WO2007004694A1 - Novel gene involved in biosynthesis of petroselinic acid and process for production of petroselinic acid - Google Patents

Abstract

Disclosed are a novel gene capable of promoting the accumulation of petroselinic acid and a process for producing petroselinic acid using the gene. A gene encoding any one of the following proteins (a), (b) and (c): (a) a protein comprising the amino acid sequence depicted in SEQ ID NO:4 or 6; (b) a protein which comprises an amino acid sequence having the deletion, substitution or addition of one or more amino acid residues in the amino acid sequence depicted in SEQ ID NO:4 or 6 and which has a petroselinoyl-ACP thioesterase activity; and (c) a protein which is encoded by DNA capable of hybridizing with DNA comprising a nucleotide sequence complementary to the nucleotide sequence depicted in SEQ ID NO:3 or 5 under stringent conditions and which has a petroselinoyl-ACP thioesterase activity.

Description

 A novel gene involved in petrothelinic acid biosynthesis, a method for producing petrothelic acid

Technical field

 The present invention relates to novel genes involved in biosynthesis of petrothelinic acid, in particular, Δ 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

 Field background technology.

 In recent years, in the field of resin materials as well, technological developments have been undertaken to produce pi-mass-derived resins from the perspective of deoiling resources and building a recycling society. .

 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. However, the use of nylon 6 and 10 is limited, and it is not widely used as a resin material.

 It is already known that dicarboxylic acids can be produced by oxidative degradation of unsaturated fatty acids, and so far hetroceric acid (the raw material of adipic acid (hexanedioic acid), the raw material monomer of nylon 6 and 6) The production technology of cis-6-octadecenoic ac id) has been studied. (Non-Patent Documents 1 to 8).

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). In the synthesis of petroceric acid, the precursor palmitoyl ACP was converted to ci s-4-hexadecenoil ACP by Δ 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. Furthermore, free petrothelinic acid is synthesized by the petrocellinol-ACP thioesterase (hereinafter referred to as PTE) and transferred to the cytoplasm. Plants that synthesize petroceric acid are thought to contain a series of biosynthetic enzymes specific to petroceric acid synthesis and their genes. Among these, Δ4-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.

 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. In order to produce petrothelic acid using recombinant technology, it is thought that many problems must be solved.

 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 6 Metab. Eng., 4, 12-21, 2002

 —Non-patent literature 7 Biochira. Biophys. Acta., 1212, 134-136, 1994

 Non-Patent Document 8 Plant Phys iol., 104, 839-844, 1994

 Non-Patent Document 9 Planta 215: 584-595 2002. Disclosure of the Invention

In view of the above-described situation, 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.

 As a result of intensive studies by the inventor in order to achieve the above-described object, Δ 4 -panoremitoinole-ACP desaturase derived from cindu (aucus caro a) belonging to the family Aceraceae is a Δ4-palmitoyl-ACP desaturase derived from Korean. As a result, a new finding was obtained that the ability to synthesize petrothelic acid was superior. In addition, the present inventor has succeeded in isolating a petroselinol-ACP thioesterase gene newly, and when this gene is used in combination with a Δ4-palmitoyl-ACP desaturase gene, the inventor has the ability to synthesize petrocellinic acid. We invented a technology that makes it approximately double.

 That is, the present invention includes the following.

 (1) A gene encoding the following protein (a), (b) or (c).

 (a) a protein comprising the amino acid sequence shown in SEQ ID NO: 2-

(b) a protein having Δ 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

 (c) It 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 has Δ4-palmityl-ACP desaturase activity Protein.

 Here, in the above (b) and (c), a protein having Δ4 ”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.

 (2) A gene encoding the following protein (a), (b) or (c).

 (a) a protein comprising the amino acid sequence shown in SEQ ID NO: 4 or 6

(b) 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.

 Here, in the above (b) and (c), the protein having petroselinol-ACP thioesterase activity is expressed simultaneously with a protein having Δ4-palmitole-ACP desaturase activity in plant cultured cells or individual plants. Ci s_4-hexadecenoic acid (ci s-4-Hexadecenoic acid) or petroselinic acid (ci s_6 -octadecenoic acid (cis -6-octadecenoic acid)) or cis-8-icosenoic acid It can be rephrased as a protein capable of increasing the amount.

 This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2005-191775 · · which is the basis of the priority of the present application. Brief Description of Drawings

 FIG. 1 is a diagram comparing the homology (%) of the amino acid sequences of various petrocellinol-ACP thioesterases and oleoyl-ACP thioesterases. In the figure, DcPTE means petroselinol-ACP thioesterase derived from carrot Da s carota, CsPTE means coriander coriand sa ti vwn, derived from petrocellino.il-ACP thioesterase, and AgPTE stands for dinore ne thum gra veolens. Original petrocellinol-ACP thioesterase, 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.

Figure 3 shows Δ 4-palmitoyl-ACP desatura from carrot Da s car o ta) FIG. 2 is a diagram showing alignment of amino acid sequences of Δ4-palmitoyl-ACP desaturase (referred to as Cs4DES) derived from the amino acid 酉 S 歹 ίΐ and coriander (referred to as Dc4DES) of Corse.

 Figure 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) This is a photograph showing the results of SDS-PAGE of oleidine-ACP thioesterase (referred to as DcOTE) histidine-labeled protein eluate from Escherichia coli one fraction at a time.

 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. BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the novel gene and the method for producing petrothelic acid according to the present invention will be described in detail with reference to the drawings.

 1. Acquisition of Δ 4-palmitoyl-ACP desaturase gene (Dc4DES gene) from carrot (Daucus carota)

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 Δ4 position in palmitoyl-ACP (palmitoyl-acyl carrier protein), which is a saturated fatty acid having 16 carbon atoms. As an example of the Dc4DES gene, a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 is shown in SEQ ID NO: 1. Manako, in the present invention, 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 Δ 4 -palmitoyl-ACP desaturase activity It may be one that encodes a protein. Here, “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.

 Furthermore, in the present invention, 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 Δ 4 -A gene encoding a protein having palmitoyl-ACP desaturase activity. Here, 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). '

 Furthermore, in the present invention, 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 Δ 4-palmitoyl- It may be a gene encoding a protein having ACP desaturase activity. Here, “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.

 Δ 4 -palmitoyl-ACP desaturase activity is a function of palmitoyl-ACP.

It means activity to desaturate the 4th position. 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. For example, by 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, 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 Δ 4-palmitoyl-ACP desaturase activity, and if these fatty acid methyl esters cannot be detected, it has Δ 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. For example, 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. By growing the resulting transformed plant into a plant body, the synthesis of petroselinic acid in the plant body can be promoted. As the specific expression promoter, it is preferable to use a seed specific expression promoter. By using a seed-specific expression promoter, petroselinic acid can be accumulated in the seed.

 For example, after growing a transformed plant into a plant body, 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. .

2. Petrocellinol-ACP thioesterase enzyme gene (PTE gene)

The presence of 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 Δ6-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.

 In the present invention, 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. . Here, “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. For example, using a mutagenesis kit (for example, Mutan-K (TAKARA) or Mutan-G (TAKARA)) using site-directed mutagenesis, or LA PCR in by TAKARA Mutation is introduced using the in vitro Mutagenesis series kit.

Furthermore, in the present invention, 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. Here, 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).

 Further, in the present invention, 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. Here, “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 μ1 For 30 minutes at 25 ° C. After the reaction solution, 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. Alternatively, mix 25 mM Tris-HCl (pH 8.0), ImM DTT, and Petrocelinol-labeled with a tritium-based Petrocelinol-ACP and water, and then pre-incubate for 5 minutes at 25 ° C. Incubate the added reaction solution 100 βΐ at 25 ° C for 30 minutes. After the reaction, 50 μ 1 isopropanol was added to stop the reaction, and then the petroceric acid produced by the enzyme reaction was separated by thin layer chromatography and added by quantifying the amount of petrothelic acid produced by a scintillation counter. The thioesterase activity of the protein to be assayed is measured.

In the present invention, the PTE gene is a gene encoding a carrot-derived PTE. However, it is meant to include a plant-derived PTE gene that biosynthesizes petroceric acid. Other than carrots, 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.

 When obtaining a PTE gene from a plant other than carrots, for example, 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. . If long nucleic acid sequences (> lOObp) are used as probes, they can also be screened by moderate stringency to obtain signals from target samples that are more than 80% homologous. it can. Also, the probe can be quite short. For example, 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.

 That is, by performing Southern hybridization using genomic DNA extracted from plants other than the probe and carrot, the PTE gene can be isolated and identified from the genomic DNA of the plant. In addition, instead of 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.

As shown in Fig. 1, it 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. There is about 30% amino acid difference between 0TE and PTE. Among them, it is shown that the polarity of some amino acids is different in the 0TE group compared to the PTE group. In the following description, “common array number X (X is a natural number)” 'means the number given to the top of the alignment in FIG. In the PTE group, the amino acids of consensus sequences 120, 125, and 373 are non-polar, while in the 0TE group, the amino acid is polar. In the PTE group, the amino acids of the common sequences Nos. 140 and 195 are polar, and in the 0TE group, the amino acid is nonpolar.

 Furthermore, it is shown that some charged amino acids are different in the 0TE group compared to the PTE group. In the PTE group, 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. In the PTE group, 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. In the PTE group, 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 presence or absence of amino acid polarity and changes in charge at these positions are thought to lead to changes in substrate selectivity. Furthermore, it is known that differences in the side chain structure of amino acids at the substrate binding site lead to changes in substrate selectivity. In the PTE group, there are differences in the side chain structure of amino acids between amino acids of consensus sequences Nos. 89, 147, 161, 177, 192, 196, 214, 217, 223, 238, 270, 287, 338 and 0 TE amino acids. Substitution of these amino acids, which differ between the 0TE and PTE groups, may alter the substrate specificity for acyl ACP.

 The PTE gene is a host plant along with the Δ 4 -palmitoyl-ACP desaturase gene.

'By introducing a functionally open cell into the cell, It has the function of greatly promoting acid synthesis.

 For example, 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 Δ4-palmitoyl-ACP desaturase gene. Alternatively, plant cells may be transformed using an expression vector comprising a PTE gene and a Δ4-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.

 For example, after growing a transformed plant to produce a 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.

Expression vector

 In the present invention, 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).

 Examples of plasmid DNA include pRS413, pRS414, pRS415, pRS416, YCp50,

YCp-type E. coli-yeast shuttle vectors such as PAUR112 or pAUR123, pYES2 or

YEp type 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, ρΒΙ Ι Ο Ι), derived from Bacillus subtilis Plasmids (eg, pUB 110, pTP5, etc.), and phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, gt10, Agtll, λZAP), ψ Χ174, M13mpl 8 or M13mpl 9 and so on. Retrotransposons include Ty factors. Examples of YAC vectors include pYACC2. Furthermore, animal viruses such as retrovirus or vaccinia virus, and insect virus vectors such as baculovirus can also be used. In the expression vector, 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. Therefore, 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. Examples of 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. In the present invention, 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. In particular, when the purpose is to accumulate synthesized petrothelic acid in the seed, it is preferable to use a seed-specific expression 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. '

 When the host is a plant, 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.

 For example, the reduced pressure infiltration method can be performed according to a known method (Shujunsha, Model Plant Experiment Protocol, 2001, 109-113 pp.). In the case of using agrobacterium, 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). According to 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.

 When the particle gun method is used, 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.

 Whether or not the gene has been integrated into the host can be confirmed by PCR, Southern hybridization, Northern hybridization, or the like. For example, 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. In addition, 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.

On the other hand, as hosts, Escherichia coli (such as Escherichia coli), Bacillus subtilis), etc. ), Belonging to the genus Rhizobium, Saccharomyces cerevisiae, Nzo Saccharose honobe

) And the like, animal cells such as COS cells and CH0 cells, and insect cells such as Sf9.

 When a bacterium such as Escherichia coli is used as a host, it is preferable that 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. For example, Escherichia coli

(Escherichia coli) DH5, Y1090, and the like. Examples of Bacillus subtilis include, but are not limited to, Bacillus subtilis 0¾cj'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. For example, 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. In the case of using yeast as a host, for example, Saccharomyces cerevisiae) Schizosaccharomyces τ ^, Nobe, Schizosaccharomyces pombe) H. pastoris (尸 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. For example, 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)].

 When animal cells are used as hosts, 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.

 When insect cells are used as hosts, Sf9 cells are used. Examples of methods for introducing the recombinant vector into insect cells include the calcium phosphate method, the lipofussion method, and the electroporation method.

Method for producing petroceric acid

 In the transformed plant described above, 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. .

There are two main methods of squeezing oil from plant tissues such as seeds and fruits of oil-producing plants. For example, to obtain fat from tissues with high fat content, such as rapeseed seeds, the tissue is coarsely crushed with a roll mill, heated to 75-85 ° C, and then compressed with an expeller or other pressing machine. Take out (squeezing method). On the other hand, raw materials with low fat content such as soybeans are extracted from tissues using a solvent such as hexane (extraction 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. Next, 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. In addition, an alcohol ester such as petroleum acid methyl ester can be obtained by adding and reacting an alcohol such as methanol to the oil.

 EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limitedly interpreted by the following examples.

[Example 1] Dc ⁴ DES gene

Plant sample

 In this example, carrot Daucus car ota L.) summer sowing fresh red 5 inch (F1 variety) was used as an experimental sample. As a gene cloning source, 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.).

Carrot RNA preparation

 About lOOmg of immature seeds and about lOOmg of leaves with different seed development stages were collected from carrot plants and ground under liquid nitrogen freezing. RNA was prepared from the obtained powdery cake using RNeasy plant mini kit manufactured by QIAGEN according to the protocol attached to the kit. ..

PCR primer design and RT-PCR for DNA fragment amplification

Corlasta, one (Coriandrum sa ti vum L.) Δ 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 Δ 9 stearoyl-ACP desaturase gene of various plants registered on GenBank. The amino acid sequence of Genetyx-Win Ver,

4. Multiple alignment analysis was performed with 0 / ATGC ver. 2.0 (manufactured by Software Development Co., Ltd.). As a result of the analysis, the following PCR primers (degenerate primers) were designed and used for RT-PCR in order to amplify the DNA fragment corresponding to the highly conserved region obtained.

PF1: 5 'CAN GAR GAR GCN CTB CCN CAN TA 3' (SEQ ID NO: 1 1)

PR1: 5 'TCV RVD AGY TTY TCN ACD ATY TT 3' (SEQ ID NO: 1 2) 9

PR2: 5 'GCN GYY KCR TGN C Y TTY TCR TC 3' (SEQ ID NO: 13)

NFO: 5 'GAN MTB CCN GAT GAN TAY TTH RTT G 3' (SEQ ID NO: 14)

NR1: 5 'CCY TCN SCN SWM AGH CCN GT 3' (SEQ ID NO: 15)

NR2: 5 'GGC ATN DVD AYY TTB WTY YTC ATC AT 3' (SEQ ID NO: 16)

 These base sequences are described in the following common mixed base sequence (IUB) notation in each country. That is, 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. For RT-PCR, 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.

 table 1

RT-PCR No. <Forward primer> <reverse primer>

 1 PF1 PR2.

 2 PF1 PR1

 3 NFO R2

 4 NFO NR1

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.

5 and 3 'RACE method and PCR

 Based on the sequence information of the DNA fragment obtained by RT-PCR, the following primers were designed and used for the 5 ′ and 3 ′ RACE methods.

 (RACE primer)

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)

 [Primers for 3 'RACE]

D2-F4: 5 'CAA ATG CCA GCT CAT GCA ATG 3'

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

It was performed at 70 ° C (5 steps every 5 ° 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.

Design of PCR primers for amplification of polypeptide coding region

 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.

4DS-F-0R1: 5 ^; ATG GCT ATG AAA TTG AAC GCC 3 '(SEQ ID NO: 2 1)

Bam-4DS-F-0R1: 5, TCT AGA GGA TCC ATG GCT ATG AAA TTG AAC GCC 3 '(SEQ ID NO: 2 2)

 4DS-R-0R: 5 'TCA TAT CAT GAT CTG' ACG GTT G 3, (SEQ ID NO: 2 3)

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. Next, using the 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, 晚 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 Ι ΐ δ 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] Identification and cloning of PTE gene

Plant sample

In 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 (Koitotron; Koito Manufacturing Co., Ltd.) were used as samples.

RNA preparation

 About 100 mg each of carrot leaves, immature seeds and ripe seeds were collected, and RNA was prepared according to the method of Example 1.

RT-PCR amplification

 0TE-like gene derived from coriander, the same celery family of carrots

BLAST search (blastn and J; and b 丄 astx; http: // www. Ncbi. Nlm. Nih. Gov / blast /) for the partial cDNA sequence of (CsOTE) (accession 'number, L20978) We listed 17 similar genes that were hit as highly homologous genes. For the deduced amino acid sequence, multiple alignment analysis was performed, and regions with high conservation were selected. In addition, a multiple alignment analysis was performed on the DNA sequence of the same gene in the same manner, and degenerate primers were designed in consideration of the frequency of codon usage in the DNA of closely related species in regions that were highly conserved at the amino acid level.

Used for RT-PCR method. The sequence is shown below. In parentheses, the base numbers corresponding to oleoyl-ACP thioesterase (AtOTE) of Arabidopsis thaliana are shown.

 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) Specifically, 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.

 As a result of RT-PCR, 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.

5 and 3, RACE method and PCR

 The database was searched again for the partial sequences obtained as a result of RT-PCR, and highly homologous genes were re-extracted. For the deduced amino acid sequence, a multiple alignment analysis was performed in the same manner, and a region with low conservation at the amino acid level was selected. Next, considering the codon usage in DNA for the region, a primer capable of specific amplification of the target gene was designed and used in the RACE method.

 [For 3 RACE]

 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)

 [For 5'-RACE]

 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)

 As a result of the RACE method, two different gene fragments were obtained during amplification. Both belonged to the Fat A cluster consisting of TE groups with specificity for unsaturated syl ACP, but one belonged to the same subcluster as the known 0TE on the molecular phylogenetic tree, whereas the other belonged to A new subcluster was formed. .

In 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).

 As a result of the RACE method, two different gene fragments were obtained. Primers for specific amplification of both were also designed. Specifically, the following 0TE gene-specific amplification primers and PTE gene-specific amplification primers were designed to re-amplify and clone the full length cDNA and polypeptide coding region based on the sequence information determined by the RACE method.

 [0TE gene specific amplification primer]

 <For 3 '-RACE>

 0TE-2F: 5 '-GCA TTC TAG GCT AGG ATT GT-3' (SEQ ID NO: 3 7)

0TE-3F: 5 1 AAG GAA GTC CTT TAT ACG-3 '(Train number 3 8)

 <5 '-RACE use>.

 OTE-ralR: 5 '-GGC GAA TCG AGA TCG AAT CT -3' (SEQ ID NO: 3 9)

0TE-m2R: 5 '-CAC CTG AGC ATT CAC CCC ATT —3' (SEQ ID NO: 40)

0TE-m3R: 5 '-CTC AAT TTC TCC GCC AAG CT-3' (SEQ ID NO: 4 1)

 [Primers for PTE gene-specific amplification]

3 For RACE>-

PTE-2F: 5'-CTT TTC CAG TCT CGG GCT TG-3 '(SEQ ID NO: 4 2)

 <For 5'-RACE>

 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)

 We also created primers with restriction enzyme sites (Bam Hl and Sacl) for introduction into the plant expression vector pBI121 as primers for amplification of the polypeptide code.

 [For estimated polypeptide code region]

XbaBam-DcPTE-OF: 5 '-TCT AGA GGA TCC ATG TTA TTG ACA ACA GGG AC -3' (sequence number 4 5) DcPTE-OF: 5 '-ATG TTA TTG ACA ACA GGG AC 1 3 "(Self Row Number 4 6)

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)

 Using these primers, carrot RNA was converted into a saddle shape and RT-PCR was performed to amplify the entire polypeptide coding region. Next, using Takara Bio DNA Ligation kit ver. 2, PCR amplified fragment and TA-Cloning (PC.R product cloning) vector

(Novagen pSTBluel) was ligated (16 ° C, 晚 reaction). Transform T0Y0B0 competent cells (. DH5) according to the attached protocol,

Transformants were selected by culturing in LB medium supplemented with X-gal and 50 μg / 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,

They are named 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. A petrocellinol-ACP thioesterase gene derived from coriander

(CsPTE gene), dill-derived petrocellinol-ACP thioesterase gene

(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.

 Construction of histidine-tagged protein expression construct

 Oleoyl- ACP thi-oest erase ^ (Plant Physio 丄., 100,

1751-1758, 1994) to estimate the mature peptide cleavage site of each gene It was set as the N-terminus of the coding region in the mature peptide expression DNA construct. In addition, the C-terminal is up to the stop codon. For 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. As a mature peptide cleavage site, 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 (Novagen cloning vector) 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.) 50/1 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.

Expression and purification of histidine-tagged proteins by E. coli

 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.

 Next, collect 20 mL of E. coli transformed with the expression construct, add 4 mL of Lys is Buffer (50 mM sodium hydrogen phosphate, 300 mM NaCl, 10 mM imidazole) and 10 mg / ml lysozyme and mix. After standing in ice for 30 minutes, the mixture was crushed by ultrasonic treatment for 10 seconds × 6 times using an ultrasonic crusher (UD-201 manufactured by T0MY).

After centrifugation at 15000 rpm and 4 ° C for 10 minutes, the supernatant is injected into a pre-equilibrated HisTrap HP column (Amersham) with a syringe and using a peristaltic pump (P-1, Araersham). Wash and elute with Buffer supplied with the kit. Elution was performed by fractionating 4 mL of ImL. Purified DcPTEa, DcOTE, and CsOTE histidine-labeled protein eluates were subjected to SDS-PAGE (fractional sodium dodecyl sulfate-polyacrylamide gel electrophoresis) for each fraction to confirm that the target protein was purified. did. The results are shown in Fig. 4.

From the results of electrophoresis shown in Fig. 4, fractions from which high-concentration proteins were eluted were selected, and protein concentrations were measured using the Lowry method (RCDC protein assembly kit, BIO-RAD). the recombinant protein of about 200 _M g each of which can be prepared.

Preparation of Asil ACP

 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.

 Table 2

'1M Tris-HCl (pH 8.0) 400 μ 1

 • 1Μ MgCl 40 μ 1

 -1Μ ATP 200 y l

 • 1 DTT 10 1

 • 20% Triton X 100 400 μ 1

 'LiCl 400 ^ 1

 -PanVera holo- ACP 1000 μ 1

 -His-tagged Acyl-ACP synthetase (0. 26 mg / mL) 48 ^ 1

 • ¾0 1502 1

After the reaction, 12 ml of water was added, and the pH was adjusted to 6.0 with acetic acid. The acyl ACP contained in the reaction solution was purified by the procedure shown in the following (A) to (M).

 (A) DEAE-Toyopearl 650C (T0S0H) 1. Fill 8 ml into an empty polypropylene column (BI0 RAD) (use lml bed volume)

 (B) Add bis Tris-HCl pH 6.0 (referred to as Buffer B) about 10 times the amount in (A) to equilibrate.

(C) Add the Asil ACP solution (pH 6.0) to the column.

 (D) Wash with 3 mL Buffer B

(E) 80% (w / w) Wash off unreacted fatty acid with 3mL of Isopropanol in Buffer B. T JP2006 / 313449

※ e ^] :. ^^^ and Buffer B are used by mixing them individually deaerated with an ultrasonic cleaner

 (F) Wash with 3 mL Buffer B

 (G) Elution with 0.5 ml of 0.6 M LiCl in Buffer B

 (H) Add 5 ml of Octyl- Sepharose (Amersham) into another empty column.

(The bed volume should be about 3ml)

 (I) Equilibrate with 10 times the amount of Buffer B in Buffer B

 (H and I operations should be performed in advance)

 (J) Elution of (G) eluate directly to (I) and apply

 (K) lOmM MES-NaOH pH6.0 (referred to as Buffer C) Wash with 5 ml

 (L) 35 ° / o (w / w) Elution with 6 mL of Isopropanol in Buffer C

 *: [Sopropanol and Buffer C are used by mixing individually degassed with an ultrasonic cleaner.

 (M) Use a vacuum centrifugal concentrator to volatilize isopropanol

Detection of thioesterase activity of purified enzyme

 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).

 After mixing 25 mM Tris-HCl (pH 8.0), ImM DTT, acyl ACP and water, pre-incubate for 5 minutes at 25 ° C (acclimate the buffer used to the reaction temperature) and add 12 / zg DcPTE, DcOTE histidine-tagged protein, and y-globulin as a control were added, respectively, so that the total amount of the reaction solution reached 100, and reacted at 25 ° C for 30 minutes. The amount of thioesterase was measured by detecting unreacted acyl ACP and free ACP as a reaction product by SDS-PAGE using 10 minutes of the reaction solution (10 μ1). The results are shown in FIG. 5 as an electrophoretogram. .

 In addition, the gel after electrophoresis is stained with CBB, and then a high-performance scanner (Pictrostat digital

400, Fuji Photo Industry Co., Ltd.) and converted to digital image data. Image analysis software Image

Analys is ver. 3.0 (Hitachi, Ltd.) Quantified. The result is shown in Fig. 6. As shown in Fig. 6, it was found that DcPTE had a significantly higher reactivity than DcOTE when Petrocelinol-ACP was used as the substrate (Fig. 6 (B)). Conversely, when oleoyl-ACP was used as the substrate (Fig. 6 (A)), the reactivity of DcOTE was higher. Therefore, we could conclude that DcPTE is a thioesterase with specificity for petroceric acid.

 Example 3

 Next, 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.

Creating a DNA construct for constant systemic expression

 Of the vectors used for transformation, Fig. 7 shows vectors for constant whole body expression. In Fig. 7, "Pnos" means Agrobacterium-derived nopaline synthase promoter, "Tnos" means Agrobacterium-derived nopaline synthase terminator, "P35S" means CaMV35S promoter, and "NPT II" means neomycin. Phosphotransferase This refers to the sputum gene.

 Specifically, 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.

Mix so that the ratio of the backbone fragment of PBI 121 to the DNA fragment to be inserted (Dc4DES gene) is 1:10, and use an equal amount of TaKaRa Ligat ion kit ver. 2 for ³⁰ minutes at 16 ° C. Ligation reaction was performed. The total amount of the reaction solution was added to 100 μl of a competent cell (E. coli strain DH5a, T0Y0B0), and transformation was performed according to the protocol specified by the manufacturer. It was applied to an LB agar medium containing 50 jug / ml kanamycin and cultured for a while.

 On the other hand, 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

(DNA fragment containing promoter and terminator) was inserted. The DcPTE gene generation unit was amplified by PCR using primers designed to add Eco RI and Dra III sites at both ends.

Production of seed-specific expression constructs

 Of the vectors used for transformation, seed-specific expression vectors are shown in FIGS. 8 (A) to (D). In FIG. 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.

 Specifically, 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.

 On the other hand, 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

(DNA fragment containing promoter and terminator) was inserted. The DcPTE gene expression unit was amplified by PCR using primers designed to add Eco RI and Dra II I sites at both ends.

Elektroporation, law into Agrobacteriwn

 The prepared plasmid was used to transform agrobacterium by electroporation (electroporation). 40 μ1 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 µ1 force. 5 (Applied to LB agar medium containing ig / ml kanamycin and 50 g / ml rifampicin, and cultured in anther culture to select transformants. Appearance of transformed colonies After performing single-colony isolation, colonies A plurality of bacteria were collected and the presence of the desired plasmid DNA was confirmed by PCR.

Preparation of Arabidopsis transformants by vacuum infiltration

 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.

 Next, Arabidopsis thaliana with reduced pressure infiltration was cultivated, and the collected seeds were used as transformed first generation seeds (T1 seeds). However, for convenience, they serve as transformed generation seeds, but not all seeds of this seed population have been transformed.

 Next, transform the first generation seeds into kanamycin-containing medium (Murashige & Skoog basic medium with 0.5g / L MES, 10g / L sucrose, 8g / L Agar, lOOmg / L carbenicin, 50mg / L kanamycin) So that it was germinated under light conditions. A transformed individual (having kanamycin resistance and growing normally) was selected after growing for about 1 to 2 weeks. 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. .

Next, approximately lOOmg of rosette leaves are collected from the first-generation transgenic plants, and liquid nitrogen is collected. Powdered under freezing. 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 (ΝΡΤΠ) 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.

 Next, the above-mentioned line in which T-DNA introduction was confirmed was continuously cultivated, and the collected seed was used as a transformed second generation seed (T2 seed). More than 15 T2 seeds were harvested from each construct and used for the following fatty acid composition analysis.

Fatty acid composition analysis protocol for Arabidopsis seeds

 In order to analyze the fatty acid composition in seeds, seed fatty acids were methyl esterified with monomethanol hydrochloride and the n-hexane extract was analyzed by GC-MS. BHT (Butylated hydroxytoluene) was added as an antioxidant for the sample. In addition, 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.

 The 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. Table 3 Protocol for qualitative analysis

(1) Sample 5tng (Fresh weight) weighing

 (2) Place in a 1.5mL eppendorf tube (PP) and add 1 tangsten beads (Qiagen)

 (3) 0.1% BHT and 5πΜ C15: 0-O e / MeOH 500 μ1 each added

 (4) Flour (mixer mill MM300, Qiagen), Freq: l / 20s, lrain.

 (5) 10% HCl / MeOH (Tokyo Kasei; stored at 4 ° C) 500μ1 added

 (6) 80 lhr (Heat block, I aki)

 (7) 1 ml of n-hexane (for analysis of phthalate ester, Tokyo Kasei)

 (8) Vortex (mixing) 5sec

 (9) Transfer the upper layer (hexane phase) to a 1.5 ml eppendolf tube (take 800 μ1 min)

 (10) Dry under reduced pressure (Concentrater 5301, eppendolf)

 (11) n-hexane (for analysis of phthalate ester, Tokyo Kasei) 100 / il added

 (12) Transfer to GC glass bottle and seal

 (13) GC / MS analysis ''

Table 4 Protocol for quantitative analysis

 (1) Sample (seed) 10 grains (Fresh weight) weighing

 (2) Place in a 1.5 mL eppendorf tube (PP) and add 1 tangsteti beads (Qiagen)

 (3) Add 0.002% BHT and 0.1 mM C15: 0-OMe / MeOH 500 1 each

 (4) Milling (mixer mill MJ1300, Qiagen), Freq: i / 20 sec, lmin.

 (5) 10% HC1 / MeOH (Sokkyo Kasei; stored at 4 ° C) Add 500 μl

 (6) 80 ° C lhr (Heat block, Iwaki)

 (7) 1 ml of n-hexane (for analysis of phthalate ester, Sokyo Kasei)

 (8) Vortex (mixing) 5sec

 (9) Transfer the upper layer (hexane phase) to 1.5ml eppendolf.tube (take 800μ1 min)

 (7) n-hexane (for phthalate analysis, Tokyo Kasei) 800 μ 1 re-added

 (8) Vortex (mixing) 5sec ·

 (9) Transfer the upper layer (hexane phase) to the l, 5tnl-marked pendolf tube (take 800μ1 minutes)

 (10) Dry under reduced pressure (Concentrater 5301, eppendolf)

 (11) n-hexane (for analysis of phthalate ester, Sokyo Kasei) 100 / z 1 added

 (12) Transfer to GC glass bottle and seal

 (13) GC / MS analysis (manufactured by HP)

The spectrum obtained by the processing according to the above protocol was prayed under the conditions shown in Table ⁵ . This is a condition in which oleic acid (C18: l, Δ9) and petrothelic acid (C18: 1, Δ6), which are positional isomers based on the double bond position, can be separated and analyzed by GC.

Table 5 Analytical instrument: HEWLETT PACKARD GC / S HP6890 series GC system

 5973 Mass Selective Detector

 Column: SUPELCO SP-2380 (inner diameter 0-25mm, lOOm)

 Separation program: Start temperature 80 ° C, temperature increase 3 ° C / min → 180 ° C (heat retention 35 minutes),

 Temperature rise at 30 / min → 240 ° C (Insulation temperature 10 minutes) Total 80 minutes.

 Analysis mode: split (split ratio 20: 1)

 Carrier gas: Helium flow rate 1 mL / min,

 Pressure 29. 9 psi, Average Velocity 20 cm / sec

Calculate the value obtained by dividing the integrated value of each peak in the Total Ion Chromatogram of the mass detector of the GC-MS system by the internal standard and the molecular weight, and using the sum as the total fatty acid material, the mole fraction (mol -%) Shows the composition of each fatty acid. In addition, it is known that 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. However, the reproducibility when analyzed under the same conditions is very high and relative. Such a comparison is sufficiently possible.

Analysis result 1

 In order to examine the effect of the Dc4DES gene obtained in the present invention on the production of petrothelinic acid, 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. As fatty acid components, we analyzed petroceric acid and cis-4-hexadecenoic acid (16: 1Δ4), which is a precursor of petroceric acid. Table 6 shows the analysis results of these monoene unsaturated fatty acids.

 Table 6 Fatty acids! : (wt% / total fatty acid content)

 Analysis plant tissue

 G16: 1 厶 4sat SE 018: 1Λ 6 people SE

 WT leaf 0 ± 0 0 ± 0

pB-4DES / T T2 leaf 4.60 0.1 0.17 ± 0.17 As a result, not synthesized in wild-type Arabidopsis Bae Toroseri phosphate is Ί. 67w t% of the total fatty acids was accumulated in leaves of transformants obtained by introducing a _P B-Dc4DES. In other words, it was shown that petrothelic acid can be efficiently produced in plant cells by using the carrot-derived Dc4DES gene (Table 6). In addition, the introduction of Coriander-derived Cs4DES gene into tobacco cultured cells, which has already been reported, has a petrothelic acid content of 2.7 wt% (Patent Document 1). More than the Cs4DES gene from Coriander

 §

It was found that the function of the Dc4DES gene derived from carrot is excellent.

Analysis result 2

 Using the rapeseed napin promoter that expresses seeds specifically, 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.

 Table 7

 Genotype Fatty acid (mol¾)

 厶 4 SE C18: 1 厶 6 SE G20: 1 A 8 SE Total SE

 Experimental value

 WT 0.00 0.00 0.D0 0,00

 pNC≤4DES T Τ2 (π = 3) 0.38 ± 0.04 0.81 ± 0.11 0.35 ± 0.05 1.54 ± 0.20

_P NDQ4DES / WT T2 (n = 3) 0.40 ± 0.03 0.89 ± 0.09 0.41 ± 0,03 1.70 Sat 0.15 pNDc4DESPTE / WT T2 (n = Q) 0.37 ± 0.01 1.37 ± 0.07 0.53 ± 0.03 2.28 ± 0.15 pNDc4DESPTE / T T3 ( n = 3) 0.42 ± 0.02 1.83 ± 0.09 0,68 ± 0.05 2.93 ± 0.15

 Literature value

 WT 0.00 0.00 0.00

 pN-Gs4DES / WT T2 n = 10) NT NT 0.6 ± 0.04

 PN-Cs4DES / fab1 T2 (n = 6) NT NT 2.4 ± 0.2

As a result, 0.81 mol% of petostellanic acid was accumulated in the seeds of the transformant introduced with _PN -Cs4DES. In contrast, the amount of petroceric acid accumulated in the seeds of the transformants into which pN-Dc4DES was introduced was 0.89 mol%, and even in the synthesis of petrothelinic acid in seeds' accumulation, the Cs4DES gene derived from coriander The function of the Dc4DES gene derived from carrot was excellent.

In the case of the transformant introduced with pN-Dc4DES-DcPTE, 1.83 mol% of petrothelic acid was accumulated. In other words, compared to the case where only the Cs4DES gene was introduced, The amount of accumulated troceric acid increased to 226%, and the introduction of DcPTE with high substrate specificity into petrocerinol-ACP promoted the production of petrothelinic acid. In other words, it was found that co-expression of the Dc4DES gene and the DcPTE gene has an effect of showing a far higher amount of accumulated petrothelic acid than when the Dc4DES gene is expressed alone. Compared to the conventional Coriander-derived Cs4DES gene used alone, a value nearly doubled was obtained. So far, no petrothelinic acid biosynthetic genes have been known that make it possible to increase the amount of petrothelic acid accumulation by co-expression with the 4DES gene.

C, there is.

 In addition, as shown in Table 6, it is possible to produce not only petroceric acid but also cis-8-icosenoic acid by expressing the Dc4DES gene alone or by co-expressing the Dc4DES gene and the DcPTE gene. I found In addition, it was confirmed that the amount of ci s-8-icosenoic acid produced could be increased when the Dc4DES gene and the DcPTE gene were co-expressed compared to when the Dc4DES gene was expressed alone. In addition, c i s-8-icosenoic acid has not been produced in plants so far.

Analysis result 3

 Furthermore, a transformed plant in which the Dc4DES gene or DcPTE gene is expressed alone,

Analysis of each fatty acid group including saturated fatty acid in a transgenic plant co-expressed with the Dc4DES gene and the DcPTE gene was performed.

 As a result, against wild-type strains and Cs 4 DES gene and Dc4DES gene-introduced transformed plants,

It was found that the saturated fatty acid content in seeds was significantly increased in transformants in which the DcPTE gene alone or the DcPTE gene and the Dc4DES gene were co-expressed (Fig. 9).

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.

Nearly doubled. 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. In addition, as shown in 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.

In the production of nylon material (dicarboxylic acid) by oxidative decomposition of plant-derived fatty acids, "the effect of increasing the saturated fatty acid content of C1 ⁸ or more in addition to petroselinic acid accumulation promoting effect" with the DcPTE gene 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. In addition, this effect can be obtained alone without using in combination with other genes such as the Dc 4 DES gene, so it can be applied to uses other than the accumulation of petroceric acid for the production of resin raw materials (saturated fatty acid production etc.) be able to. Industrial applicability

 ADVANTAGE OF THE INVENTION According to this invention, the novel gene used in order to increase the amount of the synthesis | 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.

 All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

The scope of the claims

1. A gene encoding the following protein (a), (b) or (c).

 (a) a protein comprising the amino acid sequence shown in SEQ ID NO: 2

 (b) 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: 2 and having Δ 4 -palmitoyl-ACP desaturase activity

 (c) a protein encoded by a 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 having Δ4-palmityl-ACP desaturase activity

 2. A gene encoding the following protein (a), (b) or (c).

 (a) a protein comprising the amino acid sequence shown in SEQ ID NO: 4 or 6

 (b) a protein having an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 4 or 6, and having petrocellinol-ACP tioesterase activity

 (c) a protein 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 having petro-cerinoyl-ACP tioesterase activity

 3. An expression vector having the gene of claim 1 and / or the gene of claim 2.

4. A transformant obtained by introducing the gene according to claim 1 and / or the gene according to claim 2.

 5. introducing a gene according to claim 1 and / or a gene according to claim 2 into a plant cell to produce a transformed plant;

 A method for producing petroseric acid, comprising a step of extracting petroseric acid from a tissue collected from the transformed plant.

 6. A transformed plant cell or transformed plant individual having the ability to produce petrothelic acid, characterized by introducing the gene according to claim 1 and the gene according to claim 2.

7. The gene according to claim 1 and the gene according to claim 2 are introduced. C is-4-Hexadecenoic acid An open transformed plant cell or a transformed plant individual capable of producing cis-4-Hexadecenoic acid.

 8. A transgenic plant cell or trait having the ability to produce cis-8-icosenoic acid, characterized by introducing the gene of claim 1 and the gene of claim 2 Convertible plant individual.

 9. A transformed plant cell or a transformed plant individual having the ability to increase saturated fatty acid, wherein the gene according to claim 2 is introduced.

 10. The plant cell is transformed with an expression vector having a seed-specific promoter and the gene arranged downstream of the promoter, and the petrothelic acid is extracted from the seed. Item 6. A method for producing petroceric acid according to Item 5.

 1 1. The plant cell is transformed with an expression vector having a seed-specific promoter and the gene arranged downstream of the promoter, and the ci s-4-bexadecenoic acid (cis-4-Hexadecenoi c 6. The method for producing cis_4-hexadecenoic acid according to claim 5, wherein the acid is extracted from seeds.

 1 2. The above plant cell is transformed with an expression vector having a seed-specific promoter and the above gene located downstream of the promoter, and the above cis-8-icosenoic acid (ci s-8-icosenoic acid) 6) The method for producing cis-8-icosenoic acid according to claim 5, wherein the cis-8-icosenoic acid is extracted from seeds.

 1 3. The plant cell is transformed with an expression vector having a seed-specific promoter and the gene arranged downstream of the promoter, and the saturated fatty acid is extracted from the seed. A method for producing a saturated fatty acid according to claim 5.