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WO1998046766A1 - Acides nucleiques codant une enzyme de plante jouant un role dans la synthese d'acides gras a tres longues chaines - Google Patents

Acides nucleiques codant une enzyme de plante jouant un role dans la synthese d'acides gras a tres longues chaines Download PDF

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Publication number
WO1998046766A1
WO1998046766A1 PCT/CA1998/000343 CA9800343W WO9846766A1 WO 1998046766 A1 WO1998046766 A1 WO 1998046766A1 CA 9800343 W CA9800343 W CA 9800343W WO 9846766 A1 WO9846766 A1 WO 9846766A1
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WO
WIPO (PCT)
Prior art keywords
nucleic acid
sequence
cutl
seq
leu
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Ceased
Application number
PCT/CA1998/000343
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English (en)
Inventor
Ljerka Kunst
Anthony A. Millar
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University of British Columbia
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University of British Columbia
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Filing date
Publication date
Application filed by University of British Columbia filed Critical University of British Columbia
Priority to EP98916693A priority Critical patent/EP0975767A1/fr
Priority to AU70191/98A priority patent/AU750707C/en
Priority to CA002285970A priority patent/CA2285970A1/fr
Publication of WO1998046766A1 publication Critical patent/WO1998046766A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • C12N15/8247—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 involving modified lipid metabolism, e.g. seed oil composition
    • 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
    • C12N15/8223—Vegetative tissue-specific promoters
    • 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
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • C12N15/8289—Male sterility
    • 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.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)

Definitions

  • waxes form the outermost layer of the aerial portion of the plant and are thus the first line of interaction between the plant and its environment.
  • the physical properties of this wax layer protect the plant from numerous environmental stresses. For example, the hydrophobic nature of wax prevents dehydration (nonstomatal water loss) and aids in shedding rainwater.
  • the reflective nature of wax protects the plant against UV radiation (Reicosky and Hanover, 1978). Waxes are also known to protect against acid rain (Percy and Baker, 1990) and, because they are a good solvent for organic pollutants, they are able to impede the uptake of aqueous foliar sprays (Schreiber and Schonherr, 1992).
  • the present invention provides nucleic acids (cDNAs and genomic clones) that encode a key enzyme in the synthesis of VLCFAs in plant epidermal cells.
  • the activity of this enzyme is referred to as very long chain fatty acid elongase; the activity is required for synthesis of VLCFAs of greater than 24 carbons in length. It is shown that co-suppression of the CUT1 gene in plants can disrupt VLCFA synthesis which results in plants having none of the protective wax usually found on stem surfaces.
  • such plants are conditionally male sterile: when grown under normal humidity, the plants are male sterile, but fertility can be restored by growth in an elevated humidity environment.
  • sequence identity with the Arabidopsis CUTl amino acid sequence determined by this method.
  • homologs will possess at least 75% and more preferably at least 85% and more preferably still at least 90% or 95% sequence identity over short windows of 10-20 amino acids. Methods for determining sequence identity over such short windows are described at http://www.ncbi.nlm.nih.gov/BLAST/blast FAQs.html.
  • Homologs having the sequence identities described above will, in some embodiments, also possess VLCFA elongase activity.
  • sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided.
  • the present invention provides not only the peptide homologs are described above, but also nucleic acid molecules that encode such homologs.
  • nucleic acid molecules are substantially homologous
  • stringent conditions are sequence dependent and are different under different environmental parameters. Generally, stringent conditions are selected to be about 5°C to 20°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Conditions for nucleic acid hybridization and calculation of stringencies can be found in Sambrook et al. (1989) and Tijssen (1993). Hybridization conditions and stringencies are further discussed below.
  • the length of the antisense sequence in the vector will be greater than 100 nucleotides.
  • Transcription of an antisense construct as described results in the production of RNA molecules that are the reverse complement of mRNA molecules transcribed from the endogenous CUTl gene in the plant cell. Although the exact mechanism by which antisense RNA molecules interfere with gene expression has not been elucidated, it is believed diat antisense RNA molecules bind to the endogenous mRNA molecules and thereby inhibit translation of the endogenous mRNA.
  • modification of VLCFA synthesis in plant cells can be achieved by transforming plants with CUTl nucleic acids, antisense constructs based on CUTl nucleic acid sequences or otiier variants on CUTl nucleic acid sequences.
  • CUTl cDNA and genomic sequences the creation of variants on tiiese CUTl nucleic acid sequences by standard mutagenesis techniques is now enabled.
  • T m represents the temperamre above which, under the prevailing ionic conditions, the radiolabeled probe molecule will not hybridize to its target DNA molecule.
  • the T m of such a hybrid molecule may be estimated from the following equation (Bolton and McCarthy, 1962):
  • T m 81.5°C - 16.6(log 10 [Na + ]) + 0.41(%G+C) - 0.63(% formamide) - (600/t)
  • the mutation per se need not be predetermined.
  • random mutagenesis may be conducted at die target codon or region and die expressed protein variants screened for die optimal combination of desired activity.
  • Techniques for making substitution mutations at predetermined sites in DNA having a known sequence as described above are well known. Amino acid substitutions are typically of single residues; insertions usually will be on the order of about from 1 to 10 -imino acid residues; and deletions will range about from 1 to 30 residues.
  • Plant tissue was immersed for 10 seconds in a 2:1 chloroform:methanol solution to remove surface waxes. Extracts were then evaporated to dryness under a stream of nitrogen. Waxes were dissolved in 100 ⁇ l of N.O- bis(Trimethylsilyl)trifluoroacetamide with 1% Trimethylchlorosilane (Pierce), and derivatized at 80 °C for 1 hour. Samples were analyzed in a Hewlett-Packard 5890 series II gas chromatograph equipped with a flame ionization detector, using either a DB-1 column or a DB-5 column.

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

Abstract

Molécules d'acides nucléiques codant une enzyme jouant un rôle dans l'élongation d'acides gras à très longues chaînes (VLCFA) dans des plantes. L'invention englobe un ADNc, un clone génomique et une protéine codée, ainsi que des plantes possédant une composition modifiée de VLCFA, telle que des cires épicuticulaires modifiées, et des procédés d'élaboration de ces plantes.
PCT/CA1998/000343 1997-04-14 1998-04-14 Acides nucleiques codant une enzyme de plante jouant un role dans la synthese d'acides gras a tres longues chaines Ceased WO1998046766A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP98916693A EP0975767A1 (fr) 1997-04-14 1998-04-14 Acides nucleiques codant une enzyme de plante jouant un role dans la synthese d'acides gras a tres longues chaines
AU70191/98A AU750707C (en) 1997-04-14 1998-04-14 Nucleic acids encoding a plant enzyme involved in very long chain fatty acid synthesis
CA002285970A CA2285970A1 (fr) 1997-04-14 1998-04-14 Acides nucleiques codant une enzyme de plante jouant un role dans la synthese d'acides gras a tres longues chaines

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US4383197P 1997-04-14 1997-04-14
US60/043,831 1997-04-14
US95894798A 1998-04-10 1998-04-10
US09/958,947 1998-04-10

Publications (1)

Publication Number Publication Date
WO1998046766A1 true WO1998046766A1 (fr) 1998-10-22

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CA (1) CA2285970A1 (fr)
WO (1) WO1998046766A1 (fr)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001011061A3 (fr) * 1999-08-04 2001-06-07 Univ British Columbia Regulation de la transcription embryonnaire dans des plantes
WO2001090364A3 (fr) * 2000-05-24 2002-06-13 Univ British Columbia Acide nucleique codant un enzyme biosynthetique d'acide gras possedant une chaine tres longue et appartenant a une plante
WO2001090386A3 (fr) * 2000-05-24 2002-06-20 Univ British Columbia Region regulatrice de genes promouvant une transcription precoce specifique de graines
WO2001090387A3 (fr) * 2000-05-24 2002-06-27 Univ British Columbia Region regulatrice de gene qui favorise la transcription specifique de la racine et utilisation de cette region
WO2002008403A3 (fr) * 2000-07-25 2003-01-16 Calgene Llc Sequences d'acide nucleique codantes pour la beta-cetoacyl-acp synthase et utilisation de celles-ci
US6713664B2 (en) 2000-06-08 2004-03-30 Miami University Fatty acid elongase 3-ketoacyl CoA synthase polypeptides
US6784342B1 (en) 1999-08-04 2004-08-31 The University Of British Columbia Regulation of embryonic transcription in plants
US6849435B2 (en) 2000-07-18 2005-02-01 Bayer Aktiengesellschaft Use of VLCFAE for identifying herbicidally active compounds
WO2006066859A1 (fr) * 2004-12-20 2006-06-29 Basf Plant Science Gmbh Molecules d'acides nucleiques codant des polypeptides de type kcs et procedes d'utilisation
US7301070B2 (en) 1997-04-11 2007-11-27 Calgene Llc Plant fatty acid synthases and use in improved methods for production of medium-chain fatty acids
CN113583990A (zh) * 2021-06-04 2021-11-02 西南大学 水稻全育期半矮化表型调控基因sd38及其应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005052162A1 (fr) * 2003-11-25 2005-06-09 National Research Council Of Canada Genes elongase d'acide gras (fae) et leur utilite dans l'augmentation de l'acide erucique et autres proportions d'acide gras a tres longue chaine dans l'huile de graines

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995015387A2 (fr) * 1993-11-30 1995-06-08 Calgene, Inc. Sequences d'acide nucleique codant une proteine cytoplasmique de plante impliquee dans le metabolisme d'acyle gras-coa
DE4433307A1 (de) * 1994-09-19 1996-03-21 Norddeutsche Pflanzenzucht Han Ein isoliertes Nuckleinsäurefragment und daraus abgeleitete Produkte
WO1996013582A1 (fr) * 1994-10-26 1996-05-09 Dna Plant Technology Corporation Les genes fae1 et leurs utilisations

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995015387A2 (fr) * 1993-11-30 1995-06-08 Calgene, Inc. Sequences d'acide nucleique codant une proteine cytoplasmique de plante impliquee dans le metabolisme d'acyle gras-coa
DE4433307A1 (de) * 1994-09-19 1996-03-21 Norddeutsche Pflanzenzucht Han Ein isoliertes Nuckleinsäurefragment und daraus abgeleitete Produkte
WO1996013582A1 (fr) * 1994-10-26 1996-05-09 Dna Plant Technology Corporation Les genes fae1 et leurs utilisations

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
EVENSON K. AND POST-BEITTENMILLER D.: "Fatty acid-elongation activity in rapidly expanding leek epidermis", PLANT PHYSIOLOGY, vol. 109, 1995, pages 707 - 716, XP002072592 *
NEWMAN T. ET AL.: "AC T22193", EMBL DATABASE, 27 June 1994 (1994-06-27), HEIDELBERG, XP002072575 *
NEWMAN T. ET AL.: "AC T76616", EMBL DATABASE, 25 March 1995 (1995-03-25), HEIDELBERG, XP002072576 *
SOHAL A. AND JENKINS G.: "Epidermal-specific gene expression in Brassica and Arabidopsis", PLANT PHYSIOLOGY SUPPLEMENT, vol. 111, no. 2, June 1996 (1996-06-01), pages 168, XP002072577 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7301070B2 (en) 1997-04-11 2007-11-27 Calgene Llc Plant fatty acid synthases and use in improved methods for production of medium-chain fatty acids
WO2001011061A3 (fr) * 1999-08-04 2001-06-07 Univ British Columbia Regulation de la transcription embryonnaire dans des plantes
US6784342B1 (en) 1999-08-04 2004-08-31 The University Of British Columbia Regulation of embryonic transcription in plants
WO2001090364A3 (fr) * 2000-05-24 2002-06-13 Univ British Columbia Acide nucleique codant un enzyme biosynthetique d'acide gras possedant une chaine tres longue et appartenant a une plante
WO2001090386A3 (fr) * 2000-05-24 2002-06-20 Univ British Columbia Region regulatrice de genes promouvant une transcription precoce specifique de graines
WO2001090387A3 (fr) * 2000-05-24 2002-06-27 Univ British Columbia Region regulatrice de gene qui favorise la transcription specifique de la racine et utilisation de cette region
US6713664B2 (en) 2000-06-08 2004-03-30 Miami University Fatty acid elongase 3-ketoacyl CoA synthase polypeptides
US6849435B2 (en) 2000-07-18 2005-02-01 Bayer Aktiengesellschaft Use of VLCFAE for identifying herbicidally active compounds
WO2002008403A3 (fr) * 2000-07-25 2003-01-16 Calgene Llc Sequences d'acide nucleique codantes pour la beta-cetoacyl-acp synthase et utilisation de celles-ci
US7371924B2 (en) 2000-07-25 2008-05-13 Calgene Llc Nucleic acid sequences encoding β-ketoacyl-ACP synthase and uses thereof
WO2006066859A1 (fr) * 2004-12-20 2006-06-29 Basf Plant Science Gmbh Molecules d'acides nucleiques codant des polypeptides de type kcs et procedes d'utilisation
CN113583990A (zh) * 2021-06-04 2021-11-02 西南大学 水稻全育期半矮化表型调控基因sd38及其应用

Also Published As

Publication number Publication date
AU750707C (en) 2003-05-15
AU7019198A (en) 1998-11-11
AU750707B2 (en) 2002-07-25
CA2285970A1 (fr) 1998-10-22

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