WO2013030812A1 - Semences transgéniques de soja riche en méthionine exprimant le gène de la cystathionine gamma-lyase de l'arabidopsis - Google Patents

Semences transgéniques de soja riche en méthionine exprimant le gène de la cystathionine gamma-lyase de l'arabidopsis Download PDF

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WO2013030812A1
WO2013030812A1 PCT/IB2012/054561 IB2012054561W WO2013030812A1 WO 2013030812 A1 WO2013030812 A1 WO 2013030812A1 IB 2012054561 W IB2012054561 W IB 2012054561W WO 2013030812 A1 WO2013030812 A1 WO 2013030812A1
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soybean plant
plant
seed
methionine
seeds
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PCT/IB2012/054561
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English (en)
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Shikui SONG
Wensheng HOU
Itamar GODO
Cunxiang WU
Rachel Amir
Tianfu HAN
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Gavish-Galilee Bio Applications Ltd.
Institute Of Crop Science, The Chinese Academy Of Agricultural Sciences
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Publication of WO2013030812A1 publication Critical patent/WO2013030812A1/fr

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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
    • 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
    • C12N15/8253—Methionine or cysteine
    • 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.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
    • C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
    • C12Y205/01048—Cystathionine gamma-synthase (2.5.1.48)

Definitions

  • the present invention in some embodiments thereof, relates to transgenic plants and, more particularly, but not exclusively, to transgenic soybean plants transformed with a methionine-insensitive form of the Arabidopsis cystationine gamma-synthase gene.
  • Legumes are among the most important nutritional sources of protein for human and domestic animals worldwide.
  • legumes proteins suffer from a limited level of sulfur containing essential amino acids, methionine and cysteine.
  • one of the essential amino acids is limited in the diet the nutritional quality of the food is impaired, since the other amino acids are catabolized and used as an energy source.
  • the resulting limitation of amino acids eventually leads to protein deficiency, leading to a broad range of general symptoms such as lowered resistance to diseases, decrease blood proteins and retarded mental and physical development in young children.
  • PEM Protein-Energy Malnutrition
  • WHO World Health Organization
  • Soybean is an economically important legume with an estimated 2011 world-wide production of 251 million metric tons and estimated US crop value of $35.8 billion. Soybean seeds have an average protein content of about 40%, high as compared to other seed crops. Because of their high protein content, soybeans are extensively used as a major ingredient in livestock feed with the majority of soybean meal produced used for providing an amino acid and protein source in feed for poultry, fish, pork, cattle, and other farm animals. However, soybean proteins contain only 0.5-0.67% of methionine and 0.45-0.67% of cysteine (OECD document 2001), falling short of the WHO recommendation of 3.5% for total sulfur-containing amino acids in dietary proteins.
  • Transgenic narrow leaf lupins for example, that express the sunflower seed albumin, have shown that the concentration of total methionine was doubled in mature seeds of the transgenic lupins compared with non-transgenic controls.
  • AtCGS AtCGS
  • a transgenic soybean plant expressing an exogenous cystathionine ⁇ -synthase, wherein a total methionine level in a seed of the soybean plant is elevated relative to a seed of a cultivar of the soybean plant not expressing the exogenous cystathionine ⁇ - synthase.
  • soybean plant seed of the above transgenic soybean plant the soybean plant seed being a green or a mature dry seed.
  • a processed plant product comprising a tissue of the above transgenic soybean plant, the plant product having enhanced methionine content compared to the methionine content of the processed plant product of a cultivar of said soybean plant not expressing the exogenous cystathionine ⁇ -synthase.
  • the exogenous cystathionine ⁇ - synthase expression is seed- specific.
  • green seeds of the plant have elevated total amino acid and total soluble methionine relative to total amino acid and total soluble methionine of a green seed of a cultivar of the soybean plant not expressing the exogenous cystathionine ⁇ -synthase.
  • the seeds of the plant have similar seed morphology to that of a seed of a cultivar of the soybean plant not expressing the exogenous cystathionine ⁇ -synthase.
  • the seeds of the plant have similar seed germination rate relative to a seed of a cultivar of the soybean plant not expressing the exogenous cystathionine ⁇ -synthase.
  • the dry, mature seeds of plant have elevated total nitrogen content relative to total nitrogen content of dry, mature seeds of a cultivar of the soybean plant not expressing exogenous cystathionine ⁇ - synthase.
  • the dry, mature seeds of plant have elevated total protein content relative to total protein content of dry, mature seeds of a cultivar of the soybean plant not expressing exogenous cystathionine ⁇ -synthase.
  • the dry, mature seeds of the plant have elevated soluble methionine relative to soluble methionine of dry, mature seeds of a cultivar of the soybean plant not expressing exogenous cystathionine ⁇ - synthase.
  • the dry, mature seeds of the plant have elevated total amino acids relative to total amino acids of dry, mature seeds of a cultivar of the soybean plant not expressing the exogenous cystathionine ⁇ - synthase.
  • the dry, mature seeds of the plant have elevated total methionine content relative to total methionine content of dry, mature seeds of a cultivar of the soybean plant not expressing the exogenous cystathionine ⁇ -synthase.
  • the exogenous cystathionine ⁇ - synthase is a methionine-insensitive form of Arabidopsis thaliana cystathionine ⁇ - synthase.
  • the transgenic soybean plant comprises an exogenous polynucleotide encoding the methionine-insensitive form of Arabidopsis thaliana cystathionine ⁇ -synthase transcriptionally linked to a seed-specific promoter.
  • the seed-specific promoter comprises the legumin B4 promoter.
  • the legumin B4 promoter is as set forth in SEQ ID NO: 5.
  • the exogenous polynucleotide further encodes a pea rbcS-3A chloroplast transit peptide.
  • the plant is a Jilin Small soybean cultivar.
  • the plant is a Zigong Winter soybean cultivar.
  • the seed is a mature, dry seed and the total methionine content of the soybean plant seed is at least 1.3 times to 3.0 times that of dry, mature seeds of a cultivar of the soybean plant not expressing the exogenous cystathionine ⁇ -synthase.
  • the total methionine content of the soybean plant seed is at least 1.5 times to 2.5 times that of dry, mature seeds of a cultivar of the soybean plant not expressing the exogenous cystathionine ⁇ -synthase.
  • the total methionine content of the soybean plant seed is at least 2.0 times that of dry, mature seeds of a cultivar of the soybean plant not expressing the exogenous cystathionine ⁇ -synthase.
  • the seed is a green seed and the total methionine content of the soybean plant seed is at least 1.5 times to 10 times that of green seeds of a cultivar of the soybean plant not expressing the exogenous cystathionine ⁇ -synthase. According to some embodiments of the invention, the seed is a green seed and the total methionine content of the soybean plant seed is at least 3 times to 8 times that of green seeds of a cultivar of the soybean plant not expressing the exogenous cystathionine ⁇ -synthase.
  • the seed is a green seed and the total methionine content of the soybean plant seed is at least 7 times that of green seeds of a cultivar of the soybean plant not expressing the exogenous cystathionine ⁇ - synthase.
  • nucleic acid construct comprising a polynucleotide encoding a methionine- insensitive form of Arabidopsis thaliana cystathionine ⁇ -synthase transcriptionally linked to a seed- specific promoter.
  • the seed- specific promoter is a legumanin B4 promoter as set forth in SEQ ID NO: 5.
  • the polynucleotide further comprises a nucleic acid sequence encoding a pea rbcS-3A chloroplast transit peptide.
  • a method for enhancing the total methionine level in a seed of a soybean plant comprising transforming the soybean plant with the above nucleotide construct.
  • the soybean plant is a Jilin
  • the methionine level is elevated relative to the total methionine level of the seed of a cultivar of the soybean plant not expressing the exogenous cystathionine ⁇ -synthase.
  • a method of producing a hybrid soybean plant having enhanced total methionine level in a seed of the soybean plant comprising crossing the above transgenic soybean plant with a non-identical soybean plant.
  • the non-identical soybean plant cultivar does not express a methionine-insensitive form of Arabidopsis thaliana cystathionine ⁇ -synthase.
  • a hybrid soybean plant produced by the above method, having enhanced total methionine level in a seed of the soybean plant as compared to methionine levels in a tissue of the non-identical soybean plant cultivar.
  • soybean plant produced by self-crossing the above hybrid plant.
  • a soybean crop comprising the above transgenic soybean plant.
  • a soybean crop comprising the above transgenic soybean plant.
  • a food or an animal feed comprising a tissue of the above transgenic soybean plant.
  • a food or an animal feed comprising the above soybean plant seed.
  • a processed plant product comprising a tissue of the above transgenic soybean plant, the plant product having enhanced methionine content compared to the methionine content of the processed plant product of a cultivar of the soybean plant not expressing the exogenous cystathionine ⁇ -synthase.
  • a processed plant product comprising the above soybean plant seed, the plant product having enhanced methionine content compared to the methionine content of the processed plant product of a cultivar of the soybean plant seed not expressing the exogenous cystathionine ⁇ -synthase.
  • a method of producing a soybean plant product having enhanced methionine content comprising processing the above transgenic soybean plant.
  • a method of producing a soybean plant seed product having enhanced methionine content comprising processing the above soybean plant seed.
  • all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.
  • methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control.
  • the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.
  • FIG. 1 is a scheme of the aspartate family biosynthesis pathway and methionine metabolism. Only some of the enzymes and metabolites are indicated.
  • FIG. 2 is an autoradiograph depicting Southern blot analysis of transgenic soybean plants transformed with a truncated A. thaliana cystathionine ⁇ -synthase (AtCGS) gene construct, including a glufosinate resistance selectable marker. 20 micrograms of genomic DNA from glufosinate resistant Ti plants were digested with Hind III, and separated on a PAGE. A DNA fragment from the AtCGS gene was used as a probe. The 7 left lanes (JS1-JS7) are from Jilin Small (JS, also known as Jilinxiaoli, JX) transgenic line, while lane 8 is from JS wild type seeds (JSwt, also known as JXwt).
  • JSwt Jilinxiaoli
  • the three right lanes are from Zigong Winter (ZW, also known as Zigongdongdou, ZD) cultivar, the ZWwt (also known as ZDwt) is from ZW wild type seeds. Note the absence of cross-reacting DNA sequences in the wild type genomic DNA;
  • FIGs. 3 A and 3B are immunoblots (3 A) and quantitative PCR (3B) analyses of AtCGR protein and protein expression in the seeds of selected transgenic soybean plants.
  • FIG. 3A 30 ⁇ g of protein extracts of the fresh soy seeds were separated on 12 % SDS-PAGE and elctroblotted onto nitrocellulose membrane. AtDCGS protein was detected with a rabbit anti-AtCGS primary antibody, followed by goat anti-rabbit secondary antibody. Immunodetection was conducted with an enhanced chemiluminescence kit. Gels were also stained with Coomassie blue for total protein detection (Coomassie blue).
  • FIG. 3B Total RNA was extracted from fresh soybean seeds and quantified spectrophotometrically.
  • FIG. 4 is a histogram representing the methionine content of transgenic homozygous seeds (T) of Jilin Small (JS) and Zigong Winter (ZW) expressing the AtD- CGS gene. Soluble methionine content was determined by Gas Chromatography-Mass Spectrometry (GC-MS) in the soluble fraction of green (21 days after anthesis) or mature dry seeds. The data are presented as the mean ⁇ SD of six individual seeds per line;
  • FIGs. 5A-5D represent the amino acid content of transgenic homozygous seeds of Jilin Small (JS) and Zigong Winter (ZW) expressing the AtD-CGS gene, compared to their control wild type seeds, as determined by GC-MS. Soluble amino acid content was determined by Gas Chromatography-Mass Spectrometry (GC-MS) in the soluble fraction of green (21 days after anthesis) (FIG. 5 A) or mature dry (FIG 5B) seeds of Jilin Small (JS) and Zigong Winter (ZW) transgenic (T) or wild type (WT) plants.
  • GC-MS Gas Chromatography-Mass Spectrometry
  • Total amino acid content which includes protein-bound amino acids, was determined by GC-MS in the soluble fraction of an extract of an acid hydrolysate of ground, lyophilized green (light columns) or mature dry (dark columns) seeds of JS transgenic plants (FIG. 5C). Determination of amino acids in 5A-5C was calculated using the Selected Ion Monitoring (SIM) method of GC-MS, as previously described (Golan, 2005).
  • FIG. 5D represents the amino acid content of mature dry seeds of transgenic Jilin Small (JX) and Zigong Winter (ZD) plants expressing the AtD-CGS gene, calculated using the SCAN method (total ion count).
  • FIG. 6 is a histogram representing the level of sulfate in transgenic homozygous seeds (T) of Jilin Small (JS) and Zigong Winter (ZW) expressing the AtD-CGS gene, and in the control wild type seeds.
  • Columns 1, 3, 5 and 7 represent sulfate values from control, WT seeds.
  • Columns 2, 4, 6 and 8 represent sulfate values from seeds of transgenic (T) JS or ZW plants.
  • Sulfate was measured by ion chromatography, against a S0 4 standard. The data represents the mean ⁇ SD of five individual seeds per line. Statistically significant differences (p ⁇ 0.05) are identified by asterisk;
  • FIG. 7 is a histogram representing the transcription levels of sulfate-poor protein ( ⁇ -conglycinin) and ubiquitin in homozygous green or dry mature (yellow) seeds of transgenic Jilin Small (JS) plants expressing the AtD-CGS gene, compare to control wild type Jilin Small seeds. Values represent ratio of intensity (OD) of specific ⁇ - conglycinin transcripts to ubiquitin transcripts in RT-PCR products from total soybean seed RNA.
  • JS Jilin Small (also known as Jilinxiaoli, JX).
  • WT Wild type.
  • T Transgenic. Note the reduced ⁇ -conglycinin/ubiquitin ratio in the transgenic dry (yellow) seeds, indicating reduced ⁇ -conglycinin expression;
  • FIGs. 8A and 8B are SDS-PAGE protein profiles of green or dry mature seeds of transgenic Jilin Small (JS) plants expressing the AtD-CGS gene, compare to control wild type Jilin Small seeds.
  • the whole grain protein extracts were separated by SDS- PAGE, and proteins visualized by Coomassie blue (FIG. 8A) or silver (FIG 8B) staining.
  • JS Jilin Small (also known as Jilinxiaoli, JX).
  • WT Wild type.
  • T Transgenic. Note the identical protein profiles for transgenic and wild-type seeds.
  • the present invention in some embodiments thereof, relates to transgenic plants and, more particularly, but not exclusively, to transgenic soybean plants transformed with a methionine-insensitive form of the Arabidopsis cystationine gamma-synthase gene (AtD-CGS).
  • AtD-CGS Arabidopsis cystationine gamma-synthase gene
  • the low levels of sulfur amino acids methionine and cysteine limit their nutritional quality and have required supplementation of soybean seeds and products with synthetic methionine, at great cost.
  • efforts to increase the methionine content of soybean plants and seeds have met with little success.
  • enhanced methionine soybean plants and seeds would be of great economic and social importance.
  • the present inventors have shown, for the first time, that expression of a methionine-insensitive form of the Arabidopsis cystathionines-synthase gene (AtD- CGS) in soybean plants results in increased methionine content in tissues, particularly seeds, of the transgenic plants.
  • Transgenic plants of the present invention expressing an exogenous AtD-CGS gene, exhibit increased total methionine levels, relative to similar soybean plants not expressing the exogenous AtD-CGS gene.
  • the seeds of the transgenic soybean plants are morphologically similar and germinate similarly to seeds of similar soybean plants not-expressing the exogenous AtD-CGS gene, and can be used to produce soybean seeds and soybean plant products having increased nutritional value due to the enhanced methionine and amino acid content. Additional aspects and applications of the invention are further discussed below.
  • JS and ZW were capable of being transformed and expressing the AtD-CGS sequence, and although methionine was elevated, relative to soybean cultivars not expressing the transgene, in both JS and ZW green transgenic seeds, enhanced methionine was detected only in dry mature seeds of the transgenic JS cultivar, and not in those of the transgenic ZW cultivars (see Example II).
  • transgenic soybean plant expressing an exogenous cystathionine ⁇ -synthase, wherein a total methionine level in a seed of the soybean plant is elevated relative to the same tissue of a cultivar of the soybean plant not expressing the exogenous cystathionine ⁇ -synthase.
  • the term “enhanced” or “elevated” amino acid and/or methionine level or content refers to an amino acid and/or methionine level or content at least about 1.1 times, at least about 1.3 times, at least about 1.4 times, at least about 1.5 times, at least about 1.6 times, at least about 1.7 times, at least about 1.8 times, at least about 1.9 times, at least about 2 times, at least about 2.1 times, at least about 2.3 times, at least about 2.4 times, at least about 2.5 times, at least about 2.6 times, at least about 2.7 times, at least about 2.8 times, at least about 2.9 times, at least about 3 times, at least about 3.25 times, at least about 3.50 times, at least about 3.75 times, at least about 4 times, at least about 4.25 times, at least about 4.5 times, at least about 4.75 times, at least about 5 times, at least about 5.5 times, at least about 6 times, at least about 6.5 times, at least about 7 times, at least about 7.5 times,
  • the total methionine content of the dry, mature transgenic soybean plant seed is at least 1.3 to 3.0 times that of a cultivar of the soybean plant not expressing the exogenous CGS.
  • the dry mature transgenic seeds have at least 1.5 to 2.5 times the methionine content of a cultivar of the soybean plant not expressing the exogenous CGS.
  • the dry transgenic mature seeds have at least 2.0 times the methionine content of a cultivar of the soybean plant not expressing the exogenous CGS.
  • the total methionine content of the green transgenic soybean plant seed is at least 1.5 to 10.0 times that of a cultivar of the soybean plant not expressing the exogenous CGS.
  • the green transgenic seeds have at least 3 to 8 times the methionine content of a cultivar of the soybean plant not expressing the exogenous CGS.
  • the dry transgenic mature seeds have at least 7.0 times the methionine content of a cultivar of the soybean plant not expressing the exogenous CGS.
  • the term "similar to” refers to a characteristic or parameter of a transgenic soybean plant or seed (e.g. a process, composition, duration, etc) identical to that of a non-transgenic soybean plant or seed, or differing therefrom only within a range not effecting the overall quality of the character or parameter within the soybean plant or seed.
  • plant encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, roots (including tubers), and isolated plant cells, tissues and organs.
  • the plant may be in any form including suspension cultures, embryos, meristematic regions, callus tissue, leaves, gametophytes, sporophytes, pollen, and microspores.
  • plant cell refers to plant cells which are derived and isolated from disintegrated plant cell tissue or plant cell cultures.
  • methionine level or “methionine content” refers to the level of amino acid methionine in a plant tissue, as measured by gas chromatography and/or mass spectrometry.
  • Gas-chromatography-Mass Spectrometry can be carried out in a number of methods, for example, the selected ion monitoring (SIM) method or the total ion count method (e.g. SCAN method).
  • SIM selected ion monitoring
  • soluble methionine refers to the methionine detected in a soluble fraction of an extract of the plant tissue, and does not include methionine incorporated into proteins or other insoluble plant cell fractions.
  • total methionine refers to methionine detected in a soluble fraction of plant tissue following solubilization by hydrolysis of the amino acids in the protein fractions. It will be understood that detection and measurement of other soluble and total amino acids and their content in the plant tissues can be accomplished using the same methods. Methods for measuring "nitrogen content” are well known in the art, for example, the Kjeldahl method. Methods for measuring total proteins are well known in the art, for example, estimation according to total nitrogen as measured by Kjeldahl method.
  • the term "soybean plant” refers to the species Glycine max,
  • the soybean plant is a Jilin Small (JS, also known as Jilinxiaoli, JX) cultivar or a Zigong Winter (ZW, also known as Zigongdongdou, ZD) cultivar of Glycine max.
  • Jilin Small also known as Jilinxiaoli, JX
  • ZW Zigong Winter cultivar of Glycine max.
  • Other cultivars are known in the art, for example, G. max Derry, Donegal, and Tyrone.
  • the methionine and/or amino acid content of the soybean seed of the transgenic plant is enhanced, relative to a seed of a cultivar of the soybean plant not expressing the exogenous cystathionine ⁇ -synthase gene, in all stages of seed development of the transgenic soybean plant seed.
  • the methionine and/or amino acid content of the soybean seed of the transgenic plant is enhanced in the green seeds or dry mature seeds of the transgenic soybean plant.
  • the term “green” soybean seed refers to seeds prior to seed desiccation, during a period of rapid weight gain, moisture and nutrient accumulation.
  • the "green” soybean seed is a seed removed from the pod, e.g., removed from the pod 21 days after anthesis.
  • the term “mature” and/or “dry” soybean seed refers to soybean seeds after cessation of dry weight gain. “Mature dry” or “dry mature” soybean seeds are characterized by a yellow color (as opposed to green in immature seeds) and a moisture content of 20% or less.
  • CGS cystathionines-synthase
  • methionine-insensitive cystathionines-synthase refers to a CGS enzyme lacking inhibition of enzyme activity by levels of methionine.
  • the methionine-insensitive cystathionine- ⁇ - synthase is an Arabidopsis thaliana cystathionines-synthase mutated in, or lacking, an N-terminal portion encompassing a 30 amino acid region downstream of a transit peptide sequence native to A.
  • thaliana cystathionines-synthase (corresponding to amino acids 38-68 of the native A. thaliana cystathionines-synthase polypeptide, SEQ ID NO: 6).
  • methionine-insensitive CGS the production and uses thereof are described detail in US Patent Number 7,323,338 to Amir, which is incorporated herein in its entirety.
  • the methionine-insensitive cystathionine- ⁇ - synthase is at least 60%, at least 70%, at least 80%, at least 90%, at least 95% identical to AtD-CGS (SEQ ID NO: 21).
  • AtD-CGS is encoded by the polynucleotide sequence SEQ ID NO: 4.
  • Methionine-insensitive cystathionine- ⁇ - synthase can optionally comprise sequences from homologous cystathionines-synthase sequences from other species, including but not limited to Glycine max (SEQ ID NO: 15), Zea mays (SEQ ID NO: 16), H. pylori (SEQ ID NO: 17) and E. coli (SEQ ID NO: 18).
  • exogenous cystathionines-synthase or “exogenous cystathionines-synthase'' gene refers to a heterologous cystathionines-synthase nucleic acid sequence or cystathionines-synthase amino acid sequence which may not be naturally expressed within the plant or which overexpression in the plant is desired.
  • the exogenous cystathionines-synthase gene may be introduced into the plant in a stable or transient manner, so as to produce a ribonucleic acid (RNA) molecule or a polypeptide.
  • RNA ribonucleic acid
  • exogenous cystathionines-synthase gene may comprise a nucleic acid sequence which is identical or partially homologous to an endogenous cystathionines-synthase nucleic acid sequence of the plant.
  • Nucleic acid sequences of the polypeptides of some embodiments of the invention may be optimized for expression in a specific plant host. Examples of such sequence modifications include, but are not limited to, an altered G/C content to more closely approach that typically found in the plant species of interest, and the removal of codons atypically found in the plant species commonly referred to as codon optimization.
  • an optimized gene or nucleic acid sequence refers to a gene in which the nucleotide sequence of a native or naturally occurring gene has been modified in order to utilize statistically-preferred or statistically-favored codons within the plant.
  • the nucleotide sequence typically is examined at the DNA level and the coding region optimized for expression in the plant species determined using any suitable procedure, for example as described in Sardana et al. (1996, Plant Cell Reports 15:677-681).
  • the standard deviation of codon usage may be calculated by first finding the squared proportional deviation of usage of each codon of the native gene relative to that of highly expressed plant genes, followed by a calculation of the average squared deviation.
  • a table of codon usage from highly expressed genes of dicotyledonous plants is compiled using the data of Murray et al. (1989, Nuc Acids Res. 17:477-498).
  • Codon Usage Database contains codon usage tables for a number of different species, with each codon usage table having been statistically determined based on the data present in Genbank.
  • a naturally- occurring nucleotide sequence encoding a protein of interest can be codon optimized for that particular plant species. This is effected by replacing codons that may have a low statistical incidence in the particular species genome with corresponding codons, in regard to an amino acid, that are statistically more favored.
  • one or more less- favored codons may be selected to delete existing restriction sites, to create new ones at potentially useful junctions (5' and 3' ends to add signal peptide or termination cassettes, internal sites that might be used to cut and splice segments together to produce a correct full-length sequence), or to eliminate nucleotide sequences that may negatively affect mRNA stability or expression.
  • codon optimization of the native nucleotide sequence may comprise determining which codons, within the native nucleotide sequence, are not statistically-favored with regards to a particular plant, and modifying these codons in accordance with a codon usage table of the particular plant to produce a codon optimized derivative.
  • a modified nucleotide sequence may be fully or partially optimized for plant codon usage provided that the protein encoded by the modified nucleotide sequence is produced at a level higher than the protein encoded by the corresponding naturally occurring or native gene. Construction of synthetic genes by altering the codon usage is described in for example PCT Patent Application 93/07278.
  • a “transgenic plant” refers to a plant that has incorporated a nucleic acid sequence (i.e., polynucleotides encoding cystathionine-y-synthase), including but not limited to genes that are not normally present in a host plant genome, nucleic acid sequences not normally transcribed into RNA, or any other genes or nucleic acid sequences that one desires to exogenously introduce into the wild-type plant.
  • a nucleic acid sequence i.e., polynucleotides encoding cystathionine-y-synthase
  • hybrid plant refers to a plant or a part thereof resulting from a cross between two parent plants, wherein one parent is a genetically engineered plant of the invention (transgenic plant exogenously expressing the cystathionine-y-synthase polypeptides of the present invention).
  • a cross can occur naturally by, for example, sexual reproduction, or artificially by, for example, in vitro nuclear fusion.
  • Methods of plant breeding are well-known and within the level of one of ordinary skill in the art of plant biology.
  • soybean plants can be transformed to express an exogenous methionine-insensitive cystathionine-y-synthase gene, and that the transgenic soybean plants expressing the exogenous methionine-insensitive cystathionine-y-synthase gene have enhanced methionine content.
  • a nucleic acid construct comprising a nucleic acid sequence (a polynucleotide) encoding a methionine-insensitive cystathionines-synthase polypeptide, the nucleic acid sequence being under a transcriptional control a cis-acting regulatory element.
  • a coding nucleic acid sequence is "operably linked” or “transcriptionally linked to a regulatory sequence (e.g., promoter)" if the regulatory sequence is capable of exerting a regulatory effect on the coding sequence linked thereto.
  • a regulatory sequence e.g., promoter
  • regulatory sequence means any DNA, that is involved in driving transcription and controlling (i.e., regulating) the timing and level of transcription of a given DNA sequence, such as a DNA coding for the target polypeptide, as described above.
  • a 5' regulatory region is a DNA sequence located upstream (i.e., 5') of a coding sequence and which comprises the promoter and the 5 '-untranslated leader sequence.
  • a 3' regulatory region is a DNA sequence located downstream (i.e., 3') of the coding sequence and which comprises suitable transcription termination (and/or regulation) signals, including one or more polyadenylation signals.
  • the promoter is a plant-expressible promoter.
  • plant-expressible promoter means a DNA sequence which is capable of controlling (initiating) transcription in a plant cell. This includes any promoter of plant origin, but also any promoter of non-plant origin which is capable of directing transcription in a plant cell, i.e., certain promoters of viral or bacterial origin.
  • any suitable promoter sequence can be used by the nucleic acid construct of the present invention.
  • the promoter is a constitutive promoter, a tissue- specific promoter or an inducible promoter (e.g. an abiotic stress-inducible promoter).
  • Suitable constitutive promoters include, for example, hydroperoxide lyase (HPL) promoter, CaMV 35S promoter (Odell et al, Nature 313:810-812, 1985); Arabidopsis At6669 promoter (see PCT Publication No. WO04081173A2); Arabidopsis new At6669 promoter; maize Ubi 1 (Christensen et al., Plant Sol. Biol. 18:675-689, 1992); rice actin (McElroy et al., Plant Cell 2: 163-171, 1990); pEMU (Last et al, Theor. Appl. Genet.
  • HPL hydroperoxide lyase
  • CaMV 35S promoter Odell et al, Nature 313:810-812, 1985
  • Arabidopsis At6669 promoter see PCT Publication No. WO04081173A2
  • Arabidopsis new At6669 promoter maize Ub
  • Suitable tissue-specific promoters include, but not limited to seed-preferred promoters [e.g., from seed specific genes (Simon, et al., Plant Mol. Biol. 5. 191, 1985; Scofield, et al., J. Biol. Chem. 262: 12202, 1987; Baszczynski, et al., Plant Mol. Biol. 14: 633, 1990), Brazil Nut albumin (Pearson' et al., Plant Mol. Biol. 18: 235- 245, 1992), legumin (Ellis, et al. Plant Mol. Biol. 10: 203-214, 1988), Glutelin (rice) (Takaiwa, et al., Mol. Gen. Genet.
  • endosperm specific promoters e.g., wheat LMW and HMW, glutenin-1 (Mol Gen Genet 216:81- 90, 1989; NAR 17:461-2), wheat a, b and g gliadins (EMB03: 1409-15, 1984), Barley ltrl promoter, barley Bl, C, D hordein (Theor Appl Gen 98: 1253-62, 1999; Plant J 4:343-55, 1993; Mol Gen Genet 250:750- 60, 1996), Barley DOF (Mena et al., The Plant Journal, 116(1): 53- 62, 1998), Biz2 (EP99106056.7), Synthetic promoter (Vicente-Carbajosa et al., Plant J.
  • the promoter is a seed specific promoter.
  • Seed specific promoters include, but are not limited to the napin promoter, the phaseolin promoter and the legumin B4 promoter.
  • the promoter is the legumin B4 promoter (SEQ ID NO: 5).
  • the nucleic acid construct of some embodiments of the invention can further include an appropriate selectable marker and/or an origin of replication.
  • the selectable marker is an herbicide resistance gene, for example, but not limited to the glufosinate ammonium resistance gene.
  • the nucleic acid construct of some embodiments of the invention can be utilized to stably or transiently transform plant cells.
  • stable transformation the exogenous sequence is integrated into the plant genome and as such it represents a stable and inherited trait.
  • transient transformation the exogenous polynucleotide is expressed by the cell transformed but it is not integrated into the genome and as such it represents a transient trait.
  • Agrobacterium-mediated gene transfer e.g., T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes
  • Agrobacterium-mediated gene transfer e.g., T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes
  • Klee et al. (1987) Annu. Rev. Plant Physiol. 38:467-486; Klee and Rogers in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego, Calif. (1989) p. 2- 25; Gatenby, in Plant Biotechnology, eds. Kung, S, and Arntzen, C. J., Butterworth Publishers, Boston, Mass. (1989) p.
  • the Agrobacterium system includes the use of plasmid vectors that contain defined DNA segments that integrate into the plant genomic DNA. Methods of inoculation of the plant tissue vary depending upon the plant species and the Agrobacterium delivery system. A widely used approach is the leaf disc procedure which can be performed with any tissue explant that provides a good source for initiation of whole plant differentiation. See, e.g., Horsch et al. in Plant Molecular Biology Manual A5, Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. A supplementary approach employs the Agrobacterium delivery system in combination with vacuum infiltration. The Agrobacterium system is especially viable in the creation of transgenic dicotyledonous plants.
  • the exogenous methionine-insensitive cystathionines-synthase polynucleotide is introduced into the plant by infecting the plant with a bacteria, such as using a cotelydonary-node transformation method (as described in further detail in Example I and Materials and Methods, of the Examples section which follows).
  • a bacteria such as using a cotelydonary-node transformation method (as described in further detail in Example I and Materials and Methods, of the Examples section which follows).
  • electroporation the protoplasts are briefly exposed to a strong electric field.
  • microinjection the DNA is mechanically injected directly into the cells using very small micropipettes.
  • microparticle bombardment the DNA is adsorbed on microprojectiles such as magnesium sulfate crystals or tungsten particles, and the microprojectiles are physically accelerated into cells or plant tissues.
  • Micropropagation is a process of growing new generation plants from a single piece of tissue that has been excised from a selected parent plant or cultivar. This process permits the mass reproduction of plants having the preferred tissue expressing the fusion protein.
  • the new generation plants which are produced are genetically identical to, and have all of the characteristics of, the original plant.
  • Micropropagation allows mass production of quality plant material in a short period of time and offers a rapid multiplication of selected cultivars in the preservation of the characteristics of the original transgenic or transformed plant.
  • the advantages of cloning plants are the speed of plant multiplication and the quality and uniformity of plants produced.
  • Micropropagation is a multi-stage procedure that requires alteration of culture medium or growth conditions between stages.
  • the micropropagation process involves four basic stages: Stage one, initial tissue culturing; stage two, tissue culture multiplication; stage three, differentiation and plant formation; and stage four, greenhouse culturing and hardening.
  • stage one initial tissue culturing
  • stage two tissue culture multiplication
  • stage three differentiation and plant formation
  • stage four greenhouse culturing and hardening.
  • stage one initial tissue culturing
  • the tissue culture is established and certified contaminant- free.
  • stage two the initial tissue culture is multiplied until a sufficient number of tissue samples are produced to meet production goals.
  • stage three the tissue samples grown in stage two are divided and grown into individual plantlets.
  • the transformed plantlets are transferred to a greenhouse for hardening where the plants' tolerance to light is gradually increased so that it can be grown in the natural environment.
  • transient transformation of leaf cells, meristematic cells or the whole plant is also envisaged by the present invention.
  • Transient transformation can be effected by any of the direct DNA transfer methods described above or by viral infection using modified plant viruses.
  • Viruses that have been shown to be useful for the transformation of plant hosts include CaMV, Tobacco mosaic virus (TMV), brome mosaic virus (BMV) and Bean Common Mosaic Virus (BV or BCMV). Transformation of plants using plant viruses is described in U.S. Pat. No. 4,855,237 (bean golden mosaic virus; BGV), EP-A 67,553 (TMV), Japanese Published Application No. 63-14693 (TMV), EPA 194,809 (BV), EPA 278,667 (BV); and Gluzman, Y. et al., Communications in Molecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, New York, pp. 172-189 (1988).
  • TMV Tobacco mosaic virus
  • BMV brome mosaic virus
  • BV or BCMV Bean Common Mosaic Virus Transformation of plants using plant viruses is described in U.S. Pat. No. 4,855,237 (bean golden mosaic virus; BGV), EP-A 67,553 (TMV), Japanese Published Application No. 63-
  • the virus used for transient transformations is avirulent and thus is incapable of causing severe symptoms such as reduced growth rate, mosaic, ring spots, leaf roll, yellowing, streaking, pox formation, tumor formation and pitting.
  • a suitable avirulent virus may be a naturally occurring avirulent virus or an artificially attenuated virus.
  • Virus attenuation may be effected by using methods well known in the art including, but not limited to, sub-lethal heating, chemical treatment or by directed mutagenesis techniques such as described, for example, by Kurihara and Watanabe (Molecular Plant Pathology 4:259- 269, 2003), Galon et al. (1992), Atreya et al. (1992) and Huet et al. (1994).
  • Suitable virus strains can be obtained from available sources such as, for example, the American Type culture Collection (ATCC) or by isolation from infected plants. Isolation of viruses from infected plant tissues can be effected by techniques well known in the art such as described, for example by Foster and Tatlor, Eds. "Plant Virology Protocols: From Virus Isolation to Transgenic Resistance (Methods in Molecular Biology (Humana Pr), Vol 81)", Humana Press, 1998. Briefly, tissues of an infected plant believed to contain a high concentration of a suitable virus, preferably young leaves and flower petals, are ground in a buffer solution (e.g., phosphate buffer solution) to produce a virus infected sap which can be used in subsequent inoculations.
  • a buffer solution e.g., phosphate buffer solution
  • the virus When the virus is a DNA virus, suitable modifications can be made to the virus itself. Alternatively, the virus can first be cloned into a bacterial plasmid for ease of constructing the desired viral vector with the foreign DNA. The virus can then be excised from the plasmid. If the virus is a DNA virus, a bacterial origin of replication can be attached to the viral DNA, which is then replicated by the bacteria. Transcription and translation of this DNA will produce the coat proteins which will encapsidate the viral DNA. If the virus is an RNA virus, the virus is generally cloned as a cDNA and inserted into a plasmid. The plasmid is then used to make all of the constructions. The RNA virus is then produced by transcribing the viral sequence of the plasmid and translation of the viral genes to produce the coat protein(s) which encapsidate the viral RNA.
  • a plant viral nucleic acid in which the native coat protein coding sequence has been deleted from a viral nucleic acid, a non-native plant viral coat protein coding sequence and a non-native promoter, preferably the subgenomic promoter of the non-native coat protein coding sequence, capable of expression in the plant host, packaging of the recombinant plant viral nucleic acid, and ensuring a systemic infection of the host by the recombinant plant viral nucleic acid, has been inserted.
  • the coat protein gene may be inactivated by insertion of the non-native nucleic acid sequence within it, such that a protein is produced.
  • the recombinant plant viral nucleic acid may contain one or more additional non-native subgenomic promoters.
  • Each non-native subgenomic promoter is capable of transcribing or expressing adjacent genes or nucleic acid sequences in the plant host and incapable of recombination with each other and with native subgenomic promoters.
  • Non-native (foreign) nucleic acid sequences may be inserted adjacent the native plant viral subgenomic promoter or the native and a non-native plant viral subgenomic promoters if more than one nucleic acid sequence is included.
  • the non-native nucleic acid sequences are transcribed or expressed in the host plant under control of the subgenomic promoter to produce the desired products.
  • a recombinant plant viral nucleic acid is provided as in the first embodiment except that the native coat protein coding sequence is placed adjacent one of the non-native coat protein subgenomic promoters instead of a non- native coat protein coding sequence.
  • a recombinant plant viral nucleic acid in which the native coat protein gene is adjacent its subgenomic promoter and one or more non-native subgenomic promoters have been inserted into the viral nucleic acid.
  • the inserted non-native subgenomic promoters are capable of transcribing or expressing adjacent genes in a plant host and are incapable of recombination with each other and with native subgenomic promoters.
  • Non-native nucleic acid sequences may be inserted adjacent the non-native subgenomic plant viral promoters such that the sequences are transcribed or expressed in the host plant under control of the subgenomic promoters to produce the desired product.
  • a recombinant plant viral nucleic acid is provided as in the third embodiment except that the native coat protein coding sequence is replaced by a non-native coat protein coding sequence.
  • the viral vectors are encapsidated by the coat proteins encoded by the recombinant plant viral nucleic acid to produce a recombinant plant virus.
  • the recombinant plant viral nucleic acid or recombinant plant virus is used to infect appropriate host plants.
  • the recombinant plant viral nucleic acid is capable of replication in the host, systemic spread in the host, and transcription or expression of foreign gene(s) (isolated nucleic acid) in the host to produce the desired protein.
  • the nucleic acid molecule of the present invention can also be introduced into a chloroplast genome thereby enabling chloroplast expression.
  • a technique for introducing exogenous nucleic acid sequences to the genome of the chloroplasts is known. This technique involves the following procedures. First, plant cells are chemically treated so as to reduce the number of chloroplasts per cell to about one. Then, the exogenous nucleic acid is introduced via particle bombardment into the cells with the aim of introducing at least one exogenous nucleic acid molecule into the chloroplasts. The exogenous nucleic acid is selected such that it is integratable into the chloroplast's genome via homologous recombination which is readily effected by enzymes inherent to the chloroplast.
  • the exogenous nucleic acid includes, in addition to a gene of interest, at least one nucleic acid stretch which is derived from the chloroplast's genome.
  • the exogenous nucleic acid includes a selectable marker, which serves by sequential selection procedures to ascertain that all or substantially all of the copies of the chloroplast genomes following such selection will include the exogenous nucleic acid. Further details relating to this technique are found in U.S. Pat. Nos. 4,945,050; and 5,693,507 which are incorporated herein by reference.
  • a polypeptide can thus be produced by the protein expression system of the chloroplast and become integrated into the chloroplast's inner membrane.
  • the invention also envisages expressing a plurality of exogenous polynucleotides in a single host plant to thereby achieve superior effect on methionine content and/or amino acid content of the soybean plant or seeds.
  • Expressing a plurality of exogenous polynucleotides in a single host plant can be effected by co-introducing multiple nucleic acid constructs, each including a different exogenous polynucleotide, into a single plant cell.
  • the transformed cell can then be regenerated into a mature plant using the methods described hereinabove.
  • expressing a plurality of exogenous polynucleotides in a single host plant can be effected by co-introducing into a single plant-cell a single nucleic-acid construct including a plurality of different exogenous polynucleotides.
  • Such a construct can be designed with a single promoter sequence which can transcribe a polycistronic messenger RNA including all the different exogenous polynucleotide sequences.
  • the polynucleotide sequences can be inter- linked via an internal ribosome entry site (IRES) sequence which facilitates translation of polynucleotide sequences positioned downstream of the IRES sequence.
  • IRES internal ribosome entry site
  • a transcribed polycistronic RNA molecule encoding the different polypeptides described above will be translated from both the capped 5' end and the two internal IRES sequences of the polycistronic RNA molecule to thereby produce in the cell all different polypeptides.
  • the construct can include several promoter sequences each linked to a different exogenous polynucleotide sequence.
  • the plant cell transformed with the construct including a plurality of different exogenous polynucleotides can be regenerated into a mature plant, using the methods described hereinabove.
  • expressing a plurality of exogenous polynucleotides can be effected by introducing different nucleic acid constructs, including different exogenous polynucleotides, into a plurality of plants.
  • the regenerated transformed plants can then be cross-bred and resultant progeny selected for presence or expression of methionine- insensitive cystathionines-synthase gene, or phenotype of methionine and/or amino acid content of the soybean plant or seeds as described above, using conventional plant breeding techniques.
  • a method of producing a hybrid soybean plant having enhanced total methionine level in a seed of the soybean plant comprising crossing the transgenic soybean plant of the invention with a non-identical soybean plant, for example, soybean plant cultivar that does not express a methionine-insensitive form of Arabidopsis thaliana cystathionine ⁇ - synthase, or a said non-identical soybean plant cultivar that does not have enhanced methionine and/or amino acid content.
  • a non-identical soybean plant for example, soybean plant cultivar that does not express a methionine-insensitive form of Arabidopsis thaliana cystathionine ⁇ - synthase, or a said non-identical soybean plant cultivar that does not have enhanced methionine and/or amino acid content.
  • Subsequent generations and varieties of the hybrid and pure-bred transgenic soybean plants of the present invention can be generated by in-breeding or outbreeding of the resultant plants.
  • methionine-insensitive cystathionine- ⁇ - synthase gene in the soybean plant or seeds of the present invention can be qualified using methods which are well known in the art such as those involving gene amplification Western blotting, ELISA, or at the mRNA level involving e.g., PCR or RT-PCR or Northern blot or in-situ hybridization.
  • the invention encompasses soybean plants exogenously expressing the polynucleotide(s), the nucleic acid constructs of the invention and having a phenotype of enhanced methionine and/or amino acid content of the soybean plant or seeds.
  • Plant lines exogenously expressing the polynucleotide of the invention can be screened to identify those that show the greatest increase of methionine and/or amino acid content of the soybean plant or seeds. It will be appreciated that, in addition to the enhanced methionine content, other amino acids such as alanine, valine, leucine, threonine, isoleucine, proline, glycine, apartate, phenylalanine, glutamate, tyrosine and tryptophan, individually or in combinations thereof (see FIG. 5D) can be significantly enhanced in the soybean lines exogenously expressing the polynucleotide of the invention.
  • other amino acids such as alanine, valine, leucine, threonine, isoleucine, proline, glycine, apartate, phenylalanine, glutamate, tyrosine and tryptophan, individually or in combinations thereof (see FIG. 5D) can be significantly enhanced in the soybean lines exogenously expressing the
  • the present invention is of high agricultural value for increasing phenotype of methionine and/or amino acid content of the soybean plant or seeds.
  • a food or feed e.g. forage
  • a food or feed comprising the transgenic plants or seeds or other portion thereof of the present invention.
  • the transgenic soybean plants of the present invention or parts thereof are comprised in a food or feed product (e.g., dry, liquid, paste).
  • a food or feed product is any ingestible preparation containing the transgenic plants, or parts thereof, of the present invention, or preparations made from these plants.
  • the plants or preparations are suitable for human (or animal) consumption, i.e. the transgenic soybean plants or parts thereof are more readily digested.
  • Feed products of the present invention further include a oil or a beverage adapted for animal consumption.
  • Soybeans can be used in their entireties but are commonly processed into two primary products, i.e., soybean protein (meal) and crude soybean oil. Both of these products are commonly further refined for particular uses.
  • the crude soybean oil can be broken down into glycerol, fatty acids, and sterols.
  • the soybean protein can be divided into soy flour concentrates and isolates. It will be appreciated that the transgenic soybean plants, or parts thereof, of the present invention may be used directly as feed products or alternatively may be incorporated or mixed with feed products for consumption. Furthermore, the food or feed products may be processed or used as is.
  • Examples of "food” products made from soybean include, but are not limited to, coffee creamers, margarine, mayonnaise, salad dressings, shortenings, bakery products, chocolate coatings, cereal, beer, aquaculture feed, bee feed, calf feed replacers, fish feed, livestock feed, poultry feed, and pet feed.
  • Examples of "industrial” products include, but are not limited to, binders, wood composites, anti-static agents, caulking compounds, solvents, disinfectants, fungicides, inks, paints, protective coatings, wallboard, anti-foam agents, and rubber.
  • a processed plant product comprising a soybean plant seed of the invention, the plant product having enhanced methionine content compared to the methionine content of a processed plant product of a cultivar of the soybean plant seed not expressing said exogenous cystathionine ⁇ -synthase.
  • processed plant products include, but are not limited to, any soybean plant products made from or including soy protein of the transgenic soybean plant.
  • the soybean product can further be packaged, optionally in a packaging material comprising a label indicating the high methionine and/or amino acid content of the soybean plant material.
  • transgenic soybean plants of the present invention can be grown individually, or cultivated as a crop, in a field or greenhouse or other enclosure. Commercial soybean production most commonly is carried out as a field crop, and the plants are grown in large numbers in open fields under commonly known cultivation conditions, and the soybean seeds harvested from the crop.
  • a soybean crop comprising the transgenic soybean plant of the invention.
  • transgenic soybean plants and seeds of the present invention can provide a rich source of high-sulfate protein to both animals and humans, they are suitable for cultivation in geographical regions in which both animals and humans suffer from malnutrition, such as the case with third world countries.
  • the term "about” refers to ⁇ 10 %.
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • treating includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
  • Binary plasmids comprising the AtDCGS coding sequence and Legumin B4 seed-specific promoter were prepared from a binary vector comprising the AtDCGS coding sequence and a 35S CMV promoter described by Hacham et al (Plant Journal, 2006).
  • the 35S CMV binary vector contained the 35S promoter of cauliflower mosaic virus, an ⁇ DNA sequence from the coat protein gene of tobacco mosaic virus for translation enhancement, the chloroplast transit peptide coding sequence of pea rbcS-3A (SEQ ID NO: 2) and a truncated A.
  • thaliana cystathionines-synthase coding sequence (SEQ ID NO: 4) subcloned into a binary Ti plasmid, pZPl l l, using Sphl and Smal restriction sites.
  • the 35S promoter of cauliflower mosaic virus was replaced by the seed specific promoter of Legumin B4 (SEQ ID NO: 5), using Hindlll and Noel restrictions enzymes.
  • the resulting binary vector contained the Legumin B4 promoter, the chloroplast transit peptide of pea rbcS-3A, the cDNA encoding the AtD-CGS, and a 3' terminator derived from the octopine synthetase gene of Agwbacterium tumefaciens .
  • the fragment containing all of these components was then subcloned by Hindlll and Xbal into the binary plasmid pGPTV-BAR that carries the gene for glufosinate ammonium resistance, and the plasmid was transformed into Agribacterium tumefaciens strain EHA105.
  • Agwbacterium strain EHA105 contains the plasmid pGPTV-BAR was prepared according to Paz et al. Agwbacterium was finally suspended in infection medium containing 1/10 Gamborg's B5 salts (Gamborg et al. 1968), 1/10 MS iron, 3% sucrose, 20 mM MES (pH5.4), 1/10 B5 vitamins, 3.3 mM cysteine and ImM dithiothreitol (DTT), 1.67 mg L "1 BAP, 0.25 mg L "1 GA 3 , as 200 ⁇ acetosyringone.
  • Soybean cv. Jilin Small (JS) also known as Jilinxiaoli No.1, JX
  • ZW Zigong Winter
  • ZD Zigongdongdou
  • the elongated shoots were cut down when they were about 3-4 cm tall and transferred to rooting medium comprising B5 salts, MS iron, 2% sucrose, 3 mM MES (pH5.7), B5 vitamins, 1.5 mg L "1 IBA, and 0.8% agar. When the secondary root appeared, the T 0 plants were transferred to soil.
  • the effective concentration of the glufosinate used in screening the transgenic soybean plants was confirmed by painting and spraying on the untransformed plants.
  • a concentration of 120 mg L-l glufosinate was adopted in this experiment for painting the leaves, and 250 mg L "1 for spraying on the whole plants.
  • half of the front part of the young leaf not yet reached its full size was painted with 120 mg L "1 glufosinate.
  • the T ⁇ plants were sprayed with 250 mg L "1 glufosinate when the first trifoliate fully developed.
  • the glufosinate resistance plants were examine by PCR for the insertion of AtD ⁇
  • the endogenous CGS (GmCGS) gene in soybean has homology to the exogenous AtCGS gene (67% identity). Therefore, to avoid pseudo-positive PCR result, sequence alignment was performed between the sequences of GmCGS and AtD- CGS. Primers were designed to match the sequences exist only in the AtCGS and not in the GmCGS.
  • the sequence of the sense primer for exogenous AtDCGS was of 5'- AGCAATGGTGGAA GAGTAAA-3'(SEQ ID NO: 7), while the sequence of the antisense primer was 5'-CCATACTCGAAACTCACACTC-3'(SEQ ID NO: 8).
  • AtDCGS containing positive plants screened by PCR amplification were further confirmed by Southern blot analysis.
  • Total genomic DNA was isolated from T ⁇ plants using the CTAB method described by Murray and Thompson (1980). Twenty micrograms of genomic DNA was digested with Hindlll for Southern blot analysis. The 418 bp PCR fragment containing the AtD-CGS coding region was labeled with Prime-a-Gene Labeling System (Promega, Cat # U1100) according to the manufacturer's instructions. Hybridization was performed at 65 °C for 15 hours, followed by washing in solutions of SDS (sodium dodecyl sulfate) detergent and SSC (sodium chloride and sodium citrate solution). The hybridization membrane was then exposed to X-ray film (Kodak, USA) at -80 °C which was subsequently developed.
  • SDS sodium dodecyl sulfate
  • SSC sodium chloride and sodium citrate solution
  • PCR and Southern blot-positive plants were screened for expression of the AtD- CGS protein level using western blot analysis.
  • the membrane was ponceau-S stained, blocked for lh in 5% (v/v) skim-milk, then reacted over-night with anti-AtCGS primary antibody followed by goat anti-rabbit secondary antibody.
  • the membrane was stained by ECL (Thermo Scientific). Immunodetection was conducted with an enhanced chemiluminescence kit (Pierce) in accordance with the manufacturer's instructions.
  • Primers used for amplification of 291 bp AtD-CGS fragment were: forward 5 -CAAGTTGGG GATCACTGTCAC-3 SEQ ID NO: 9), reverse 5 -
  • CCAGCAAGAACATCATTGTGTCC-3 SEQ ID NO: 10 For ⁇ -conglycinin amplification of an 828 bp fragment were: forward 5 -GCGGG AG AGCC A TACTTACC-3 SEQ ID NO: 11), reverse 5 -CGCCTGCAAGGAAGTTCCTC-3 (SEQ ID NO: 12).
  • RT-PCR products were separated on 1% agarose EtBr gel, photographed with Fuji Film thermal Imaging System FTI-500 and the bands' intensity were quantified using the Biolmage Intelligent Quantifier.
  • Free amino acids were extracted from a sample of seeds. Detection of amino acids was performed by GC-MS as previously described (Golan et al., 2005). 25 mg of dry seeds were ground in 1 ml of methanol. After 10 min of centrifugation (4°C, top speed), the supernatants were collected, and 700 ⁇ of chloroform and 375 DDW were added to the supernatant. After 30 min of centrifugation at 3,000 g, 450 ⁇ from the upper water phase was collected, dried and dissolved in 140 ⁇ of 20 mg/ml methoxyaminhydrochlorid in piridin.
  • the amino acid sample was analyzed with GC-MS using a SCAN method (total ions count). Peak areas of methionine as well as other amino acids are uniquely and sensitively detected by this method, such that only selected m/z values are detected in the analysis.
  • NRC National Research Council
  • Lipids were determined by the Soxhlet method, essentially as described by Kim et al (Kim, 2006). 2 grams of soybean seeds were ground to powder with an analytical grinder AIO(IKA), the powder placed in a dried paper wrap in the extraction cylinder of the Soxhelt apparatus with anhydrous ether and incubated overnight. The ether was transferred to an extraction tube and fresh ether added to the extraction cylinder. The solvent was then boiled and refluxed in the extractor at 70-80 °C for 6 hours. The sample wrap with remaining sample was dried in a dessicator and then overnight in an oven at 105 °C for 2 hours, weighed and the lipid content calculated. Results are expressed as percent of total lipids (g/100 gram dry seeds).
  • Total proteins were determined according to Kjeldahl et al (Kim et al, 2006). For each sample, 25 soybean seeds were ground to a powder by an analytical grinder A10 (IKA). 200 mg soybean powder was mixed with 0.5 g CuS0 4 and 5 g K 2 S0 4 in a burette, followed by gentle addition of 18 ml H 2 S0 4 . Mixtures were then heated to 340- 370 °C for 2 hours for digestion, with cleaning of the exhaust gas by a scrubber. After cooling to room temperature, protein content was determined in the Kjeldahl Apparatus K-370 (BUCHI). Total proteins were estimated from the total nitrogen according to the equation described in AOAC, 1984 (Williams, 1984).
  • a methionine- insensitive AtD-CGS gene was expressed in transgenic soybean plants.
  • a construct with the AtD-CGS cDNA under the control of a strong, constitutive plant- specific promoter is prepared.
  • the promoter, the AtD-CGS cDNA, a chloroplast transit peptide and terminator were inserted into a binary vector carrying a selective marker gene such as glufosinate ammonium resistance.
  • Soybean plants such as cv Jilin Small (JS) (also known as Jilinxiaoli No.l, JX) and Zigong Winter (ZW) (also known as Zigongdongdou, ZD) are transformed via A. tumefaciens (strain EHA105), and cultivated in the presence of the selective factor, such as glufosinate ammonium (basta).
  • the resistant soybean plants from each of the transformed cultivars are screened using PCR to confirm the insertion of the AtD-CGS gene, and positive lines (inserts detected) are selected.
  • AtDCGS AtDCGS gene in seeds
  • a construct with the AtD-CGS cDNA under the control of the seed- specific promoter of Legumin B4 was prepared.
  • the promoter, the AtD-CGS cDNA, the chloroplast transit peptide and terminator were inserted into the binary vector pGPTV-BAR carrying the gene for glufosinate ammonium resistance.
  • Soybean cv Jilin Small (JS) also known as Jilinxiaoli No.1, JX
  • ZW Zigong Winter
  • ZD Zigongdongdou
  • tumefaciens strain EHA105
  • strain EHA105 tumefaciens
  • tumefaciens strain EHA105
  • Thirty independent basta-resistant soybean plants from each of the two cultivars were screen using PCR to confirm the insertion of the AtD-CGS gene.
  • Southern blot was performed.
  • FIG. 2 shows the results obtained from several of the detected lines (JS1-JS7, ZW1 and ZW2), showing that most of these lines have one copy of AtD-CGS gene, and confirming no cross-reactive species in the untransformed wild type cultivars.
  • AtD-CGS expression levels were determined in the transgenic green seeds (21 + 2 days old seeds), which corresponds to the reserve accumulation stage, characterized by expression of seed storage proteins and accumulation of seed reserves.
  • Western blot analysis using rabbit anti-AtCGS antibodies While no cross -reactivity was observed between these antibodies and the endogenous soybean CGS, a protein band at the expected size of AtD-CGS (50 kDa) was detected in seven lines from the JS (also known as JX) cultivar lines and six lines of the ZW (also known as ZD) cultivar lines. One line from each cultivar that has the highest expression level of AtD-CGS protein, was then self pollinated to form homozygous lines. Southern blot analysis carried out on the selected JS and ZW homozygous lines showed that these lines carried a single AtD-CGS gene (Fig. 2). The further studies were preformed on these two exemplary lines.
  • Transgenic soybean seeds expressing AtD-CGS accumulate significantly higher level of soluble methionine than wild type seeds: To assess whether high expression level of AtD-CGS affects methionine content, the level of soluble methionine was measured in the selected transgenic seeds from T 2 progenies. The level of soluble methionine increased in green-developing seeds of the transgenic line of JX-173 from 110 + 13 in the wild type to 763 + 79 nmol/g fresh weight (FW) (about 7-fold increase). Similar elevation was also observed in ZD- 104 transgenic line, in which the methionine content increased from 130 + 14 in wild type seeds to 719 + 51 nmol/g FW in the transgenic line (6-fold increase) (Fig. 4A, Table 1).
  • Table 1 Contents of soluble amino acids (nmole/g FW) in green-developing seeds of transgenic lines expressing the AtD-CGS gene (T) and in the wild type (WT) seeds, in two cultivars Jilinxiaoli No. 1 (JX) and Zigongdongdou (ZD). The data are presented as the mean ⁇ SD of five individual seeds per line. Statistically significant differences (p ⁇ 0.05) are identified by asterisk.
  • Threonine level had significantly increased in the green-developing seeds of JX-173 (from 460 to 656 nmol/g FW; 1.4-fold), but not in ZD-104 seeds (Table 1).
  • the level of methionine was determined in mature-dry seeds of the two transgenic lines.
  • JS and ZW also known as JX and ZD transgenic lines
  • the level of soluble methionine increased about 2-folds in comparison to the wild type seeds (FIG.4A, FIG. 5D, Table 2).
  • Table 2 Contents of soluble amino acids (nmole/g FW) in dry mature seeds of transgenic (T) and wild type (WT) seeds in two cultivars Jilinxiaoli No. 1 (JX) and Zigongdongdou (ZD).
  • the transgenic seeds express the AtD-CGS gene.
  • the data are presented as the mean ⁇ SD of six individual seeds per line. Statistically significant differences (p ⁇ 0.05) are identified by asterisk.
  • Transgenic Jilin Small also known as Jilinxiaoli No.1, JX
  • ZW Zigong Winter
  • ZD Zigongdongdou
  • Table 3 Contents of total amino acids (nmole/g FW) in dry mature seeds of transgenic (T) and wild type (WT) seeds in two cultivars Jilinxiaoli No.1 (JX) and Zigongdongdou (ZD).
  • the transgenic seeds express the AtD-CGS gene.
  • the data are presented as the mean ⁇ SD of six individual seeds per line. Statistically significant differences (p ⁇ 0.05) are identified by asterisk.
  • Glycine 2328 i 269 3076 + : 187* 50274 2577 4688 ⁇ 611
  • Methionine 116 d 35 254 + :54* 2974 :96 313: b 108
  • Tyrosine 54 + 48 164 b44 14404 :626 1917+ 1533
  • Tryptophan 40 + 42 74 + 20 426: 204 617 ⁇ 490
  • JX-173 has significantly higher (24.5%) content of total amino acids per g dry seeds.
  • the significantly higher level of amino acids in this line suggests that when methionine synthesis is enhanced, most of the soluble amino acids are able to incorporate into proteins.
  • FIG. 6 Comparison of sulfate (FIG. 6) and low sulfur proteins (FIG. 7) between the transgenic and wild type soybean cultivars shows that while the sulfate content of the dry mature JS transgenic seeds was similar to that of the wild type dry mature seeds, there was a significant reduction in the expression of the low- sulfur protein ⁇ - conglycinin (FIG 7, yellow).
  • the content of total nitrogen and total protein, increase in the transgenic seeds expressing AtD-CGS The increase in the total amount of protein-incorporated amino acids in JX-173 suggests that the seeds of this line have higher protein content. To verify this, the total nitrogen contents were measured and the levels of total proteins calculated. The increase in total amino acids was accompanied with a significant increase in the contents of total nitrogen and estimated protein levels in mature-dry seeds of the transgenic JX-173 soybean plants (elevation of 5.19%). High elevation in the total nitrogen and proteins contents (by 3.71%) was also observed in ZD-104 line, although their total amino acids did not increase (Table 1). Without wishing to be limited to a single hypothesis, these results suggest that methionine availability limits the protein synthesis in JX-173 soybean seeds, and as its level increases, it promotes the incorporation of other amino acids into proteins, which levels increase.
  • JX-173 transgenic seeds also had a 4% increase in total reducing sugars, which represents the starch content, and ZD- 104 increased by about 9.4%, although such elevation was not observed in the two other transgenic lines of ZD.
  • ZD- 104 increased by about 9.4%, although such elevation was not observed in the two other transgenic lines of ZD.
  • the transgenic soybean lines expressing AtD-CGS have normal seed morphology and normal germination rate: Transgenic soybean seeds expressing AtD-CGS gene and having higher total methionine content exhibited normal seed morphology.
  • AtD-CGS seed-specific expression of AtD-CGS
  • germination rate fifty transgenic seeds from each cultivar as well as their corresponding wild type seeds were planted in soil under growth-chamber conditions. No alteration in germination rates between the transgenic lines and wild type seeds were observed.
  • Cis-analysis of a seed protein gene promoter the conservative RY repeat CATGCATG within the legumin box is essential for tissue-specific expression of a legumin gene. Plant J 2, 233-239.
  • Soluble methionine enhances accumulation of a 15 kDa zein, a methionine-rich storage protein, in transgenic alfalfa but not in transgenic tobacco plants. J Exp Bot 56, 2443-2452.
  • L-Cysteine increases Agrobacterium-mediated T-DNA delivery into soybean cotyledonary-node cells. Plant Cell Rep 20, 706-711.
  • serine acetlytransferase produced large increases in O-acetylserine and free cysteine in developing seeds of a grain legume. J Exp Bot 61, 721-733.

Abstract

L'invention concerne des plants de soja transgéniques, transformés au moyen d'une forme insensible à la méthionine du gène de la cystathionine gamma-lyase de l'Arabidopsis (AtD-CGS) et des procédés pour leur production. L'expression du gène AtD-CGS dans les plants de soja transgéniques a pour conséquence une teneur accrue en méthionine et acides aminés dans les semences de plants transgéniques. L'invention concerne également des semences de plants de soja transgéniques possédant des teneurs accrues en méthionine et acides aminés qui peuvent être utilisées pour produire des semences et des produits à base de semences bénéficiant d'une plus grande valeur nutritive grâce à la teneur accrue en méthionine et acides aminés.
PCT/IB2012/054561 2011-09-04 2012-09-04 Semences transgéniques de soja riche en méthionine exprimant le gène de la cystathionine gamma-lyase de l'arabidopsis WO2013030812A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110684811A (zh) * 2019-11-13 2020-01-14 浙江工业大学 一种提高甲硫氨酸产量的方法
WO2023215735A3 (fr) * 2022-05-02 2023-12-28 The Curators Of The University Of Missouri Méthodes et compositions pour augmenter le contenu d'acides aminés et de protéines dans des plantes

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000055303A2 (fr) * 1999-03-18 2000-09-21 Rutgers, The State University Plantes transgeniques possedant des proprietes de saveur accruees

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000055303A2 (fr) * 1999-03-18 2000-09-21 Rutgers, The State University Plantes transgeniques possedant des proprietes de saveur accruees

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
FUJIWARA TOM ET AL.: "Improvement of methionine content in soybean through metabolic regulation", SOY PROTEIN RESEARCH (JAPAN), vol. 9, 31 December 2006 (2006-12-31), pages 13 - 16 *
FUJIWARA TOM ET AL.: "Improvement of methionine contents in soybean through regulation of metabolism", SOY PROTEIN RESEARCH (JAPAN), vol. 8, 31 December 2005 (2005-12-31), pages 8 - 10 *
HACHAM Y. ET AL.: "In vivo internal deletion in the N-terminus region of Arabidopsis cystathionine gamma-synthase results in CGS expression that is insensitive to methionine", PLANT JOURNAL, vol. 45, no. 6, 31 March 2006 (2006-03-31), pages 955 - 967 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110684811A (zh) * 2019-11-13 2020-01-14 浙江工业大学 一种提高甲硫氨酸产量的方法
CN110684811B (zh) * 2019-11-13 2021-06-29 浙江工业大学 一种提高甲硫氨酸产量的方法
WO2023215735A3 (fr) * 2022-05-02 2023-12-28 The Curators Of The University Of Missouri Méthodes et compositions pour augmenter le contenu d'acides aminés et de protéines dans des plantes

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