CA2280196A1 - Chimeric genes and methods for increasing the lysine content of the seeds of plants - Google Patents

Chimeric genes and methods for increasing the lysine content of the seeds of plants Download PDF

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Publication number
CA2280196A1
CA2280196A1 CA002280196A CA2280196A CA2280196A1 CA 2280196 A1 CA2280196 A1 CA 2280196A1 CA 002280196 A CA002280196 A CA 002280196A CA 2280196 A CA2280196 A CA 2280196A CA 2280196 A1 CA2280196 A1 CA 2280196A1
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seq
nucleic acid
plant
chimeric gene
ketoglutarate reductase
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CA002280196A
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CA2280196C (en
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Saverio Carl Falco
Raymond Ervin Mcdevitt Iii
Sabine Ursula Epelbaum
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EIDP Inc
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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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/823—Reproductive tissue-specific promoters
    • C12N15/8234—Seed-specific, e.g. embryo, endosperm
    • 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
    • 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
    • C12N15/8254—Tryptophan or lysine
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Nutrition Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Developmental Biology & Embryology (AREA)
  • Pregnancy & Childbirth (AREA)
  • Reproductive Health (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Pretreatment Of Seeds And Plants (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Chimeric genes are disclosed. One chimeric gene encodes a plant lysine ketoglutarate reductase and a second chimeric gene encodes lysine-insensitive dihydrodipicolinic acid synthase (DHDPS) which is operably linked to a plant chloroplast transit sequence, all operably linked to plant seed-specific regulatory sequences. Methods for their use to produce increased levels of lysine in the seeds of transformed plants are provided.

Claims (20)

1. An isolated nucleic acid fragment comprising a nucleic acid sequence encoding all or part of lysine ketoglutarate reductase.
2. The nucleic acid fragment of Claim 1 wherein the nucleic acid sequence encodes a polypeptide essentially similar to the polypeptide described by SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:112, SEQ ID NO:117, SEQ ID
NO:118, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130 or SEQ ID NO:132.
3. The nucleic acid fragment of Claim 1 comprising a nucleic acid sequence wherein the nucleic acid sequence is essentially similar to that of SEQ
ID NO:110, SEQ ID NO:111, SEQ ID NO:115, SEQ ID NO:116, SEQ ID
NO:119, SEQ ID NO:120, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129 or SEQ ID NO:131.
4. The nucleic acid fragment of Claim 1 comprising a nucleic acid sequence of SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:115, SEQ ID
NO:116, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129 or SEQ ID NO:131.
5. The nucleic acid fragment of Claim 1 wherein the nucleic acid sequence encodes a polypeptide as set forth in SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:112, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:121, SEQ ID
NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130 or SEQ ID NO:132.
6. A chimeric gene comprising the isolated nucleic acid fragment of Claim 1 encoding lysine ketoglutarate reductase or a subfragment thereof, operably linked to suitable seed-specific regulatory sequences wherein said chimeric gene reduces lysine ketoglutarate reductase activity in seeds of plants transformed with the chimeric gene.
7. The chimeric gene according to Claim 6 wherein the isolated nucleic acid fragment comprises a nucleic acid sequence or subsequence thereof essentially similar to chat of SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:123, SEQ ID
NO:125, SEQ ID NO:127, SEQ ID NO:129 or SEQ ID NO:131.
8. A plant cell wherein lysine ketoglutarate reductase activity is reduced due to a mutation in a gene encoding lysine ketoglutarate reductase.
9. A plant cell transformed with the chimeric gene of Claim 6 or 7 wherein said transformed plant cell has reduced lysine ketoglutarate reductase activity.
10. A plant seed wherein lysine ketoglutarate reductase activity is reduced due to a mutation in a gene encoding lysine ketoglutarate reductase.
11. A plant seed transformed with the chimeric gene of Claim 6 or 7 wherein said transformed plant seed has reduced lysine ketoglutarate reductase activity.
12. The plant cell according to Claim 9 wherein said plant cell is selected from the group of plants consisting of Arabidopsis, corn, soybean, rapeseed, wheat and rice.
13. The plant seed according to Claim 11 wherein said plant cell is selected from the group of plants consisting of Arabidopsis, corn, soybean, rapeseed, wheat and rice.
14. A method for reducing lysine ketoglutarate reductase activity in a plant seed which comprises:
(a) transforming plant cells with the chimeric gene of claim 6 or 7;
(b) regenerating fertile mature plants from the transformed plant cells obtained from step (a) under conditions suitable to obtain seeds;
(c) screening progeny seed of step (b) for reduced lysine ketoglutarate reductase activity; and (d) selecting those lines whose seeds contain reduced lysine ketoglutarate reductase activity.
15. Seed obtained from the plant of Claim 14.
16. A nucleic acid fragment comprising (a) a first chimeric gene of Claim 6 or 7 and (b) a second chimeric gene wherein a nucleic acid fragment encoding dihydrodipicolinic acid synthase which is substantially insensitive to inhibition by lysine is operably linked to a plant chloroplast transit sequence and to a plant seed-specific regulatory sequence.
17. A plant comprising in its genome a first chimeric gene of Claim 6 or 7 wherein said gene reduces lysine ketoglutarate reductase activity in seeds of transformed plants and a second chimeric gene wherein a nucleic acid fragment encoding dihydrodipicolinic acid synthase which is substantially insensitive to inhibition by lysine is operably linked to a plant chloroplast transit sequence and to a plant seed-specific regulatory sequence.
18. A plant comprising in its genome the nucleic acid fragment of Claim 16.
19. Seed obtained from the plant of Claim 17 comprising in its genome the first and second chimeric genes.
20. Seed obtained from the plant of Claim 18 comprising in its genome the nucleic fragment of Claim. 16.
CA2280196A 1997-03-27 1998-03-27 Chimeric genes and methods for increasing the lysine content of the seeds of plants Expired - Fee Related CA2280196C (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US82462797A 1997-03-27 1997-03-27
US08/824,627 1997-03-27
PCT/US1998/006051 WO1998042831A2 (en) 1997-03-27 1998-03-27 Chimeric genes and methods for increasing the lysine content of the seeds of plants

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CA2280196A1 true CA2280196A1 (en) 1998-10-01
CA2280196C CA2280196C (en) 2012-05-15

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EP (1) EP0973880A2 (en)
JP (1) JP2001502923A (en)
KR (1) KR20010005645A (en)
CN (1) CN1253584A (en)
AU (1) AU747997B2 (en)
BR (1) BR9811256A (en)
CA (1) CA2280196C (en)
HU (1) HUP0002305A3 (en)
ID (1) ID22486A (en)
PL (1) PL336042A1 (en)
TR (1) TR199902349T2 (en)
WO (1) WO1998042831A2 (en)

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US20070079396A1 (en) * 2005-10-03 2007-04-05 Malvar Thomas M Transgenic plant seed with increased lysine
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Also Published As

Publication number Publication date
WO1998042831A3 (en) 1998-12-10
HUP0002305A2 (en) 2000-09-28
KR20010005645A (en) 2001-01-15
AU6780198A (en) 1998-10-20
CA2280196C (en) 2012-05-15
HUP0002305A3 (en) 2002-09-30
BR9811256A (en) 2000-07-18
CN1253584A (en) 2000-05-17
TR199902349T2 (en) 2000-01-21
PL336042A1 (en) 2000-06-05
JP2001502923A (en) 2001-03-06
ID22486A (en) 1999-10-21
AU747997B2 (en) 2002-05-30
EP0973880A2 (en) 2000-01-26
WO1998042831A2 (en) 1998-10-01

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