleic acid molecule configured to silence threonine aldolase expression.
  • the present invention further relates to animal feed and foodstuff containing the plant seeds having the nucleic acid molecule configured to silence threonine aldolase expression.
  • the present invention also relates to a method of making a mutant plant having an increased level of seed threonine compared to that of a non-mutant plant.
  • This method involves providing at least one cell of a non-mutant plant containing a gene encoding a functional threonine aldolase.
  • the at least one cell of a non-mutant plant is treated under conditions effective to inactivate the gene, thereby yielding at least one mutant plant cell containing an inactivated threonine aldolase gene.
  • the at least one mutant plant cell is propagated into a mutant plant, which has an increased level of seed threonine compared to that of the non-mutant plant.
  • the present invention further relates to a nucleic acid construct having a nucleic acid molecule encoding a tlireonine aldolase.
  • the construct also includes a mitochondrial targeting sequence, a 5' DNA promoter sequence, and a 3' terminator sequence.
  • the nucleic acid molecule encoding a threonine aldolase, the mitochondrial targeting sequence, the promoter, and the terminator are operatively coupled to permit transcription of the nucleic acid molecule.
  • Another aspect of the present invention relates to expression vectors, host cells, plant cells, plants, and plant seeds transformed with the nucleic acid molecule a encoding a threonine aldolase.
  • the present invention further relates to ariimal feed and foodstuff containing the plant seeds transformed with the nucleic acid molecule a encoding a threonine aldolase.
  • Another aspect of the present invention is a method of increasing threonine content in a plant by providing a transgenic plant or plant seed transformed with a nucleic acid construct having a nucleic acid molecule encoding a threonine aldolase, a mitochondrial targeting sequence, a 5' DNA promoter sequence, and a 3' terminator sequence. The method involves growing the transgenic plant or a transgenic plant grown from the transgenic plant seed under conditions effective to increase the tl ⁇ eonine content of the transgenic plant or the plant grown from the transgenic plant seed.
  • the present invention also relates to a method of increasing isoleucine content in a plant.
  • This method involves providing a non-wild-type plant having increased threonine content compared to a corresponding wild-type plant.
  • the non- wild-type plant is transformed with a nucleic acid construct under conditions effective to yield a transgenic plant that overexpresses an enzyme that functions to catalyze biosynthesis of isoleucine from threonine.
  • the nucleic acid construct used for this method includes a nucleic acid molecule encoding an enzyme that functions to catalyze biosynthesis of isoleucine from threonine, a 5' DNA promoter sequence, and a 3' terminator sequence.
  • the nucleic acid molecule, the promoter, and the terminator are operatively coupled to permit transcription of the nucleic acid molecule.
  • the method involves growing the transgenic plant under conditions effective to increase the isoleucine content of the transgenic plant.
  • the present invention also relates to a method of identifying a plant having a high seed threonine content.
  • This method involves providing a candidate plant.
  • the candidate plant is analyzed for the presence, in its genome, of a gene encoding a non-functional threonine aldolase. The presence of the gene indicates that the candidate plant has high seed threonine content.
  • Tlireonine and certain other essential amino acids are present in lower than optimal amounts in grains that are widely used as feed for farm animals and also make up the majority of the diet of some human populations (Coruzzi and Last, "Amino Acids," Biochemistry and Molecular Biology of Plants 358-410 (2000)).
  • Crop plants with such suboptimal levels of dietary amino acids include com (lysine, tryptophan, and methionine), rice (lysine, isoleucine, and tlireonine), wheat (lysine), potato (isoleucine, methionine, and cysteine) and soy (methionine and threonine).
  • Animal feed is often supplemented with relatively expensive chemically synthesized versions of these five amino acids.
  • essential amino acids are attractive targets for nutritional improvement through biotechnology.
  • a general increase in seed protein would raise the nutritional value of grain crops. Since amino acids are the building blocks of proteins, elevated seed amino acid levels, or import of amino acids into the seeds is a prerequisite to increasing overall seed protein content.
  • Altering threonine aldolase activity in plants can be used in various ways for plant improvement. For example, reduction of threonine aldolase enzymatic activity can be used to increase threonine content. As can be observed in the HST2 mutant (discussed herein infra), reduction of threonine aldolase activity can be used to increase seed threonine content. Mutations similar to the HST2 mutation described herein infra could be identified in crop plants. In addition, RNA interference (RNAi) could be used to decrease the level of threonine aldolase to increase threonine levels.
  • RNA interference RNA interference
  • RNAi constructs could be used to increase levels of threonine in seeds, but not alter the amino acid metabolism of other parts of the plants.
  • tlireonine aldolase is a homotetramer (Ogawa et al., "Serine Hydroxymethyltransferase and Tlireonine Aldolase: Are They Identical?," Int J Biochem Cell Biol 32:289-301 (2000))
  • tlireonine aldolase to improve crops includes expression of threonine aldolase in mitochondria to increase threonine content.
  • the reaction catalyzed by tlireonine aldolase is reversible.
  • the reaction could proceed in the direction of threonine.
  • Arabidopsis threonine aldolases (described herein infra) are predicted to be cytosolic enzymes.
  • the highest glycine content, or at least the highest metabolic flux through glycine, in the plant is likely to be in the mitochondria, where the conversion of glycine to serine by serine hydroxymethyltransferase occurs during photorespiration (Figure 7).
  • Expression of threonine aldolase linked to a mitochondrial targeting sequence could be used to redirect some of this flux toward threonine instead of serine. This would increase the level of free threonine in the plant.
  • Arabidopsis threonine aldolase, or any other threonine aldolase could be targeted to the mitochondria of a crop plant in this manner.
  • tlireonine aldolase to improve crops involves increased threonine as a prerequisite for increasing isoleucine content.
  • Threonine serves as a precursor for the biosynthesis of isoleucine ( Figure 8).
  • Figure 8 a prerequisite for increasing seed isoleucine content is the production of increased amounts of threonine.
  • Alteration of threonine aldolase activity by mutations or RNA interference could be used in conjunction with modification or upregulation of later steps in the pathway leading to isoleucine (e.g. acetolactate synthase) to increase the seed levels of this amino acid.
  • Figures 1 A-1C show comparisons of amino acid measurement by
  • HPLC-MS/MS (white bars) and HPLC/FD (black bars);
  • Each bar represents the mean and standard deviation of 10 samples.
  • Figure 2 shows sample mass chromato grams of free amino acids in
  • FIG. 1 shows the fold increase over wild type in seed amino acid content for 43 mutants affected in the production of Asp-derived amino acids. Each bar represents the average of 3 to 8 mutant measurements compared to the average amino acid content of 7 to 24 wild type plants that were grown together and assayed by HPLC-MS/MS in the same microtiter plate. Error bars represent the standard error of the difference of the mutant and wild type means for each line.
  • FIG. 4 shows correlation of seed Ile and Leu contents of Col-0, Ler, and mutants. Mutant data points are the average of 3 to 8 measurements; wild-type Col-0 and Ler the average of 32 measurements. Mutants are the same as the Ile + Leu mutants in Figure 4.
  • Figure 5 shows distribution of seed Thr content in segregating F 2 lines derived from a cross between mutant line HST2 and wild-type Ler. Bars representing seed Thr content of four Col-0 and four Ler plants are indicated on the right. The Y— axis scale represents Thr peak areas divided by the peak area of a Val-d8 standard added to each sample.
  • FIG. 6 shows that cloned Arabidopsis Thr aldolase relieves the Gly auxotrophy of yeast strain YM13.
  • the top half of each plate is the W3031 B wild-type GLY1; SHM1; SHM2 haploid strain; the bottom half is the isogenic Gly auxotroph strain YM13 glyl; shml; shm2.
  • Figure 7 shows pathways and locations of enzymes involved in photorespfration. If threonine aldolase were expressed in the mitochondria (it is normally in the cytosol), some of the glycine that is converted to serine by serine hydroxymethyltransferase would be converted to threonine.
  • Figure 8 shows the pathway leading to isoleucine, leucine, and valine from threonine pyruvate, including sited of feedback inhibition.
  • TD threonine deaminase
  • ALS acetolactate synthase.
  • Increased threonine levels are a prerequisite for having increased isoleucine levels in the plant.
  • Increased production of threonine through manipulation of threonine aldolase could be combined with downstream enzymes to increase isoleucine content.
  • Figure 9 shows biosynthetic pathways leading from aspartate to threonine, methionine, lysine, and isoleucine, including known mechanisms of feedback inhibition.
  • AK aspartate kinase
  • DHDPS dihydrodipicolinate synthase
  • HSD homeserine dehydrogenase
  • TS threonine synthase
  • TD threonine deaminase
  • LKR lysine ketoglutarate reductase
  • SAMS s-adenosylmethionine synthase
  • ALS acetolactate synthase
  • CGS cystathionine gamma-synthase.
  • the present invention relates to a nucleic acid construct having a nucleic acid molecule configured to silence threonine aldolase expression.
  • the construct also includes a 5' DNA promoter sequence and a 3' terminator sequence.
  • the nucleic acid molecule, the promoter, and the terminator are operatively coupled to permit transcription of the nucleic acid molecule.
  • a suitable nucleic acid molecule configured to silence threonine aldolase expression can include, for example, any nucleic acid molecule that silences expression of any threonine aldolase in a plant. Examples of threonine aldolases from various plants are described below. [0034] One example of a threonine aldolase is from Arabidopsis thaliana
  • This Arabidopsis thaliana threonine aldolase has an amino acid sequence of SEQ ID NO:l (GenBank gi] 18390920) as follows: 1 MVMRSVDLRS DTVTRPTDAM REAMCNAEVD DDV GYDPTA RRLEE ⁇ MAKM 51 MGKEAA FVP SGTMGNLISV MVHCDVRGSE VILGDNCHIH VYENGGISTI 101 GGVHPKTVKN EEDGTMDLEA IEAAIRDPKG STFYPSTRLI CLENTHANSG
  • This protein is encoded by a gene found on chromosome 1 of Arabidopsis thaliana and has a nucleotide sequence of SEQ ID NO:2 (GenBank gi
  • This protein is encoded by a gene found on chromosome 3 of Arabidopsis thaliana and has a nucleotide sequence of SEQ ID NO: 4 (GenBank gi
  • a tlireonine aldolase from rice (Oiyza sativ ) has an amino acid sequence of SEQ ID NO:5 (GenBank gi
  • This protein is encodedby a gene having the nucleotide sequence ofSEQ ID NO:6 (GenBank gi
  • a threonine aldolase from corn has an amino acid sequence ofSEQ IDNO:7 (based uponthe Open Reading Frame Finder (GenBank, NIH) graphical analysis ofGenBank gi
  • GLKEVKGLRVDAGSVETNMVFIDIEEGTKTRAEKICKYMEERGILVMQESSSRMRW LHHQISASDVQYALSCFQALAVKGVQNEMG This protein is encoded by a gene having the nucleotide sequence of SEQ ID NO:8 (GenBank gi
  • the threonine aldolase from soybean has an amino acid sequence of SEQ ID NO:9 (GenBank gi
  • MVTRIVDLRSDTVTKPTEAMRAAMASAEVDDDV GYDPTAFRLETEMAKTMGKEAAL FVPSGTMGNLVSVLVHCDVRGSEVI GDNCHINIFENGGIATIGGVHPRQVKNNDDG TMDIDLIEAAIRDPMGELFYPTTKLICLENTHANSGGRCLSVEYTDRVGELAKKHGL KLHIDGARIFNASVALGVPVDRLVQAADSVSVCLSKGIGAPVGSVIVGSKNFIAKAR RLRKTLGGGMRQIGLLCAAALVALQENVGKLESDHKKARL ADGLNEVKGLRVDACS VETNMVFIDIEEGTKTRAEKICKYMEERGI VMQESSSRMRWLHHQISASDVQYAL SCFQQALAVKGVQKEMGN
  • This protein is encoded by a gene having the nucleotide sequence of SEQ ID NO: 10 (GenBank gi
  • This protein is encoded by a gene having the nucleotide sequence of SEQ ID NO: 12 (GenBank g ⁇
  • This protein is encoded by a gene having the nucleotide sequence of SEQ ID NO: 14 (GenBank gi
  • a threonine aldolase from cotton has an amino acid sequence of SEQ ID NO: 17 (based on the Open Reading Frame Finder (GenBank, NIH) graphical anaylsis of GenBank gi
  • This protein is encoded by a gene having the nucleotide sequence of SEQ ID NO: 18 (GenBank gi
  • the nucleic acid molecule configured to silence threonine aldolase is effective in silencing expression of a threonine aldolase polypeptide having an amino acid sequence that is at least 70 percent identical (or alternatively 75 percent, 80 percent, 85 percent, 90 percent, or 95 percent identical) to a threonine aldolase amino acid sequence of, for example, SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:ll, SEQ ID NO: 13, SEQ ID NO: 15, and/or SEQ ID NO: 17.
  • the nucleic acid molecule configured to silence threonine aldolase is effective in silencing expression of a trrreonine aldolase polypeptide having an amino acid sequence that is at least 70 percent identical (or alternatively 75 percent, 80 percent, 85 percent, 90 percent, or 95 percent identical) to at least 100 amino acid residues of a threonine aldolase amino acid sequence of, for example, SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:ll, SEQ ID NO:13, SEQ ID NO:15, and/or SEQ ID NO: 17.
  • the nucleic acid molecule configured to silence tlireonine aldolase is effective in silencing expression of a threonine aldolase that is encoded by an encoding nucleic acid molecule having a nucleotide sequence that is at least 70 percent identical (or alternatively 75 percent, 80 percent, 85 percent, 90 percent, or 95 percent identical) to a nucleotide sequence of, for example, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, and/or SEQ ID NO:18.
  • the nucleic acid molecule configured to silence threonine aldolase is effective in silencing expression of a threonine aldolase polypeptide that is encoded by an encoding nucleic acid molecule having a nucleotide sequence that is at least 70 percent identical (or alternatively 75 percent, 80 percent, 85 percent, 90 percent, or 95 percent identical) to at least 300 nucleotide bases of a nucleotide sequence of, for example, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO:16, and/or SEQ ID NO:18.
  • the nucleic acid construct of the present invention can include a nucleic acid molecule that includes a dominant negative mutation and encodes a non- functional threonine aldolase. This construct is suitable in suppression or interference of endogenous mRNA encoding threonine aldolase.
  • the nucleic acid construct of the present invention can include a nucleic acid molecule that is positioned in the nucleic acid construct to result in suppression or interference of endogenous mRNA encoding threonine aldolase.
  • the nucleic acid construct of the present invention can include a nucleic acid molecule that encodes threonine aldolase and is in sense orientation.
  • the nucleic acid construct of the present invention can include a nucleic acid molecule that is an antisense form of a threonine aldolase encoding nucleic acid molecule.
  • the nucleic acid construct of the present invention can include a nucleic acid molecule including a first segment encoding a threonine aldolase, a second segment in an antisense form of a threonine aldolase encoding nucleic acid molecule, and a third segment linking the first and second segments.
  • the DNA promoter of the nucleic acid construct of the present invention can include, for example, a constitutive plant promoter or an inducible plant promoter.
  • the DNA promoter of the nucleic acid construct of the present invention can be, for example, a tissue-specific promoter and/or an organ- specific promoter.
  • the present invention further relates to expression vectors, host cells, plant cells, plants, and plant seeds having a nucleic acid molecule configured to silence threonine aldolase expression.
  • the present invention further relates to animal feed and foodstuff containing the plant seeds having the nucleic acid molecule configured to silence threonine aldolase expression.
  • Another aspect of the present invention is a method of increasing threonine content in a plant. This method involves providing a transgenic plant or plant seed transformed with a nucleic acid construct having a nucleic acid molecule configured to silence threonine aldolase expression, a 5' DNA promoter sequence, and a 3' terminator sequence. The method involves growing the transgenic plant or a transgenic plant grown from the transgenic plant seed under conditions effective to increase the threonine content of the transgenic plant or the plant grown from the transgenic plant seed.
  • the transgenic plant or transgenic plant grown from the transgenic plant seed is obtained by providing a nucleic acid construct having a nucleic acid molecule configured to silence threonine aldolase expression (examples of which are described herein), a 5' DNA promoter sequence; and a 3' terrninator sequence, where the nucleic acid molecule, the promoter, and the terminator are operatively coupled to permit transcription of the nucleic acid molecule.
  • a plant cell is then transformed with the nucleic acid construct to yield a transgenic plant with increased threonine content (compared to a non-transgenic plant).
  • the method can further involve propagating plants from the transformed plant cell.
  • Suitable methods for fransforrning the plant can include, for example, Agrobacterium-mGdiated transformation, vacuum infiltration, biolistic transformation, electroporation, micro-injection, polyethylene-mediated transformation, and/or laser- beam transformation. The various aspects of this method are described in more detail infra.
  • the nucleic acid construct results in interference of tlireonine aldolase expression by sense or co-suppression in which the nucleic acid molecule of the construct is in a sense (5'- 3') orientation.
  • Co- suppression has been observed and reported in many plant species and may be subject to a transgene dosage effect or, in another model, an interaction of endogenous and transgene transcripts that results in aberrant mRNAs (Senior, "Uses of Plant Gene Silencing,” Biotechnology and Genetic Engineering Reviews 15:79-119 (1998); Waterhouse et al., "Exploring Plant Genomes by RNA-Induced Gene Silencing,” Nature Review: Genetics 4: 29-38 (2003), which are hereby incorporated by reference in their entirety).
  • a construct with the nucleic acid molecule in the sense orientation may also give sequence specificity to RNA silencing when inserted into a vector along with, a construct of both sense and antisense nucleic acid orientations as described infra (Wesley et al., "Construct Design for Efficient, Effective and High- Throughput Gene Silencing in Plants,” Plant Journal 27(6) 581-590 (2001), which is hereby incorporated by reference in its entirety).
  • the nucleic acid construct results in interference of threonine aldolase expression by the use of antisense suppression in which the nucleic acid molecule of the construct is an antisense (3' ⁇ 5') orientation.
  • antisense RNA to down-regulate the expression of specific plant genes is well known (van der Krol et al., Nature, 333:866- 869 (1988) and Smith et al., Nature, 334:724-726 (1988), which are hereby incorporated by reference in their entirety).
  • Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (Weintraub, "Antisense RNA and DNA,” Scientific American 262:40 (1990), which is hereby incorporated by reference in its entirety). In the target cell, the antisense nucleic acids hybridize to a target nucleic acid and interfere with transcription, and/or RNA processing, transport, translation, and/or stability.
  • one aspect of the present invention involves a construct which contains the nucleic acid molecule of the present invention being inserted into the construct in antisense orientation.
  • Interference of threonine aldolase expression is also achieved in the present invention by the generation of double-stranded RNA ("dsRNA") through the use of inverted-repeats, segments of gene-specific sequences oriented in both sense and antisense orientations.
  • sequences in the sense and antisense orientations are linked by a tliird segment, and inserted into a suitable expression vector having the appropriate 5' and 3' regulatory nucleotide sequences operably linked for transcription.
  • the expression vector having the modified nucleic acid molecule is then inserted into a suitable host cell or subject.
  • the third segment linking the two segments of sense and antisense orientation may be any nucleotide sequence such as a fragment of the ⁇ - glucuronidase ("GUS”) gene.
  • a functional (splicing) intron of tlireomne aldolase may be used for the third (linking) segment, or, in yet another aspect of the present invention, other nucleotide sequences without complementary components in the threonine aldolase gene may be used to link the two segments of sense and antisense orientation (Chuang et al., "Specific and Heritable Genetic Interference by Double- Stranded RNA in Arabidopsis thaliana," Proc.
  • the sense and antisense segments may be oriented either head-to-head or tail-to-tail in the construct.
  • hpRNA hairpin RNA
  • dsRNA hairpin RNA
  • Another aspect of the present invention involves using hairpin RNA (“hpRNA”) which may also be characterized as dsRNA. This involves RNA hybridizing with itself to form a hairpin structure that comprises a single-stranded loop region and a base-paired stem.
  • a linker may be used between the inverted repeat segments of sense and antisense sequences to generate hairpin or double-stranded RNA, the use of intron- free hpRNA can also be used to achieve silencing of tlireonine aldolase expression.
  • a plant may be transformed with constructs encoding both sense and antisense orientation molecules having separate promoters and no third segment linking the sense and antisense sequences (Chuang et al., "Specific and Heritable Genetic Interference by Double-Stranded RNA in Arabidopsis thaliana," Proc.
  • tlireonine aldolase nucleotide sequences of the present invention may be inserted into any of the many available expression vectors and cell systems using reagents that are well known in the art.
  • Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gtl 1, gt WES.tB, Charon 4, and plasmid vectors such as pG-Cha, p35S-Cha, pBR322, pBR325, pACYC177, pACYC1084, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKClOl, SV 40, pBluescript II SK+/- or KS +/- (see "Stratagene Cloning Systems” Catalog (1993) from Stratagene, La Jolla, CA, which is hereby incorporated by reference in its entirety), pQE, pIH821, pGEX, pET series (see Studier et. al., "Use of T7 RNA Polymerase to Direct Expression of Cloned Genes," Gene Expression
  • viral vectors such as lamb
  • Recombinant molecules can be introduced into cells via transformation, particularly transduction, conjugation, mobilization, or electroporation.
  • the DNA sequences are cloned into the vector using standard cloning procedures in the art, as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY:Cold Spring Harbor Press (1989), and Ausubel et al., Current Protocols in Molecular Biology, New York, N.Y:John Wiley & Sons (1989), which are hereby incorporated by reference in their entirety.
  • the various nucleic acid sequences may normally be inserted or substituted into a bacterial plasmid.
  • Any convenient plasmid may be employed, which will be characterized by having a bacterial replication system, a marker which allows for selection in a bacterium, and generally one or more unique, conveniently located restriction sites.
  • Numerous plasmids referred to as transformation vectors, are available for plant transformation. The selection of a vector will depend on the preferred transformation technique and target species for transformation. A variety of vectors are available for stable transformation using Agrobacterium tumefaciens, a soilborne bacterium that causes crown gall.
  • Crown gall are characterized by tumors or galls that develop on the lower stem and main roots of the infected plant. These tumors are due to the transfer and incorporation of part of the bacterium plasmid DNA into the plant chromosomal DNA.
  • This transfer DNA (T-DNA) is expressed along with the normal genes of the plant cell.
  • the plasmid DNA, pTi, or Ti-DNA, for "tumor inducing plasmid,” contains the vir genes necessary for movement of the T-DNA into the plant.
  • the T- DNA carries genes that encode proteins involved in the biosynthesis of plant regulatory factors, and bacterial nutrients (opines).
  • the T-DNA is delimited by two 25 bp imperfect direct repeat sequences called the "border sequences.”
  • the border sequences By removing the oncogene and opine genes, and replacing them with a gene of interest, it is possible to transfer foreign DNA into the plant without the formation of tumors or the multiplication oi Agrobacterium tumefaciens. Fraley et al., "Expression of Bacterial Genes in Plant Cells," Proc. Nat'l Acad. Sci. 80:4803-4807 (1983), which is hereby incorporated by reference in its entirety. [0060] Further improvement of this technique led to the development of the binary vector system. Bevan, "Binary Agrobacterium Vectors for Plant Transformation,” Nucleic Acids Res.
  • control elements or "regulatory sequences” are also incorporated into the vector-constmct. These include non-translated regions of the vector, promoters, and 5' and 3' untranslated regions which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. Tissue-specific and organ-specific promoters can also be used.
  • a constitutive promoter is a promoter that directs expression of a gene throughout the development and life of an organism.
  • Examples of some constitutive promoters that are widely used for inducing expression of transgenes include the nopaline synthase (NOS) gene promoter, from Agrobacterium tumefaciens (U.S. Patent No. 5,034,322 to Rogers et al., which is hereby incorporated by reference in its entirety), the cauliflower mosaic virus (CaMV) 35S and 19S promoters (U.S. Patent No.
  • An inducible promoter is a promoter that is capable of directly or indirectly activating transcription of one or more DNA sequences or genes in response to an inducer. In the absence of an inducer, the DNA sequences or genes will not be transcribed.
  • the inducer can be a chemical agent, such as a metabolite, growth regulator, herbicide, or phenolic compound, or a physiological stress directly imposed upon the plant such as cold, heat, salt, toxins, or through the action of a pathogen or disease agent such as a virus or fungus.
  • a plant cell containing an inducible promoter may be exposed to an inducer by externally applying the inducer to the cell or plant such as by spraying, watering, heating, or by exposure to the operative pathogen.
  • An example of an appropriate inducible promoter is a glucocorticoid-inducible promoter (Schena et al., "A Steroid-Inducible Gene Expression System for Plant Cells," Proc. Natl. Acad. Sci.
  • transgene-encoded protein is induced in the transformed plants when the transgenic plants are brought into contact with nanomolar concentrations of a glucocorticoid, or by contact with dexamethasone, a glucocorticoid analog.
  • Scbena et al. "A Steroid-Inducible Gene Expression System for Plant Cells," Proc. Natl. Acad. Sci. USA 88:10421-5 (1991); Aoyama et al., "A Glucocorticoid-Mediated Transcriptional Induction System in Transgenic Plants," Plant J.
  • inducible promoters include promoters that function in a tissue specific manner to regulate the gene of interest within selected tissues of the plant. Examples of such tissue specific or developmentally regulated promoters include seed, flower, fruit, or root specific promoters as are well known in the field (U.S. Patent No. 5,750,385 to Shewmaker et al., which is hereby incorporated by reference in its entirety).
  • tissue specific or developmentally regulated promoters include seed, flower, fruit, or root specific promoters as are well known in the field (U.S. Patent No. 5,750,385 to Shewmaker et al., which is hereby incorporated by reference in its entirety).
  • One of the promoters suitable in the present invention is the tuber-specific granule bound starch synthase (GBSS) promoter.
  • tissue- and organ-specific promoters have been developed for use in genetic engineering of plants (Potenza et al., “Targeting Transgene Expression in Research, Agricultural, and Environmental Applications: Promoters used in Plant Transformation," In Vitro Cell. Dev. Biol. Plant 40:1-22 (2004), which is hereby incorporated by reference in its entirety).
  • promoters include those that are floral-specific (Annadana et al., "Cloning of the Chrysanthemum UEP1 Promoter and Comparative Expression in Florets and Leaves of Dendranthema grandiflora " Transgenic Res.
  • the nucleic acid construct of the present invention also includes an operable 3' regulatory region, selected from among those which are capable of providing correct transcription termination and polyadenylation of mRNA for expression in the host cell of choice, operably linked to a modified trait nucleic acid molecule of the present invention.
  • operable 3' regulatory region selected from among those which are capable of providing correct transcription termination and polyadenylation of mRNA for expression in the host cell of choice, operably linked to a modified trait nucleic acid molecule of the present invention.
  • 3' regulatory regions are known to be operable in plants. Exemplary 3' regulatory regions include, without limitation, the nopaline synthase ("nos") 3' regulatory region (Fraley et al., "Expression of Bacterial Genes in Plant Cells," Proc. Nat 'I Acad. Sci.
  • nucleic acid constructs of the present invention can be ligated together to produce the expression systems which contain the nucleic acid constructs of the present invention, using well known molecular cloning techniques as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition Cold Spring Harbor, NY:Cold Spring Harbor Press (1989), and Ausubel et al. Current Protocols in Molecular Biology, New York, N.Y:John Wiley & Sons (1989), which are hereby incorporated by reference in their entirety. [0068] Once the nucleic acid construct of the present invention has been prepared, it is ready to be incorporated into a host cell.
  • another aspect of the present invention relates to a recombinant host cell containing one or more of the nucleic acid constructs.
  • this method is carried out by transforuiing a host cell with a nucleic acid construct of the present invention under conditions effective to achieve transcription of the nucleic acid molecule in the host cell.
  • This is achieved with standard cloning procedures known in the art, such as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Springs Laboratory, Cold Springs Harbor, New York (1989), which is hereby incorporated by reference in its entirety.
  • Suitable host cells include, but are not limited to, bacteria, virus, yeast, mammalian cells, insect, plant, and the like.
  • the host cells are either a bacterial cell or a plant cell.
  • Methods of transformation may result in transient or stable expression of the nucleic acid under control of the promoter.
  • a nucleic acid construct of the present invention is stably inserted into the genome of the recombinant plant cell as a result of the transformation, although transient expression can serve an important purpose, particularly when the plant under investigation is slow-growing.
  • Plant tissue suitable for transformation includes leaf tissue, root tissue, meristems, zygotic and somatic embryos, callus, protoplasts, tassels, pollen, embryos, anthers, and the like. The means of transformation chosen is that most suited to the tissue to be transformed.
  • Transient expression in plant tissue can be achieved by particle bombardment (Klein et al., "High- Velocity Microprojectiles for Delivering Nucleic Acids Into Living Cells," Nature 327:70-73 (1987), which is hereby incorporated by reference in its entirety), also known as biolistic transformation of the host cell, as disclosed in U.S. Patent Nos.
  • Biologically active particles e.g., dried bacterial cells containing the vector and heterologous DNA
  • Other variations of particle bombardment now known or hereafter developed, can also be used.
  • An appropriate method of stably introducing the nucleic acid construct into plant cells is to infect a plant cell with Agrobacterium tumefaciens or Agrobacterium rhizogenes previously transformed with the nucleic acid construct.
  • the Ti (or RI) plasmid of Agrobacterium enables the highly successful transfer of a foreign nucleic acid molecule into plant cells.
  • a variation of Agrobacterium transformation uses vacuum infiltration in which whole plants are used (Senior, "Uses of Plant Gene Silencing," Biotechnology and Genetic Engineering Reviews 15:79-119 (1998), which is hereby incorporated by reference in its entirety).
  • nucleic acid molecule may also be introduced into the plant cells by electroporation (Fromm et al., Proc. Natl. Acad. Sci. USA 82:5824 (1985), which is hereby incorporated by reference in its entirety).
  • plant protoplasts are electroporated in the presence of plasmids containing the expression cassette.
  • Electroporated plant protoplasts reform the cell wall, divide, and regenerate.
  • Other methods of transformation include polyethylene-mediated plant transformation, micro-injection, physical abrasives, and laser beams (Senior, "Uses of Plant Gene Silencing," Biotechnology and Genetic Engineering Reviews 15:79-119 (1998), which is hereby incorporated by reference in its entirety).
  • the precise method of transformation is not critical to the practice of the present invention. Any method that results in efficient transformation of the host cell of choice is appropriate for practicing the present invention. [0074] After transformation, the transformed plant cells must be regenerated.
  • Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts or a petri plate containing explants is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted. Alternatively, embryo formation can be induced in the callus tissue. These embryos germinate as natural embryos to form plants.
  • the culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is usually reproducible and repeatable.
  • transformed cells are first identified using a selection marker simultaneously introduced into the host cells along with the nucleic acid construct of the present invention.
  • selection markers include, without limitation, markers encoding for antibiotic resistance, such as the neomycin phosphotransferae II ("nptll") gene which confers kanamycin resistance (Fraley et al., Proc. Natl. Acad. Sci. USA 80:4803-4807 (1983), which is hereby incorporated by reference in its entirety), and the genes which confer resistance to gentamycin, G418, hygromycin, streptomycin, spectinomycin, tetracycline, chloramphenicol, and the like.
  • nptll neomycin phosphotransferae II
  • Cells or tissues are grown on a selection medium containing the appropriate antibiotic, whereby generally only those transformants expressing the antibiotic resistance marker continue to grow.
  • Other types of markers are also suitable for inclusion in the expression cassette of the present invention.
  • a gene encoding for herbicide tolerance such as tolerance to sulfonylurea is useful, or the dhfr gene, which confers resistance to metriotrexate (Bourouis et al., EMBO J. 2: 1099- 1104 (1983), which is hereby incorporated by reference in its entirety).
  • reporter genes which encode for enzymes providing for production of an identifiable compound are suitable.
  • uidA a gene from Escherichia coli that encodes the ⁇ -glucuronidase protein, also known as GTJS (Jefferson et al., "GUS Fusions: ⁇ Glucuronidase as a Sensitive and Versatile Gene Fusion Marker in Higher Plants," EMBO J. 6:3901-3907 (1987), which is hereby incorporated by reference in its entirety).
  • GTJS ⁇ -glucuronidase protein
  • enzymes providing for production of a compound identifiable by luminescence, such as luciferase, are useful.
  • the selection marker employed will depend on the target species; for certain target species, different antibiotics, herbicide, or biosynthesis selection markers are preferred.
  • transgenic plants Plant cells and tissues selected by means of an inhibitory agent or other selection marker are then tested for the acquisition of the transgene (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New Yori Cold Spring Harbor Press (1989), which is hereby incorporated by reference in its entirety).
  • the transgene can be transferred to other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
  • transgenic plants of this type are produced, the plants themselves can be cultivated in accordance with conventional procedure so that the nucleic acid construct is present in the resulting plants.
  • transgenic seeds are recovered from the transgenic plants. These seeds can then be planted in the soil and cultivated using conventional procedures to produce transgenic plants. The component parts and fruit of such plants are encompassed by the present invention.
  • the present invention can be utilized in conjunction with a wide variety of plants or their seeds. Suitable plants are any plant with a threonine aldolase gene, including dicots and monocots. Plants can include: rice, corn, soybean, canola, potato, wheat, mung bean, alfalfa, barley, rye, cotton, sunflower, peanut, sweet potato, bean, pea, chicory, lettuce, endive, cabbage, bmssel sprout, beet, parsnip, turnip, cauliflower, broccoli, radish, spinach, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, melon, citrus, strawberry, grape, raspberry, pineapple, tobacco, tomato, sorghum, sugarcane, banana, Arabidopsis thaliana, Saintpaulia, petunia, pelargonium, poinsettia, chrysanthemum, carnation, and zinnia.
  • the present invention also relates to expression vectors, host cells, plant cells, plants, and plant seeds having a nucleic acid molecule configured to silence threonine aldolase expression.
  • the present invention further relates to animal feed and foodstuff containing the plant seeds having the nucleic acid molecule configured to silence tlireonine aldolase expression.
  • the present invention also relates to a method of making a mutant plant having an increased level of seed threonine compared to that of a non-mutant plant.
  • This method involves providing at least one cell of a non-mutant plant containing a gene encoding a functional threonine aldolase.
  • the at least one cell of a non-mutant plant is treated under conditions effective to inactivate the gene, thereby yielding at least one mutant plant cell containing an inactivated tlireonine aldolase gene.
  • the at least one mutant plant cell is propagated into a mutant plant, which has an increased level of seed tlireonine compared to that of the non-mutant plant.
  • the functional threonine aldolase can be as described herein supra.
  • the treating step inolves subjecting the at least one cell of the non-mutant plant to a chemical mutagenizing agent under conditions effective to yield at least one mutant plant cell contarning an inactive tfaeonine aldolase gene.
  • Suitable chemical mutagenizing agents can include, for example, ethylmethanesulfonate.
  • the treating step inolves subjecting the at least one cell of the non-mutant plant to a radiation source under conditions effective to yield at least one mutant plant cell contarning an inactive threonine aldolase gene.
  • Suitable radiation sources can include, for example, sources that are effective in producing ultraviolet rays, gamma rays, or fast neutrons.
  • the treating step inolves inserting an inactivating nucleic acid molecule into the gene encoding the functional threonine aldolase or its promoter under conditions effective to inactivate the gene.
  • Suitable deactivating nucleic acid molecules can include, for example, a transposable element.
  • transpo sable elements include, without limitation, an Activator (Ac) transposon, a Dissociator (Ds) transposon, or a Mutator (Mu) transposon.
  • Ac Activator
  • Ds Dissociator
  • Mo Mutator
  • the treating step involves subjecting the at least one cell of the non-mutant plant to Agrobacterium transformation under conditions effective to insert an Agrobacterium T-DNA sequence into the gene, thereby inactivating the gene.
  • Suitable Agrobacterium T-DNA sequences can include, for example, those sequences that are carried on a binary transformation vector of AC 106, pAC161, pGABIl, pADISl, pCSAHO, pDAPlOl, derivatives ofpBLN19, or pCAMBIA plasmid series.
  • the treating step inolves transforming the at least one cell of the non-mutant plant by site- directed mutagenesis of the threonine aldolase gene or its promoter under conditions effective to yield at least one mutant plant cell containing an inactive threonine aldolase gene.
  • the treating step can also involve mutagenesis by homologous recombination of the threonine aldolase gene or its promoter, targeted deletion of a portion of the threonine aldolase gene sequence or its promoter, and/or targeted insertion of a nucleic acid sequence into the tlireonine aldolase gene or its promoter.
  • the various plants that can be used in this method are the same as those described supra with respect to the method of increasing threonine content in a plant.
  • Another aspect of the present invention relates to mutant plants produced by this method, as well as mutant plant seeds produced by growing the mutant plant under conditions effective to cause the mutant plant to produce seed.
  • the present invention further relates to a nucleic acid construct having a nucleic acid molecule encoding a tlireonine aldolase.
  • the construct also includes a mitochondrial targeting sequence, a 5' DNA promoter sequence, and a 3' terminator sequence.
  • the nucleic acid molecule encoding a tlireonine aldolase, the mitochondrial targeting sequence, the promoter, and the terminator are operatively coupled to permit transcription of the nucleic acid molecule.
  • Suitable nucleic acid molecules encoding threonine aldolases include, for example, those described herein supra.
  • Suitable mitochondrial targeting sequences can be mitochondrial targeting leader sequences including, for example, a soybean alternative oxidase gene (GenBank gi
  • the DNA promoter (of the nucleic acid constmct containing the nucleic acid molecule encoding a threoriine aldolase) can include, for example, a constitutive plant promoter or an inducible plant promoter (as described in more detail supra).
  • the DNA promoter of this nucleic acid constmct can be, for example, a tissue- specific promoter (as described in more detail supra).
  • the present invention further relates to expression vectors, host cells, plant cells, plants, and plant seeds having a nucleic acid constmct including a nucleic acid molecule encoding a threonine aldolase, a mitochondrial targeting sequence, a 5' DNA promoter sequence, and a 3' terminator sequence.
  • the present invention further relates to arrimal feed and foodstuff containing the plant seeds having this nucleic acid construct.
  • Another aspect of the present invention is a method of increasing threonine content in a plant by providing a transgenic plant or plant seed transformed with a nucleic acid construct having a nucleic acid molecule encoding a threonine aldolase, a mitochondrial targeting sequence, a 5' DNA promoter sequence, and a 3' terminator sequence.
  • the method involves growing the transgenic plant or a transgenic plant grown from the transgenic plant seed under conditions effective to increase the tlireonine content of the transgenic plant or the plant grown from the transgenic plant seed.
  • the step of providing a transgenic plant or plant seed transformed with the nucleic acid construct involves providing the constmct and transforming a plant cell with the construct.
  • Suitable methods for transforming the plant can include, for example, Agrobacterium-med ⁇ ated transformation, vacuum infiltration, biolistic transformation, electroporation, micro- injection, polyethylene-mediated transformation, and/or laser-beam transformation.
  • the present invention also relates to a method of increasing isoleucine content in a plant. This method involves providing a non-wild-type plant having increased threonine content compared to a corresponding wild-type plant.
  • the non- wild-type plant is transformed with a nucleic acid construct under conditions effective to yield a transgenic plant that overexpresses an enzyme that functions to catalyze biosynthesis of isoleucine from threoriine.
  • the nucleic acid construct used for this method includes a nucleic acid molecule including an enzyme that functions to catalyze biosynthesis of isoleucine from threonine, a 5' DNA promoter sequence, and a 3' terminator sequence.
  • the nucleic acid molecule, the promoter, and the terminator are operatively coupled to permit transcription of the nucleic acid molecule.
  • the method involves growing the transgenic plant under conditions effective to increase the isoleucine content of the transgenic plant.
  • Suitable enzymes for use in this method include, for example, threonine deaminase and acetolactate synthase.
  • non- feedback-inhibited forms of the threonine deaminase or acetolactate synthase can be used.
  • the non-wild-type plant is a mutant plant containing a gene encoding a nonfunctional threonine aldolase.
  • the non-wild-type plant is a plant transformed with a tfreonine aldolase silencing nucleic acid constmct.
  • a suitable threonine aldolase silencing nucleic acid constmct can include a nucleic acid molecule configured to silence threonine aldolase expression, a 5' DNA promoter sequence, and a 3' terminator sequence, where the nucleic acid molecule, the promoter, and the terminator are operatively coupled to permit transcription of the nucleic acid molecule. Examples of these threonine silencing nucleic acid constructs are described herein supra.
  • the present invention also relates to a method of identifying a plant having a high seed threonine content.
  • This method involves providing a candidate plant.
  • the candidate plant is analyzed for the presence, in its genome, of a gene encoding a non- functional threonine aldolase. The presence of the gene indicates that the candidate plant has high seed tlireonine content.
  • Example 1 Application of a High-Throughput HPLC-MS/MS Assay to Arabidopsis Mutant Screening; Evidence That Threonine Aldolase Plays a Role in Seed Nutritional Quality
  • Map-based cloning demonstrated that one mutant has increased seed Thr content due to a mutation in Thr aldolase.
  • the methodology described below will allow similar mutant screens to be performed in other genetically tractable plants, and direct monitoring of nutritionally valuable molecules in conventional and molecular plant breeding.
  • Col-0, Ler and RI lines were obtained from the Arabidopsis Biological Resource Center (www.arabidopsis.org). Mutagenized Col-0 and Ler seeds were purchased from Lehle Seeds (Round Rock, TX). Generation of mutant populations has been described previously (Van Eenennaam et al., "Engineering Vitamin E Content: From Arabidopsis Mutant to Soy Oil," Plant Cell 15:3007-3019 (2003), which is hereby incorporated by reference in its entirety).
  • Plants were grown in Conviron (Winnipeg, Canada) growth chambers in standard nursery flats (approximately 20 cm by 40 cm) using Metromix 200 potting soil (Scotts, Marysville, OH) at 23 °C under continuous cool white fluorescent light with an intensity of 150 micromols m "2 sec "1 photo synthetic photon flux density (PPFD). Plants were fertilized twice weekly with a dilute solution of Peters 20-20-20 fertilizer (Scotts, Marysville, OH), adjusted to a 50 ppm nitrogen concentration. A maximum of 96 M 2 lines were planted from each of 110 independent seed pools. Since each pool contained an average of 8 seeds from each of 1000 Mi lines, the chances of planting sibling M 2 seeds were low.
  • PPFD photo synthetic photon flux density
  • YM13 W3031B shml::HIS3 shm2::LEU2 glyl::URA3 were kindly supplied by A. Bognar (Univerity of Toronto).
  • Yeast strains were grown on YPD medium (1% yeast extract, 2% peptone, 2% dextrose) or on SD medium (0.67% Bacto-yeast nitrogen bas, 2% dextrose) supplemented, where appropriate, with 750 ⁇ g/ml Gly, 30 ⁇ g/ml Leu, and 20 ⁇ g ml each uracil, Trp, adenine, and His.
  • 2,3,4,4,4,5,5,5-d8 (Aldrich) was added to each tube and samples were reconstituted by letting them dissolve at for 30 min at 4°C.
  • approximately 100 mg of Arabidopsis leaf material was placed into 1.4 ml tubes in a 96-well microtiter plate (Matrix). The tissue was frozen in liquid nitrogen and lyophilized, resulting in approximately 10 mg of dried leaf material. Further processing of these samples was the same as described above for Arabidopsis seeds.
  • the sample injection volume was 5 ⁇ l, and a Gilson (Middleton, WI) 215 liquid handler equipped with an 819 injector was used to load the samples.
  • Mobile phases used were A. 0.05% acetic acid in water and B. 90% acetonitrile, 0.05% acetic acid in water.
  • the total HPLC run time was 1.5 min, using the following gradient: 0 to 0.6 min 100% A, 1 nil/min; 0.61 to 1.0 min, 90% B, 1 to 3 ml/min; 1.1 to 1.4 min, 100% A, 3 ml min, 1.4 to 1.5 min, 100% A, 3 to 1 nil/min.
  • Amino acids were detected using a Micromass Triple Quadrupole Quattro Ultima mass spectrometer (Waters, Milford, MA) using an electro spray positive mode, 3.0 kV capillary voltage, 25 V cone voltage, 120°C source temperature, and 300°C desolvation temperature. Collection times, collision energies and masses of observed ions for each amino acid were as described in Table 1. Table 1. Collection times, collision energy and masses of observed ions
  • Example 6 Ile/Leu Separation Method
  • the instmmentation described above for the 19 amino acid separation was used, but the HPLC-MS/MS protocol was changed. Mobile phases were: A. 0.05% acetic acid in water and B. 100% acetonitrile, 0.05% acetic acid.
  • the total HPLC run time was 4 min using the following gradient: 0 to 2 min, 100% A, 1.5 niFmin; 2 to 3 min, 10% A, 90% B, 1.5 ml/min; 3 to 4 min, 100% A, 2 ml/min.
  • Ile and Leu were detected using electrospray positive mode, 3.5 kV capillary voltage, 25 V cone voltage, 120°C source temperature, 250°C desolvation temperature, and 10 eV collision energy.
  • the mass/charge ratio of the detected parent and daughter ions were 132.21 and 86.60, respectively.
  • Example 7 Lys/Gln Separation Method
  • the instramentation described above for the 19 amino acid separation was used, but the HPLC-MS/MS protocol was changed.
  • Mobile phases were: A. 0.05% acetic acid in water and B. 100% acetonitrile, 0.05% acetic acid.
  • the total HPLC run time was 1.5 min using the following gradient: 0 to 0.6 min, 100% A, 1 ml/min; 0.6 to 1.2 min, 100% B, 2 ml/min; 1.2 to 1.5 min, 100% A, 2 ml/min.
  • Ile and Leu were detected using electrospray positive mode, 3.5 kV capillary voltage, 25 V cone voltage, 120°C source temperature, 250°C desolvation temperature, and 15 eV collision energy.
  • the mass/charge ratio of the detected parent and daughter ions were 147.11 and 84.6, respectively.
  • Chromosomal DNA was amplified by PCR and was sequenced using an ABI3700 sequencer (Applied Biosystems, Foster City, CA) as described previously (Jander et al., "Ethylmethanesulfonate Saturation Mutagenesis in Arabidopsis to Determine Frequency of Herbicide Resistance," Plant Physiol 131:139-146 (2003); Van Eenennaam et al., “Engineering Vitamin E Content: From Arabidopsis Mutant to Soy Oil,” Plant Cell 15:3007-3019 (2003), which are hereby incorporated by reference in their entirety).
  • SNPs single nucleotide polymorphisms
  • A1G08630 was amplified from the full-length cDNA clone U148646 (ABRC) using the primers AGTCGGATCCATGGTGATGAGAAGTGTGGATCT (SEQ ID NO: 19) and AGCTCTGCAGTTAGGTTCGGCTTGGTTCCTG (SEQ ID NO:20) and was cloned into the tetracycline-repressible yeast vector pCM185 (Gari et al., "A Set of Vectors with a Tetracycline-Regulatable Promoter System for Modulated Gene Expression in Saccharomyces cerevisiae," Yeast 13:837-848 (1997), which is hereby incorporated by reference in its entirety) using the restriction enzymes BamHI and Pstl.
  • the mutant cDNA was amplified using the same primers as for the wild-type cDNA and was cloned into pCM185.
  • Yeast cultures (40 ml) were grown overnight in SD medium supplemented with 20 ⁇ g/ml adenine, 30 ⁇ g/ml leucine, 40 ⁇ g/ml tryptophan, 20 ⁇ g/ml uracil, 20 ⁇ g/ml histidine, and 750 ⁇ g/ml glycine.
  • Grade yeast extracts were prepared by glass bead disruption (Ausubel et al., "Cunent Protocols in Molecular Biology," Hoboken: John Wiley and Sons, Inc. (1998), which is hereby incorporated by reference in its entirety). Extracts were desalted using Econo-Pac 10DG columns (Bio-Rad, Hercules, CA).
  • Thr aldolase reactions were carried out in 200 ⁇ l total volume with 100 mM Hepes/NaOH, pH 8.0, 50 ⁇ M pyridoxal-P, and 0, 1, 2, 5, 10, or 50 mM Thr as the substrate (Liu et al., "Gene Cloning, Biochemical Characterization and Physiological Role of a Thermostable Low-Specificity L-Threonine Aldolase from Escherichia coli," Eur J Biochem 255:220-226 (1998a), which is hereby incorporated by reference in its entirety).
  • Arabidopsis seed extracts are shown in Figure 2 and the masses measured for each amino acid are listed in Table 1 (supra).
  • the levels of free Cys and Gly in Arabidopsis seeds are to low too be quantified reliably by HPLC-MS/MS.
  • Lys and Gin are separated by about 0.14 min in the same mass chromatogram, and Gin is considerably more abundant than Lys in Arabidopsis seeds.
  • the tail of the Gin peak can adversely affect the quantitation of Lys in this assay.
  • Leu and Ile are not separated by this method.
  • Example 13 Mutant Screen
  • the 1.5-min HPLC-MS/MS assay described above was sufficiently fast and reliable to use for a large-scale screen for Arabidopsis mutants with altered arnino acid content. Although the assay detects both leaf and seed amino acids with high efficiency, a mutant screen was chosen to be performed for alteration of free arnino acids in seeds. Two major factors were considered in this decision: the level of biological variability was quite a bit lower in seeds than leaves, presumably reflecting well characterized diurnal regulation of amino acid levels in green tissue (Coruzzi et al., "Amino Acids," In Buchanan et al., eds., Biochemistry and Molecular Biology of Plants, Rockville, MD: Am. Soc. Plant Physiol. Press, pp. 358-410 (2000), which is hereby incorporated by reference in its entirety), and seeds of crop plants are the main targets of efforts to alter amino acid levels.
  • a total of 322 lines showed significantly increased amino acid levels (three standard deviations above the mean) in two different extractions of the same seed samples.
  • at least one of the Asp- derived arnino acids (Ile, Lys, Met, and Thr) was increased in 103 mutant lines.
  • These mutants were of particular interest due to the essential and sometimes limiting role of Asp-derived amino acids in human and animal diets.
  • all of the 103 mutants affected in Asp-derived amino acids were grown together with wild-type plants in a replicated planting. Seeds were harvested and subjected to amino acid analysis by HPLC-MS/MS.
  • the 25% of F 2 lines with the lowest levels of seed Thr were genotyped with five markers on each of the five chromosomes, and genetic linkage of the HST2 mutation was found to two markers on chromosome 1.
  • Genotyping with additional markers nanowed the position of the HST2 mutation to a region of chromosome 1 between markers CER474622 on BAG T21E18 and CER432610 on BAG F7G19 (see www.arabidopsis.org/Cereon).
  • Analysis of the sequence in this region identified a Thr aldolase homologue (AT1G08630; EC 4.1.2.5) as a candidate gene.
  • Thr aldolase catalyzes the reversible reaction Thr ⁇ Gly + acetaldehyde in mammals and microbes
  • Liu et al. "Glyl Gen of Saccharomyces cerevisiae Encodes a Low-Specificity L-Threonine Aldolase that Catalyzes Cleavage of L- ⁇ //o-Threonine and L-Threonine to Glycine: Expression of the Gene in Escherichia coli and Purification and Characterization of the Enzyme," Eur J Biochem 245:289-293 (1997); Liu et al., “Gene Cloning, Biochemical Characterization and Physiological Role of a Thermostable Low-Specificity L-Threonine Aldolase from Escherichia coli " Eur J Biochem 255:220-226 (1998a); Liu et al., " Gene Cloning, Nucleotide Sequencing, Purification, and
  • AT1G08630 encodes an Arabidopsis Thr aldolase was strengthened by transformation of cDNA clones into yeast strain YM13 (glyl; shml; shm2), which is a Gly auxotroph due to mutations in Thr aldolase and both yeast Ser hydroxymethyltransferases (SHMT, EC 2.1.2.1) (McNeil et al., "Cloning and Molecular Characterization of Three Genes, Including Two Genes Encoding Serine Hydroxymethyltransferases, Whose Inactivation is Required to Render Yeast Auxotrophic for Glycine," J iol Chem 269:9155-9165 (1994); Monschau et al., "Identification of Saccharomyces Cerevisia
  • Genome Initiative "Analysis of the Genome Sequence of the Flowering Plant,” Arabidopsis thaliana. Nature 408:796-815 (2000), which is hereby incorporated by reference in its entirety) identified another likely Thr aldolase (AT3G04520) on chromosome 3, with 70% amino acid sequence identity to the chromosome 1 gene (AT1G08630).
  • AT3G04520 Thr aldolase
  • AT1G08630 and AT3G04520 genes are proposed to be named THAI and THA2 (rHreonine aldolase), respectively.
  • the mutation that was identified in line ⁇ ST2 is designated the thal-1 allele.
  • Example 16 High-Throughput HPLC-MS/MS Assay for Screening Arabidopsis Mutants Yields Evidence That Threonine Aldolase Plays a Role in Seed Nutritional Quality [0126]
  • the HPLC-MS/MS assay described herein allows determination of 18 amino acids from plant tissue in 1.5 minutes, which is 15 to 60- fold faster than traditional derivatization and reverse-phase HPLC. With the previous method, it would have been very time-consuming to screen 10,000 mutant families for altered seed arnino acid content.
  • mutants were identified with increased levels of Ile, Lys, Met or Thr.
  • the direct screening method employed could yield mutants affected in a variety of mechanisms, including altered biochemical or genetic regulation of pathway enzymes (including allosteric effects), increased amino acid transport, loading into the developing seeds, or reduced catabolism.
  • mutant screen described herein is fundamentally different from previous approaches that have been used to identify plant amino acid genes.
  • most previously known plant amino acid mutants were identified by selections using toxic amino acid analogues in seedling screens, toxic mixtures of arnino acids on seedlings, or transgenic expression of enzymes with altered regulation, direct screerring for free arnino acid levels in seeds was performed.
  • mapping and sequencing of the AT1G08630 Thr aldolase gene from high seed Thr mutant HST2 (thai -I) identified a Glyl 14Arg missense mutation.
  • a recessive thal-1 mutation might produce a 3:1 high: low threonine segregation ratio, because pooled F 3 seeds from individual F 2 plants were assayed.
  • a heterozygous F 2 parent would produce seeds that individually segregate in a 1 :2: 1 Mendelian manner, resulting in pooled F 3 seeds that have elevated Thr levels compared to wild type, even though only 25% of all seeds are homozygous mutant.
  • the purified maize and mung bean enzymes were reported to cleave L-allo- Thr (a non-natural substrate) and L-Ser, but not L-Thr.
  • substrate specificity has been found for several purified SHMT enzymes including rat, rabbit, human, and E. coli (Ogawa et al., "Serine Hydroxymethyltransferase and Tlireonine Aldolase: Are They Identical? IntJ Biochem Cell Biol 32:289-301 (2000), which is hereby incorporated by reference in its entirety).
  • Thr aldolases do not have the capacity to bind tetrahydro folate, wliich is necessary for the SHMT Ser cleavage reaction (Kielkopf et al., "X-ray Structures of Threonine Aldolase Complexes: Structural Basis of Substrate Recognition," Biochemistry 41:11711- 11720 (2002), which is hereby incorporated by reference in its entirety), and the Vma Km values for cleavage of Thr by Thr aldolases are much higher than those by SHMTs (Ogawa et al., "Serine Hydroxymethyltransferase and Threonine Aldolase: Are They Identical?
  • Thr aldolase may serve to remove excess Thr.
  • Thr aldolase may function as an alternate pathway for Gly biosynthesis in plants, in addition to the Gly produced during photorespiration and from 3-phosphoglycerate as a branch off of glycolysis (Bourguignon et al., "Serine and Glycine Metabolism in Higher Plants," In Singh, B.K., ed., Plant Amino Acids, Basel: Marcel Dekker, pp. 111-146 (1999), which is hereby incorporated by reference in its entirety).
  • a third possible role for plant Thr aldolase is that the reaction Thr — Gly + acetaldehyde functions in the production of acetyl-CoA, which can be produced from acetaldehyde by the sequential action of acetaldehyde dehydrogenase and acetyl-CoA synthase.
  • Thr aldolase might help to catalyze Thr biosynthesis, or perhaps reduce the amount of acetaldehyde produced in plants emerging from anoxic stress (Kursteiner et al., "The Pyruvate Decarboxylasel Gene of Arabidopsis is Required During Anoxia but not Other Environmental Stresses," Plant Physiol 132:968-978 (2003); Tsuji et al., 'Induction of Mitochondrial Aldehyde Dehydrogenase by Submergence Facilitates Oxidation of Acetaldehyde During Re- Aeration in Rice," FEBS Lett 546:369-373 (2003), which are hereby incorporated by reference in their entirety).
  • the high-speed HPLC-MS/MS assay described herein can be easily applied to other plant species, animals, or microbes to determine amino acid content. Modifications of the tissue extraction and HPLC-MS/MS detection protocols would allow extension of this arnino acid assay to a more general metabolite profiling method. By using this assay for an Arabidopsis mutant screen, a mutation that causes high seed Thr levels by reducing the activity of Thr aldolase has been identified.
  • TILLING Total Induced Localized Lesions IN Genomes
  • Greene et al. "Spectrum of Chemically Induced Mutations from a Large-Scale Reverse-Genetic Screen in Arabidopsis," Genetics 164:731 -740 (2003); Till et al., “Large-Scale Discovery of Induced Point Mutations with High- Throughput Tilling,” Genome Res 13:524-530 (2003), which are hereby incorporated by reference in their entirety
  • This method is being applied for targeted identification of mutations in rice (see, e.g., USDA-ARS project # 5306-21000-016-00) and maize (see, e.g., NSF award #0321510).
  • Step 1 a mutagenized population of rice plants is generated by chemical mutagenesis, or is otherwise acquired.
  • Step 2 likely threonine aldolases (for instance gi
  • PCR primers are designed using the CODDLE program to amplify segments of about 1000 base pairs from the rice genome and end-label the products (Greene et al., "Spectrum of Chemically Induced Mutations from a Large- Scale Reverse-Genetic Screen in Arabidopsis," Genetics 164:731-740 (2003); Till et al., “Large-Scale Discovery of Induced Point Mutations with High-Throughput Tilling,” Genome Res 13:524-530 (2003), which are hereby incorporated by reference in their entirety).
  • Step 4 primers from Step 3 threonine aldolase segments from pooled samples of DNA from the mutagenized rice population, CEL 1 nuclease is used to cleave single base pair DNA mismatches, and cleaved products are visualized on polyacrylamide gels (Greene et al., "Spectrum of Chemically Induced Mutations from a Large-Scale Reverse-Genetic Screen in Arabidopsis," Genetics 164:731-740 (2003); Till et al., “Large-Scale Discovery of Induced Point Mutations with High- Throughput Tilling," Genome Res 13:524-530 (2003), which are hereby incorporated by reference in their entirety).
  • Step 5 individual plants from the pools that show mutations in Step 4 are re-assayed to identify the ones that carry mutations.
  • Step 6 threonine aldolase is sequenced to determine the site of the mutations.
  • Step 7 enzymatic assays and threonine measurements on the mutant plants are used to determine whether tlrreoriine aldolase has been inactivated.
  • the approach outlined above can be applied to identify threonine aldolase mutations in rice or any other crop plant. Not only knockout mutations that completely abolish enzymatic activity, but also missense mutations that merely reduce enzymatic activity can be identified.
  • RNA interference RNA interference
  • RNA self-complementary, doublestranded "hairpin” RNA can be used to efficiently silence endogenous plant genes (Wesley et al., "Constmct Design for Efficient, Effective and High-Throughput Gene Silencing in Plants," Plant J 27:581-590 (2001), which is hereby incorporated by reference in its entirety).
  • Threonine aldolase in soybean or other plants could be silenced by the introduction of such constmcts into the plants by methods such as particle bombardment or Agrobacterium-mediated transformation. Through reduction of threonine aldolase activity, these plants would have a higher tlireonine content.
  • transgenic soybean plants with reduced threonine aldolase expression could be generated in the following manner: Step 1, soybean threonine aldolase genes (for instance gi
  • RNA silencing constmcts are generated by cloning the soybean DNA into binary plant transformationvectors such as pHANNIB AL or pHELLSGATE (Wesley et al., "Constmct Design for Efficient, Effective and High- Throughput Gene Silencing in Plants," Plant J 27:581-590 (2001), which is hereby incorporated by reference in its entirety).
  • PCR primers that would be used to amplify various crop plant threonine aldolase DNA sequences with appropriate restriction enzyme sites (Xhol + Kpnl and Xbal + C , respectively) for cloning into restriction sites of the the pHANNIBAL vector for plant transformation and gene silencing (Wesley et al., "Constmct Design for Efficient, Effective and High-Throughput Gene Silencing in Plants," Plant J 27:581-590 (2001), which is hereby incorporated by reference in its entirety) are listed below.
  • Primer2 (Forward) (SEQ ID NO:23): 5 '-ATGCATGCTCTAGATTCGTGCCGTCCGGCACCATGGCCAACCTC-3'
  • Primer2 (Forward) (SEQ ID NO:27): 5 '—ATGCATGCTCTAGAGAGGCGGAGATGGCCGCGGTCATGGGCAAG-3'
  • Primer2 (Forward) (SEQ ID NO:35): 5 ' -ATGCATGCTCTAGAGAAAGAGGAACAGCTAATTCATAAAATTTA-3 '
  • Primer2 (Forward) (SEQ ID NO:39): 5 ' -ATGCATGCTCTAGACTCCTCCCCACTCATCTTCTCTGGCCCAAC-3 '
  • Primer2 (Forward) (SEQ ID NO:43): 5 ' -ATGCATGCTCTAGACTTCCAACATTACATGATATACAAGCGACA- Primer2 (Reverse) (SEQ ID NO:44):
  • Primer2 (Forward) (SEQ ID NO:47): 5 ' -ATGCATGCTCTAGAGACGAGCATCCTTCTTCATTTTGCACAGGT-3 '
  • PCR primers for cloning crop plant threonine aldolase DNA sequences into the recombination sites of the pHELLSGATE vector for plant transformation and gene silencing are listed below.
  • Primer2 (SEQ ID NO:58): 5 ' -GGGGACCACTTTGTACAAGAAAGCTGGGTGGTGGTGGGATAATGCAGCGCCCCCGCCCC-3 '
  • Primer2 (SEQ ID NO:60):
  • Primerl (SEQ ID NO:61): 5' -GGGGACAAGTTTGTACAAAAAAGCAGGCTGACGAGCATCCTTCTTCATTTTGCACAGGT-3 '
  • Step 3 vectors from Step 2 are used to generate transgenic soybeans by Agrobacterium-mediated transfonnation (Liu et al., "Efficient Agrobacterium Tumefaciens-Mediated Transformation of Soybeans Using an Embryonic Tip Regeneration System," Planta 219:1042-1049 (2004), which is hereby incorporated by reference in its entirety) or other methods.
  • Step 4 enzymatic assays and tlireonine measurements on the transgenic plants are used to determine whether threonine aldolase has been inactivated.
  • Inactivation of threonine aldolase by RNA interference could be used in a similar manner in other crop species. Different levels of silencing efficiency can result in an allelic series of plants with different levels of threonine aldolase activity.
  • Example 19 Identification of a Transposon Insertion in Maize (Zea mays) Threonine Aldolase to Increase Threonine Content
  • Plant genes can be inactivated by insertion of transposons or
  • Agrobacterium T-DNA sequences Such insertions could also be used to inactivate threonine aldolase, thereby decreasing the level of activity of this enzyme and increasing the amount of free threonine in the plant.
  • Activator Ac
  • two commonly used transposable elements used for mutagenesis are Activator (Ac) (Brutnell and Conrad, “Transposon Tagging Using Activator (Ac) in Maize," Methods Mol Biol 236:157- 176 (2003); Kolkman et al., “Distribution of Activator (Ac) Throughout the Maize Genome for Use in Regional Mutagenesis," Genetics advance online publication:(2004), which are hereby incorporated by reference in their entirety),
  • Step 3 a line with a nearby Activator (Ac) transposon insertion is identified in available collections (Kolkman et al., "Distribution of Activator (Ac) Throughout the Maize Genome for Use in Regional Mutagenesis," Genetics advance online publication: (2004), which is hereby incorporated by reference in its entirety).
  • Step 4 Ac transposition is induced and, since Ac has a propensity to transpose to linked sites in the genome, some fraction of these transposition events will result in insertions in the threonine aldolase gene.
  • Step 5 insertions in the threonine aldolase gene are identified by PCR using primers complementary to threonine aldolase and the Ac transposons.
  • Step 6 knockout of threonine aldolase is confirmed by enzymatic activity assays and by measurement of plant threonine levels. Suitable primers complementary to maize threonine aldolase and the ends of the Ac transposon are described below.
  • T- DNA insertion vector pROK2 (Alonso et al., "Genome- Wide Insertional Mutagenesis of Arabidopsis Thaliana,” Science 301 :653-657 (2003), which is hereby incorporated by reference in its entirety) is used to make a T-DNA insertion library in a given plant
  • primers complementary to the ends of the T-DNA and complementary to plant threonine aldolase sequences would be used to amplify the insertion boundary by PCR. This amplified sequence would be verified by DNA sequencing. Primers specific for the ends of the pROK2 T-DNA and for crop plant threonine aldolase genes that could be used for this amplification are listed below.
  • Plant threonine aldolase-specific primers for amplification of insertion boundaries Plant threonine aldolase-specific primers for amplification of insertion boundaries:
  • Reverse primer (SEQ ID NO:78): 5 ' -GGGTAACATATTTGAAAGCTAGGATCTCGA-
  • Canola (Brassica napus):
  • threonine aldolases function as homotetramers (Ogawa et al., "Serine Hydroxymethyltransferase and Threomne Aldolase: Are They Identical?,” Int J Biochem Cell Biol 32:289-301 (2000), which is hereby incorporated by reference in its entirety), it may be possible to isolate a dominant negative mutation in a plant threonine aldolase, overproduce this mutant enzyme in an otherwise wild-type plant, inactivate wild-type threonine aldolase that forms a tetramer with the mutant enzyme, and thereby increase threonine content in the plant.
  • Step 1 wild-type plant tlireonine aldolase is cloned into a yeast vector with an inducible promoter, for instance the GAL1/GAL10 galactose-inducible promoter in a plasmid such as pBM150 (Steel et al., "A Screen for Dominant
  • Step 2 the plasmid constmct from Step 1 is used to rescue the glycine auxotrophy of yeast strain YM13 (McNeil et al., "Cloning and Molecular Characterization of Three Genes, Including Two Genes Encoding Serine Hydroxymethyltransferases, Whose Inactivation Is Required to Render Yeast Auxotrophic for Glycine," J Biol Chem 269:9155-9165 (1994); Monschau et al., "Identification of Saccharomyces Cerevisiae Glyl as a Threonine Aldolase: A Key Enzyme in Glycine Biosynthesis," FEMS Microbiol Lett 150:55-60 (1997); Woldman and Appling, "A General Method for Determining
  • Step 3 wild-type plant threonine aldolase is cloned into a different vector with a different regulatable promoter, for instance pCM185 (Gari et al., "A Set of Vectors with a Tetracycline-Regulatable Promoter System for Modulated Gene Expression in Saccharomyces Cerevisiae,” Yeast 13:837-848 (1997), which is hereby incorporated by reference in its entirety), which has a tetracycline-repressible promoter.
  • pCM185 a different regulatable promoter
  • Step 4 the 5 construct from Step 3 is chemically mutagenized, the yeast strain in Step 2 is transformed, and colonies are selected on yeast minimal medium in the presence of tetracycline and the absence of glycine.
  • Step 5 the colonies from Step 4 are replica- plated onto yeast minimal medium without tetracycline (to induce production of the mutated threonine aldolase) and without glycine, to identify colonies that are unable0 to grow under these conditions. This selection would identify mutated threonine aldolase whose production inactivates the wild-type threonine aldolase produced from the construct described in Steps 1 and 2.
  • Step 6 the mutant threonine aldolase gene from Step 5 is sequenced to identify the mutation(s).
  • Step 7 the mutant threonine aldolase from Step 5 is cloned into a plant transformation vector, for instance one of 5 the pCAMBIA T-DNA vectors (Roberts et al., "A Comprehensive Set of Modular Vectors for Advanced Manipulations and Efficient Transformation of Plants by Both Agrobacterium and Direct DNA Uptake Methods, www.Cambia.Org/” (2003), which is hereby incorporated by reference in its entirety), which can be used for Agrobacterium-mediated transformation, and is used to make a transgenic plantO overexpressing the mutant enzyme.
  • a plant transformation vector for instance one of 5 the pCAMBIA T-DNA vectors (Roberts et al., "A Comprehensive Set of Modular Vectors for Advanced Manipulations and Efficient Transformation of Plants by Both Agrobacterium and Direct DNA Uptake Methods, www.Cambia.Org/” (2003), which is hereby incorporated by reference in its entirety), which can be used for Agrobacterium-
  • a transgenic plant can be generated by particle bombardment (Taylor and Fauquet, "Microparticle Bombardment as a Tool in Plant Science and Agricultural Biotechnology," DNA Cell Biol 21 :963-977 (2002), which is hereby incorporated by reference in its entirety) or other methods.
  • Step 8 inactivation of the wild-type enzyme in the plant is verified by5 enzymatic assays and determination of increased threonine content.
  • Example 21 Expression of Wild Type Plant Threonine Aldolase in Mitochondria to Increase Threonine Content
  • the threonine aldolase reaction is reversible, depending on the relativeO substrate concentrations. Therefore, by expressing threonine aldolase in a part of the plant with high glycine concentrations or glycine flux might result in the production of higher levels of threonine.
  • Mitochondria are organelles in which glycine is converted to serine by the action of serine hydroxymethyltransferase ( Figure 7). Based on sequence comparisons, threonine aldolase appears to be a cytosolic enzyme in plants.
  • threonine aldolase with a mitochondrial targeting sequence might allow the enzyme to function in threonine synthesis.
  • Plant mitochondrial targeting sequences are known and have been used to target foreign proteins to mitochondria in transgenic plants (Ambard-Bretteville et al., "Discrete Mutations in the Presequence of Potato Formate Dehydrogenase Inhibit the in Vivo Targeting of Gfp Fusions into Mitochondria," Biochem Biophys Res Commun 311 :966-971 (2003); Guda et al., “Mitopred: A Web Server for the Prediction of Mitochondrial Proteins," Nucleic Acids Res 32:W372-374 (2004); Hwang et al., "Novel Targeting Signals Mediate the Sorting of Different Isoforms of the Tail- Anchored Membrane Protein Cytochrome B5 to Either Endoplasmic Reticulum or Mitochondria," Plant Cell 16:3002-3019
  • Step 1 An example of one method by which threonine aldolase could be targeted to mitochondria is: Step 1, a threonine aldolase homolog in the plant of interest is identified by comparison to known, functional threonine aldolases from Arabidopsis or microorganisms.
  • Step 2 the threonine aldolase gene is fused to a mitochondrial targeting sequence — for instance, that of potato formate dehydrogenase (Ambard-Bretteville et al., "Discrete Mutations in the Presequence of Potato Formate Dehydrogenase Inhibit the in Vivo Targeting of Gfp Fusions into Mitochondria," Biochem Biophys Res Commun 311 :966-971 (2003), which is hereby incorporated by reference in its entirety) — and is cloned into a plant transformation vector.
  • a mitochondrial targeting sequence for instance, that of potato formate dehydrogenase (Ambard-Bretteville et al., "Discrete Mutations in the Presequence of Potato Formate Dehydrogenase Inhibit the in Vivo Targeting of Gfp Fusions into Mitochondria," Biochem Biophys Res Commun 311 :966-971 (2003), which is hereby
  • Step 3 a plant is genetically transformed (e.g., by using Agrobacterium transformation, particle bombardment, or other transformation methods) with the constmct of a mitochondrial targeting sequence fused to threonine aldolase.
  • Step 4 threonine aldolase enzymatic activity and tlireonine levels are measured in the transformed plant. Similar methods to that described above could be used to target tlireonine aldolase to other plant organelles that might have high glycine levels, for instance the peroxisome (Figure 7).
  • mitochondrial targeting peptides include, for example, the following: (1) Potato formate dehydrogenase: ATGGCGATGAGTCGTGTAGCTTCTACAGCAGCTCGTGCTATTACT TCACCTTCATCCTTAGTTTTTACC (SEQ ID NO: 85)
  • Example 22 Reduction of Plant Threonine Aldolase Activity, Combined with Increased Production of Isoleucine Biosynthetic Enzymes or Feedback-Insensitive Isoleucine Biosynthetic Enzymes, to Increase Isoleucine Content
  • Threonine serves as a direct precursor for the amino acid isoleucine
  • Figure 8 which is also a target for plant genetic engineering. Plants that have high threonine levels as a result of modification of threonine aldolase expression could be firrther engineered to have high isoleucine content. At least two of the enzymes (threonine aldolase and acetolactate synthase) acting between threonine and isoleucine are subject to feedback inhibition by downstream metabolites (Figure 8).
  • the levels of isoleucine could be greatly increased in a threonine aldolase- altered plants by expressing feedback-insensitive versions of threonine deaminase (Garcia and Mourad, "A Site-Directed Mutagenesis Interrogation of the Carboxy- Terrninal End of Arabidopsis Thaliana Threonine Dehydratase/Deaminase Reveals a Synergistic Interaction between Two Effector-Binding Sites and Contributes to the Development of a Novel Selectable Marker," Plant Mol Biol 55:121-134 (2004); Mourad and King, "L-O-Methylthreonine-Resistant Mutant of Arabidopsis Defective in Isoleucine Feedback Regulation," Plant Physiol 107:43-52 (1995), which are hereby incorporated by reference in their entirety) and/or acetolactate synthase (Hervieu and Vaucheret, "A Single Amino Acid Change in Acetolac
  • plants with elevated isoleucine levels could be engineered in the following manner: Step 1, plants with altered expression of threomne aldolase are created as described supra in Examples 17 to 21, or by other methods. Step 2, genes encoding threomne deaminase and/or acetolactate synthase are cloned into plasmid vectors, and mutations conferring feed-back insensitivity are created by site-directed mutagenesis. Step 3, mutated threonine deaminase and/or acetolactate synthase are cloned into plant transformation vectors.
  • Step 4 plants are transformed to express mutated threonine deaminase and/or acetolactate synthase by Agrobacterium-mediated transformation, particle bombardment or other methods.
  • Step 5 transformants with the desired phenotype are identified by enzymatic activity assays and isoleucine measurements.

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CN115109769A (zh) * 2022-05-20 2022-09-27 华东理工大学 一种l-苏氨酸醛缩酶突变体以及在l-丝氨酸合成中的应用
CN117264976A (zh) * 2023-09-19 2023-12-22 河北省农林科学院棉花研究所（河北省农林科学院特种经济作物研究所） 一种棉花细胞壁强度调控基因及其应用
CN117264976B (zh) * 2023-09-19 2024-03-19 河北省农林科学院棉花研究所（河北省农林科学院特种经济作物研究所） 一种棉花细胞壁强度调控基因及其应用

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