WO2007004694A1 - Gène innovant impliqué dans la biosynthèse de l'acide pétrosélinique et procédé de production d'acide pétrosélinique - Google Patents

Gène innovant impliqué dans la biosynthèse de l'acide pétrosélinique et procédé de production d'acide pétrosélinique Download PDF

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WO2007004694A1
WO2007004694A1 PCT/JP2006/313449 JP2006313449W WO2007004694A1 WO 2007004694 A1 WO2007004694 A1 WO 2007004694A1 JP 2006313449 W JP2006313449 W JP 2006313449W WO 2007004694 A1 WO2007004694 A1 WO 2007004694A1
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gene
acid
seq
protein
amino acid
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PCT/JP2006/313449
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Japanese (ja)
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Yukio Okamura
Yoko Yamanaka
Nobuhiko Muramoto
Ikuo Nishida
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Toyota Jidosha Kabushiki Kaisha
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Priority to CN2006800241478A priority Critical patent/CN101213298B/zh
Priority to AU2006266760A priority patent/AU2006266760B2/en
Priority to CA2614062A priority patent/CA2614062C/fr
Priority to JP2007523443A priority patent/JP4840360B2/ja
Publication of WO2007004694A1 publication Critical patent/WO2007004694A1/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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • 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
    • 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.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0083—Miscellaneous (1.14.99)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids

Definitions

  • a novel gene involved in petrothelinic acid biosynthesis a method for producing petrothelic acid
  • the present invention relates to novel genes involved in biosynthesis of petrothelinic acid, in particular, ⁇ 4 -palmitoinole-ACP desaturase gene derived from carrot, and petrocerinoyl-ACP thioesterase gene derived from plant.
  • the present invention relates to a process for producing petrothelic acid using
  • Nylon a type of engineering plastic, is synthesized by polymerizing aminocarboxylic acid or diamine and dicarboxylic acid as raw materials, but most of the raw monomers are produced from fossil resources in the chemical industry.
  • Sebacic acid (1, 10-decanedioic acid) is used as a biomass-derived nylon raw material, which is produced by cleaving castor oil extracted from castor with a caustic alkali. It becomes a raw material.
  • nylon 6 and 10 is limited, and it is not widely used as a resin material.
  • Non-Patent Documents 1 to 8 The production technology of cis-6-octadecenoic ac id has been studied.
  • Coriander and carrots of the Apiaceae plants contain more than 80% of petroceric acid in the oil components of seeds, but they are not suitable for the production of betrothelic acid due to low seed yield.
  • Plant fatty acids are newly synthesized in plastids (chloroplasts).
  • chloroplasts the precursor palmitoyl ACP was converted to ci s-4-hexadecenoil ACP by ⁇ 4-palmitoyl-ACP desaturase (hereinafter referred to as 4DES). Later, the chain length was extended by the prokaryotic fatty acid synthase complex to be petrocellinoyl-ACP.
  • PTE petrocellinol-ACP thioesterase
  • Plants that synthesize petroceric acid are thought to contain a series of biosynthetic enzymes specific to petroceric acid synthesis and their genes.
  • ⁇ 4-palmitoyl-ACP desaturase derived from coriander The gene was cloned, and originally a transgenic plant was introduced in which the gene was introduced into Arabidopsis thaliana, which does not produce petrothelinic acid. Accumulated amount was only about 1% in seed oil.
  • Non-patent Document 8 Although PTE has been shown to have enzyme activity (Non-patent Document 8), the gene has not yet been cloned.
  • Plant fat production and its constituent fatty acid composition involve not only fatty acid biosynthesis in plastids, but also transport of fatty acid derivatives in the cytoplasm and triacylglycerol synthesis in the endoplasmic reticulum membrane.
  • fatty acid derivatives in the cytoplasm and triacylglycerol synthesis in the endoplasmic reticulum membrane.
  • Patent Literature 1 United States Patent 5, 430, 134
  • Patent Document 2 International Publication No. 94/01565
  • Non-Patent Document 1 Proc. Natl. Acad. Sci. USA, 89, 11184-11188,
  • Non-Patent Document 2 Plant J., 17 (6), 679-688, 1999.
  • Non-Patent Document 3 Prog. Lipid Res., 33 (1/2), 155-163, 1994
  • Non-Patent Document 4 Plant Phys iol., 124, 681-692, 2000
  • Non-Patent Document 5 Plant Mol. Biol., 47, 507-518, 2001
  • Non-Patent Document 8 Plant Phys iol., 104, 839-844, 1994
  • Non-Patent Document 9 Planta 215: 584-595 2002. Disclosure of the Invention
  • the present invention can promote the accumulation of petrothelic acid. It is an object of the present invention to provide a novel gene and a method for producing petrothelic acid using the gene.
  • ⁇ 4 -panoremitoinole-ACP desaturase derived from cindu (aucus caro a) belonging to the family Aceraceae is a ⁇ 4-palmitoyl-ACP desaturase derived from Korean.
  • cindu aucus caro a
  • Aceraceae is a ⁇ 4-palmitoyl-ACP desaturase derived from Korean.
  • the present inventor has succeeded in isolating a petroselinol-ACP thioesterase gene newly, and when this gene is used in combination with a ⁇ 4-palmitoyl-ACP desaturase gene, the inventor has the ability to synthesize petrocellinic acid.
  • the present invention includes the following.
  • a protein having ⁇ 4-palmitoyl-ACP desaturase activity including an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2
  • a protein having ⁇ 4 ”palmitoyl-ACP desaturase activity is expressed by cis-4-hexadecenoic acid ( Can increase the accumulation of cis-4-Hexadecenoic aci d) or petroselinic acid (cis-6-octadecenoic acid) or cis_8-icosenoic acid It can be paraphrased as protein.
  • a protein having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 4 or 6, and having petrocellinol-ACP tiosterase activity (c) A protein having a petro-cerinoyl-ACP thioesterase activity, encoded by DNA that is hybridized under stringent conditions to DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 3 or 5.
  • the protein having petroselinol-ACP thioesterase activity is expressed simultaneously with a protein having ⁇ 4-palmitole-ACP desaturase activity in plant cultured cells or individual plants.
  • Ci s_4-hexadecenoic acid ci s-4-Hexadecenoic acid
  • petroselinic acid ci s_6 -octadecenoic acid (cis -6-octadecenoic acid)
  • cis-8-icosenoic acid It can be rephrased as a protein capable of increasing the amount.
  • FIG. 1 is a diagram comparing the homology (%) of the amino acid sequences of various petrocellinol-ACP thioesterases and oleoyl-ACP thioesterases.
  • DcPTE means petroselinol-ACP thioesterase derived from carrot Da s carota
  • CsPTE means coriander coriand sa ti vwn, derived from petrocellino.il-ACP thioesterase
  • AgPTE stands for dinore ne thum gra veolens.
  • CsOTE means oleoinole-ACP thioesterase from coriandrum (Coriandrum sa ti vum)
  • DcOTE means reoil-ACP thioesterase from carrot Daucus car ota) .
  • FIG. 2-1 shows the alignment of the amino acid sequences of various betacellinoyl-ACP thioesterases and oleoyl-ACP thioesterases.
  • Fig. 2-2 shows the alignment of amino acid sequences of various betacellinoyl-ACP thioesterases and oleoreyl-ACP thioesterases.
  • FIG. 2 is a diagram showing alignment of amino acid sequences of ⁇ 4-palmitoyl-ACP desaturase (referred to as Cs4DES) derived from the amino acid ⁇ S ⁇ ⁇ and coriander (referred to as Dc4DES) of Corse.
  • Cs4DES ⁇ 4-palmitoyl-ACP desaturase
  • Dc4DES coriander
  • FIG. 4 shows purified petroselinol-derived ACP thioesterase from carrot Daucus carot (referred to as DcPTE), oleoyl-ACP thioesterase from coriandrum sa ti vum (referred to as CsOTE) and carrot Daucus carota)
  • DcPTE oleoyl-ACP thioesterase from coriandrum sa ti vum
  • CsOTE oleoyl-ACP thioesterase from coriandrum sa ti vum
  • DcOTE oleoyl-ACP thioesterase from coriandrum sa ti vum
  • FIG. 5 is a photograph showing the results of SDS-PAGE after reacting purified ATP with DcPTE and DcOTE histidine-labeled protein eluate using asil ACP as a substrate.
  • FIG. 6 is a characteristic diagram showing the results of quantifying the bands included in the photograph shown in FIG. 5 using image analysis software.
  • FIG. 7 is a block diagram schematically showing the constant systemic expression vector prepared in the example.
  • FIG. 8 is a block diagram schematically showing the seed-specific development vector prepared in the example.
  • FIG. 9 shows the results of fatty acid composition analysis in the seeds of the transformed plant.
  • the Dc4DES gene encodes a protein (Dc4DES) containing the amino acid sequence shown in SEQ ID NO: 2.
  • Dc4DES is a protein with active individuals that does not contain the ⁇ 4 position in palmitoyl-ACP (palmitoyl-acyl carrier protein), which is a saturated fatty acid having 16 carbon atoms.
  • a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 is shown in SEQ ID NO: 1.
  • the Dc4DES gene comprises an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2, and has ⁇ 4 -palmitoyl-ACP desaturase activity It may be one that encodes a protein.
  • “a plurality of amino acids” means 2 to 150, preferably 2 to 80, more preferably 2 to 40.
  • the region to be deleted, substituted or added is not particularly limited. For example, the region from 1 to 99 or 270 to 386, preferably from 1 to 99 or 301 to 386 in the amino acid sequence shown in SEQ ID NO: 2. The region is more preferably the 1st to 53rd region or the 381st to 386th region.
  • the Dc4DES gene is composed of an amino acid sequence having a homology of 50% or more, preferably 70% or more, more preferably 90% or more in the amino acid sequence represented by SEQ ID NO: 2, and ⁇ 4 -A gene encoding a protein having palmitoyl-ACP desaturase activity.
  • the numerical value of the above homology is obtained by executing a command of the Maxim matching method, for example, using DNA analysis (Hitachi Software Engineering), which is sequence analysis software.
  • the parameters at that time are the default settings (initial settings). '
  • the Dc4DES gene is encoded by DNA that hybridizes under stringent conditions to DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 1, and ⁇ 4-palmitoyl- It may be a gene encoding a protein having ACP desaturase activity.
  • “hyperpridation under stringent conditions” means, for example, that after heating at 42 ° C. in a solution of 6 XSSC, 0.5% SDS, and 50% formamide, 0. 1 Indicates that a positive hybridization signal is still observed even under conditions of washing at 68 ° C in a solution of 1 XSSC and 0.5% SDS.
  • ⁇ 4 -palmitoyl-ACP desaturase activity is a function of palmitoyl-ACP.
  • the presence or absence of this activity can be determined by analyzing the DNA fragment encoding the protein to be assayed without ac is 4-hexadecenoic acid, petroceric acid, ci S-8-icosenoic acid such as Arabidopsis thaliana. Cis-4-hexadecenoic acid, petroceric acid, cis-8- in the lipids of the introduced plant. It can be assayed by measuring the presence or absence of icosenoic acid.
  • ci s- Transform plant cells that do not accumulate 4-hexadecenoic acid, petroceric acid, or cis-8-icosenoic acid extract the lipids of the produced transformed plant cells or the seeds of the regenerated plants.
  • Fatty acid methyl ester is obtained by treating the extracted lipids with methanolic hydrochloric acid, etc., and the amount of methyl ester of petrocelic acid, the amount of fatty acid methyl ester of cis-4-hexadecenoic acid, the amount of fatty acid methyl ester of cis-8-icosenoic acid Is measured by gas chromatography or the like. If these fatty acid methyl esters can be detected, the protein to be assayed has ⁇ 4-palmitoyl-ACP desaturase activity, and if these fatty acid methyl esters cannot be detected, it has ⁇ 4-palmitoyl-ACP desaturase activity. I can say no.
  • the Dc4DES gene has a function of promoting the synthesis of petroselinic acid in the cell by being introduced into the host plant cell in a functional manner.
  • a plant cell is transformed using an expression vector in which the Dc4DES gene is arranged downstream of a constitutive expression promoter or a specific expression promoter, that is, at a position controllable by the promoter. To do.
  • the specific expression promoter it is preferable to use a seed specific expression promoter.
  • petroselinic acid can be accumulated in the seed.
  • petroseric acid is pulverized in tissues such as seeds and mixed with a hydrochloric acid-methanol solution to methyl esterize petroceric acid.
  • Hexane extraction can be performed and the hexane extract can be detected with a gas chromatograph-mass spectrometer (GC-MS) instrument. Based on the results of detection using a GC-MS instrument, the ability to promote the synthesis of petrothelic acid can be analyzed. .
  • GC-MS gas chromatograph-mass spectrometer
  • PTE gene is indicated in coriander (Gofiandrum sa ti vium). What has been excised but not isolated and cloned is a newly isolated and cloned gene in the present invention.
  • the PTE gene is a gene that encodes thioesterase (PTE), which is highly specific for the acyl ACP (acyl carrier protein) having a double bond at the ⁇ 6-position such as petrocellinol-ACP. PTE is involved in the production of free petrothelic acid.
  • Examples of the PTE gene include a gene encoding a carrot-derived PTE containing the amino acid sequence shown in SEQ ID NO: 4 or 6.
  • the gene encoding the protein consisting of the amino acid sequence shown in SEQ ID NO: 4 is shown in SEQ ID NO: 3, and the gene encoding the protein consisting of the amino acid sequence shown in SEQ ID NO: 6 is shown in SEQ ID NO: 5.
  • Carrot-derived PTE gene and PTE are referred to as DcPTE gene and DcPTE, respectively.
  • the DcPTE gene includes an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 4 or 6, and may encode a protein having activity.
  • a plurality of amino acids means 2 to 188, preferably 2 to 64, more preferably 2 to 44.
  • the region to be deleted, substituted or added is not particularly limited. For example, the region of 1 to 70 ′ or 311 to 375 in the amino acid sequence shown in SEQ ID NO: 4, preferably 1 to 57 or 368 to 375. Area, more preferably the 1st to 32nd areas.
  • the PTE gene encoding a protein containing an amino acid sequence into which the amino acid sequence shown in SEQ ID NO: 4 or 6 is substituted, deleted or inserted is a known technique such as the Kunkel method or the Gapped duplex method or the like. It can be obtained by adopting a method according to this and introducing a desired mutation into the nucleotide sequence shown in SEQ ID NO: 3 or 5.
  • a mutagenesis kit for example, Mutan-K (TAKARA) or Mutan-G (TAKARA)
  • LA PCR in by TAKARA Mutation is introduced using the in vitro Mutagenesis series kit.
  • the DcPTE gene is composed of an amino acid sequence having a homology of 50% or more, preferably 70% or more, more preferably 90% or more in the amino acid sequence represented by SEQ ID NO: 4 sentence 6; And Petrocellinoir -ACP It may be a gene encoding a protein having sterase activity.
  • the numerical value of the homology is obtained, for example, by executing a command of the Maxim matching method using DNASIS (Hitachi Soft-to-Air Engineering) which is a sequence analysis software. In this case, the parameters are set to the default settings (initial settings).
  • the DcPTE gene is encoded by DNA that hybridizes under stringent conditions to DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 3 or 5, and Petrocellinol-ACP It may be a gene encoding a protein having esterase activity.
  • “hybridizes under stringent conditions” means, for example, 0.1% after heating at 42 ° C. in a solution of 6 XSSC, 0.5% SDS and 50% formamide. This indicates that a positive hypritized signal is still observed even under conditions of washing at 68 ° C. in a solution of XSSC, 0.5% SDS.
  • Petrocellinol-ACP thioesterase activity refers to the activity of degrading petroselinol-ACP into petroselinic acid and ACP. The activity is 25 mM
  • Tris-HCl (pH 8.0), ImM DTT, Petrocerinol-ACP and water are mixed, preincubated for 5 minutes at 25 ° C, and the reaction solution containing 15 / _ig protein to be assayed 100 ⁇ 1 For 30 minutes at 25 ° C.
  • unreacted petrolinoyl-ACP and reaction product free ACP are separated by SDS-PAGE.
  • the gel after electrophoresis is stained with CBB, and then the amount of free ACP, which is the reaction product, is quantified with a densitometer by SDS-PAGE, and the thiesterase activity of the added protein to be assayed is measured.
  • the PTE gene is a gene encoding a carrot-derived PTE.
  • a plant-derived PTE gene that biosynthesizes petroceric acid.
  • plants that biosynthesize petroceric acid include Corianda-"" y Corian drum sa tivium), /, Seri Petroseli um crispurn) "Inore ⁇ Anethum graveolens), etc., Ivy, Hedera helix, ralia ela td), and the like.
  • all or part of the 187th to 1128th region in the base sequence of the DcPTE gene shown in SEQ ID NO: 3 or 5 can be used as a probe.
  • long nucleic acid sequences > lOObp
  • they can also be screened by moderate stringency to obtain signals from target samples that are more than 80% homologous. it can.
  • the probe can be quite short.
  • oligonucleotides may be used, but should be at least about 10, preferably at least about 15, and more preferably 20 nucleotides. When short regions are used for probes, a higher degree of sequence identity is required than for longer probes.
  • the PTE gene can be isolated and identified from the genomic DNA of the plant.
  • genomic DNA mRNA extracted from the plant was synthesized as a cocoon.
  • the cDNA can also be used to isolate and identify the PTE gene of the plant. Based on the nucleotide sequence of the DcPTE gene shown in SEQ ID NO: 3 or 5, isolated from coriander (Cor Ian drum sativium) and vinore, Arw thwn graveolens), the sequences of the 7 DcPTE homologous genes were sequenced respectively. The numbers 7 and 9 are shown.
  • the amino acid sequence (CsPTE) deduced from the coriander-derived PTE gene shown in SEQ ID NO: 7 is shown in SEQ ID NO: 8, and the amino acid sequence (AgPTE) deduced from the dill-derived PTE gene shown in SEQ ID NO: 9 is sequenced The number 10 is shown.
  • Figure 1 shows the amino acid homology (%) between proteins in the thioesterase of various plants.
  • Fig. 1 shows high similarity in the PTE group including DcPTE, CsPTE and AgPTE. It also shows high homology between DcOTE and CsOTE. In contrast, the PTE group and the 0TE group show relatively low homology. Therefore, this It can be said that a novel protein whose amino acid homology with PTE proteins included in these PTE groups exceeds 80% has a very high probability of being included in PTE groups. That is, the PTE gene according to the present invention has a homology of 80 ° / with the amino acid sequence shown in SEQ ID NO: 4, 6, 8 or 10. DNA encoding a protein containing an amino acid sequence exceeding the above will also be included.
  • Figure 2 shows the alignment of the amino acid sequences of DcPTE, CsPTE, AgPTE, DcOTE, and CsOTE.
  • 0TE and PTE There is about 30% amino acid difference between 0TE and PTE.
  • “common array number X (X is a natural number)” 'means the number given to the top of the alignment in FIG.
  • the amino acids of consensus sequences 120, 125, and 373 are non-polar, while in the 0TE group, the amino acid is polar.
  • the amino acids of the common sequences Nos. 140 and 195 are polar, and in the 0TE group, the amino acid is nonpolar.
  • the amino acids of the common sequences Nos. 149 and 246 are uncharged amino acids, while in the 0TE group, the amino acids are positively charged.
  • the amino acid of the common sequence No. 244 is negatively charged, while in the 0TE group, the amino acid is an uncharged amino acid.
  • the amino acid of the 270th common sequence is positively charged, while in the 0TE group, the amino acid is an amino acid having no charge.
  • the PTE gene is a host plant along with the ⁇ 4 -palmitoyl-ACP desaturase gene.
  • an expression vector is constructed in which the PTE gene is arranged downstream of a constitutive expression promoter or a specific expression promoter, that is, at a position controllable by the promoter.
  • the expression vector is used to transform plant cells (see 1. above) obtained by transforming the ⁇ 4-palmitoyl-ACP desaturase gene.
  • plant cells may be transformed using an expression vector comprising a PTE gene and a ⁇ 4-palmitoyl-ACP desaturase gene arranged downstream of a constitutive expression promoter or a specific expression promonitor. In any case, by cultivating the resulting transformed plant to give a plant body, it is possible to promote the synthesis of petolic acid and serine acid in the plant body.
  • petrothelic acid is pulverized in tissue such as seeds and mixed with a hydrochloric acid-methanol solution to methyl esterize petrothelic acid, Extract with hexane and detect the hexane extract with a gas chromatograph-mass spectrometer (GC-MS) instrument. Based on the results of detection by GC-MS, the ability to promote the synthesis of petrothelic acid can be analyzed.
  • tissue such as seeds
  • a hydrochloric acid-methanol solution to methyl esterize petrothelic acid
  • Extract with hexane Extract with hexane and detect the hexane extract with a gas chromatograph-mass spectrometer (GC-MS) instrument.
  • GC-MS gas chromatograph-mass spectrometer
  • the expression vector has the Dc4DES gene and / or the PTE gene described in 1 and 2 above.
  • the expression vector is not particularly limited as long as it is a plasmid vector, or a chromosomal transfer type vector that can be integrated into the genome of the host organism, for example, plasmid DNA, butteriophage DNA, retrotransposon. DNA and artificial chromosome DNA (YAC).
  • plasmid DNA examples include pRS413, pRS414, pRS415, pRS416, YCp50,
  • YCp-type E. coli-yeast shuttle vectors such as PAUR112 or pAUR123, pYES2 or
  • E. coli-yeast shuttle vector such as YEpl3, Yip type E. coli-yeast shuttle vector such as pRS403, pRS404, pRS405, pRS406 pAURlOl or pAUR135, plasmid derived from E.
  • coli pBR322, pBR325, pUC18, pUC19, pUC118, pUC119, pTVl
  • Wind pTV119N pBluescript, pHSG298, pHSG396 or ColE-based plasmids such as Trc99A, pACYC177 Or pl 5A-based plasmids such as pACYC184, pSClOl-based plasmids such as pMW118, pMW119, pMW218 or pMW219), agrobacterium-derived plasmids (eg, ⁇ ⁇ ⁇ ⁇ ), derived from Bacillus subtilis Plasmids (eg, pUB 110, pTP5, etc.), and phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, gt10, Agtll, ⁇ ZAP), ⁇ ⁇ 174, M13mpl 8 or M13mpl 9 and so on.
  • Retrotransposons include Ty factors.
  • Examples of YAC vectors include pYACC2.
  • animal viruses such as retrovirus or vaccinia virus, and insect virus vectors such as baculovirus can also be used.
  • the Dc4DES gene and / or the PTE gene must be incorporated into the vector in such a state that they can be expressed.
  • the expressible state means that these Dc4DES gene and / or PTE gene and promoter are linked so that the host organism into which the Dc4DES gene and / or PTE gene is introduced is expressed under the control of a predetermined promoter. Means to be incorporated into a vector.
  • the vector in addition to the Dc4DES gene and / or PTE gene, the vector includes a promoter and terminator, a cis-element such as an enhancer if necessary, a splicing signal, a poly A addition signal, a selection marker, a ribosome binding sequence (SD sequence) Etc. can be connected.
  • the selection marker include an ampicillin resistance gene, an antibiotic resistance gene such as a kanamycin resistance gene and a hygromycin resistance gene, and a herbicide resistance gene such as a bialaphos resistance gene.
  • the promoter contained in the expression vector is not particularly limited, and examples thereof include a constitutive expression promoter, a tissue-specific expression promoter, and a stimulus-inducible promoter.
  • seed-specific expression promoters include rapeseed napin A promoter, Arabidopsis FAE1 promoter, oleosin promoter 1, soybean-derived glutelin B 1 promoter, ama stearoyl-ACP desaturase (SAD) promoter, etc. be able to.
  • Transformants-Transformants can be produced using the expression vectors described above. That is, A transformant can be prepared by introducing the above-described expression vector into a host so that the Dc4DES gene and / or the PTE gene contained in the vector can be expressed.
  • the host is not particularly limited. For example, there are plants belonging to celery family, eggplant family, Brassicaceae family, Gramineae family, legume family, rose family, chrysanthemum family, lily family, dianthus family, cucurbitaceae family, convolvulaceae family, akaza family. In particular, it is desirable to use plants of the family Apiaceae or Aphranaceae. '
  • the transformed plant can be obtained as follows.
  • Plants to be transformed in the present invention include whole plants, plant organs (eg, leaves, petals, stems, roots, seeds, etc.), plant tissues (eg, epidermis, phloem, soft tissue, xylem, vascular bundle, It means any of a palisade tissue, a spongy tissue, etc.) or a plant cultured cell.
  • the expression vector can be introduced into a plant by a usual transformation method, for example, a reduced pressure infiltration method (agro-acterium method), a particle gun method, a PEG method, an electroporation method, or the like.
  • the reduced pressure infiltration method can be performed according to a known method (Shujunsha, Model Plant Experiment Protocol, 2001, 109-113 pp.).
  • the expression vector is introduced into an appropriate agrobacterium, for example, Agrobac teriwn twnefacien LBA4404 strain, and this strain is introduced into the leaf disk method (inner shrine).
  • Hirofumi Plant Genetic Manipulation Manual, 1990, 27-31pp, Kodansha Scientific, Tokyo), etc., it is possible to infect a sterile cultured leaf of a host (for example, tobacco) to obtain a transformed plant.
  • the plant body, the plant organ, and the plant tissue itself may be used as they are, or may be used after preparing a section, or may be used after preparing a protoplast.
  • the sample thus prepared can be processed using a gene transfer apparatus (eg, PDS-1000 (BI0-RAD)). Treatment conditions are different from plants or samples (normally, but at a pressure of about 450 to 2000 psi and a distance of about 3 to 12 cm.
  • Tumor tissues, shoots, hairy roots, etc. obtained as a result of transformation can be used as they are for cell culture, tissue culture or organ culture, and can be appropriately used by using conventionally known plant tissue culture methods. Concentration of plant hormones (auxin, site power Inine, gibberellin, abscisic acid, ethylene, brassinolide, etc.) can be regenerated into plants.
  • telomere length is a region of DNA sequence.
  • DNA is prepared from transformants and PCR is performed by designing DNA-specific primers. PCR can be performed under the same conditions as those used to prepare the plasmid. After that, the amplified product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., stained with bromide zyme, SYBR Green solution, etc., and the amplified product is detected as a single band. It can be confirmed that it has been transformed.
  • amplification products can be detected by performing PCR using primers previously labeled with a fluorescent dye or the like. Furthermore, it is possible to adopt a method in which the amplification product is bound to a solid phase such as a microplate and the amplification product is confirmed by fluorescence or enzyme reaction.
  • Escherichia coli such as Escherichia coli
  • Bacillus subtilis Bacillus subtilis
  • Belonging to the genus Rhizobium, Saccharomyces cerevisiae, Nzo Saccharose honobe animal cells such as COS cells and CH0 cells, and insect cells such as Sf9.
  • the recombinant vector is capable of autonomous replication in the bacterium, and at the same time is composed of a ribosome binding sequence, the gene of the present invention, and a transcription termination sequence.
  • Escherichia coli a bacterium such as Escherichia coli
  • Bacillus subtilis examples include, but are not limited to, Bacillus subtilis 03 ⁇ 4cj'JZ).
  • the method for introducing a recombinant vector into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria.
  • a method using calcium ions [Cohen, SN et al .: Proc. Natl. Acad. Sci., USA, 69: 2110 (1972)], electroporation method and the like can be mentioned.
  • yeast for example, Saccharomyces cerevisiae
  • Schizosaccharomyces ⁇ ⁇ , Nobe, Schizosaccharomyces pombe) H. pastoris ( ⁇ ioris) and the like are used.
  • the method for introducing a recombinant vector into yeast is not particularly limited as long as it is a method for introducing DNA into yeast.
  • the Electroporation Yoshi method [Becker, DM et al .: Methods. Enzymol., 194: 182 (1990)]
  • Spheroplast method [Hinnen, A. et al .: Proc. Natl. Acad. Sci., USA, 75: 1929 (1978)]
  • lithium acetate method [Itoh, H .: J. Bacteriol., 153: 163 (1983)].
  • monkey cells COS-7, Vero, Chinese hamster ovary cells (CH0 cells), mouse L cells, rat GH3, and human FL cells are used.
  • Examples of the method for introducing a recombinant vector into an animal cell include an electro-poration method, a calcium phosphate method, and a ribofunction method.
  • Sf9 cells When insect cells are used as hosts, Sf9 cells are used.
  • methods for introducing the recombinant vector into insect cells include the calcium phosphate method, the lipofussion method, and the electroporation method.
  • the synthesis of petroselinic acid can be promoted by the function of the introduced Dc4DES gene and / or PTE gene.
  • Petroselinic acid synthesized and accumulated in the transformed plant can be extracted using a conventionally known method. .
  • the fats and oils produced by these processes are a mixture of fatty acids (including petrothelic acid) and esters such as glycerin, and include triacylglycerol, diacylglycerol, monoacylglycerol, phospholipids, and the like.
  • fatty acids can be obtained by hydrolyzing these fats and oils. Obtained fatty acids By separating and purifying the mixture, it is possible to obtain highly pure petroceric acid.
  • an alcohol ester such as petroleum acid methyl ester can be obtained by adding and reacting an alcohol such as methanol to the oil.
  • carrot Daucus car ota L.) summer sowing fresh red 5 inch (F1 variety) was used as an experimental sample.
  • F1 variety a fixed species having the same homologous gene sequence is desirable, but F1 varieties were used for the convenience of experiments (prepared samples that flowered early).
  • Carrot seeds were purchased from Ota seedlings.
  • a sample of a ginjin plant was cultivated under conditions of 25 ° C, 16 hours of sunshine, and 60% humidity in an artificial meteorological device (Kitotron, manufactured by Koito Manufacturing Co., Ltd.).
  • Corlasta one (Coriandrum sa ti vum L.) ⁇ 4 palmitoyl— ACP desaturase (Cs4DES) at BLAST (htt P : // w w. Ncbi. Nlra. Nih. Gov / BLAST /) in NCBI site ) Homologous genes of), and the polypeptides encoded by the obtained Cs4DES gene (GenBank accession number M93115) and the ⁇ 9 stearoyl-ACP desaturase gene of various plants registered on GenBank.
  • the amino acid sequence of Genetyx-Win Ver The amino acid sequence of Genetyx-Win Ver,
  • PR2 5 'GCN GYY KCR TGN C Y TTY TCR TC 3' (SEQ ID NO: 13)
  • NR2 5 'GGC ATN DVD AYY TTB WTY YTC ATC AT 3' (SEQ ID NO: 16)
  • R represents A or G
  • Y represents C or T
  • M represents A or C
  • K represents G or T
  • S represents G or C
  • W represents A or T H represents A or T or C
  • B represents G or T or C
  • V represents G or A or C
  • D represents G or A or T
  • N represents A or C or G or T Indicates.
  • RT-PCR was performed using the PCR primer pairs shown in Table 1.
  • QIAGEN's One-step RT-PCR kit was used. The reaction solution composition was carried out using an Eppendorf thermal cycler (master cycler's gradient) according to the protocol attached to the kit. The firing temperature was 50 ° C to 70 ° C (5 steps every 5 ° C).
  • RT-PCR was performed at 50 ° C for 30 minutes, 94 ° C for 15 minutes, followed by a cycle of 94 ° C for 1 minute, 50-70 ° C for 1 minute, and 72 ° C for 1 minute 30 seconds. The treatment was performed for 40 cycles, followed by treatment at 72 ° C for 15 minutes. After the reaction, 4 ° C was maintained.
  • the reaction solution after PCR was electrophoresed with TAE buffer using agarose gel. After electrophoresis, the agarose gel was stained with ethimumubu amide to confirm the target fragment. The target fragment was cut out with a scalpel together with the gel, and eluted and purified from the gel using a QIAGEN QIAquick Gel extract ion kit. The base sequence of the purified PCR product was confirmed using an ABI DNA sequencer (3100 Genetic Analyzer). For the sequence reaction, ABI BigDye Terminator Cycle Sequencing, FS kit (ver. 3.0) was used. The experimental procedure was performed according to the ABI manual. The primers listed in Table 1 were used for determining the base sequence.
  • D2-R2 5 'GCG GTT CTC CTC AGC AGT C 3' (SEQ ID NO: 17)
  • D2-R3 5 'GTT GGC ATG GGA GAT GAA TG 3' (SEQ ID NO: 1 8)
  • D2-F5 5 'CAG CAG ATT GGA GTC TAC TC 3' (SEQ ID NO: 20)
  • the 5 ′ RACE method and the 3 ′ RACE method were performed using Roche ⁇ / 3 ′ RACE Kit. PCR was performed using Ex Taq Hot Start Version manufactured by Takara Bio. The composition of the reaction solution was in accordance with the protocol attached to the kit. An Eppendorf thermal cycler (Master Ikura I. Gradient) was used. Annealing temperature from 50 ° C
  • PCR conditions were 94 ° C for 15 minutes, followed by 30 cycles of 94 ° C for 1 minute, 50-70 ° C for 1 minute and 72 ° C for 1 minute 30 seconds, and then 72 ° C. Treated with C for 15 minutes. After the reaction, 4 ° C was maintained.
  • the PCR product was purified and the nucleotide sequence was determined in the same manner as described above.
  • the following primers were designed to amplify and clone the entire polypeptide coding region based on the sequence information determined by the RACE method.
  • Polypeptide coding region Amplification PCR primers with the following restriction enzyme sites (BamHI and SacI) for introduction into the plant expression vector pBI121 were also prepared.
  • Sac- 4DS- R-0R1 5 'TCT AGA CGA GCT CTC ATA TCA TGA TCT GAC GGT TG 3' (Column number 2 4)
  • PCR was performed using these primers to amplify the entire polypeptide coding region.
  • DNA Ligat ion kit ver. 2 from Takara Bio
  • the DNA fragment of the polypeptide coding region and the TA-Cloning (for cloning PCR products) vector (Novagen pSTBlue) ligation (16 ° C, ⁇ reaction).
  • a T0Y0B0 cell (.co'DH5) was transformed according to the attached protocol, cultured in LB medium supplemented with IPTG, X-gal, and 50 g / ral kanamycin, and a transformant was selected.
  • Plasmid DNA was prepared using QIAGEN's Plasmid mini kit from the cells obtained by liquid culture in LB medium supplemented with 50 / g / ml kanamycin. The inserted fragment was confirmed by gel electrophoresis to obtain a plasmid DNA in which the target fragment was expected to be subcloned.
  • Sequencing primers are primers targeting the T7 and Ml3 sequences present at both ends of the pSTBlue vector cloning site (Takara Bio BcaBEST Sequencing Primer T7; 5 'TAC TAC GAC TCA CTA TAG GG 3 '(SEQ ID NO: 2 5) and M13 Primer M4; 5' GTT TTC CCA GTC ACG AC 3 ', (SEQ ID NO: 2 6)) were used.
  • the nucleotide sequence of the Dc4DES gene isolated in this Example and the amino acid sequence of Dc4DES are shown in SEQ ID NOs: 1 and 2, respectively.
  • the obtained base sequence was analyzed and edited using Genetyx-Win Ver. 4.0 / ATGC ver. 2 (manufactured by Software Development Co., Ltd.). '.
  • the nucleotide sequence homology (i dent i ty) between the Dc4DES gene and the previously reported coriander-derived gene (Cs4DES) is 88. 0 in the polypeptide coding region (presumed) sequence (Dc4DES gene ⁇ ⁇ ⁇ bp, Cs4DES gene 1158 bp). %Met. Amino acid sequence homology
  • the (identity) was 90.2% in the polypeptide coding region predicted amino acid sequence (Dc4DES gene 386aa, Cs4DES gene 385aa).
  • the alignment of the Dc4DES amino acid sequence and the Cs4DES amino acid sequence is shown in FIG. In FIG. 3, the upper row is the Dc4DES amino acid sequence and the lower row is the Cs4DES amino acid sequence.
  • Example 2 an F1 variety of carrot Daucus caro ta was used as an experimental sample. We used summer sowing fresh red 5 inch, Yangshu 5 inch, and fixed Kuroda 5 inch. Nin. Gin seeds were purchased from Ota seedlings. As a gene cloning source, a fixed species with the same homologous gene sequence is desirable, but F1 varieties were also used for the convenience of the experiment (a sample that had flowered early) was prepared. The same celery family plants, coriander (Corianirum sa ti vi um) and ai / grass (Anetnum gra veolens cv. Mammo th) were cultivated and used.
  • Plants grown at 25 ° C, 16 hours of sunshine, and 50% humidity in an artificial meteorological instrument were used as samples.
  • RNA was prepared according to the method of Example 1.
  • TE-PF3 (515-536): 5 '-RTG GNA CNM GRG KRR ATT GGA T —3' (SEQ ID NO: 2 7)
  • TE-PF2 (415-438): 5 '— CTB ATW TGG GTB ACD DMN MGN ATG -3 "(SEQ ID NO: 2 8)
  • TE-PF1 (235-257): 5 '— GAR RAY GGN YWN TCB TAY AMR GA -3' (SEQ ID NO: 2 9)
  • TE-PR1 (886-915): 5-TGR CAY TCN CKY CKR TAR TC -3 "(SEQ ID NO: 30)
  • TE-PR2 (787-809): 5 '-ACR TTR TTN ACR TGY TKR TTC AT-3' (SEQ ID NO: 3 1)
  • TE-PRO 1041-1061: 5 '-GTD SKN CMV CKR TTK AKY TC -3 "(SEQ ID NO: 3 2)
  • the above primers were used using the QIAGEN One-step RT-PCR kit. set RT-PCR amplification was performed using the mated pair.
  • the reaction solution composition was carried out using an Eppendorf thermal cycler (master cycler gradient) according to the standard protocol attached to the kit. Since this device can perform reactions with any temperature gradient (up to 12 steps) on the heat block, the annealing temperature, which is important for amplification efficiency and specificity, ranges from 50 ° C to 70 ° C (5 This was done in 5 steps per ° C).
  • PCR conditions were 30 minutes at 50 ° C, 15 minutes at 94 ° C, then 40 cycles of 94 ° C for 1 minute, 50-70 ° C for 1 minute and 72 ° C for 1 minute 30 seconds. Then, it was treated at 72 ° C for 15 minutes. After the reaction, 4 ° C was maintained.
  • a specific amplification product (about 500 bp) was obtained by combining TE-PF2 and TE-PR1.
  • the base sequence of this amplified product was decoded, and molecular phylogenetic analysis was performed using the partial sequence. As a result, it was found to belong to the 0TE cluster.
  • the database was searched again for the partial sequences obtained as a result of RT-PCR, and highly homologous genes were re-extracted.
  • a multiple alignment analysis was performed in the same manner, and a region with low conservation at the amino acid level was selected.
  • a primer capable of specific amplification of the target gene was designed and used in the RACE method.
  • TE-D1F 5 '-TAG CAA GTG GGT GAT GAT-3' (SEQ ID NO: 3 3)
  • TE-D3F 5 '-GTT TTC TGC CCC AAA ACA CC 1 3' (SEQ ID NO: 3 4)
  • TE-D2R 5 '-TAT TCA TCT CGA ACA TCA T-3' (SEQ ID NO: 3 5)
  • TE-D1R 5 '-ATC ATC ACC CAC TTG CTA -3' (SEQ ID NO: 3 6)
  • Example 2 determination of the base sequence and analysis / editing of the obtained base sequence were performed in the same manner as in Example 1. Furthermore, molecular phylogenetic tree analysis Using the DNA data bank gene analysis service (http://www.ddbj.nig.ac.jp/Wel come-j.html), the Clustal W program (base Sequence ⁇ Multiple alignment of amino acid sequences and phylogenetic tree creation program).
  • 0TE-2F 5 '-GCA TTC TAG GCT AGG ATT GT-3' (SEQ ID NO: 3 7)
  • OTE-ralR 5 '-GGC GAA TCG AGA TCG AAT CT -3' (SEQ ID NO: 3 9)
  • 0TE-m3R 5 '-CTC AAT TTC TCC GCC AAG CT-3' (SEQ ID NO: 4 1)
  • PTE-2F 5'-CTT TTC CAG TCT CGG GCT TG-3 '(SEQ ID NO: 4 2)
  • PTE-4R 5 '-GGA AGC AAC TCA TCG TCG TCT GT -3' (SEQ ID NO: 4 3)
  • PTE-2R 5 '-CAA GCC CGA GAC TGG AAA AG-3' (SEQ ID NO: 4 4)
  • Sac-DcPTE-6R 5 '-TCT AGA CGA GCT CCT AGT TTA AAC AGT ACA CTG-3' (Urban U number 4 7)
  • DcPTE-6R 5'-CTA GTT TAA ACA GTA CAC TG-3 '(SEQ ID NO: 4 8)
  • carrot RNA was converted into a saddle shape and RT-PCR was performed to amplify the entire polypeptide coding region.
  • PCR amplified fragment and TA-Cloning PC.R product cloning
  • Transformants were selected by culturing in LB medium supplemented with X-gal and 50 ⁇ g / ml kanamycin. Plasmid DNA was prepared from the cells obtained by liquid culture in LB medium supplemented with 50; g / ml kanamycin using the QIAGEN Plasmid mini kit. The inserted fragment was confirmed by gel electrophoresis to obtain a plasmid DNA in which the target fragment was expected to be subcloned. In this example, two types of Dc4PTE genes were isolated. The two isolated Dc4PTE genes are identified as Dc4PTEa,
  • Dc4PTEb their base sequences are shown in SEQ ID NOs: 3 and 5, respectively, and the two amino acid sequences of Dc4PTE are shown in SEQ ID NOs: 4 and 6, respectively.
  • the DcPTE gene was cloned as described above. Similar to the cloning of the DcPTE gene, cloning of the PTE gene was also performed for coriander and dill.
  • AgPTE gene was isolated. Their base sequences are shown in SEQ ID NOs: 7 and 9, respectively, and the amino acid sequences of two types of Dc4PTE are shown in SEQ ID NOs: 8 and 10, respectively. next,
  • the activity of the petroselinol-ACP thioesterase of the enzyme encoded by the DcPTE gene was examined. Specifically, as shown below, histidine-labeled recombinant protein was prepared using E. coli and the enzyme activity was analyzed.
  • Oleoyl- ACP thi-oest erase ⁇ Plant Physio ⁇ ., 100,
  • the DcPTEa gene a mature peptide cl eavage site was estimated between the base sequence encoding the 32nd amino acid and the base sequence encoding the 33rd amino acid.
  • the DcOTE gene and the CsOTE gene were estimated as the mature peptide cleavage site between the base sequence encoding the 51st amino acid and the base sequence encoding the 52nd amino acid.
  • DcPTEa, DcOTE, and CsOTE cDNAs cloned into Bluel were amplified by PCR using a vertical type, and the product was mixed with the expression vector for histidine-tagged protein PQE-30 UA (QIAGEN)
  • the amount of TaKaRa Ligat ion kit ver. 2 was added and subcloned by ligation reaction for 30 minutes at 16 ° C.When subcloning this vector, the recombinant protein with 6XHis added to the N-terminus was transferred in E. coli. preparation can.
  • the reaction mixture JM109 strain having lacl q mutation, Takara Bio Inc.
  • E. coli combination competent cells the total amount of and added pressure, the manufacturer specifies protocol Thus it was transformed operation.
  • Resulting transformants from grown in 50 zg / ml ampicillin containing LB agar medium) was prepared plasmid.
  • the prepared histidine-tagged protein expression DNA construct was transformed into E. coli (JM109 strain) (or stored before plasmin preparation. Use glycerol stock for IJ) and Overnight Express Autoinduction System 1 (Merck). Expression was induced by culturing overnight in LB medium supplemented with 50 ig / ral ampicillin.
  • Facil ACP used as a substrate was prepared as follows. First, a hexane solution (lOOmM) of fatty acid (oleic acid, petrothelic acid) was prepared, and 6.2 was put into a 10 ml glass test tube and dried with a nitrogen gas. Then, the reaction liquid of the following Table 2 was added, and it was made to react for 60 minutes at 37 degreeC on a heat block.
  • Buffer B Add bis Tris-HCl pH 6.0 (referred to as Buffer B) about 10 times the amount in (A) to equilibrate.
  • ⁇ and Buffer B are used by mixing them individually deaerated with an ultrasonic cleaner
  • the histidine-labeled protein purified by the above procedure was assayed for thioesterase activity using isyl ACP (oleoyl ACP, petroselinol-ACP) as a substrate. Prior to the reaction, the protein concentration of both solutions was measured by the Lowry method (RCDC protein assembly kit, manufactured by BIO-RAD).
  • a transformed plant into which the Dc4DES gene cloned in Example 1 and the DcPTE gene cloned in Example 2 were introduced was prepared, and the ability to synthesize petroceric acid in the transformed plant was examined. That is, a transformed plant into which the Dc4DES gene was introduced alone, a transformed plant into which the DcPTE gene was introduced alone, and a transformed plant into which both the Dc4DES gene and the DcPTE gene were introduced were prepared.
  • Fig. 7 shows vectors for constant whole body expression.
  • Pnos means Agrobacterium-derived nopaline synthase promoter
  • Tnos means Agrobacterium-derived nopaline synthase terminator
  • P35S means CaMV35S promoter
  • NPT II means neomycin. Phosphotransferase This refers to the sputum gene.
  • the GUS gene contained in the plant expression vector pBI121 (Clontech) was replaced with the cDNA sequence of the Dc4DES gene.
  • the DNA fragment encoding the 0RF region of the Dc4DES gene was amplified by PCR using primers designed to add Bam HI at the 5 ′ end and Sac I sequence at the 3 ′ end.
  • This PCR product was mixed with pSTBluel (Novagen cloning vector), an equal amount of TaKaRa Ligation kit ver. 2 was added, and a ligation reaction was performed at 16 ° C for 30 minutes. The total amount of the reaction solution was added to a 50 ⁇ 1 competent cell (co i DH5, manufactured by TOYOBO), and the transformation was performed according to the protocol specified by the manufacturer.
  • a plasmid was prepared from the obtained transformant (50; grown on LB agar medium containing g / ml kanamycin). The obtained plasmid was treated with restriction enzymes (Bam HI and Sac I). Next, restriction enzyme treatment (Bam HI and Sac I) was similarly performed to excise the GUS gene linked to the downstream of the CaMV35S promoter in PBI 121. These restriction enzyme digests The product was subjected to 0.8% agarose gel electrophoresis, and the QIAquick gel extraction kit and Geneclean II (BIO 101) manufactured by QIAGEN were used. Each was separated and purified.
  • the vector for co-expression of the Dc4DES gene and the DcPTE gene (Fig. 6 (D)) is located near the LB of the T-DNA in the expression vector for Dc4DES expression shown in Fig. 6 (A).
  • DcPTE gene expression unit between unique Eco RI and Dra III sites is located near the LB of the T-DNA in the expression vector for Dc4DES expression shown in Fig. 6 (A).
  • the DcPTE gene generation unit was amplified by PCR using primers designed to add Eco RI and Dra III sites at both ends.
  • FIGS. 8 (A) to (D) seed-specific expression vectors are shown in FIGS. 8 (A) to (D).
  • “Pnap” means the napin A promoter derived from rapeseed (B. campestris cv. Kizakinona tane). The promoter is known to be used by Monsanto to control rapeseed oil content.
  • a seed-specific expression vector was prepared by replacing the CaMV35S promoter in the vector shown in FIG. 7 with a rapeseed napin A promoter.
  • the GUS gene was replaced with the cDNA sequence of Dc4DES gene, DcPTE gene or Cs4DES.
  • the vector for co-expression of the Dc4DES gene and the DcPTE gene (Fig. 8 (D)) is a unique protein near the LB of T-DNA in the expression vector for Dc4DES expression shown in Fig. 8 (A).
  • DcPTE gene expression unit between the Eco RI site and Dra III site is a unique protein near the LB of T-DNA in the expression vector for Dc4DES expression shown in Fig. 8 (A).
  • the DcPTE gene expression unit was amplified by PCR using primers designed to add Eco RI and Dra II I sites at both ends.
  • the prepared plasmid was used to transform agrobacterium by electroporation (electroporation).
  • 40 ⁇ 1 of ⁇ robac erii ⁇ tumefaciens (LBA4404 strain) prepared by a conventional method was dissolved, 5 1 (25 ⁇ g) of DNA solution was added, and the mixture was allowed to stand on ice for 1-2 minutes. Subsequently, it was placed in an ice-cooled BI0-RAD cuvette (0.2 cm) and a pulse current of 1.25 kV and 10 F was applied using a Shimadzu gene introduction device. Immediately after cooling, the S0C medium was incubated at 28 ° C for 1 hour with 460 ⁇ 1 force.
  • Arabidopsis thaliana et al. (Colombia) was transformed by the vacuum infiltration method.
  • the vacuum infiltration method was carried out according to Shujunsha, model plant experimental protocol, 2001, 109-113 pp.
  • T1 seeds transformed first generation seeds
  • kanamycin-containing medium Morashige & Skoog basic medium with 0.5g / L MES, 10g / L sucrose, 8g / L Agar, lOOmg / L carbenicin, 50mg / L kanamycin
  • a transformed individual having kanamycin resistance and growing normally
  • Individuals whose true leaves were normally developed were transplanted again to the same medium, grown for about 1 to 2 weeks, and reselected.
  • the obtained line was used as a transformed first generation plant (T1 plant). Force Namicin resistant strains were transplanted to non-sterile vermiculites and cultivated in a non-sterile environment. .
  • DNA was prepared using the QIAGEN DNA preparation kit (DNeasy plant mini kit) according to the standard protocol attached to the kit.
  • PCR amplification was performed using Ex Taq DNA polymerase manufactured by Takara Bio, using PCR primers targeting the drug resistance gene ( ⁇ ) in T-DNA and the introduced fatty acid synthesis system gene.
  • the obtained PCR amplification product fragment was electrophoresed in a TAE buffer using a 0.8% agarose gel and then stained with ethidium promide to confirm the amplification of the target fragment. The presence or absence of gene transfer was determined by the presence or absence of amplification.
  • T2 seed More than 15 T2 seeds were harvested from each construct and used for the following fatty acid composition analysis.
  • seed fatty acids were methyl esterified with monomethanol hydrochloride and the n-hexane extract was analyzed by GC-MS.
  • BHT Butylated hydroxytoluene
  • a methanol solution of methyl ester (SIGMA) of pentadecanoic acid (C15: 0) not contained in vegetable oil as an internal standard is added directly to the seed after weighing and used to correct experimental errors when preparing samples for analysis. did.
  • n-hexane used for the extraction was phthalate analytical grade n-hexane (Tokyo Kasei). This eliminates the effects of phthalates that behave like fatty acids during GC analysis.
  • Table 3 shows the protocol for qualitative analysis and Table 4 shows the protocol for quantitative analysis.
  • Carrier gas Helium flow rate 1 mL / min
  • the mole fraction (mol -%) Shows the composition of each fatty acid.
  • the intensity of fragments detected by a mass spectrometer differs depending on the quality of the substance and does not simply reflect the molecular weight.
  • the reproducibility when analyzed under the same conditions is very high and relative. Such a comparison is sufficiently possible.
  • a transformed plant with a constant systemic expression was produced using a pB-4DES construct that expresses the Dc4DES gene under the control of the CaMV35S promoter. Leaves were collected from this transformed plant, oil and fat components were extracted, and fatty acid composition analysis was performed by the method described above.
  • fatty acid components we analyzed petroceric acid and cis-4-hexadecenoic acid (16: 1 ⁇ 4), which is a precursor of petroceric acid. Table 6 shows the analysis results of these monoene unsaturated fatty acids.
  • transgenic plants expressing Dc4DES gene, Cs4DES gene, and DcPTE gene that express seeds specifically are prepared, and the oil and fat components are extracted from these seeds and fatty acids are extracted by the method described above.
  • a compositional analysis was performed. Table 7 shows the analysis results of monoene unsaturated fatty acids. In Table 7, “NT” stands for not tested.
  • the content of stearic acid (C18: 0), icosanoic acid (C20: 0), and docosanoic acid (C22: 0) in plant seeds with the DcPTE gene introduced is the same as the content in plant seeds without the DcPTE gene introduced.
  • the DcPTE genes are classified as a group of tioesterase genes called Fat A, which has specificity for unsaturated fatty acids-ACP, based on a molecular phylogenetic analysis based on the deduced amino acid sequence.
  • Fat A tioesterase genes
  • Example 2 it can be expected that the effect of increasing unsaturated fatty acid content can be expected from the results of showing substrate specificity for betacellinoyl ACP, but it is difficult to predict the production effect of saturated fatty acid. This It turned out to be a gene that had an exceptionally remarkable effect that could not be predicted by a vendor.
  • nylon material dicarboxylic acid
  • the effect of increasing the saturated fatty acid content of C1 8 or more in addition to petroselinic acid accumulation promoting effect is very advantageous It is considered to be a property. That is, by increasing the saturated fatty acid content, the content of unsaturated fatty acids other than petroceric acid (causing oxidative degradation and causing impurities to be generated) will be lowered.
  • combination in manufacture of petroselinic acid can be provided, and the manufacturing method of the novel petroselinic acid using the said gene can be provided. If a gene involved in the production of petrothelic acid according to the present invention is used, for example, petroselinic acid can be accumulated in large amounts in plant seeds.

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Abstract

La présente invention concerne un gène innovant capable de favoriser l'accumulation d'acide pétrosélinique et un procédé de fabrication d'acide pétrosélinique à l'aide du gène. L'invention concerne un gène codant pour l'une quelconque des protéines (a), (b) et (c) : (a) protéine comprenant la séquence d'acides aminés décrite sous les SEQ ID NO : 4 ou 6 ; (b) protéine dont la séquence d'acides aminés comprend la délétion, la substitution ou l'addition d'un ou plusieurs résidus d'acides aminés dans la séquence d'acides aminés décrite sous les SEQ ID NO : 4 ou 6 et qui présente une activité pétrosélinoyl-ACP thioestérase ; et (c) protéine codée par un ADN capable de s'hybrider avec un ADN comprenant une séquence nucléotidique complémentaire de la séquence nucléotidique décrite sous les SEQ ID NO : 3 ou 5 dans des conditions stringentes et qui présente une activité pétrosélinoyl-ACP thioestérase.
PCT/JP2006/313449 2005-06-30 2006-06-29 Gène innovant impliqué dans la biosynthèse de l'acide pétrosélinique et procédé de production d'acide pétrosélinique WO2007004694A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CN2006800241478A CN101213298B (zh) 2005-06-30 2006-06-29 参与岩芹酸生物合成的新基因和用于生产岩芹酸的方法
AU2006266760A AU2006266760B2 (en) 2005-06-30 2006-06-29 Novel genes involved in petroselinic acid biosynthesis and method for producing petroselinic acid
CA2614062A CA2614062C (fr) 2005-06-30 2006-06-29 Gene innovant implique dans la biosynthese de l'acide petroselinique et procede de production d'acide petroselinique
JP2007523443A JP4840360B2 (ja) 2005-06-30 2006-06-29 ペトロセリン酸生合成に関与する新規遺伝子、ペトロセリン酸の製造方法

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JP2005191775 2005-06-30
JP2005-191775 2005-06-30

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WO2007004694A1 true WO2007004694A1 (fr) 2007-01-11

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JP (1) JP4840360B2 (fr)
CN (1) CN101213298B (fr)
AU (1) AU2006266760B2 (fr)
CA (2) CA2614062C (fr)
WO (1) WO2007004694A1 (fr)

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JPH10510438A (ja) * 1995-09-29 1998-10-13 カルジーン，インコーポレーテッド 植物ステアロイル−ａｃｐチオエステラーゼ配列および植物種子油のステアリン酸塩含量を増大させる方法
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GB9918022D0 (en) * 1999-07-30 1999-09-29 Unilever Plc Skin care composition

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US5430134A (en) * 1992-08-07 1995-07-04 Michigan State University Method for production of petroselinic acid and OMEGA12 hexadecanoic acid in transgenic plants
JPH10505237A (ja) * 1994-08-31 1998-05-26 イー・アイ・デユポン・ドウ・ヌムール・アンド・カンパニー カノラ及びダイズのパルミトイル−ａｃｐチオエステラーゼ遺伝子のヌクレオチド配列、並びにダイズ及びカノラ植物の油の脂肪酸量の調節におけるこれらの使用
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Also Published As

Publication number Publication date
CA2700545C (fr) 2012-08-28
CN101213298A (zh) 2008-07-02
JP4840360B2 (ja) 2011-12-21
AU2006266760A1 (en) 2007-01-11
CA2614062C (fr) 2011-11-15
CA2614062A1 (fr) 2007-01-11
JPWO2007004694A1 (ja) 2009-01-29
CA2700545A1 (fr) 2007-01-11
AU2006266760B2 (en) 2011-12-15
CN101213298B (zh) 2012-04-18

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