EP0769061A1 - Genes chimeres et procede d'augmentation de la teneur en threonine de graines de plantes - Google Patents

Genes chimeres et procede d'augmentation de la teneur en threonine de graines de plantes

Info

Publication number
EP0769061A1
EP0769061A1 EP95925537A EP95925537A EP0769061A1 EP 0769061 A1 EP0769061 A1 EP 0769061A1 EP 95925537 A EP95925537 A EP 95925537A EP 95925537 A EP95925537 A EP 95925537A EP 0769061 A1 EP0769061 A1 EP 0769061A1
Authority
EP
European Patent Office
Prior art keywords
seeds
gene
plant
plants
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP95925537A
Other languages
German (de)
English (en)
Inventor
Saverio Carl Falco
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
EIDP Inc
Original Assignee
EI Du Pont de Nemours and Co
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by EI Du Pont de Nemours and Co filed Critical EI Du Pont de Nemours and Co
Publication of EP0769061A1 publication Critical patent/EP0769061A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1217—Phosphotransferases with a carboxyl group as acceptor (2.7.2)

Definitions

  • lysine Human food and animal feed, derived from many grains, are deficient in essential amino acids, such as lysine, the sulfur amino acids methionine and cysteine, threonine and tryptophan.
  • threonine is the third most limiting amino acid, after lysine and the sulfur amino acids, for the dietary requirements of many animals.
  • Galili and co-workers have reported on the introduction of an E. coli lysC gene that encodes a lysine-sensitive AK enzyme into tobacco cells via transformation [Galili et al. (1992) Eur. Patent Appl. 91119328.2; Shaul et al. (1992) Plant Physiol. i 00:1157-1163]. Expression of the E. coli enzyme resulted in small increases in the levels of free threonine in d e leaves and seeds of transformed plants, but effects on the total tlireonine content were too small to be detected. Falco isolated a mutant of the E. coli lysC gene, which encoded a lysine- insensitive AK.
  • nucleic acid fragments refers to a large molecule which can be single- stranded or double-stranded, composed of monomers (nucleotides) containing a sugar, phosphate and either a purine or pyrimidine.
  • a "nucleic acid fragment” is a fraction of a given nucleic acid molecule.
  • End-product inhibition or “feedback inhibition” refers to a biological regulatory mechanism wherein the catalytic activity of an enzyme in a biosynthetic pathway is reversibly inhibited by binding to one or more of the end-products of the pathway when the concentration of the end-product(s) reaches a sufficiently high level, thus slowing the biosynthetic process and preventing over-accumulation of the end-product.
  • E. coli (Katinka et al. (1980) Proc. Natl. Acad. Sci. USA 77:5730-5733], the metL gene of E. coli (Zakin et al. (1983) /. Biol. Chem. 23 ⁇ °:3028-3031], the lysC gene of E. c ⁇ 7[Cassan et al. (1986) /. Biol. Chem. 2(5 :1052-1057], and the HOM3 gene of 5. cerevisiae [Rafalski et al. (1988) J. Biol. Chem. 2(55:2146-2151].
  • the thrA gene of E. coli encodes a bifunctional protein, AKI-HDHI.
  • promoters of seed-specific genes also maintain their temporal and spatial expression pattern in transgenic plants.
  • Such examples include linking either the Phaseolin or Arabidopsis 2S albumin promoters to the Brazil nut 2S albumin coding sequence and expressing such combinations in tobacco, Arabidopsis, or Brassica napus [Altenbach et al., (1989) Plant Mol. Biol. 25:513-522; Altenbach et al., (1992) Plant Mol. Biol. 25:235-245; De Clercq et al.,
  • nucleic acid fragment of the invention will be the heterologous promoters from several extensively- characterized soybean seed storage protein genes such as those for the Kunitz trypsin inhibitor [Jofuku et al. (1989) Plant Cell 2:1079-1093; Perez-Grau et al. (1989) Plant Cell 2:1095-1109], glytinin [Nielson et al. (1989) Plant Cell 2:313-328], ⁇ -conglycinin [Harada et al. (1989) Plant Cell 2:415-425].
  • primers add an Nco I site which includes a translation initiation codon at the amino terminus of the AKII-HDHII protein.
  • restriction site and additional codon GCC coding for alanine, was also added to the amino terminus of the protein.
  • the primers also add a Kpn I site immediately following the translation stop codon.
  • PCR was performed using a Perkin-Elmer Cetus kit according to the instructions of the vendor on a thermocycler manufactured by the same company. The primers were at a concentration of 10 ⁇ M and the thermocy cling conditions were:
  • Embryogenic callus was arranged in a circular area of about 4 cm in diameter in the center of a 100 X 20 mm petri dish containing N6-1.0 medium supplemented with 0.25M sorbitol and 0.25M mannitol.
  • the tissue was placed on this medium for 4-6 h prior to bombardment as a pretreatment and remained on the medium during the bombardment procedure.
  • the petri dish containing the tissue was placed in the chamber of the PDS-1000/He. The air in the chamber was then evacuated to a vacuum of 28-29 inch of Hg.
  • phaseolin 5' region/cts/metL/phaseolin 3' region A seed-specific expression cassette composed of the promoter and transcription terminator from the gene encoding the ⁇ subunit of the seed storage protein phaseolin from the bean Phaseolus vulgaris [Doyle et al. (1986) /. Biol. Chem. 261:9228-9238] was used for expression in transformed soybean.
  • the phaseolin cassette includes about 500 nucleotides upstream (5') from the translation initiation codon and about 1650 nucleotides downstream (3') from the translation stop codon of phaseolin.
  • the seed expression cassette, phaseolin 5' region/cts/metL/phaseolin 3' region, (Example 4) was isolated as an approximately 4.5 kb Hind HI fragment from pBT766 ( Figure 6). This fragment was inserted into a unique Hind WL site of the vector carrying the marker gene creating plasmid pBT767.
  • To 50 ⁇ L of a 60 mg/mL 1 ⁇ gold particle suspension was added (in order); 5 ⁇ L DNA (1 ⁇ g/ ⁇ L), 20 ⁇ l spermidine (0.1 M), and 50 ⁇ L CaCl2 (2.5 M). The particle preparation was agitated for three minutes, spun in a microfuge for 10 seconds and the supernatant removed.
  • the DNA-coated particles were then washed once in 400 ⁇ L 70% ethanol and resuspended in 40 ⁇ L of anhydrous ethanol.
  • the DNA/particle suspension was sonicated three times for one second each. Five ⁇ L of the DNA-coated gold particles were then loaded on each macro carrier disk.
  • MOLECULE TYPE DNA (genomic)

Landscapes

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

Abstract

Gène chimère codant une aspartokinase-homosérine déshydrogénase (AK-HDH) bifonctionnelle insensible aux rétroactions, qui est lié de manière active à une séquence de transit de chloroplaste de plante et à des séquences régulatrices spécifiques des graines de plantes. Ledit gène chimère est transformé dans des plantes, des quantités accrues de thréonine libre s'accumulant dans les graines.
EP95925537A 1994-07-08 1995-07-06 Genes chimeres et procede d'augmentation de la teneur en threonine de graines de plantes Withdrawn EP0769061A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US27291794A 1994-07-08 1994-07-08
US272917 1994-07-08
PCT/US1995/008501 WO1996001905A1 (fr) 1994-07-08 1995-07-06 Genes chimeres et procede d'augmentation de la teneur en threonine de graines de plantes

Publications (1)

Publication Number Publication Date
EP0769061A1 true EP0769061A1 (fr) 1997-04-23

Family

ID=23041823

Family Applications (1)

Application Number Title Priority Date Filing Date
EP95925537A Withdrawn EP0769061A1 (fr) 1994-07-08 1995-07-06 Genes chimeres et procede d'augmentation de la teneur en threonine de graines de plantes

Country Status (8)

Country Link
EP (1) EP0769061A1 (fr)
AU (1) AU2963695A (fr)
BR (1) BR9510174A (fr)
CA (1) CA2192550A1 (fr)
HU (1) HUT77112A (fr)
MX (1) MX9606384A (fr)
WO (1) WO1996001905A1 (fr)
ZA (1) ZA955629B (fr)

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CA2292778A1 (fr) 1997-06-06 1998-12-10 E.I. Du Pont De Nemours And Company Enzymes de biosynthese d'acides amines d'origine vegetale
US7368633B2 (en) 1997-06-06 2008-05-06 E.I. Du Pont De Nemours And Company Plant amino acid biosynthetic enzymes
DE69840650D1 (fr) * 1997-06-12 2009-04-23 Du Pont
US7053282B1 (en) * 1998-02-09 2006-05-30 Pioneer Hi-Bred International, Inc. Alteration of amino acid compositions in seeds
US7964774B2 (en) 2008-05-14 2011-06-21 Monsanto Technology Llc Plants and seeds of spring canola variety SCV384196
GB2462645A (en) * 2008-08-15 2010-02-17 Advanced Technologies Modification of plant threonine production by aspartate kinase
CA2758824A1 (fr) 2009-04-17 2010-10-21 Basf Plant Science Company Gmbh Promoteur vegetal apte a agir dans l'endosperme et ses utilisations
MX2011013224A (es) 2009-06-09 2012-06-01 Pioneer Hi Bred Int Promotor de endospermo temprano y metodos de uso.
US8778672B2 (en) 2009-10-26 2014-07-15 Pioneer Hi Bred International Inc Somatic ovule specific promoter and methods of use
WO2013096810A1 (fr) 2011-12-21 2013-06-27 The Curators Of The University Of Missouri Variété de soja s05-11482
WO2013096818A1 (fr) 2011-12-21 2013-06-27 The Curators Of The University Of Missouri Variété de soja s05-11268
US9006515B2 (en) 2012-01-06 2015-04-14 Pioneer Hi Bred International Inc Pollen preferred promoters and methods of use
EP2800816A1 (fr) 2012-01-06 2014-11-12 Pioneer Hi-Bred International Inc. Promoteur spécifique d'ovule et méthodes d'utilisation
US8835720B2 (en) 2012-04-26 2014-09-16 Monsanto Technology Llc Plants and seeds of spring canola variety SCV967592
US8878009B2 (en) 2012-04-26 2014-11-04 Monsanto Technology, LLP Plants and seeds of spring canola variety SCV318181
US8859857B2 (en) 2012-04-26 2014-10-14 Monsanto Technology Llc Plants and seeds of spring canola variety SCV259778
WO2014059155A1 (fr) 2012-10-11 2014-04-17 Pioneer Hi-Bred International, Inc. Promoteurs de cellules de garde et leur utilisation
AU2014241045B2 (en) 2013-03-13 2017-08-31 Pioneer Hi-Bred International, Inc. Glyphosate application for weed control in brassica
CN108998471B (zh) 2013-03-14 2022-11-04 先锋国际良种公司 用以防治昆虫害虫的组合物和方法
US10023877B2 (en) 2013-03-15 2018-07-17 Pioneer Hi-Bred International, Inc. PHI-4 polypeptides and methods for their use
EA030896B1 (ru) 2013-08-16 2018-10-31 Пайонир Хай-Бред Интернэшнл, Инк. Инсектицидные белки и способы их применения
ES2937045T3 (es) 2013-09-13 2023-03-23 Pioneer Hi Bred Int Proteínas insecticidas y métodos para su uso
CN117903266A (zh) 2014-02-07 2024-04-19 先锋国际良种公司 杀昆虫蛋白及其使用方法
CA2955828A1 (fr) 2014-08-08 2016-02-11 Pioneer Hi-Bred International, Inc. Promoteurs d'ubiquitine et introns, et procedes d'utilisation
US20170247719A1 (en) 2014-09-17 2017-08-31 Pioneer Hi-Bred International, Inc. Compositions and methods to control insect pests
BR112017007932A2 (pt) 2014-10-16 2018-01-23 Du Pont proteínas inseticidas e métodos para uso das mesmas
US20170359965A1 (en) 2014-12-19 2017-12-21 E I Du Pont De Nemours And Company Polylactic acid compositions with accelerated degradation rate and increased heat stability
WO2016186986A1 (fr) 2015-05-19 2016-11-24 Pioneer Hi Bred International Inc Protéines insecticides et leurs procédés d'utilisation
BR112017027382A2 (pt) 2015-06-16 2018-08-28 Pioneer Hi-Bred International, Inc. elemento de silenciamento, construto de dna, construto de expressão, cassete de expressão, célula hospedeira, composição, célula vegetal, planta ou parte de planta, semente transgênica, método para controlar um inseto-praga de planta, kit para controlar insetos-praga
CN116003550A (zh) 2015-08-06 2023-04-25 先锋国际良种公司 植物来源的杀昆虫蛋白及其使用方法
DK3341483T3 (da) 2015-08-28 2020-03-16 Pioneer Hi Bred Int Ochrobactrum-medieret transformation af planter
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Also Published As

Publication number Publication date
AU2963695A (en) 1996-02-09
HUT77112A (hu) 1998-03-02
CA2192550A1 (fr) 1996-01-25
ZA955629B (en) 1997-01-06
WO1996001905A1 (fr) 1996-01-25
BR9510174A (pt) 1997-11-04
MX9606384A (es) 1997-03-29

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