JP4840360B2 - A novel gene involved in biosynthesis of petrothelic acid, a method for producing petrothelic acid - Google Patents

Description

  The present invention relates to novel genes involved in petroselinic acid biosynthesis, in particular, a carrot-derived Δ4-palmitoyl-ACP desaturase gene, and a plant-derived petrocerinoyl-ACP thioesterase gene. It relates to a manufacturing method.

In recent years, technology development for producing resins derived from biomass has also been promoted from the viewpoint of removing petroleum resources and building a recycling society in resin materials.
Nylon, a kind of engineering plastic, is synthesized by polymerizing aminocarboxylic acid or diamine and dicarboxylic acid as raw materials, but most of the raw material monomers are produced from fossil resources in the chemical industry. Sebacic acid (1,10-Decandioic Acid) is used as a biomass-derived nylon raw material, which is produced by cleaving castor oil extracted from castor with a caustic alkali. Become. 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 decomposition of unsaturated fatty acids, and petroceric acid (cis-6), which is a raw material for adipic acid, which is a raw material monomer for nylon 6,6, has been known so far. -Octodecenoic acid) production technology has been studied. (Non-patent documents 1 to 8).
Coriander, carrots, and the like of the Apiaceae plants contain 80% or more of petroceric acid in the oil component of seeds, but are not suitable for the production of petroceric acid due to low seed yield. Plant fatty acids are newly synthesized in plastids (chloroplasts). The synthesis of petrothelic acid was carried out by converting the precursor palmitoyl ACP into cis-4-hexadecenoyl ACP by Δ4-palmitoyl-ACP desaturase (hereinafter referred to as 4DES), and then extending the chain length by a prokaryotic fatty acid synthase complex. To petrocerinoyl-ACP. Furthermore, free petroceric acid is synthesized by petrocellinoyl-ACP thioesterase (hereinafter referred to as PTE) and transferred to the cytoplasm. Plants that synthesize petroceric acid are thought to have a series of biosynthetic enzymes specific to petroceric acid synthesis and their genes, among which the coriander-derived Δ4-palmitoyl-ACP desaturase gene has been cloned, Originally, a transgenic plant was introduced in which a gene was introduced into Arabidopsis thaliana, which is a plant that does not produce petroceric acid. Although the accumulation of petroceric acid was confirmed, the accumulated amount was only 1% in the seed oil. %.
Moreover, although the presence of enzyme activity has been shown for PTE (Non-Patent Document 8), the gene has not yet been cloned.
Plant fat production and its constituent fatty acid composition involve not only the fatty acid biosynthesis system in plastids but also the transport of fatty acid derivatives in the cytoplasm and the triacylglycerol synthesis system in the endoplasmic reticulum membrane. It is considered that many problems must be solved in order to produce petroceric acid using a replacement technique.
United States Patent 5,430,134 International Publication No. 94/01565 Proc. Natl. Acad. Sci. USA, 89, 11184-11188, 1992. Plant J.M. 17 (6), 679-688, 1999. Prog. Lipid Res. 33 (1/2), 155-163, 1994 Plant Physiol. , 124, 681-692,2000 Plant Mol. Biol. 47, 507-518, 2001 Metab. Eng. , 4, 12-21, 2002 Biochim. Biophys. Acta. 1212, 134-136, 1994 Plant Physiol. , 104, 839-844, 1994 Planta 215: 584-595 2002.

The present invention has been made in view of the above-described circumstances, and an object thereof is to provide a novel gene capable of promoting the accumulation of petrothelic acid 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, Δ4-palmitoyl-ACP desaturase derived from carrot (Daucus carota) belonging to the family Aceraceae is synthesized by petrothelic acid as compared with Δ4-palmitoyl-ACP desaturase derived from coriander. New findings such as excellent performance were obtained. In addition, the present inventor succeeded in isolating a petrocellinoyl-ACP thioesterase gene newly, and when this gene is used in combination with a Δ4-palmitoyl-ACP desaturase gene, the ability to synthesize petroceric acid is approximately doubled. Invented the technology to make it.
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) 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: 2, and Δ4-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-palmitoyl-ACP desaturase activity Here, in the above (b) and (c), the protein having Δ4-palmitoyl-ACP desaturase activity is expressed by cis-4-hexadecenoic acid (cis-4-hexadecenoic acid) when expressed in plant cultured cells or plant individuals. acid) or petrosseri Acid (cis-6- octadecenoic acid (cis-6-octadecenoic acid)) or cis-8- accumulated amount of icosenoic acid (cis-8-icosenoic acid) can be referred to as protein capable of increasing.
(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, and (b) 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 petrocellinoyl A protein having ACP thioesterase activity (c) 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 petroselinoyl -Protein having ACP thioesterase activity In the above (b) and (c), the protein having petroselinoyl-ACP thioesterase activity means Δ4-palmitoyl-ACP desaturase activity in plant cultured cells or individual plants. By co-expressing a protein with Cis-4-hexadecenoic acid (cis-4-hexadecenoic acid) or cis-6-octadecenoic acid) when expressed with a protein having Δ4-palmitoyl-ACP desaturase activity alone In other words, the protein can increase the amount of cis-8-icosenoic acid.
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.

FIG. 1 is a diagram comparing the homology (%) of amino acid sequences of various petrocellinoyl-ACP thioesterases and oleoyl-ACP thioesterases. In the figure, DcPTE means petrocerinoyl-ACP thioesterase derived from carrot (Daucus carota), CsPTE means petrocellinoyl-ACP thioesterase derived from coriander sativum, and AgPTE stands for dil ) Derived petrocerinoyl-ACP thioesterase, CsOTE refers to oleoyl-ACP thioesterase derived from Coriander sativum, and DcOTE refers to oleoyl-ACP thioesterase derived from carrot (Daucus carota) To do.
FIG. 2-1 is a diagram showing an alignment of amino acid sequences of various petrocellinoyl-ACP thioesterases and oleoyl-ACP thioesterases.
FIG. 2-2 is a diagram showing alignment of amino acid sequences of various petrocerinoyl-ACP thioesterases and oleoyl-ACP thioesterases.
FIG. 3 shows an alignment of the amino acid sequence of Δ4-palmitoyl-ACP desaturase (referred to as Dc4DES) derived from carrot (Daucus carota) and the amino acid sequence of Δ4-palmitoyl-ACP desaturase (referred to as Cs4DES) derived from coriandrum sativum. FIG.
FIG. 4 shows purified petroselinoyl-ACP thioesterase (referred to as DcPTE) from carrot (Daucus carota), oleoyl-ACP thioesterase (referred to as CsOTE) from coriander (Corandrum sativum) and carrot (Daucus carota). ) Derived oleoyl-ACP thioesterase (referred to as DcOTE) from histidine-labeled protein eluate from E. coli.
FIG. 5 is a photograph showing the results of SDS-PAGE after reaction with purified A-conjugated histidine-labeled protein eluate of DcPTE and DcOTE using acyl 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 a constant systemic expression vector prepared in the example.
FIG. 8 is a block diagram schematically showing the seed-specific expression vector prepared in the example.
FIG. 9 is a diagram showing the results of fatty acid composition analysis in the seeds of a transformed plant.

  Hereinafter, the novel gene and the method for producing petroseric acid according to the present invention will be described in detail with reference to the drawings.
1. Acquisition of Δ4-palmitoyl-ACP desaturase gene (Dc4DES gene) derived from carrot (Daucus carota)
  The Dc4DES gene encodes a protein (Dc4DES) comprising the amino acid sequence shown in SEQ ID NO: 2. Dc4DES is a protein having an activity of desaturating the Δ4 position in palmitoyl-ACP (palmitoyyl-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.
  In the present invention, the Dc4DES 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: 2, and encodes a protein having Δ4-palmitoyl-ACP desaturase activity. It may be. 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, but for example, the 1-99th or 270-386th region in the amino acid sequence shown in SEQ ID NO: 2, preferably the 1-99th or 301-386th region, More preferably, it is the 1st to 53rd or 381 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 Δ4- It may be a gene encoding a protein having palmitoyl-ACP desaturase activity. Here, the numerical value of the homology is obtained by executing a command of the Maxim matching method, for example, using DNASIS (Hitachi Software Engineering) which is sequence analysis software. The parameters at that time are 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 has Δ4-palmitoyl-ACP desaturase activity. It may be a gene encoding a protein having Here, “hybridizes under stringent conditions” means, for example, heating in a solution of 6 × SSC, 0.5% SDS and 50% formamide at 42 ° C., then 0.1 × SSC, This shows that a positive hybridization signal is still observed even under the condition of washing at 68 ° C. in a 0.5% SDS solution.
  The Δ4-palmitoyl-ACP desaturase activity means an activity that desaturates the Δ4 position in palmitoyl-ACP. For the presence or absence of the activity, a DNA fragment encoding the protein to be assayed is introduced into a functioning host plant cell that does not accumulate cis-4-hexadecenoic acid, petroceric acid, cis-8-icosenoic acid such as tobacco or Arabidopsis thaliana. It can be assayed by measuring the presence or absence of cis-4-hexadecenoic acid, petrothelic acid, cis-8-icosenoic acid in the lipids of the introduced plant. For example, cis-4-hexadecenoic acid such as tobacco and Arabidopsis, using an expression vector in which the Dc4DES gene is arranged downstream of the constitutive expression promoter or the specific expression promoter, that is, at a position controllable by the promoter, Plant cells that do not accumulate petroceric acid, cis-8-icosenoic acid are transformed. The lipids of the seeds of the produced transformed plant cells or plant bodies regenerated therefrom are extracted. Fatty acid methyl ester is obtained by treating the extracted lipid with methanolic hydrochloric acid, etc., and the amount of petroselinic acid methyl ester, cis-4-hexadecenoic acid fatty acid methyl ester, and cis-8-icosenoic acid fatty acid methyl ester contained in this gas are gasses. Measure by chromatography. If these fatty acid methyl esters can be detected, it can be said that the protein to be assayed has Δ4-palmitoyl-ACP desaturase activity, and if these fatty acid methyl esters cannot be detected, it does not have Δ4-palmitoyl-ACP desaturase activity.
  The Dc4DES gene has a function of promoting petroselinic acid synthesis in a cell by introducing it into a functioning host plant cell. 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. By cultivating the obtained transformed plant to obtain 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 with a tissue such as a seed and mixed with a hydrochloric acid-methanol solution to methyl esterize petroselinic acid and extract with hexane. The hexane extract can be detected by a gas chromatograph-mass spectrometer (GC-MS) apparatus. From the result of detection by the GC-MS apparatus, the ability to promote the synthesis of petrothelic acid can be analyzed.
2. Petrocellinoyl-ACP thioesterase enzyme gene (PTE gene)
  The PTE gene is a newly isolated and cloned gene in the present invention in Coriander sativum, although its presence has been pointed out but not isolated or cloned. The PTE gene is a gene encoding thioesterase (PTE) having high specificity for acyl ACP (acyl carrier protein) having a double bond at Δ6 position, such as petrocellinoyl-ACP. PTE is involved in the production of free petroselinic 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. A gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 4 is shown in SEQ ID NO: 3, and a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 6 is shown in SEQ ID NO: 5. The carrot-derived PTE gene and PTE are referred to as DcPTE gene and DcPTE, respectively.
  In the present invention, the DcPTE gene may include 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 1st to 70th or 311 to 375th region, preferably the 1st to 57th or 368 to 375th region in the amino acid sequence shown in SEQ ID NO: 4, More preferably, it is the 1st to 32nd region.
  The PTE gene encoding a protein containing an amino acid sequence in which substitution, deletion, or insertion is introduced into the amino acid sequence shown in SEQ ID NO: 4 or 6 is a known method such as the Kunkel method or Gapped duplex method, or the like. It can be obtained by adopting the method and introducing a desired mutation into the base sequence shown in SEQ ID NO: 3 or 5. For example, using a mutagenesis kit (for example, Mutan-K (manufactured by TAKARA) or Mutan-G (manufactured by TAKARA)) using site-directed mutagenesis or the like, or LA PCR in vitro Mutagenesis of TAKARA Mutation is introduced using a 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 or 6. It may be a gene encoding a protein having petrocellinoyl-ACP thioesterase activity. Here, the numerical value of the homology is obtained by executing a command of the Maxim matching method, for example, using DNASIS (Hitachi Software Engineering) which is sequence analysis software. The parameters at that time are default settings (initial settings).
  Furthermore, 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 petrocellinoyl-ACP. It may be a gene encoding a protein having thioesterase activity. Here, “hybridizes under stringent conditions” means, for example, heating in a solution of 6 × SSC, 0.5% SDS and 50% formamide at 42 ° C., then 0.1 × SSC, This shows that a positive hybridization signal is still observed even under the condition of washing at 68 ° C. in a 0.5% SDS solution.
  The petrocellinoyl-ACP thioesterase activity means the activity of decomposing petrocellinoyl-ACP into petrothelic acid and ACP. This activity is obtained by mixing 25 mM Tris-HCl (pH 8.0), 1 mM DTT, petrocellinoyl-ACP and water, pre-incubating at 25 ° C. for 5 minutes, and adding 15 μg of the protein to be assayed. 100 μl is reacted at 25 ° C. for 30 minutes. After the reaction solution, unreacted petrocellinoyl-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 as a reaction product is quantified with a densitometer by SDS-PAGE to measure the thioesterase activity of the added protein to be assayed. Alternatively, 25 mM Tris-HCl (pH 8.0), 1 mM DTT, and petrocellinoyl-ACP labeled with tritium are mixed with water and then pre-incubated at 25 ° C. for 5 minutes. 100 μl of the added reaction solution is reacted at 25 ° C. for 30 minutes. After the reaction, 50 μl of isopropanol was added to stop the reaction, and then the petroceric acid generated by the enzyme reaction was separated by thin layer chromatography, and the amount of petrothelic acid generated by scintillation counter was quantified to add the thioesterase of the protein to be assayed. Measure activity.
  Further, in the present invention, the PTE gene is not limited to a gene encoding carrot-derived PTE, and means that it includes a plant-derived PTE gene that biosynthesizes petroseric acid. As plants for biosynthesis of petroceric acid, other than carrots, coriander (Coriandrum sativium), parsley (Peteroselium crispum), dill (Anetum graveolens), etc. A plant etc. can be mentioned.
  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 a long nucleic acid sequence (> 100 bp) is used as a probe, it can also be screened with moderate to high stringency to obtain a signal from a target sample that is 80% or more homologous sequence. Also, the probe may 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 using short regions 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. The PTE gene of the plant can be isolated and identified by using cDNA synthesized using mRNA extracted from the plant as a template instead of genomic DNA.
  Based on the base sequence of the DcPTE gene shown in SEQ ID NO: 3 or 5, the base sequences of DcPTE homologous genes isolated from Coriander sativium and Dill (Anetum graveolens) are shown in SEQ ID NOs: 7 and 9, respectively. 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 SEQ ID NO: 10 shows. Moreover, the amino acid homology (%) between each protein in the thioesterase of various plants is shown in FIG.
  As shown in FIG. 1, high homology is shown in the PTE group including DcPTE, CsPTE and AgPTE. Moreover, high homology is also shown between DcOTE and CsOTE. On the other hand, the PTE group and the OTE group show relatively low homology. Therefore, 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 includes a DNA encoding a protein containing an amino acid sequence whose homology with the amino acid sequence shown in SEQ ID NO: 4, 6 or 8, 10 exceeds 80%. .
  Further, FIG. 2 shows an alignment diagram of amino acid sequences of DcPTE, CsPTE, AgPTE, DcOTE, and CsOTE. There is an approximately 30% amino acid difference between OTE and PTE. Among them, it is shown that the polarity of some amino acids is different in the OTE group compared to the PTE group. In the following description, “common array X (X is a natural number)” means a 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 nonpolar, while in the OTE group, the amino acids are polar. In the PTE group, the amino acids of the common sequences Nos. 140 and 195 are polar, whereas in the OTE group, the amino acid is nonpolar.
  Furthermore, it is shown that some charged amino acids are different in the OTE 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 OTE group, the amino acids are positively charged. Further, in the PTE group, the amino acid of the common sequence 244 is negatively charged, while in the OTE group, the amino acid is an amino acid having no charge. In the PTE group, the amino acid of the common sequence 270 is positively charged, while in the OTE group, the amino acid is an amino acid having no charge.
  It is thought that the presence or absence of amino acid polarity and changes in charge at these positions 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 is a difference in amino acid side chain structure between the amino acids of consensus sequences Nos. 89, 147, 161, 177, 192, 196, 214, 217, 223, 238, 270, 287, 338 and OTE. Substituting those amino acids that differ between the OTE and PTE groups may modify the substrate specificity for acyl ACP.
  The PTE gene has a function of greatly promoting petroceric acid synthesis in the cell by introducing it into the host plant cell together with the Δ4-palmitoyl-ACP desaturase gene.
  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 a plant cell obtained by transforming the Δ4-palmitoyl-ACP desaturase gene (see 1. above). Alternatively, plant cells may be transformed using an expression vector in which the PTE gene and Δ4-palmitoyl-ACP desaturase gene are arranged downstream of the constitutive expression promoter or the specific expression promoter. In either case, petroselinic acid synthesis in the plant can be promoted by growing the resulting transformed plant into a plant.
  For example, after growing a transformed plant into a plant body, petroseric acid is pulverized with a tissue such as a seed and mixed with a hydrochloric acid-methanol solution to methyl esterize petroselinic acid and extract with hexane. The hexane extract can be detected by a gas chromatograph-mass spectrometer (GC-MS) apparatus. From the result of detection by the GC-MS apparatus, the ability to promote the synthesis of petrothelic acid can be analyzed.
Expression vector
  In the present invention, the expression vector is the above-mentioned 1. And 2. Which has the Dc4DES gene and / or the PTE gene described in 1. above. The expression vector is not particularly limited as long as it is a plasmid type vector or a chromosome introduction type vector that can be integrated into the genome of the host organism. For example, plasmid DNA, bacteriophage DNA, retrotransposon DNA, artificial chromosome DNA (YAC: yeast) artifical chromosome).
  Examples of plasmid DNA include YCp type E. coli-yeast shuttle vectors such as pRS413, pRS414, pRS415, pRS416, YCp50, pAUR112 or pAUR123, YEp type E. coli-yeast shuttle vectors such as pYES2 or YEp13, pRS403, pRS404, pRS405, pRS406, YIp-type E. coli-yeast shuttle vectors such as pAUR101 or pAUR135, plasmids derived from E. coli (pBR322, pBR325, pUC18, pUC19, pUC118, pUC119, pTV118N, pTV119N, pBluescript, pHSG298, pHSG396 or pTrcC17pCoCY17) P15A system such as Plasmids such as Smid, pMW118, pMW119, pMW218 or pMW219), Agrobacterium-derived plasmids (eg, pBI101), Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, etc.), etc. λ phage (Charon 4A, Charon 21A, EMBL3, EMBL4, λgt10, λgt11, λZAP), φX174, M13mp18, M13mp19 and the like. Examples of retrotransposons include Ty factor. 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, it is necessary that the Dc4DES gene and / or the PTE gene be incorporated into the vector in such a state that they can be expressed. An expressible state is a vector in which these Dc4DES gene and / or PTE gene and a promoter are linked so that the Dc4DES gene and / or PTE gene is expressed under the control of a predetermined promoter in a host organism into which the Dc4DES gene and / or PTE gene is introduced. Means to be incorporated into Therefore, in addition to the Dc4DES gene and / or PTE gene, a promoter and terminator, a cis element such as an enhancer, a splicing signal, a poly A addition signal, a selection marker, a ribosome binding sequence (SD sequence) and the like are linked to the vector. be able to. Examples of selectable markers include antibiotic resistance genes such as ampicillin resistance gene, kanamycin resistance gene and hygromycin resistance gene, and herbicide resistance genes such as 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 stimulation-inducible promoter. Among them, when it is intended to accumulate synthesized petroceric acid in the seed, it is preferable to use a seed-specific expression promoter. As the seed-specific expression promoter, rapeseed-derived napin A promoter, Arabidopsis-derived FAE1 promoter, oleosin promoter, soybean-derived glutelin B1 promoter, flax stearoyl-ACP desaturase (SAD) promoter, and the like can be used.
Transformant
  A transformant can be prepared using the expression vector 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 PTE gene contained in the vector can be expressed. The host is not particularly limited. For example, plants belonging to the celery family, solanaceous family, cruciferous family, gramineous family, legume family, rose family, asteraceae family, lily family, dianthus family, cucurbitaceae family, convolvulaceae family, red department family, etc. In particular, it is desirable to use a celery family or a cruciferous plant as a host.
  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, A fence-like tissue, a spongy tissue, etc.) or a plant cultured cell is meant.
  An expression vector can be introduced into a plant by a conventional transformation method, for example, a reduced pressure infiltration method (Agrobacterium 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.). When Agrobacterium is used, an expression vector is introduced into an appropriate Agrobacterium such as Agrobacterium tumefaciens LBA4404, and this strain is introduced by the leaf disk method (Hirofumi Uchimiya, plant gene manipulation). Manual, 1990, 27-31pp, Kodansha Scientific, Tokyo, etc.) can be used to infect a sterile cultured leaf piece of a host (for example, tobacco) to obtain a transformed plant.
  When the particle gun method is used, a plant body, a plant organ, and a plant tissue itself may be used as they are, or may be used after preparing a section, or a protoplast may be prepared and used. The sample thus prepared can be processed using a gene introduction apparatus (for example, PDS-1000 (BIO-RAD)). The treatment conditions vary depending on the plant or sample, but are usually performed at a pressure of about 450 to 2000 psi and a distance of about 3 to 12 cm.
  Tumor tissue, 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. The plant can be regenerated by administration of a concentration of plant hormones (auxin, cytokinin, gibberellin, abscisic acid, ethylene, brassinolide, etc.).
  Whether or not the gene has been incorporated into the host can be confirmed by PCR, Southern hybridization, Northern hybridization, or the like. For example, DNA is prepared from the transformant, PCR is performed by designing a DNA-specific primer. PCR can be performed under the same conditions as those used for preparing the plasmid. Thereafter, the amplification product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., stained with ethidium bromide, SYBR Green solution, etc., and the amplification product is detected as one band, It can be confirmed that it has been transformed. Moreover, PCR can be performed using a primer previously labeled with a fluorescent dye or the like to detect an amplification product. Furthermore, it is possible to employ 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, examples of the host include Escherichia such as Escherichia coli, Bacillus subtilis such as Bacillus subtilis, Pseudomonas putida such as Pseudomonas pt. Examples include bacteria belonging to the genus, yeasts such as Saccharomyces cerevisiae and Schizosaccharomyces pombe, and animal cells such as COS cells and CHO cells, and Sf9. Cell.
  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 comprises a ribosome binding sequence, the gene of the present invention, and a transcription termination sequence. Examples of E. coli include Escherichia coli DH5α and Y1090. Examples of Bacillus subtilis include, but are not limited to, Bacillus subtilis. 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, S .; N. et al. : Proc. Natl. Acad. Sci. USA, 69: 2110 (1972)], electroporation method and the like.
  When yeast is used as a host, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris 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 method [Becker, D. et al. M.M. et al. : Methods. Enzymol. 194: 182 (1990)], spheroplast method [Hinnen, A. et al. et al. : Proc. Natl. Acad. Sci. USA, 75, 1929 (1978)], lithium acetate method [Itoh, H. et al. : J. Bacteriol. , 153: 163 (1983)].
  When animal cells are used as hosts, monkey cells COS-7, Vero, Chinese hamster ovary cells (CHO cells), mouse L cells, rat GH3, human FL cells and the like are used. Examples of methods for introducing a recombinant vector into animal cells include an electroporation method, a calcium phosphate method, and a lipofection method.
  When insect cells are used as hosts, Sf9 cells and the like are used. Examples of the method for introducing a recombinant vector into insect cells include the calcium phosphate method, lipofection method, electroporation method and the like.
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. Petroceric acid synthesized and accumulated in the transformed plant can be extracted using a conventionally known technique.
  The method of squeezing oils and fats from plant tissues such as seeds and fruits of oil-producing plants can be broadly divided into a pressing method and an extraction method. For example, a method of obtaining fat from a tissue having a high fat content such as rapeseed seeds is obtained by crushing and compressing the tissue with a roll mill or the like and heating to 75 to 85 ° C., and then pressing with a pressing machine such as an expeller to take out the fat ( Squeezing method). On the other hand, a raw material having a low fat content such as soybean is extracted from a tissue using a solvent such as hexane (extraction method). The fats and oils produced by these steps 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. By separating and purifying the resulting fatty acid mixture, it is possible to obtain high purity petroceric acid. Moreover, alcohol esters, such as a petroselinic acid methyl ester, can be obtained by adding and reacting alcohol, such as methanol, to fats and oils.
  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.

Cloning of Dc4DES gene
Plant Sample In this example, carrot (Daucus carota 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 variety was used for the convenience of the experiment (a sample that flowered early) was prepared. Carrot seeds were purchased from Ota seedlings. A carrot plant that was cultivated under conditions of 25 ° C., 16 hours of sunshine, and 60% humidity in an artificial meteorological device (Koitron, manufactured by Koito Seisakusho) was used as a sample.
Preparation of carrot RNA About 100 mg of immature seeds and about 100 mg of leaves with different seed development stages were collected from carrot plants and ground under liquid nitrogen freezing. From the obtained pulverized product, RNA was prepared according to the protocol attached to the kit using RNeasy plant mini kit manufactured by QIAGEN.
PCR primer design and RT-PCR for DNA fragment amplification
Searched for homologous genes collected from BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) in the NCBI site by coriander (Coriandrum sativum L.) Δ4 palmitoyl-ACP desaturase (Cs4DES) Subsequently, the amino acid sequence of the polypeptide encoded by the obtained Cs4DES gene (GenBank accession number M93115) and the Δ9 stearoyl-ACP desaturase gene of various plants registered on Gen Bank is Genetyx-Win Ver. 4.0 / ATGC ver. Multiple alignment analysis was performed with 2.0 (Software Development). As a result of the analysis, the following PCR primers (degenerate primers) were designed and used for RT-PCR in order to amplify the obtained DNA fragment corresponding to the highly conserved region.
PF1: 5 ′ CAN GAR GAR GCN CTB CCN CAN TA 3 ′ (SEQ ID NO: 11)
PR1: 5 ′ TCV RVD AGY TTY TCN ACD ATY TT 3 ′ (SEQ ID NO: 12)
PR2: 5 ′ GCN GYY KCR TGN CKY TTY TCR TC 3 ′ (SEQ ID NO: 13)
NF0: 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)
In addition, these base sequences are described in the following notation method for common base sequences (IUB) 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 Show.
RT-PCR was performed using the PCR primer pairs shown in Table 1. One-step RT-PCR kit manufactured by QIAGEN was used for RT-PCR. The reaction solution composition was carried out using an Eppendorf thermal cycler (master cycler gradient) according to the protocol attached to the kit. The annealing temperature was 50 ° C. to 70 ° C. (5 steps every 5 ° C.). RT-PCR was performed at 50 ° C. for 30 minutes and at 94 ° C. for 15 minutes, followed by 40 cycles of 94 ° C. for 1 minute, 50-70 ° C. for 1 minute and 72 ° C. for 1 minute 30 seconds, Then, it processed at 72 degreeC for 15 minutes. After the reaction, 4 ° C. was maintained.
The reaction solution after PCR was electrophoresed with TAE buffer using an agarose gel. The agarose gel after electrophoresis was stained with ethidium bromide 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 QIAquick Gel extraction kit manufactured by QIAGEN. The base sequence of the purified PCR product was confirmed using an ABI DNA sequencer (3100 Genetic Analyzer). For the sequencing reaction, ABI BigDye Terminator Cycle Sequencing, FS kit (ver. 3.0) was used. The experimental procedure was carried out according to the manual made by ABI. In addition, the primer of Table 1 was used for the determination of a base sequence.
5 'and 3' RACE methods and PCR
Based on the sequence information of the DNA fragment obtained by RT-PCR, the following primers were designed and used for 5 'and 3' RACE methods.
[Primers for 5 'RACE]
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: 18)
[3'RACE primer]
D2-F4: 5 'CAA ATG CCA GCT CAT GCA ATG 3' (SEQ ID NO: 19)
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 a Roche 5 ′ / 3 ′ RACE Kit. PCR was performed using Ex Taq Hot Start Version manufactured by Takara Bio. The reaction solution composition followed the protocol attached to the kit. An Eppendorf thermal cycler (master cycler gradient) was used. The annealing temperature was 50 ° C. to 70 ° C. (5 steps every 5 ° C.). PCR conditions were 94 ° C for 15 minutes, then 30 cycles of 94 ° C for 1 minute, 50-70 ° C for 1 minute and 72 ° C for 1 minute 30 seconds, followed by 72 ° C for 15 minutes. . After the reaction, 4 ° C. was maintained.
Purification of the PCR product and determination of the base sequence were performed in the same manner as described above.
Design of PCR Primer for Amplifying 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. The following primers with restriction enzyme sites (BamH I and Sac I) for introduction into the plant expression vector pBI121 were also prepared as PCR primers for polypeptide coding region amplification.
4DS-F-OR1: 5 ′ ATG GCT ATG AAA TTG AAC GCC 3 ′ (SEQ ID NO: 21)
Bam-4DS-F-OR1: 5 ′ TCT AGA GGA TCC ATG GCT ATG AAA TTG AAC GCC 3 ′ (SEQ ID NO: 22)
4DS-R-OR: 5 ′ TCA TAT CAT GAT CTG ACG GTT G 3 ′ (SEQ ID NO: 23)
Sac-4DS-R-OR1: 5 ′ TCT AGA CGA GCT CTC ATA TCA TGA TCT GAC GGT TG 3 ′ (SEQ ID NO: 24)
PCR was performed using these primers to amplify the entire polypeptide coding region. Next, DNA Ligation kit ver. 2 was used for ligation (16 ° C., overnight reaction) of the DNA fragment of the polypeptide coding region and the TA-Cloning (PCR product cloning) vector (pSTBlue 1 from Novagen). A TOYOBO competent cell (E. coli DH5α) was transformed according to the attached protocol, cultured in LB medium supplemented with IPTG, X-gal, and 50 μg / ml kanamycin, and a transformant was selected. Plasmid DNA was prepared from the cells obtained by liquid culture in an LB medium supplemented with 50 μg / ml kanamycin by picking up the emerged colonies using a plasmid mini kit manufactured by QIAGEN. The inserted fragment was confirmed by gel electrophoresis to obtain plasmid DNA in which the target fragment was expected to be subcloned.
Sequencing primers were primers targeting T7 and M13 sequences present at both ends of the cloning site of pSTBlue1 vector (Takara Bio BcaBEST Sequencing Primer T7; 5 ′ TAA TAC GAC TCA CTA TAG GG 3 ′ (SEQ ID NO: 25) And M13 Primer M4; 5 ′ GTT TTC CCA GTC ACG AC 3 ′ (SEQ ID NO: 26)). 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 Genetyx-Win Ver. 4.0 / ATGC ver. 2 (Software Development Co., Ltd.) was used for analysis and editing.
The nucleotide sequence identity between the Dc4DES gene and the previously reported coriander-derived gene (Cs4DES) was 88.0% in the polypeptide coding region (presumed) sequence (Dc4DES gene 1161 bp, Cs4DES gene 1158 bp). Further, the amino acid sequence identity was 90.2% in the polypeptide coding region deduced amino acid sequences (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.

Identification and cloning of the PTE gene
In plant sample Example 2, as an experimental sample, F1 varieties of carrots (Daucus carota L.), summer sowing fresh red 5 inch, Yangshu 5 inch, and fixed type Shin Kuroda 5 inch were used. Carrot seeds were purchased from Ota seedlings. As a gene cloning source, a fixed species having the same homologous gene sequence is desirable, but F1 varieties were also used for the convenience of the experiment (a sample that flowered early) was prepared. In addition, coriander (Corillarum sativium) and dill (Anetum graveolens cv. Mammoth), which are the same celery family, were also cultivated and used.
A plant body cultivated at 25 ° C., 16 hours of sunshine, and 50% humidity in an artificial meteorological apparatus (Koitron, Koito Seisakusho) was used as a sample.
Preparation of RNA About 100 mg each of carrot leaves, immature seeds, and mature seeds were collected, and RNA was prepared according to the method of Example 1.
BLAST search (blastn and blastx; http: //www.ncbi.) For a partial cDNA sequence (accession number, L20978) of an OTE-like gene (CsOTE) derived from coriander, the same family of celery family as RT-PCR amplified carrots. nlm.nih.gov/blast/) and a list of 17 similar genes hit as highly homologous genes. Multiple alignment analysis was further performed on the deduced amino acid sequence, and a highly conserved region was selected. In addition, multiple alignment analysis was performed on the DNA sequence of the same gene in the same way, and degenerate primers were designed in consideration of the codon usage in closely related DNA for regions that were highly conserved at the amino acid level and used for RT-PCR. did. The sequence is shown below. The numbers in parentheses indicate base numbers corresponding to oleoyl-ACP thioesterase (AtOTE) of Arabidopsis thaliana.
TE-PF3 (515-536): 5′-RTG GNA CNM GRG KRR ATT GGA T-3 ′ (SEQ ID NO: 27)
TE-PF2 (415-438): 5′-CTB ATW TGG GTB ACD DMN MGN ATG-3 ′ (SEQ ID NO: 28)
TE-PF1 (235-257): 5′-GAR RAY GGN YWN TCB TAY AMR GA-3 ′ (SEQ ID NO: 29)
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: 31)
TE-PR0 (1041-1061): 5′-GTD SKN CMV CKR TTK AKY TC-3 ′ (SEQ ID NO: 32)
Specifically, RT-PCR amplification was performed using the above-described primer combination pair using a QIAGEN One-step RT-PCR kit. 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 the reaction by setting an arbitrary (up to 12 steps) temperature gradient on the heat block, the annealing temperature important for amplification efficiency and specificity is 50 ° C to 70 ° C (every 5 ° C). 5 steps). PCR conditions were 50 ° C. for 30 minutes, 94 ° C. for 15 minutes, and then 94 cycles of 94 ° C. for 1 minute, 50-70 ° C. for 1 minute and 72 ° C. for 1 minute 30 seconds, followed by 72 cycles. Treated for 15 minutes at ° C. After the reaction, 4 ° C. was maintained.
As a result of RT-PCR, a specific amplification product (about 500 bp) was obtained by the combination of TE-PF2 and TE-PR1. When the base sequence of this amplified product was decoded and a molecular phylogenetic tree analysis was performed using the partial sequence, it was found to belong to the OTE cluster.
5 'and 3' RACE methods and PCR
The database was searched again for the partial sequences obtained as a result of RT-PCR, and genes with high homology 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, in consideration of 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: 33)
TE-D3F: 5′-GTT TTC TGC CCC AAA ACA CC-3 ′ (SEQ ID NO: 34)
[For 5'-RACE]
TE-D2R: 5'-TAT TCA TCT CGA ACA TCA T-3 '(SEQ ID NO: 35)
TE-D1R: 5′-ATC ATC ACC CAC TTG CTA-3 ′ (SEQ ID NO: 36)
As a result of the RACE method, two different gene fragments were obtained in the amplification. Both belonged to the Fat A cluster consisting of TEs with specificity for unsaturated acyl ACPs, but one belonged to the same subcluster as the known OTE on the molecular phylogenetic tree, while the other was new Sub-clusters were 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, the molecular phylogenetic tree analysis is performed by using the gene analysis service (http://www.ddbj.nig.ac.jp/Welcome-j.html) of the National Institute of Genetics, Japan DNA Data Bank, and the Clustal W program ( (Multiple alignment of base sequence / amino acid sequence and phylogenetic tree creation program).
Since two different gene fragments were obtained as a result of the RACE method, primers for specific amplification of both were also designed. That is, the following OTE gene-specific amplification primer and PTE gene-specific amplification primer were designed to re-amplify and clone the full length of the cDNA and polypeptide coding region based on the sequence information determined by the RACE method.
[Primers for OTE gene-specific amplification]
<For 3'-RACE>
OTE-2F: 5′-GCA TTC TAG GCT AGG ATT GT-3 ′ (SEQ ID NO: 37)
OTE-3F: 5′-AAG GAA GTC CTT TAT ACG-3 ′ (SEQ ID NO: 38)
<For 5'-RACE>
OTE-m1R: 5′-GGC GAA TCG AGA TCG AAT CT-3 ′ (SEQ ID NO: 39)
OTE-m2R: 5'-CAC CTG AGC ATT CAC CCC ATT-3 '(SEQ ID NO: 40)
OTE-m3R: 5′-CTC AAT TTC TCC GCC AAG CT-3 ′ (SEQ ID NO: 41)
[Primers for PTE gene-specific amplification]
<For 3'-RACE>
PTE-2F: 5′-CTT TTC CAG TCT CGG GCT TG-3 ′ (SEQ ID NO: 42)
<For 5'-RACE>
PTE-4R: 5′-GGA AGC AAC TCA TCG TCG TCT GT-3 ′ (SEQ ID NO: 43)
PTE-2R: 5′-CAA GCC CGA GAC TGG AAA AG-3 ′ (SEQ ID NO: 44)
In addition, a primer added with restriction enzyme sites (BamH1 and SacI) for introduction into the plant expression vector pBI121 was also prepared as a primer for amplifying the polypeptide code.
[For putative polypeptide coding region]
XbaBam-DcPTE-OF: 5′-TCT AGA GGA TCC ATG TTA TTG ACA ACA GGG AC-3 ′ (SEQ ID NO: 45)
DcPTE-OF: 5′-ATG TTA TTG ACA ACA GGG AC-3 ′ (SEQ ID NO: 46)
Sac-DcPTE-6R: 5′-TCT AGA CGA GCT CCT AGT TTA AAC AGT ACA CTG-3 ′ (SEQ ID NO: 47)
DcPTE-6R: 5′-CTA GTT TAA ACA GTA CAC TG-3 ′ (SEQ ID NO: 48)
Using these primers, RT-PCR was performed using carrot RNA as a template to amplify the entire polypeptide coding region. Next, DNA Ligation kit ver. 2 was used to ligate the PCR-amplified fragment and TA-Cloning (PCR product cloning) vector (pSTBlue 1 from Novagen) (reaction overnight at 16 ° C.). A TOYOBO competent cell (E. coli DH5α) was transformed according to the attached protocol, cultured in LB medium supplemented with IPTG, X-gal, and 50 μg / ml kanamycin, and a transformant was selected. Plasmid DNA was prepared from the cells obtained by liquid culture in an LB medium supplemented with 50 μg / ml kanamycin by picking up the emerged colonies using a plasmid mini kit manufactured by QIAGEN. The inserted fragment was confirmed by gel electrophoresis to obtain 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 named Dc4PTEa and Dc4PTEb, their base sequences are shown in SEQ ID NOs: 3 and 5, respectively, and the amino acid sequences of the two types 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, the cloning of the PTE gene was also attempted for coriander and dill. As a result, the petrocerinoyl-ACP thioesterase gene derived from coriander (CsPTE gene), the petrocellinoyl-ACP thioesterase gene derived from 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 petroselinoyl-ACP thioesterase activity 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.
Preparation of expression constructs for histidine-tagged proteins Based on the knowledge of known safflower-derived oleoyl-ACP thioesterase (Plant Physiol., 100, 1751-1758, 1994), the type of maturity peptide cleavage site is estimated, Set as the N-terminus of the coding region in the construct. In addition, the C-terminal side is up to the stop codon. For the DcPTEa gene, a distance between the base sequence encoding the 32nd amino acid and the base sequence encoding the 33rd amino acid was estimated as a “mature peptide cleavage site”. For the DcOTE gene and the CsOTE gene, a gap between the base sequence encoding the 51st amino acid and the base sequence encoding the 52nd amino acid was estimated as a “mature peptide cleavage site”.
The region was amplified by PCR using DcPTEa, DcOTE, and CsOTE cDNA cloned in pST Blue1 (Novagen cloning vector) as a template. The product was mixed with an expression vector for histidine-tagged protein pQE-30UA (manufactured by QIAGEN), and an equal amount of TaKaRa Ligation kit ver. 2 was added and a ligation reaction was performed at 16 ° C. for 30 minutes for subcloning. By subcloning into this vector, a recombinant protein with 6XHis added to the N-terminus can be prepared in E. coli. The total amount of the reaction solution was added to 50 μl of E. coli competent cells (JM109 strain with lacI ^q mutation, manufactured by Takara Bio Inc.), and the transformation operation was performed according to the protocol specified by the manufacturer. A plasmid was prepared from the obtained transformant (grown on an LB agar medium containing 50 μg / ml ampicillin).
Expression and purification of histidine-tagged protein by Escherichia coli The prepared histidine-tagged protein expression DNA construct was transformed into E. coli (JM109 strain) (or using a glycerol stock stored before plasmid preparation), and the Overnight Express Automation System 1 (Merck) ) And 50 μg / ml ampicillin added to the LB medium overnight to induce expression.
Next, 20 mL of E. coli transformed by the expression construct was collected, 4 mL of Lysis Buffer (50 mM sodium hydrogen phosphate, 300 mM NaCl, 10 mM imidazole) and 10 mg / ml lysozyme were added, mixed, and mixed in ice for 30 minutes. Then, the mixture was crushed by ultrasonic treatment for 10 seconds × 6 times using an ultrasonic crushing device (UD-201 manufactured by TOMY).
After centrifugation at 15000 rpm and 4 ° C. for 10 minutes, the supernatant is injected into a HisTrapHP column (Amersham) that has been equilibrated in advance with a syringe, and a kit is used using a peristaltic pump (P-1, manufactured by Amersham). The sample was washed and eluted with the buffer attached. Elution was fractionated in 4 mL total volume of 1 mL.
When the purified histidine-labeled protein eluates of DcPTEa, DcOTE, and CsOTE were subjected to SDS-PAGE (fractional sodium dodecyl sulfate-polyacrylamide gel electrophoresis) one fraction at a time, it was confirmed that the target protein was purified. confirmed. The results are shown in FIG.
From the results of electrophoresis shown in FIG. 4, fractions from which high-concentration proteins were eluted were selected, and protein concentrations were measured by the Lowry method (RCDC protein assay kit, BIO-RAD). 200 μg of recombinant protein could be prepared.
Preparation of acyl ACP Acyl ACP used as a substrate was prepared as follows. First, a hexane solution (100 mM) of fatty acid (oleic acid, petroceric acid) was prepared, 6.2 μl was placed in a 10 ml glass test tube and dried with nitrogen gas. Then, the reaction liquid of the following Table 2 was added, and it was made to react at 37 degreeC on a heat block for 60 minutes.
After the reaction, 12 ml of water was added, and the pH was adjusted to 6.0 with acetic acid. Acyl ACP contained in the reaction solution was purified by the procedures shown in the following (A) to (M).
(A) 1.8 ml of DEAE-Toyopearl 650C (TOSOH) is packed into an empty polypropylene column (manufactured by BIO RAD) (referred to as 1 ml bed volume).
(B) Add bis Tris-HCl pH 6.0 (referred to as Buffer B) about 10 times the amount of (A) and equilibrate.
(C) Add acyl ACP solution (pH 6.0) to the column (D) Wash with 3 mL of Buffer B (E) Wash off unreacted fatty acid with 3 mL of 80% (w / w) Isopropanol in Buffer B.
* Isopropanol and Buffer B are mixed with degassed using an ultrasonic cleaner. (F) Washed with 3 mL of Buffer B (G) Eluted with 5 ml of 0.6 M LiCl in Buffer B (H) Put 4.5 ml of Octyl-Sepharose (manufactured by Amersham) into the column (bet volume is about 3 ml).
(I) Equilibrate with Buffer B 10 times the amount of (H) (H and I operations are performed in advance)
(J) The eluate of (G) is directly eluted and applied to (I) (K) Washed with 5 ml of 10 mM MES-NaOH pH 6.0 (referred to as Buffer C) (L) 35% (w / w) Isopropanol in Elution with 6 mL of Buffer C * Use Isopropanol and Buffer C after mixing with degassed with an ultrasonic cleaner individually (M) Use a vacuum centrifugal concentrator to volatilize isopropanol
Detection of thioesterase activity of purified enzyme The histidine-labeled protein purified by the above-described procedure was measured for thioesterase activity using acyl ACP (oleoyl ACP, petrocellinoyl-ACP) as a substrate. Prior to the reaction, the protein concentration of both solutions was measured by the Lowry method (RCDC protein assay kit, manufactured by BIO-RAD).
After mixing 25 mM Tris-HCl (pH 8.0), 1 mM DTT, acyl ACP and water, preincubation was performed at 25 ° C. for 5 minutes (acclimation of buffers to be used to the reaction temperature), and 12 μg of DcPTE and DcOTE were added. Histidine-labeled protein and γ-globulin were added as a control group, respectively, so that the total amount of the reaction solution became 100 μl, and the reaction was performed at 25 ° C. for 30 minutes. The thioesterase activity was measured by detecting unreacted acyl ACP and free ACP as a reaction product by SDS-PAGE using 10 minutes (10 μl) of the reaction solution. The result is shown in FIG. 5 as an electrophoresis photograph.
In addition, the gel after electrophoresis was stained with CBB, converted into digital image data using a high-performance scanner (Pictrostat digital 400, Fuji Photo Industry), and image analysis software Image Analysis ver. The concentration of acyl ACP and free ACP bands was quantified at 3.0 (Hitachi). The result is shown in FIG. As shown in FIG. 6, when petrocellinoyl-ACP was used as a substrate (FIG. 6B), it was found that DcPTE has a clearly higher reactivity than DcOTE. Conversely, when oleoyl-ACP was used as a substrate (FIG. 6A), the reactivity of DcOTE was higher. Therefore, it could be concluded that DcPTE is a thioesterase with specificity for petroceric acid.

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.
Preparation of DNA construct for constant systemic expression Among vectors used for transformation, a vector for constant systemic expression is shown in FIG. In FIG. 7, “Pnos” means a nopaline synthase promoter derived from Agrobacterium, “Tnos” means a nopaline synthase terminator derived from Agrobacterium, “P35S” means a CaMV35S promoter, and “NPT II” means neomycin. Refers to the phosphotransferase II 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 ORF region of the Dc4DES gene was amplified by PCR using primers designed to add BamHI at the 5 ′ end and Sac I sequence at the 3 ′ end. This PCR product was mixed with pSTBlue1 (Novagen cloning vector), and an equal amount of TaKaRa Ligation kit ver. 2 was added and ligation reaction was performed at 16 ° C. for 30 minutes. The total amount of the reaction solution was added to 50 μl of competent cells (E. coli DH5α, manufactured by TOYOBO), and transformation was performed according to the protocol specified by the manufacturer. A plasmid was prepared from the obtained transformant (grown on an LB agar medium containing 50 μg / ml kanamycin). The resulting plasmid was treated with restriction enzymes (Bam HI and Sac I). Next, restriction enzyme treatment (Bam HI and Sac I) was similarly performed in order to excise the GUS gene linked downstream of the CaMV35S promoter in pBI121. These restriction enzyme digestion products were subjected to 0.8% agarose gel electrophoresis, and QIAquick gel extraction kit and Geneclean II (BIO 101) manufactured by QIAGEN were used to remove the DNA fragment containing Dc4DES gene and the backbone of pBI121 (GUS gene was removed) Were separated and purified.
The mixture was mixed so that the ratio of the backbone fragment of pBI121 and the DNA fragment to be inserted (Dc4DES gene) was 1:10, and an equal amount of TaKaRa Ligation kit ver. The ligation reaction was performed at 16 ° C. for 30 minutes using 2. The total amount of the reaction solution was added to 100 μl of competent cells (E. coli strain DH5α, TOYOBO), and transformation was performed according to the manufacturer's designated protocol. The solution was applied to an LB agar medium containing 50 μg / ml kanamycin and cultured overnight.
On the other hand, the vector for co-expressing the Dc4DES gene and the DcPTE gene (FIG. 6 (D)) is unique in the vicinity of LB of T-DNA in the expression vector for Dc4DES expression shown in FIG. 6 (A). It was prepared by inserting an expression unit (DNA fragment containing a promoter and terminator) of the DcPTE gene between the Eco RI site and the Dra III site. The DcPTE gene expression unit was amplified by PCR using primers designed to add Eco RI site and Dra III site 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 a napin A promoter derived from rapeseed (B. campestriscv. Kizakinonatane). The promoter is known to be used by Monsanto for controlling 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. In addition, the GUS gene was replaced with the cDNA sequence of Dc4DES gene, DcPTE gene or Cs4DES.
On the other hand, the vector for co-expressing the Dc4DES gene and the DcPTE gene (FIG. 8 (D)) is unique in the vicinity of the LB of T-DNA in the expression vector for Dc4DES expression shown in FIG. 8 (A). It was prepared by inserting an expression unit (DNA fragment containing a promoter and terminator) of the DcPTE gene between the Eco RI site and the Dra III site. The DcPTE gene expression unit was amplified by PCR using primers designed to add Eco RI site and Dra III site at both ends.
Gene introduction into Agrobacterium by electroporation Agrobacterium was transformed by electroporation (electroporation) using the prepared plasmid. 40 μl of Agrobacterium tumefaciens (LBA4404 strain) competent cell prepared by a conventional method was dissolved, 5 μl (25 μg) of the 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 BIO-RAD cuvette (0.2 cm) and a pulse current of 1.25 kV and 10 μF was applied using a Shimadzu gene introduction device. Immediately after that, 460 μl of cooled SOC medium was added and cultured at 28 ° C. for 1 hour. A transformant was selected by applying to an LB agar medium containing 50 μg / ml kanamycin and 50 μg / ml rifampicin and culturing overnight. After single-colony isolation was performed on the appeared transformed colonies, a plurality of colonies were picked and the presence of the target plasmid DNA was confirmed by PCR.
Preparation of Arabidopsis Transformant by Vacuum Infiltration Method Arabidopsis thaliana et. Columbia was transformed by the vacuum infiltration method. The vacuum infiltration method is described in Shujunsha, Model Plant Experiment Protocol, 2001, 109-113 pp. Performed according to.
Next, Arabidopsis thaliana with reduced pressure infiltration was cultivated, and the collected seed was used as a transformed first generation seed (T1 seed). However, these are treated as transformed generation seeds for convenience, but not all seeds of this seed population have been transformed.
Next, transformed first generation seeds in a medium containing kanamycin (Murashige & Skog basic medium supplemented with 0.5 g / L MES, 10 g / L sucrose, 8 g / L Agar, 100 mg / L carbericillin, 50 mg / L kanamylin) And germinated under light conditions. A transformed individual (having kanamycin resistance and growing normally) was selected after growing for about 1 to 2 weeks. Individuals in which the 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). The kanamycin resistant line was transplanted to non-sterile vermiculite and cultivated by acclimatization to a non-sterile environment.
Next, about 100 mg of rosette leaves were collected from the transformed first generation plants and pulverized under liquid nitrogen freezing. DNA was prepared using a DNA preparation kit (DNeasy plant mini kit) manufactured by QIAGEN according to the standard protocol attached to the kit. PCR amplification was performed using Ex Taq DNA polymerase from Takara Bio, using PCR primers targeting the drug resistance gene (NPTII) in T-DNA and the introduced fatty acid synthesis gene.
The obtained PCR amplification product fragment was electrophoresed with TAE buffer using 0.8% agarose gel and then stained with ethidium bromide 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-described 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). 15 or more T2 seeds were harvested for 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 hydrochloric acid-methanol, and the n-hexane extract was analyzed by GC-MS. In addition, BHT (Butylated hydrogentoluene) 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 was directly added to the seed after weighing, and used for correcting experimental errors when preparing samples for analysis.
As the n-hexane used for extraction, phthalate analytical grade n-hexane (Tokyo Kasei) was used. Thereby, the influence of the phthalate ester which shows the same behavior as a fatty acid during GC analysis can be excluded.
The protocol for qualitative analysis is shown in Table 3, and the protocol for quantitative analysis is shown in Table 4.
The spectrum obtained by the treatment according to the above protocol was analyzed under the conditions shown in Table 5. This condition is a condition in which oleic acid (C18: 1, Δ9) and petroceric acid (C18: 1, Δ6), which are positional isomers based on the double bond position, can be separated and analyzed by GC.
A 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 is calculated, and the sum is used as the total fatty acid, and the molar fraction (mol- %) Shows the composition of each fatty acid. It is known that the fragment intensity detected by the mass spectrometer varies depending on the substance and does not simply reflect the molecular weight. However, the reproducibility and quantitative performance when analyzed under the same conditions are very high, and relative Comparisons are well possible.
Analysis result 1
In order to investigate the effect of the Dc4DES gene obtained in the present invention on the production of petrothelinic acid, a constantly whole body expression type transformed plant was prepared using the pB-4DES construct expressing 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. In addition, as a fatty-acid component, it analyzed about cis-4-hexadecenoic acid (16: 1 (DELTA) 4) which is a petroceric acid and the precursor of a petroceric acid. Table 6 shows the analysis results of these monoene unsaturated fatty acids.
As a result, 7.67 wt% of all fatty acids accumulated in the leaves of the transformant into which pB-Dc4DES was introduced with petrothelic acid that was not synthesized in wild-type Arabidopsis thaliana. That is, it was shown that petroceric acid can be efficiently produced in plant cells by using the carrot-derived Dc4DES gene (Table 6). Moreover, since the petroseric acid content in the case where the Cs4DES gene derived from coriander has been introduced into a cultured cell of tobacco that has already been reported is 2.7 wt% (patent document 1), in the synthesis and accumulation of petrothelinic acid, it is derived from coriander. It was found that the function of the carrot-derived Dc4DES gene is superior to that of the Cs4DES gene.
Analysis result 2
Using the rapeseed napin promoter that expresses seeds specifically, transgenic plants expressing Dc4DES gene, Cs4DES gene, and DcPTE gene are produced specifically, and oil and fat components are extracted from these seeds by the method described above. Fatty acid composition analysis was performed. Table 7 shows the analysis results of the monoene unsaturated fatty acid. In Table 7, “NT” is an abbreviation for “not tested”.
As a result, 0.81 mol% of petroceric acid was accumulated in the seeds of the transformant introduced with pN-Cs4DES. On the other hand, the amount of petroceric acid accumulated in the seeds of the transformant introduced with pN-Dc4DES was 0.89 mol%, and the carrot was also more carrot than Coriander-derived Cs4DES gene in the synthesis and accumulation of petrothelic acid in the seeds. The function of the derived Dc4DES gene was excellent.
In the case of the transformant introduced with pN-Dc4DES-DcPTE, 1.83 mol% of petroceric acid was accumulated. That is, compared to the case where only the Cs4DES gene was introduced, the amount of accumulated petrothelinic acid increased to 226%, and the introduction of DcPTE having high substrate specificity into petroselinoyl-ACP produced petroselinic acid. Was promoted. That is, it was found that co-expression of the Dc4DES gene and the DcPTE gene has an effect of showing a much higher amount of accumulated petroceric acid than when the Dc4DES gene is expressed alone. Moreover, when compared with the case where the Coriander-derived Cs4DES gene, which is a conventional technique, was used alone, a value nearly doubled was obtained. So far, no petrothelinic acid biosynthetic gene has been known, which makes it possible to increase the amount of petrothelic acid accumulation by co-expression with the 4DES gene, which is the first discovery in the world.
Moreover, 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 by expression or by co-expressing the Dc4DES gene and the DcPTE gene. I found Moreover, when the Dc4DES gene and the DcPTE gene were co-expressed, it was confirmed that the amount of cis-8-icosenoic acid produced could be increased compared to the case where the Dc4DES gene was expressed alone. In addition, cis-8-icosenoic acid has not been produced in plants so far.
Analysis result 3
Furthermore, each fatty acid composition including saturated fatty acid was analyzed in a transformed plant in which the Dc4DES gene or the DcPTE gene was expressed alone and in a transformed plant in which the Dc4DES gene and the DcPTE gene were co-expressed.
As a result, the saturated fatty acid content in the seeds was significantly increased in the transformant in which the DcPTE gene alone or the DcPTE gene and the Dc4DES gene were co-expressed with respect to the wild strain and the Cs4DES gene-transformed plant. (Fig. 9). The content of stearic acid (C18: 0), icosanoic acid (C20: 0), and docosanoic acid (C22: 0) in plant seeds into which the DcPTE gene has been introduced is twice that in plant seeds into which the DcPTE gene has not been introduced. It was close. The DcPTE gene is classified as a group of thioesterase genes called Fat A having specificity for unsaturated fatty acid-ACP based on molecular phylogenetic tree analysis based on the deduced amino acid sequence. In addition, as shown in Example 2, the results showed that the substrate specificity of Peterserinoyl ACP is shown, but although the effect of increasing the unsaturated fatty acid content can be expected, the effect of increasing the production of saturated fatty acids is difficult to predict. It turned out to be a gene that had an exceptionally remarkable effect that could not be predicted by a vendor.
When producing nylon raw materials (dicarboxylic acids) by oxidative degradation of plant-derived fatty acids, the “effect of increasing saturated fatty acid content of C18 or higher in addition to the effect of promoting the accumulation of petrothelic acid” possessed by the DcPTE gene is a very advantageous property. It is thought that it becomes. 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) is lowered. Moreover, since this effect can be obtained independently without using in combination with other genes such as Dc4DES gene, 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.). .

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 a petroceric acid can be provided, and the manufacturing method of the novel petroceric 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.
[Sequence Listing]

Claims (8)

  A gene encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 4 or 6.   An expression vector having the gene according to claim 1.   A transformant into which the gene according to claim 1 has been introduced.   4. The transformant according to claim 3, wherein a carrot-derived Δ4-palmitoyl ACP desaturase gene encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 2 has been introduced.   A transformed plant cell or a transformed plant having the ability to produce petroselinic acid, wherein a carrot-derived Δ4-palmitoyl ACP desaturase gene encoding a protein comprising the gene according to claim 1 and the amino acid sequence shown in SEQ ID NO: 2 is introduced. individual.   A transformed plant cell or a transformed plant individual having the ability to increase saturated fatty acid, wherein the gene according to claim 1 is introduced. Introducing the gene according to claim 1 into a plant cell to produce a transformed plant;
And a step of extracting a saturated fatty acid from the tissue collected from the transformed plant.   The method for producing a saturated fatty acid according to claim 7, wherein the transformed plant is further introduced with a carrot-derived Δ4-palmitoyl ACP desaturase gene encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 2.