AU2006266760B2 - Novel genes involved in petroselinic acid biosynthesis and method for producing 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

DESCRIPTION Novel Genes Involved in Petroselinic Acid Biosynthesis and Method for Producing Petroselinic Acid Technical Field The present invention relates to novel genes involved in petroselinic acid biosynthesis. The present invention particularly relates to a carrot-derived A4-palmitoyl-ACP desaturase gene and a plant-derived petroselinoyl-ACP thioesterase gene. The present invention further relates to a method for producing petroselinic acid using these novel genes. Background Art In recent years, technical development has been underway in the field of resin materials for production of resins derived from biomasses in view of the establishment of recycling society not dependent on oil resources. Nylon, which is a type of engineering plastic, is synthesized from aminocarboxylic acid, or via polymerization of diamine with dicarboxylic acid as a raw material. Most of raw material monomers are produced from the fossil resources via chemical engineering techniques. Sebacic acid (1,10-decanedioic acid) is used as a biomass-derived raw material for nylon. This sebacic acid is produced by cleaving castor oil extracted from a castor-oil plant using caustic alkali and is then used as a raw material for nylon 6,10. However, the applications of nylon 6,10 are limited, and thus nylon 6,10 is not broadly used as a resin material. It is known that dicarboxylic acid can be produced by oxidative decomposition of unsaturated fatty acid. Technology for producing petroselinic acid (cis-6-octadecenoic acid) to be used as a raw material for adipic acid (hexanedioic acid) which is a raw material monomer for nylon 6,6, has been examined to date (Non-patent documents I to 8). - 1- Umbellifers including coriander, carrot, and the like contain petroselinic acid that accounts for 80% or more of the fat and oil content of seeds. However, they are inappropriate for production of petroselinic acid since their seed yields are low. Plant fatty acid is newly synthesized in plastids (chloroplasts). Petroselinic acid is synthesized as follows. Palmitoyl ACP, which is a precursor is converted to cis-4-hexadecenoil ACP by A4-palmitoyl-ACP desaturase (hereinafter, referred to as 4DES), and then the chain length is elongated by a prokaryotic fatty acid synthase complex to give petroselinoyl-ACP. Free petroselinic acid is then synthesized by petroselinoyl-ACP thioesterase (hereinafter, referred to as PTE), and it is then transported to the cytoplasm. It is thought that a series of biosynthesis system enzymes and genes thereof that are specific to petroselinic acid synthesis are present in plants that synthesize petroselinic acid. Of these genes, a coriander-derived A4-palmitoyl-ACP desaturase gene has been cloned and introduced into Arabidopsis thaliana, which is a plant originally producing no petroselinic acid, so as to produce a transformed plant. Although accumulation of petroselinic acid could have been confirmed, the amount of accumulation accounts for only approximately 1% of the fat and oil content in seeds. Regarding PTE, the presence of its enzyme activity has been demonstrated (Non-patent document 8), but the gene thereof has remained uncloned. Not only a fatty acid biosynthesis system in plastids, but also intracytoplasmic transport of fatty acid derivatives and a triacylglycerol synthesis system in endoplasmic reticulum membranes are involved in fat and oil production and the composition of constitutive fatty acids in plants. For petroselinic acid production using genetic recombination technology, many problems to be addressed remain. Patent document I United States Patent No. 5,430,134 Patent document 2 International Publication No. 94/01565 Non-patent document 1 Proc. Natl. Acad. Sci. U.S.A., 89, 11184-11188, 1992 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 Physiol., 124, 681-692, 2000 -2- Non-patent document 5 Plant Mol. Biol., 47, 507-518, 2001 Non-patent document 6 Metab. Eng., 4, 12-21, 2002 Non-patent document 7 Biochim. Biophys. Acta., 1212, 134-136, 1994 Non-patent document 8 Plant Physiol., 104, 839-844, 1994 5 Non-patent document 9 Planta 215: 584-595 2002. Disclosure of the Invention In one embodiment, the present invention provides novel genes capable of promoting the accumulation of petroselinic acid and to provide a method for 10 producing petroselinic acid using such genes. As a result of intensive studies, the present inventors have acquired new findings, such as the fact that A4-palmitoyl-ACP desaturase derived from carrot (Daucus carota) belonging to the family Umbelliferae is superior to A4-palmitoyl-ACP desaturase derived from coriander in terms of petroselinic 15 acid synthesis ability. Moreover, the present inventors have succeeded for the first time in isolation of a petroselinoyl-ACP thioesterase gene and have invented technology that involves using the gene in combination with a A4-palmitoyl-ACP desaturase gene, so as to approximaly double petroselinic acid synthesis ability. The present invention encompasses the followings. 20 (1) A gene encoding the following protein (a), (b), or (c): (a) a protein that comprises the amino acid sequence shown in SEQ ID NO: 2; (b) a protein that comprises an amino acid sequence shown in SEQ ID NO: 2 including deletion, substitution, or addition of one or a plurality of amino acids and has A4-palmitoyl-ACP desaturase activity; or 25 (c) a protein that is encoded by a DNA hybridizing under stringent conditions to a DNA comprising a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1 and has A4-palmitoyl-ACP desaturase activity. -3 - In (b) and (c) above, "a protein that has A4-palmitoyl-ACP desaturase activity" can be rephrased as a protein capable of increasing the amount of cis-4-hexadecenoic acid, petroselinic acid (cis-6-octadecenoic acid), or cis-8-icosenoic acid accumulated through its expression in a cultured plant cell or 5 plant. (2) A gene encoding the following protein (a), (b), or (c): (a) a protein that comprises the amino acid sequence shown in SEQ ID NO: 4 or 6; (b) a protein that comprises an amino acid sequence shown in SEQ ID NO: 4 or 6 10 including deletion, substitution, or addition of one or a plurality of amino acids and has petroselinoyl-ACP thioesterase activity; or (c) a protein that is encoded by a DNA hybridizing under stringent conditions to a DNA comprising a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 3 or 5 and has petroselinoyl-ACP thioesterase 15 activity. In (b) and (c) above, "a protein that has petroselinoyl-ACP thioesterase activity" can be rephrased as a protein capable of increasing the amount of cis-4-hexadecenoic acid, petroselinic acid (cis-6-octadecenoic acid), or cis-8-icosenoic acid through co-expression of this protein with a protein having 20 A4-palmitoyl-ACP desaturase activity in a cultured plant cell or plant, where the amount obtained via co-expression is greater than that obtained via expression of only the protein having A4-palmitoyl-ACP desaturase activity. The present invention also provides an isolated gene encoding a protein that comprises the amino acid sequence shown in SEQ ID No:4 or 6. -4- The invention also provides an isolated gene comprising the sequence shown in SEQ ID NO:3 or 5. The invention also provides a transformed plant cell or transformed plant, into which a A4-palmitoyl-ACP desaturase gene derived from Daucus carota 5 shown in SEQ ID NO:l, or encoding a protein that comprises the amino acid sequence shown in SEQ ID NO: 2 is introduced. The invention also provides a transformed plant cell or a transformed plant having an improved productivity of saturated fatty acid, into which a gene according to the invention is introduced. 10 The invention also provides a method for producing saturated fatty acid, comprising steps of: introducing a gene according to the invention into a plant cell to produce a transformed plant; and extracting saturated fatty acid from tissues collected from the 15 transformed plant. This description includes part or all of the contents as disclosed in the description and/or drawings of Japanese Patent Application No. 2005-191775, which is a priority document of the present application. Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a 20 stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any 25 or all of these matters form part of the prior art base or were common general - 4A knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application. Brief Description of the Drawings Fig. 1 shows a comparison of various types of petroselinoyl-ACP 5 thioesterase and oleoyl-ACP thioesterase in terms of amino acid sequence homology (%). In Fig. 1, DcPTE denotes carrot (Daucus carota)-derived petroselinoyl-ACP thioesterase, CsPTE denotes coriander (Coriandrum sativum)-derived petroselinoyl-ACP thioesterase, AgPTE denotes dill (Aneihum graveolens)-derived petroselinoyl-ACP thioesterase, CsOTE denotes coriander 10 (Coriandrum sativum)-derived oleoyl-ACP thioesterase, and DcOTE denotes carrot (Daucus carota)-derived oleoyl-ACP thioesterase. - 4B - Fig. 2-1 shows alignment of the amino acid sequences of various types of petroselinoyl-ACP thioesterase and oleoyl-ACP thioesterase. Fig. 2-2 shows alignment of the amino acid sequences of various types of petroselinoyl-ACP thioesterase and oleoyl-ACP thioesterase. Fig. 3 shows alignment of the amino acid sequence of carrot (Daucus carota)-derived A4-palmitoyl-ACP desaturase (referred to as Dc4DES) and the amino acid sequence of coriander (Coriandrun sativun)-derived A4-palmitoyl-ACP desaturase (referred to as Cs4DES). Fig. 4 is a photograph showing the results of performing SDS-PAGE for each fraction of histidine-labeled protein eluates of purified carrot (Daucus carota)-derived petroselinoyl-ACP thioesterase (referred to as DcPTE), coriander (Coriandrum sativum)-derived oleoyl-ACP thioesterase (referred to as CsOTE), and carrot (Daucus carota)-derived oleoyl-ACP thioesterase (referred to as DcOTE) with the use of Escherichia coli. Fig. 5 shows photographs showing the results of performing reaction using acyl ACP as a substrate in the histidine-labeled protein eluates of purified DcPTE and DcOTE followed by SDS-PAGE. Fig. 6 shows characteristic graphs showing the results of quantification of bands contained in the photographs shown in Fig. 5 using image analysis software. Fig. 7 is a configuration figure schematically showing a vector for continuous systemic expression prepared in Examples. Fig. 8 is a configuration figure schematically showing vectors for seed-specific expression prepared in Examples. Fig. 9 shows the results of fatty acid composition analysis performed for the seeds of transformed plants. Preferred Embodiments of the Invention Hereafter, novel genes and a method for producing petroselinic acid according to the present invention are described in detail with reference to drawings thereof. -5- 1. Isolation of carrot (Daucus carota)-derived A4-palmitoyl-ACP desaturase gene (Dc4DES gene) A Dc4DES gene encodes a protein (Dc4DES) comprising the amino acid sequence shown in SEQ ID NO: 2. Dc4DES is a protein having activity of unsaturating palmitoyl-ACP (palmitoyl-acyl carrier protein) that is a C16 saturated fatty acid at position A4. As an example of the Dc4DES gene, a gene encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 2 is shown in SEQ ID NO: 1. Furthermore, in the present invention, the Dc4DES gene may be a gene encoding a protein that comprises an amino acid sequence shown in SEQ ID NO: 2 including deletion, substitution, or addition of one or a plurality of amino acids and has A4-palmitoyl-ACP desaturase activity. Here, the term "a plurality of amino acids" means 2 to 150, preferably 2 to 80, and more preferably 2 to 40 amino acids. Examples of a region to be subjected to deletion, substitution, or addition include, but are not particularly limited to, a region between amino acids 1 and 99 or between amino acids 270 and 386, preferably a region between amino acids 1 and 99 or between amino acids 301 and 386, and more preferably a region between amino acids I and 53 or between amino acids 381 and 386 in the amino acid sequence shown in SEQ ID NO: 2. Furthermore, in the present invention, the Dc4DES gene may be a gene encoding a protein that comprises an amino acid sequence having 50% or more, preferably 70% or more, further preferably 90% or more homology with the amino acid sequence shown in SEQ ID NO: 2 and has A4-palmitoyl-ACP desaturase activity. The above numerical values representing homology can be found by performing commands of a maximum matching method, for example, using DNASIS (Hitachi Software Engineering Co., Ltd.) that is sequence analysis software. Parameters to be used herein are default (initial setting) parameters. Furthermore the Dc4DES gene in the present invention may be a gene encoding a protein that is encoded by a DNA hybridizing under stringent conditions to a DNA comprising a nucleotide sequence complementary to the nucleotide sequence shown in -6- SEQ ID NO: 1 and has A4-palmitoyl-ACP desaturase activity. Here the term "hybridizing under stringent conditions" means that even under conditions where heating is performed at 42*C in a solution (6xSSC, 0.5% SDS, and 50% formamide) followed by washing at 68*C in a solution (0.1xSSC and 0.5% SDS), for example, positive hybridization signals are continuously observed. The term "A4-palmitoyl-ACP desaturase activity" means activity of unsaturating palmitoyl-ACP at position A4. The presence or the absence of such activity can be tested as follows. A DNA fragment encoding a protein to be tested is introduced into cells of host plants (e.g, tobacco or Arabidopsis thaliana) that do not accumulate cis-4-hexadecenoic acid, petroselinic acid, and cis-8-icosenoic acid, so that the gene can function. The presence or the absence of cis-4-hexadecenoic acid, petroselinic acid, and cis-8-icosenoic acid in the lipid of plant bodies into which the DNA fragment has been introduced is measured. For example, cells of plants (e.g., tobacco or Arabidopsis thaliana) that do not accumulate cis-4-hexadecenoic acid, petroselinic acid, and cis-8-icosenoic acid are transformed with the use of an expression vector prepared by locating the Dc4DES gene downstream of a persistent expression promoter or a specific expression promoter; that is, at a position so that the gene can be controlled by the relevant promoter. Lipid of the thus prepared transformed plant cells or the seeds of plant bodies regenerated from the cells is extracted. The thus extracted lipid is treated with methanol hydrochloric acid or the like to give fatty acid methyl esters. The amount of petroselinic acid methyl ester, the amount of cis-4-hexadecenoic acid fatty acid methyl ester, and the amount of cis-8-icosenoic acid fatty acid methyl ester contained therein are measured by gas chromatography or the like. If these fatty acid methyl esters can be detected, it can be said that the tested protein has A4-palmitoyl-ACP desaturase activity. If these fatty acid methyl esters cannot be detected, it can be said that the tested protein lacks A4-palmitoyl-ACP desaturase activity. The Dc4DES gene has a function of promoting petroselinic acid synthesis within host plant cells into which the gene has been introduced into the cells so that the gene can function. For example, plant cells are transformed using an expression vector -7constructed by locating the Dc4DES gene downstream of a persistent expression promoter or a specific expression promoter; that is, at a position so that the gene can be controlled by the relevant promoter. The thus obtained transformed plants are grown to plant bodies, so that petroselinic acid synthesis in the plant bodies can be promoted. A seed-specific expression promoter is preferably used as such a specific expression promoter. With the use of such seed-specific expression promoter, petroselinic acid can be accumulated in seeds. Petroselinic acid can be detected as follows. For example, transformed plants are grown to plant bodies and then tissue of seeds or the like is crushed. The product is then mixed with a hydrochloric acid-methanol solution for methylesterification of petroselinic acid contained in the tissue, followed by hexane extraction. The hexane extract can be subjected to detection using a gas chromatography-mass spectrometry (GC-MS) apparatus. As a result of detection using the GC-MS apparatus, the ability of promoting petroselinic acid synthesis can be analyzed. 2. Petroselinoyl-ACP thioesterase enzyme gene (PTE gene) The presence of a PTE gene in coriander (Coriandrun sativium) has been suggested, but the gene has been neither isolated nor cloned. Such PTE gene has been isolated and cloned for the first time in the present invention. The PTE gene is a gene encoding thioesterase (PTE) with high specificity to acyl ACP (acyl carrier protein) having a double bond at position A6, such as petroselinoyl-ACP. PTE is involved in free petroselinic acid synthesis. An example of the PTE gene is a gene encoding carrot-derived PTE comprising the amino acid sequence shown in SEQ ID NO: 4 or 6. Specifically, a gene encoding the protein comprising the amino acid sequence shown in SEQ ID NO: 4 is shown in SEQ ID NO: 3. A gene encoding the protein comprising the amino acid sequence of SEQ ID NO: 6 is shown in SEQ ID NO: 5. In addition, carrot-derived PTE gene and PTE are referred to as a DcPTE gene and DcPTE, respectively. The DcPTE gene in the present invention may be a gene encoding a protein that comprises an amino acid sequence derived from the amino acid sequence shown in SEQ -8- ID NO: 4 or 6 by deletion, substitution, or addition of one or a plurality of amino acids and has activity. Here, the term "a plurality of amino acids" means 2 to 188, preferably 2 to 64, and more preferably 2 to 44 amino acids. Examples of a region to be subjected to deletion, substitution, or addition include, but are not particularly limited to, a region between amino acids I to 70 or between amino acids 311 to 375, preferably a region between amino acids 1 and 57 or between amino acids 368 and 375, and more preferably a region between amino acids 1 and 32 in the amino acid sequence shown in SEQ ID NO: 4. Furthermore, a PTE gene encoding a protein comprising an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 4 or 6 by introduction of substitution, deletion, or insertion can be obtained by introducing a desired mutation into the nucleotide sequence shown in SEQ ID NO: 3 or 5 with the use of a known technique such as the Kunkel or the Gapped duplex method or via a technique in accordance therewith. Mutation can be introduced with the use of a mutagenesis kit utilizing site-specific mutagenesis (e.g., Mutan-K (TAKARA) or Mutan-G (TAKARA)) or the LA PCR in vitro Mutagenesis Series Kit (TAKARA), for example. Furthermore, the DcPTE gene in the present invention may be a gene encoding a protein that comprises an amino acid sequence having 50% or more, preferably 70% or more, and more preferably 90% or more homology with the amino acid sequence shown in SEQ ID NO: 4 or 6 and has petroselinoyl-ACP thioesterase activity. The above numerical values of homology can be found by performing commands of a maximum matching method, for example, using DNASIS (Hitachi Software Engineering Co., Ltd.) that is sequence analysis software. Parameters to be used herein are default (initial setting) parameters. Moreover, the DcPTE gene in the present invention may also be a gene encoding a protein that is encoded by a DNA hybridizing under stringent conditions to a DNA comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 3 or 5 and has petroselinoyl-ACP thioesterase activity. Here the term "hybridizing under stringent conditions" means that even under conditions in which heating is -9performed at 42*C in a solution (6xSSC, 0.5% SDS, and 50% formamide) followed by washing at 68*C in a solution (O.IxSSC and 0.5% SDS), for example, positive hybridization signals are continuously observed. The term "petroselinoyl-ACP thioesterase activity" means activity of decomposing petroselinoyl-ACP into petroselinic acid and ACP. Such activity can be determined as follows. 25 mM Tris-HCl (pH8.0), 1 mM DTT, petroselinoyl-ACP, and water are mixed and then preincubation is performed at 25*C for 5 minutes. 15 pg of a protein to be tested is added to 100 p.1 of the reaction solution, followed by 30 minutes of reaction at 25*C. After reaction, unreacted petroselinoyl-ACP and free ACP that is a reaction product are separated by SDS-PAGE. After electrophoresis, gel is subjected to CBB staining and then the amount of free ACP (separated by SDS-PAGE) that is the reaction product is quantified using a densitometer. Thus the thioesterase activity of the protein added was determined. Alternatively, 25 mM Tris-HCI (pH8.0), 1 mM DTT, petroselinoyl-ACP having a petroselinoyl group labeled with tritium, and water are mixed and then preincubation is performed at 25*C for 5 minutes. A protein to be tested is added to 100 pl of the reaction solution, followed by 30 minutes of reaction at 25*C. After reaction, 50 pl of isopropanol is added to stop reaction. Petroselinic acid that is subsequently generated by enzyme reaction is separated by thin-layer chromatography. The amount of the thus generated petroselinic acid is quantified using a scintillation counter. Thus the thioesterase activity of the protein added is determined. Furthermore, an example of the PTE gene in the present invention is not limited to a gene encoding carrot-derived PTE. Examples of the PTE gene include PTE genes derived from plants that undergo biosynthesis of petroselinic acid. Examples of plants that undergo biosynthesis of petroselinic acid include, in addition to carrot, umbellifers such as coriander (Coriandrum sativium), parsley (Petroselium crispun), and dill (Anethum graveolens) and plants of the family Araliaceae such as ivy (Hedera helix) and aralia (Aralia elata). When a PTE gene is obtained from plants other than carrot, the whole or a -10portion of the region between nucleotides 187 and 1128 in the nucleotide sequence of the DcPTE gene shown in SEQ ID NO: 3 or 5 can be used as a probe, for example. When a long nucleic acid sequence (>100 bp) is used as a probe, screening can also be performed under middle- or higher-level stringency conditions in order to obtain signals from a target sample having a sequence with 80% or higher homology. Furthermore, a very short probe may also be used herein. For example, an oligonucleotide that may also be used herein should have a length of at least approximately 10, preferably at least approximately 15, and more preferably 20 nucleotides. When a short region is used as a probe, sequence identity is required at a level higher than that required for a long probe. Specifically, a PTE gene can be isolated and identified from the genomic DNA extracted from plants other than carrot via Southern hybridization using the above-mentioned probe and the genomic DNA. In addition, the PTE gene of such a plant can also be isolated and identified using cDNA synthesized using mRNA extracted from the plant as a template, instead of using such a genomic DNA. Based on the nucleotide sequence of the DcPTE gene shown in SEQ ID NO: 3 or 5, the nucleotide sequences of the DcPTE homologous genes isolated from coriander (Coriandrum sativium) and dill (Anethum graveolens), respectively, are shown in SEQ ID NO: 7 and 9, respectively. In addition, the amino acid sequence (CsPTE) predicted from the coriander-derived PTE gene shown in SEQ ID NO: 7 is shown in SEQ ID NO: 8. The amino acid sequence (AgPTE) predicted from the dill-derived PTE gene shown in SEQ ID NO: 9 is shown in SEQ ID NO: 10. Moreover, amino acid homologies (%) among thioesterase proteins of various plants are shown in Fig. 1. As in Fig. 1, members (including DcPTE, CsPTE, and AgPTE) of the PTE group share high homologies. DcOTE shows high homology with CsOTE. In contrast, the PTE group shows relatively low homology with the OTE group. Therefore, it can be said that a novel protein having more than 80% amino acid homology with PTE proteins included in the PTE group is highly likely included in the PTE group. Hence, examples of the PTE gene according to the present invention also include a DNA encoding a -11 protein that comprises an amino acid sequence having more than 80% homology with the amino acid sequence shown in SEQ ID NO: 4, 6, 8, or 10. Moreover, Fig. 2 shows alignment of the amino acid sequences of DcPTE, CsPTE, AgPTE, DcOTE, and CsOTE. An approximately 30% amino acid difference is present between OTE and PTE. In particular, some amino acids in the OTE group differ from the same in the PTE group in terms of polarity. In the following explanation, the term "(amino acid) X in common sequence (X is a natural number)" means a number assigned on the upper row of alignment in Fig. 2. Amino acids 120, 125, and 373 in the common sequence in the PTE group are nonpolar, whereas these amino acids in the OTE group are polar. Moreover, amino acids 140 and 195 of the common sequence in the PTE group are polar, whereas these amino acids in the OTE group are nonpolar. Furthermore, some charged amino acids in the OTE group differ from the same in the PTE group. Amino acids 149 and 246 of the common sequence in the PTE group are not charged, whereas these amino acids are positively charged in the OTE group. Furthermore, amino acid 244 of the common sequence in the PTE group is negatively charged, whereas these amino acids are not charged in the OTE group. Furthermore, amino acid 270 of the common sequence in the PTE group is positively charged, whereas the amino acid in the OTE group is not charged. It is considered that the presence or the absence of polarity and changes in charge of amino acids at these positions result in changes in substrate selectivity. Moreover, it is known that differences in side chain structures of amino acids at substrate-binding sites cause changes in substrate selectivity. Amino acids 89, 147, 161, 177, 192, 196, 214, 217, 223, 238, 270, 287, and 338 of the common sequence in the PTE group differ from these amino acids in the OTE group in terms of amino acid side chain structure. Substitution of these amino acids that are different between the OTE group and the PTE group can possibly alter substrate specificity to acyl ACP. The PTE gene has a function of significantly promoting petroselinic acid synthesis in host plant cells when the PTE gene is introduced together with a -12- A4-palmitoyl-ACP desaturase gene into the host plant cells in a form so that the PTE gene can function. For example, an expression vector is constructed by locating the PTE gene downstream a persistent expression promoter or a specific expression promoter; that is, arranging the PTE gene at a position so that the gene can be controlled by the relevant promoter. Plant cells (see 1. above) transformed with a A4-palmitoyl-ACP desaturase gene is further transformed with the thus constructed expression vector. Alternatively, plant cells may also be transformed using an expression vector constructed by locating the PTE gene and the A4-palmitoyl-ACP desaturase gene downstream of a persistent expression promoter or a specific expression promoter. In both cases, the thus obtained transformed plants are grown to plant bodies and then petroselinic acid synthesis in the plant bodies can be promoted. Petroselinic acid can be detected as follows. For example, transformed plants are grown to plant bodies and then tissue of seeds or the like is crushed. The product is then mixed with a hydrochloric acid-methanol solution for methylesterification of petroselinic acid contained in the tissue, followed by hexane extraction. The hexane extract can be subjected to detection using a gas chromatography-mass spectrometry (GC-MS) apparatus. As a result of detection using the GC-MS apparatus, the ability of promoting petroselinic acid synthesis can be analyzed. Expression vector In the present invention, an expression vector contains the Dc4DES gene and/or the PTE gene explained in 1 and 2 above. The expression vector to be used herein is not particularly limited, as long as it is a plasmid-type vector or a type of vector to be introduced into chromosome (that can be incorporated into the genome of a host organism). Examples of such vector include plasmid DNAs, bacteriophage DNAs, retrotransposon DNAs, and artificial chromosome DNAs (YAC: yeast artificial chromosome). Examples of such plasmid DNAs include YCp Escherichia coli-yeast shuttle vectors (e.g., pRS413, pRS414, pRS415, pRS416, YCp50, pAUR112, and pAUR123), - 13- YEp Escherichia coli-yeast shuttle vectors (e.g., pYES2 and YEp13), YIp Escherichia coli-yeast shuttle vectors (e.g., pRS403, pRS404, pRS405, pRS406, pAURIOI, and pAUR135), Escherichia coli-derived plasmids (e.g., ColE plasmids such as pBR322, pBR325, pUC18, pUC19, pUCII8, pUC119, pTV118N, pTV119N, pBluescript, pHSG298, pHSG396, and pTrc99A, p15Aplasmids such as pACYC177 and pACYC184, and pSCIOI plasmids such as pMWI18, pMW119, pMW218, and pMW219), Agrobacterium-derived plasmids (e.g., pBI101), and Bacillus subtilis-derived plasmids (e.g., pUB110 and pTP5). Examples of such phage DNAs include k phages (e.g., Charon4A, Charon21A, EMBL3, EMBL4, Xgtl0, %gtl, and XZAP), pX174, M13mpl8, and Ml3mp19. Examples of retrotransposons include Ty factors. Examples of YAC vectors include pYACC2 and the like. Furthermore, animal viruses such as retrovirus and vaccinia virus and insect virus vectors such as baculovirus can also be used. Incorporation of the Dc4DES gene and/or the PTE gene into such an expression vector is required under conditions that enable expression of the gene. Such "conditions that enable expression of the gene" means that the Dc4DES gene and/or PTE gene is incorporated into a vector via its ligation to a promoter, so that the Dc4DES gene and/or PTE gene can be expressed under the control of a predetermined promoter in a host organism into which the gene is introduced. Accordingly, a promoter and a terminator, and if desired, a cis element such as an enhancer, a splicing signal, a polyA addition signal, a selection marker, a ribosome binding sequence (SD sequence), and the like can be ligated to the vector, in addition to the Dc4DES gene and/or PTE gene,. In addition, examples of such a selection marker include antibiotic resistance genes such as an ampicillin resistance gene, a kanamycin resistance gene, a hygromycin resistance gene, and herbicide resistance genes such as a Bialaphos resistance gene. Examples of a promoter that is contained in an expression vector to be used in the present invention include, but are not particularly limited to, a persistent expression promoter, a tissue specific expression promoter, and a promoter inducible via stimulation. Of these examples, the use of a seed-specific expression promoter is preferable in order to accumulate synthesized petroselinic acid within seeds. As such seed-specific -14expression promoter, a rapeseed-derived napin A promoter, an Arabidopsis thaliana-derived FAEI promoter, an oleosin promoter, a soybean-derived glutelin B1 promoter, a flax stearoyl-ACP desaturase (SAD) promoter, or the like can be used. Transformant Transformants can be prepared using the above-described expression vector. Specifically, transformants can be prepared by introducing the above-described expression vector into hosts so that the Dc4DES gene and/or PTE gene contained in the vector can be expressed. Examples of the families to which host plants belong include, but are not particularly limited to, the family Umbelliferae, the family Solanaceae, the family Brassicaceae, the family Gramineae, the family Leguminosae, the family Rosaceae, the family Asteraceae, the family Liliaceae, the family Caryophyllaceae, the family Cucurbitaceae, the family Convolvulaceae, and the family Chenopodiaceae. Plants belonging to the family Umbelliferae or the family Brassicaceae are particularly desired host plants. When hosts are plants, transformed plants can be obtained as described below. Subject plants to be transformed in the present invention mean any of the whole plant bodies, plant organs (e.g., leaves, petals, stems, roots, and seeds), plant tissue (e.g., epidermis, phloems, parenchymas, xylems, vascular bundles, palisade tissue, and spongy tissue), and cultured plant cells. An expression vector can be introduced into a plant with the use of a general transformation method such as a vacuum infiltration method (Agrobacterium method), a particle gun method, a PEG method, an electroporation method or the like. For example, a vacuum infiltration method can be performed according to a known technique (Shujunsha, Experimental Protocols for Model Plants, 2001, pp. 109-113). When Agrobacterium is used, an expression vector is introduced into appropriate Agrobacterium (e.g., Agrobacterium tuinefaciens LBA4404 strain). Sterile cultured leaf sections of a host (e.g., tobacco) are infected with the strain according to a leaf disc method (written by Hirofumi Uchimiya, Genetic Engineering Manuals for Plants, 1990, pp. 27-31, Kodansha Scientific Ltd., Tokyo), for example. Hence, -15transformed plants can also be obtained. Furthermore, when a particle gun method is employed, plant bodies, plant organs, plant tissue itself may be directly used. Alternatively, sections or protoplasts may be prepared therefrom and then used. The thus prepared samples can be treated with a gene introduction apparatus (e.g., PDS-1000 (BIO-RAD laboratories)). Treatment conditions differ depending on plants or samples. Treatment is generally performed at a pressure approximately between 450 psi and 2000 psi and with a distance approximately between 3 cm and 12 cm. Tumor tissue, shoots, hairy roots and the like that are obtained as a result of transformation can be directly used for cell culture, tissue culture, or organ culture. Moreover, such tumor tissue, shoots, hairy roots and the like can be regenerated into plant bodies with the use of a conventionally known plant tissue culture method that involves administration of a plant hormone (e.g., auxin, cytokinin, gibberellin, abscisic acid, ethylene, and brassinolide) with an appropriate concentration. Whether or not a gene is incorporated into a host can be confirmed by a PCR method, a Southern hybridization method, a Northern hybridization method, or the like. For example, a DNA is prepared from a transformant, a DNA-specific primer is designed, and then PCR is performed. PCR can be performed under the above conditions similar to those employed for preparation of a plasmid. Subsequently, the amplification product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, or capillary electrophoresis, followed by staining using ethidium bromide, SYBR Green liquid, or the like. The amplification product is then detected in the form of a single band, so that successful transformation can be confirmed. Moreover, PCR is performed using a primer that is labeled in advance with a fluorescent dye or the like, so that the amplification product can also be detected. Furthermore, a method that can also be employed herein involves binding an amplification product to a solid phase such as a microplate and then confirming the amplification product by a fluorescence reaction, an enzyme reaction, or the like. Meanwhile, examples of hosts include bacteria belonging to the genus -16- Escherichia (e.g., Escherichia coli), the genus Bacillus (e.g., Bacillus subtilis), the genus Pseudomonas (e.g., Pseudomonas putida), or the genus Rhizobium (e.g., Rhizobium meliloti), yeast such as Saccharoinyces cerevisiae and Schizosaccharonyces ponbe, animal cells such as COS cells and CHO cells, and insect cells such as Sf9. When bacteria such as Escherichia coli are used as hosts, a recombination vector is preferably autonomously replicable within bacteria and at the same time, the vector is preferably composed of a ribosome binding sequence, the gene of the present invention, and a transcription termination sequence. Examples of Escherichia coli include Escherichia coli DH5a and Escherichia coli Y1090. An example of Bacillus subtilis is Bacillus subtilis, but is not limited thereto. A method for introducing a recombination vector into bacteria is not particularly limited, as long as it is a method for introducing DNA into bacteria. Examples of such a method include a method using calcium ions [Cohen, S.N. et al.: Proc. Natl. Acad. Sci., U.S.A., 69: 2110(1972)] and an electroporation method. When yeast is used as a host, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, or the like is used. A method for introducing a recombination vector into yeast is not particularly limited, as long as it is a method for introducing DNA into yeast. Examples of such a method include an electroporation method [Becker, D.M. et al.: Methods. Enzymol., 194: 182 (1990)], a spheroplast method [Hinnen, A. et al.: Proc. Natl. Acad. Sci., U.S.A., 75: 1929 (1978)], and a lithium acetate method [Itoh, H.:J. Bacteriol., 153:163(1983)]. When animal cells are used as hosts, monkey cells (e.g., COS-7 and Vero), Chinese hamster ovary cells (CHO cells), mouse L cells, rat GH3, human FL cells or the like are used. Examples of a method for introducing a recombination 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 or the like are used. Examples of a method for introducing a recombination vector into insect cells include a calcium phosphate method, a lipofection method, and an electroporation method. - 17- Method for producing petroselinic acid Petroselinic acid synthesis can be promoted in the above transformed plants because of the function of the introduced Dc4DES gene and/or PTE gene. Petroselinic acid synthesized and accumulated in the transformed plants can be extracted using a conventionally known technique. Methods for pressing and extracting fats and oils from the plant tissue (such as seeds or fruits of a type of plant producing oil) are largely classified into two methods: a squeeze technique and an extraction method. A method for obtaining fat and oil from tissue with a high fat and oil content, such as rapeseed seeds involves performing primary crushing and pressing tissue with a roll mill or the like, heating at a temperature between 75*C and 85*C, pressing and squeezing with a press such as an expeller, and then extracting fats and oils (squeeze technique). Meanwhile, in the case of a raw material with a low fat and oil content, such as soybean, fats and oils are extracted from tissue using a solvent such as hexane (extraction method). Fats and oils produced via these steps are mixtures of fatty acids (containing petroselinic acid) and esters such as glycerin. Hence, triacylglycerol, diacylglycerol, monoacylglycerol, phospholipids, and the like are contained herein. Next, such fat and oil are subjected to hydrolysis, so that fatty acids can be obtained. The thus obtained fatty acid mixture is separated and purified, so that very pure petroselinic acid can be obtained. Moreover, alcohol such as methanol is added to fats and oils and then reaction is performed, an alcohol ester such as petroselinic acid methyl ester can be obtained. Hereafter, the present invention is described in greater detail with reference to examples, although the technical scope of the present invention is not construed by the following examples in a limited manner. [Example 1] Cloning of Dc4DES gene Plant samples In this example, carrot (Daucus carota L., Natsu-maki (summer-seeding) senko gosun, F1 variety) was used as an experimental sample. A purebred variety having the - 18same homologous gene sequence is desired as a gene cloning source. For convenience of experimentation (samples that had bloomed early could be prepared), plants of the Fl variety were used. Carrot seeds were purchased from OTA SEED Co., Ltd. Seeds were grown within a temperature-controlled chamber (Koito-toron, Koito Manufacturing Co., Ltd.) under conditions of 25*C, 16 hours of exposure to sunshine, and humidity of 60%. The thus cultivated carrot plant bodies were used as samples. Preparation of carrot RNA Approximately 100 mg of immature seeds that differ in development stage and approximately 100 mg of leaves were collected from the carrot plant bodies. The seeds and leaves were each crushed under liquid nitrogen freezing. From the thus obtained crushed products, RNA was prepared using an RNeasy plant mini kit (QIAGEN) according to the protocols provided for the kit. Design of PCR primers for amplification of DNA fragments and RT-PCR BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) within the NCBI site was searched for a gene homologous to that of A4 palmitoyl-ACP desaturase (Cs4DES) of coriander (Coriandrum sativum L.), which was then collected. The amino acid sequence of a polypeptide encoded by the thus obtained Cs4DES gene (GenBank accession number M93115) and the amino acid sequences of polypeptides encoded by A9 stearoyl-ACP desaturase genes of various types of plants registered in the Gen Bank were subjected to multiple alignment analysis using Genetyx-Win Ver. 4.0/ATGC ver. 2.0 (Software Development). As a result of analysis, the following PCR primers (degenerate primers) were designed and used for RT-PCR in order to amplify DNA fragments corresponding to the thus obtained highly-conserved regions. PFl: 5' CAN GAR GAR GCN CTB CCN CAN TA 3' (SEQ ID NO: 11) PRi: 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) NFO: 5' GAN MTB CCN GAT GAN TAY TTH RTT G 3' (SEQ ID NO: 14) NRI: 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) -19- In addition, these nucleotide sequences are recorded based on the following International Union of Biochemistry (IUB) notation. Specifically, R denotes A or G; Y denotes C or T; M denotes A or C; K denotes G or T; S denotes G or C; W denotes A or T; H denotes A or T or C; B denotes G or T or C; V denotes G or A or C; D denotes G or A or T; and N denotes A or C or G or T. RT-PCR was performed using PCR primer pairs listed in Table 1. A one-step RT-PCR kit (QIAGEN) was used for RT-PCR. The composition of a reaction solution was prepared according to protocols provided for the kit. RT-PCR was performed using a thermal cycler (Master Cycler Gradient, Eppendorf) at annealing temperatures ranging from 50*C to 70*C (5 reaction stages at annealing temperatures of 50*C, 55*C, 60*C, 65*C, and 70*C). Treatment was performed under RT-PCR conditions consisting of 50*C for 30 minutes and 94*C for 15 minutes and 40 cycles of treatment each consisting of 94*C for 1 minute, 50*C to 70*C for 1 minute, and 72*C for 1 minute and 30 seconds, followed by 72*C for 15 minutes. After reaction, 4*C was maintained. Table 1 RT-PCR No. < fonvard primer > < reverse primer > 1 PF1 PR2 2 PF1 PR1 3 NFO NR2 4 NFO NR1 Each reaction solution after PCR was subjected to electrophoresis using agarose gel and TAE buffer. After electrophoresis, agarose gel was stained with ethidium bromide and then target fragments were confirmed. Portions corresponding to target fragments were excised together with gel using a scalpel. The relevant portions were eluted and purified from gel using a QlAquick Gel extraction kit (QIAGEN). The nucleotide sequences of the thus purified PCR products were confirmed using a DNA sequencer (3100 Genetic Analyzer, ABI). Sequencing reaction was performed using an ABI BigDye Terminator Cycle Sequencing FS kit (ver.3.0). Experimental protocols -20 were used according to the manuals of ABI. In addition, the nucleotide sequences were determined using the primers listed in Table 1. 5' and 3' RACE method and PCR The following primers were designed based on the sequence information of DNA fragments obtained by RT-PCR and then 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) [Primers for 3' RACE] 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 implemented using a 5'/3' RACE Kit (Roche). Moreover, PCR was performed using Ex Taq Hot Start Version (TAKARA BIO INC.). The composition of the reaction solution was prepared according to the protocols included in the kit. A thermal cycler (Master cycler gradient, Eppendorf) was used. The annealing temperature employed herein ranges from 50*C to 70*C (5 reaction stages with annealing temperatures of 50*C, 55*C, 60*C, 65*C, and 70*C). Treatment was performed under PCR conditions consisting of 94*C for 15 minutes and 30 cycles of treatment each consisting of 94*C for 1 minute, 50 0 C to 70 0 C for 1 minute, and 72 0 C for 1 minute and 30 seconds, followed by 72 0 C for 15 minutes. After reaction, 4*C was maintained. Purification of PCR products and determination of nucleotide sequences were performed in the same manner as in the above-mentioned methods. Design of PCR primers for amplification of polypeptide coding regions The following primers were designed to perform amplification and cloning of all polypeptide-coding regions based on the sequence information determined by the RACE method. As PCR primers for amplification of polypeptide coding regions, the following primers were also prepared by adding restriction enzyme sites (BanH I and Sac I) for introduction into a plant expression vector pBIl21. - 21 - 4DS-F-ORI: 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) All polypeptide coding regions were amplified by PCR with the use of these primers. Next, the DNA fragment of each polypeptide coding region was ligated to a vector (for cloning of PCR products) (pSTBluel, Novagen) for TA-Cloning at 16*C (overnight reaction) using a DNA Ligation kit ver. 2 (TAKARA BIO INC.). Competent cells (E.coli DH5ca, TOYOBO) were transformed according to the protocols included in the kit and then cultured in LB medium supplemented with IPTG, X-gal, and 50 ptg/ml kanamycin. Thus, transformants were selected. Colonies that had appeared were picked up and then subjected to liquid culture in LB medium supplemented with 50 tg/ml kanamycin. Plasmid DNAs were prepared from the thus obtained microbial bodies using a Plasmid mini kit (QIAGEN). Fragment insertion was confirmed by gel electrophoresis and then plasmid DNA (expected to contain the target fragment that had been subcloned therein) was obtained. As primers for sequencing, primers (BcaBEST Sequencing Primer T7 (TAKARA BIO INC.); 5' TAA TAC GAC TCA CTA TAG GG 3' (SEQ ID NO: 25) and M13 Primer M4 (TAKARA BIO INC.); 5'GTT TTC CCA GTC ACG AC 3' (SEQ ID NO: 26)) targeting the T7 sequence and M13 sequence, respectively, existing at both ends of the cloning site in a pSTBluel vector 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 thus obtained nucleotide sequence was analyzed and edited using Genetyx-Win Ver. 4.0/ ATGC ver. 2 (Software Development). The nucleotide sequence homology (identity) between the Dc4DES gene and a previously reported coriander-derived gene (Cs4DES) was 88.0% in terms of the -22polypeptide coding region (predicted) sequence (Dc4DES gene: 1161 bp; and Cs4DES gene: 1158 bp). Furthermore, the amino acid sequence homology (identity) between the genes was 90.2% in terms of the predicted amino acid sequence of the polypeptide coding region (Dc4DES gene: 386aa; and Cs4DES gene: 385aa). Fig. 3 shows alignment of the Dc4DES amino acid sequence and the Cs4DES amino acid sequence. In Fig. 3, the Dc4DES amino acid sequence is shown in the upper row and the Cs4DES amino acid sequence is shown in the lower row. [Example 2] Identification and cloning of PTE gene Plant samples In Example 2, F1 varieties of carrot (Daucus carota L., Natsu-maki (summer-seeding) senko gosun, Yoshun gosun, and Shin Kuroda Gosun (purebred variety)) were used as experimental samples. Carrot seeds were purchased from OTA SEED Co., Ltd. A purebred variety having the same homologous gene sequence is desired as a gene cloning source. For convenience of experimentation (samples that had bloomed early could be prepared), plants of F1 variety were also used. Furthermore, the same umbelllifers, coriander (Corianlrum sativium) and dill (Anethum graveolens cv. mammoth) were also cultivated and used. Plant bodies were cultivated in a temperature-controlled chamber (Koito-toron; Koito Manufacturing Co., Ltd.) under conditions of 25*C, 16 hours of exposure to sunshine, and humidity of 50% and then the cultivated plant bodies were used as samples. RNA preparation Approximately 100 mg each of carrot leaves, immature seeds, and mature seeds was collected and then RNA was prepared according to the method in Example 1. RT-PCR amplification BLAST search (blastn and blastx; http://www.ncbi.nlm.nih.gov/blast/) was performed for a partial cDNA sequence (accession No. L20978) of an OTE-like gene (CsOTE) derived from coriander that is also an umbellifer as in the case of carrot. 17 types of analogous gene that had been found as genes having high homology were listed - 23 up. The predicted amino acid sequences of them were further subjected to multiple alignment analysis. Highly-conserved regions were selected. The DNA sequences of the same gene were further similarly subjected to multiple alignment analysis. Degenerate primers were designed for regions highly conserved at the amino acid level based on the codon usage frequency in DNA of the related species. The thus designed primers were used for the RT-PCR method. The primer sequences are as shown below. In addition, numerical figures in parentheses represent corresponding nucleotide Nos. based on Arabidopsis thaliana oleoyl-ACP thioesterase (AtOTE). 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-PRI (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-PRO (1041-1061): 5'-GTD SKN CMV CKR TTK AKY TC-3' (SEQ ID NO: 32) Specifically, RT-PCR amplification was performed using a One-step RT-PCR kit (QIAGEN) and the above primer pairs. The composition of the reaction solution was prepared according to the standard protocols included in the kit. RT-PCR was performed using a thermal cycler (Master Cycler Gradient, Eppendorf). With the use of the apparatus, any temperature gradient (12 reaction stages at maximum) can be set on a heat block and then reaction can be performed. Hence, RT-PCR reaction was performed at annealing temperatures required for amplification efficiency and specificity ranging from 50*C to 70*C (5 reaction stages with annealing temperatures of 50*C, 55*C, 60*C, 65*C, and 70*C). The reaction was performed under PCR conditions consisting of 50*C for 30 minutes, 94*C for 15 minutes, and 40 cycles of treatment each consisting of 94*C for 1 minute, 50*C to 70*C for 1 minute, and 72*C for 1 minute and 30 seconds, followed by 72*C for 15 minutes. After reaction, 4*C was maintained. As a result of RT-PCR, a specific amplification product (approximately 500 bp) could be obtained with the use of the combination of TE-PF2 and TE-PRI. The nucleotide sequence of the amplification product was identified. The molecular -24phylogenetic tree was analyzed using the partial sequences, so that the product was found to belong to the OTE cluster. 5' and 3' RACE method and PCR The database was searched again for the partial sequences obtained as a result of RT-PCR. Genes having high homology were extracted again. The predicted amino acid sequences of the genes were similarly subjected to multiple alignment analysis. Regions low-conserved at the amino acid level were selected. Next, primers enabling specific amplification of target genes were designed for the regions based on the codon usage frequency in DNA and then used for the RACE method. [for 3'-RACE] TE-D IF: 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-DIR: 5'-ATC ATC ACC CAC TTG CTA-3' (SEQ ID NO: 36) As a result of the RACE method, two different types of gene fragment were obtained by amplification. Both gene fragments belonged to a Fat A cluster composed of a TE group having specificity to unsaturated acyl ACP. One of these fragments belonged to a subcluster same as that of the known OTE on the molecular phylogenetic tree and the other forms a new subcluster. Moreover, in Example 2, determination of nucleotide sequences and analysis and edition of the obtained nucleotide sequences were performed in a manner similar to that in Example 1. Furthermore, the molecular phylogenetic tree analysis was performed with the use of the gene analysis service (http://www.ddbi.nip.ac.jp/Welcome-i.html) of the DNA Data Bank of Japan, the National Institute of Genetics (using Clustal W program (a program for multiple nucleotide sequence.amino acid sequence alignment and construction of phylogenetic trees). Two different types of gene fragment were obtained as a result of the RACE method. Primers for specific amplification of the two gene fragments were also -25designed. Specifically, the following primers for OTE-gene-specific amplification and primers for PTE-gene-specific amplification were designed for re-amplification and cloning of the full-length 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-mlR: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) Furthermore, as primers for amplification of polypeptide coding regions, the following primers were also prepared by adding restriction enzyme sites (BamH I and Sac I) for introduction into a plant expression vector pBl 121. [for predicted polypeptide coding regions] 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) RT-PCR was performed using these primers and carrot RNA as a template, so -26that all polypeptide-coding regions were amplified. Next, a PCR amplification fragment was ligated to a vector (for cloning of PCR products) (pSTBluel, Novagen) for TA-cloning at 16*C (overnight reaction) using a DNA Ligation kit ver. 2 (TAKARA BIO INC.). Competent cells (E.coli DH5a, TOYOBO) were transformed according to the protocols included in the kit and then cultured in LB medium supplemented with IPTG, X-gal, and 50 pg/ml kanamycin. Thus, transformants were selected. Colonies that had appeared were picked up and then subjected to liquid culture in LB medium supplemented with 50 pg/ml kanamycin. Plasmid DNAs were prepared from the thus obtained microbial bodies using a Plasmid mini kit (QIAGEN). Insertion of the fragments was confirmed by gel electrophoresis and then plasmid DNAs predicted to have the target fragments that had been subcloned therein were obtained. In this example, two types of Dc4PTE gene were isolated. The two thus isolated types of Dc4PTE gene were named Dc4PTEa and Dc4PTEb. Their nucleotide sequences are shown in SEQ ID NOS: 3 and 5, respectively. The amino acid sequences of the two types of Dc4PTE are shown in SEQ ID NOS: 4 and 6, respectively. As described above, the DcPTE genes were cloned. Similar to the cloning of the DcPTE genes, cloning of PTE genes had been attempted for coriander and dill. A coriander-derived petroselinoyl-ACP thioesterase gene (CsPTE gene) and a dill-derived petroselinoyl-ACP thioesterase gene (AgPTE gene) were isolated. Their nucleotide sequences are shown in SEQ ID NOS: 7 and 9, respectively. The amino acid sequences of the two types of Dc4PTE are shown in SEQ ID NO: 8 and 10, respectively. Next, petroselinoyl-ACP thioesterase activity of enzymes that are encoded by the DcPTE genes was examined. Specifically, histidine-labeled recombinant proteins were prepared using Escherichia coli as described below. The enzyme activity was then analyzed. Preparation of expression constructs of histidine-labeled proteins Based on the findings concerning known Safflower-derived oleoyl-ACP thioesterase (Plant Physiol., 100, 1751-1758, 1994), the mature peptide cleavage site of each gene was estimated and then determined as the N-terminus of a coding region in a DNA construct for mature peptide expression. Moreover, the C terminal side ends at -27the stop codon. Regarding the DcPTEa genes, the mature peptide cleavage site was estimated to be located between the nucleotide sequence encoding amino acid 32 and the nucleotide sequence encoding amino acid 33. Regarding the DcOTE gene and the CsOTE gene, the mature peptide cleavage site was estimated to be located between the nucleotide sequence encoding amino acid 51 and the nucleotide sequence encoding amino acid 52. The region was amplified by the PCR method using DcPTEa, DcOTE, or CsOTE cDNA cloned into pST Bluel (cloning vector, Novagen) as a template. The products were each mixed with an expression vector pQE-30 UA (QIAGEN) for a histidine-labeled protein, an equivalent amount of TaKaRa Ligation kit ver.2 was added to the product, followed by 30 minutes of ligation reaction at 16*C for subcloning. Subcloning into the vector makes it possible to prepare a recombinant protein in which 6XHis has been added to the N terminus using Escherichia coli. The total volume of the reaction solution was added to 50 pl of Escherichia coli competent cells (JM109 strain having lacI" mutation, TAKARA BIO INC.). Transformation was then performed according to the protocols specified by the manufacturer. Plasmids were prepared from the thus obtained transformants (grown in LB agar medium supplemented with 50 pig/ml ampicillin). Histidine-labeled protein expression in Escherichia coli and purification of the protein Escherichia coli (JM109 strain) was transformed (or using a glycerol stock stored prior to plasmid preparation) with the thus prepared DNA constructs for histidine-labeled protein expression. E. coli was cultured overnight in LB medium supplemented with Overnight Express Autoinduction System 1 (Merck) and 50 pig/ml ampicillin, so that expression was induced. Next, 20 mL of Escherichia coli transformed with each expression construct was collected. 4 mL of a lysis buffer (50 mM sodium hydrogenphosphate, 300 mM NaCl, and 10 mM imidazole) and 10 mg/ml lysozyme were added to and then mixed with the solution. The resultant was allowed to stand in ice for 30 minutes. The product was disrupted by ultrasonication (10 seconds x 6) using an ultrasonicator (UD-201, TOMY). -28- After 10 minutes of centrifugation at 15000 rpm and 4*C, the supernatant was injected into a HisTrap HP column (Amersham) that had been equilibrated in advance using a syringe. With the use of a perister pump (P-1, Amersham), the resultant was washed and eluted with a buffer included in the kit. Elution was performed for I mL each of the solution so that the total of 4 mL of the solution was fractionated. SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) was performed for each fraction of the purified histidine-labeled protein eluates of DcPTEa, DcOTE, and CsOTE. Thus, purification of the target proteins was confirmed. Fig. 4 shows the results. Based on the results of electrophoresis in Fig. 4, fractions in which the proteins had been eluted at high concentrations were selected. The concentrations of the proteins were measured by the Lowry method (RCDC protein assay kit, BIO-RAD). Hence, recombinant proteins (approximately 200 pg each) could be prepared. Preparation of acyl ACP Acyl ACP to be used as a substrate was prepared as follows. First, a hexane solution (100 mM) of fatty acid (oleic acid, petroselinic acid) was prepared. 6.2 pl of the solution was added to a 10 ml glass test tube and then dried and solidified with nitrogen gas. Subsequently, a reaction solution as shown in Table 2 below was added, followed by 60 minutes of reaction at 37*C on a heat block. Table 2 * 1 M Tris-HCI(pH 8.0) 400 pl -1 M MgCl 40 p * 1 MATP 200 pl -1 M DTT 10 pl * 20% Triton X-100 400 pl - 4 M LiCl 400 p1 * Holo-ACP (PanVera) 1000 [1 * His-tagged Acyl-ACP synthetase (0.26 mg/mL) 48 p.1 * H 2 0 1502 pl After reaction, 12 ml of water was added and then the solution was adjusted at pH6.0 using acetic acid. Acyl ACP contained in the reaction solution was purified by -29the following steps (A) to (M). (A) Fill (1 ml of bed volume) an unfilled polypropylene column (BIO-RAD LABORATORIES) with 1.8 ml of DEAE-Toyopearl 650C (TOSOH). (B) Equilibrate bis Tris-HCl pH6.0 (referred to as Buffer B) through addition of (A) in an amount 10 times greater than that of (A). (C) Add an acyl ACP solution (pH 6.0) to the column. (D) Wash with 3 mL of Buffer B. (E) Wash off unreacted fatty acid using 3 mL of 80% (w/w) isopropanol in Buffer B. XIsopropanol and Buffer B used herein had been prepared by deaerating each of them with an ultrasonic cleaning system and then mixing them. (F) Wash with 3 mL of Buffer B (G) Elute with 5 ml of 0.6M LiCl in Buffer B (H) Add 4.5 ml of Octyl-Sepharose (Amersham) to another unfilled column (to a bed volume of approximately 3 ml) (I) Equilibrate with Buffer B in an amount 10 times greater than that of (H). (perform steps H and I in advance) (J) Directly elute and apply the eluate of (G) to (I). (K) Wash with 5 ml of 10 mM MES-NaOH pH6.0 (referred to as Buffer C) (L) Elute with 6 mL of 35% (w/w) isopropanol in Buffer C -X-Isopropanol and Buffer C used herein had been prepared by deaerating each of them with an ultrasonic cleaning system and then mixing them. (M) Volatilize isopropanol using a vacuum centrifuge. Detection of thioesterase activity of purified enzymes Thioesterase activity of the histidine-labeled proteins purified via the above steps was determined using acyl ACP (oleoyl ACP or petroselinoyl-ACP) as a substrate. Before reaction, the protein concentrations in both solutions were measured by the Lowry method (RCDC protein assay kit, BIO-RAD). 25 mM Tris-HCl (pH 8.0), 1 mM DTT, acyl ACP, and water were mixed, followed by 5 minutes of preincubation (buffers used herein had been allowed to stand to -30reach reaction temperatures) at 25*C. 12 pg of the histidine-labeled protein (DcPTE or DcOTE) or y-globulin (as a control group) was added so that the total amount of the reaction solution was 100 pl, followed by 30 minutes of reaction at 25*C. Through detection of unreacted acyl ACP and free ACP (reaction product) by SDS-PAGE using one-tenth of the amount of the reaction solution (10 pl), thioesterase activity was determined. Fig. 5 shows the results in the form of electrophoresis photographs. After CBB staining of gel that had been subjected to electrophoresis, digital image data were produced using a high performance scanner (Pictrostat digital 400, FUJIFILM). Band concentrations of acyl ACP and free ACP were quantified using image analysis software, Image Analysis ver. 3.0 (Hitachi). Fig. 6 shows the results. As shown in Fig. 6, it was revealed that when petroselinoyl-ACP was used as a substrate (Fig. 6(B)), DcPTE clearly had higher reactivity than that of DcOTE. In contrast, when oleoyl-ACP was used as a substrate (Fig. 6(A)), DcOTE had higher reactivity. Therefore, it could be concluded that DcPTE is thioesterase having specificity to petroselinic acid. [Example 3] Next, transformed plants were produced by introducing the Dc4DES gene cloned in Example I and the DcPTE gene cloned in Example 2. The ability of petroselinic acid synthesis in the transformed plants was examined. Specifically, transformed plants in which the Dc4DES gene alone had been introduced, transformed plants in which the DcPTE gene alone had been introduced, and transformed plants in which the Dc4DES gene and the DcPTE gene were introduced together were produced. Preparation of DNA constructs for continuous systemic expression Among vectors that were used for transformation, Fig. 7 shows a vector for continuous systemic expression. In Fig. 7, "Pnos" denotes an Agrobacterium-derived nopaline synthase promoter, "Tnos" denotes an Agrobacterium-derived nopaline synthase terminator, "P35S" denotes a CaMV35S promoter, and "NPT II" denotes a neomycin phosphotransferase II gene. Specifically, a GUS gene contained in a plant expression vector pBI121 -31- (Clontech) was substituted with the cDNA sequence of the Dc4DES gene. A DNA fragment encoding the ORF region of the Dc4DES gene was amplified by the PCR method using primers designed so as to add a Bam HI sequence to the 5' terminal side and Sac I sequence to the 3' terminal side. The PCR product was mixed with pSTBluel (cloning vector, Novagen) and then an equivalent amount of TaKaRa Ligation kit ver.2 was added to the mixture, followed by 30 minutes of ligation reaction at 16*C. The total amount of the reaction solution was added to 50 pl of competent cells (E.coli DH5ca, TOYOBO) and then transformation was performed according to the protocols specified by the manufacturer. Plasmids were prepared from the thus obtained transformants (grown in LB agar medium supplemented with 50 pg/ml kanamycin). The thus obtained plasmids were treated with restriction enzymes (Bam HI and Sac I). Next, to excise the GUS gene ligated downstream of a CaMV35S promoter within pBIl21, treatment with restriction enzymes (Bam HI and Sac I) was performed similarly. Products digested with these restriction enzymes were subjected to 0.8 % agarose gel electrophoresis. DNA fragments containing the Dc4DES gene and the pBI 121 backbone (corresponding to a portion from which the GUS gene had been removed) were each isolated and purified using QIAquick gel extraction kit (QIAGEN) and Geneclean II (BIO 101). A pBIl21 backbone fragment and a DNA fragment (Dc4DES gene) to be subjected to insertion were mixed at a ratio of I : 10. Ligation reaction was performed for 30 minutes at 16*C using an equivalent amount of TaKaRa Ligation kit ver.2. The total amount of the reaction solution was added to 100 pl of competent cells (E.coli strain DH5a, TOYOBO) and then transformation was performed according to the protocols specified by the manufacturer. LB agar medium containing 50 ptg/ml kanamycin was coated with the resultant, followed by overnight culture. A vector (Fig. 6(D)) for co-expression of the Dc4DES gene and the DcPTE gene was constructed by inserting an expression unit (a DNA fragment containing a promoter and a terminator) of the DcPTE gene between a unique Eco RI site and Dra III site located in the vicinity of LB of T-DNA in an expression vector for Dc4DES expression -32as shown in Fig. 6(A). The expression unit of the DcPTE gene was amplified by the PCR method using primers that had been designed to add an Eco RI site and a Dra III site to both ends. Preparation of DNA constructs for seed-specific expression Among vectors used for transformation, vectors for seed-specific expression are shown in Fig. 8(A) to (D). In Fig. 8(A) to (D), "Pnap" denotes a rapeseed (B. campestris cv Kizakinonatane)-derived napin A promoter. It is known that Monsanto Company uses this promoter for controlling rapeseed fat and oil content. Specifically, the vectors for seed-specific expression were constructed by substituting the CaMV35S promoter in the vector shown in Fig. 7 with the rapeseed-derived napin A promoter. Furthermore, the GUS gene was substituted with the Dc4DES gene, DcPTE gene, or the cDNA sequence of Cs4DES. Meanwhile, the vector (Fig. 8(D)) for co-expression of the Dc4DES gene and the DcPTE gene was constructed by inserting an expression unit (a DNA fragment containing a promoter and a terminator) of the DcPTE gene between the unique Eco RI site and Dra III site in the vicinity of LB of T-DNA in the expression vector for Dc4DES expression as shown in Fig. 8(A). The expression unit of the DcPTE gene was amplified by the PCR method using primers that had been designed to add Eco RI and Dra III sites to both ends. Gene introduction into Agrobacterium by electroporation method Agrobacterium was transformed by an electroporation method using the thus prepared plasmids. 40 pl of competent cells of Agrobacterium tumefaciens (LBA4404 strain) that had been prepared by a conventional method was dissolved and then 5 pl (25 ig) of a DNA solution was added. The solution was allowed to stand on ice for 1 to 2 minutes. Subsequently, the solution was put into an ice-cooled cuvette (0.2 cm, BIO-RAD). A pulse current (1.25 kV and 10 tF) was applied using a gene introduction apparatus (Shimadzu). 460 pl of immediately-cooled SOC medium was added and then the resultant was cultured at 28'C for 1 hour. LB agar medium containing 50 pig/ml kanamycin and 50 pg/ml rifampicin was coated with the solution, followed by overnight - 33 culture. Thus, transformants were selected. Single-colony isolation was performed for transformant colonies that had appeared. A plurality of colonies were then picked up and then the presence of the target plasmid DNAs was confirmed by the PCR method. Preparation of Arabidopsis thaliana transformants by vacuum infiltration Arabidopsis thaliana (Arabidopsis thaliana et.Columbia) was transformed by a vacuum infiltration method. The vacuum infiltration method was performed according to Experimental Protocols for Model Plants, Shujunsha, 2001, pp. 109-113. Next, Arabidopsis thaliana subjected to vacuum infiltration was cultivated. Seeds were then collected and were determined to be 1 " generation transformant seeds (TI seeds). However, these seeds were handled as transformant generation seeds, for convenience. Nevertheless, not all seeds of this seed population had been transformed. Next, 1 " generation transformant seeds were seeded in medium supplemented with kanamycin (Murashige & Skoog base medium supplemented with 0.5 g/L MES, 10 g/L sucrose, 8 g/L agar, 100 mg/L carbenicillin, and 50 mg/L kanamycin). Seeds were caused to bud under light conditions. The plants were grown for approximately I to 2 weeks and then transformed individual plants (that had grown normally because of their kanamycin resistance) were selected. The individual plants that had developed their leaves normally were transplanted again in the same medium and then grown for approximately 1 to 2 weeks for re-selection. Plant bodies of the thus obtained line were determined to be 1 st generation transformant plant bodies (TI plants). Plants of the kanamycin-resistant line were transplanted in unsterilized vermiculite so that they could become acclimatized to a non-sterilized environment, followed by cultivation. Next, approximately 100 mg of rosette leaves were collected from the 1 " generation transformant plant bodies and then crushed under liquid nitrogen freezing. DNA was prepared using a DNA preparation kit (DNeasy plant mini kit, QIAGEN) according to the standard protocols included in the kit. PCR amplification was performed using PCR primers targeting a drug resistance gene (NPTII) within T-DNA and the introduced fatty acid synthesis system genes and Ex Taq DNA polymerase (TAKARA BIO INC.). -34- The thus obtained fragments (PCR amplification products) were subjected to electrophoresis using 0.8% agarose gel and a TAE buffer. Ethidium bromide staining was then performed, so that amplification of the target fragments was confirmed. The presence or the absence of the introduced genes was determined based on the presence or the absence of amplification. Next, plants of a line for which the above described introduction of T-DNA had been confirmed were continuously cultivated. Seeds collected from the plants were determined to be 2 nd generation transformant seeds (T2 seeds). T2 seeds of 15 or more lines were harvested per construct and then used for the following fatty acid composition analysis. Protocols for analysis of fatty acid composition of Arabidopsis thaliana seeds To perform analysis of fatty acid composition in seeds, fatty acid in seeds was methyl-esterified with hydrochloric acid-methanol and then the n-hexane extract was analyzed by GC-MS. In addition, BHT (Butylated hydroxytoluene) was added as an antioxidant for samples. Furthermore, as an internal standard, a methanol solution of methyl ester (SIGMA) of pentadecanoic acid (C15:0) not contained in plant oil was directly added to seeds after weighing the seeds. The resultant was used for correction of experimental errors occurring at the time of preparation of samples for analysis. n-hexane (Tokyo Chemical Industry Co., Ltd.) of a grade suitable for phthalate analysis was used for extraction. This can eliminate the effects of phthalate, which shows a behavior similar to that of fatty acid during GC analysis. Table 3 shows protocols for qualitative analysis and Table 4 shows protocols for quantitative analysis. Table 3 Protocols for qualitative analysis (1) Weigh 5 mg of a sample (fresh weight) (2) Add the sample to a 1.5-mL eppendorf tube (PP) and then add one tangsten bead (Qiagen) (3) Add 0.1% BHT and 5 mM C15:0-OMe /MeOH (500 pl each) (4) Crush (mixer mill MM300, Qiagen) the resultant under conditions of Freq:1/20s and - 35 - 1 minute. (5) Add 500 pl1 of 10% HCl/MeOH (Tokyo Chemical Industry, stored at 4*C) (6) Maintain at 80*C for 1 hour (Heat block, Iwaki) (7) Add I ml of n-hexane (for phthalate analysis, Tokyo Chemical Industry) (8) Vortex (mixing) for 5 seconds (9) Transfer the upper layer (hexane phase, 800 pl) to a 1.5-ml eppendolf tube (10) Dry and solidify under reduced pressure (Concentrator 5301, Eppendolf) (11) Add 100 pl of n-hexane (for phthalate analysis, Tokyo Chemical Industry) (12) Transfer the resultant into a GC glass tube vial and then seal the vial (13) GC/MS analysis ****************** ********* ************* *** *********** **** ***** ***** **** Table 4 Protocols for quantitative analysis ***** ** ****** **** ** *** ******** *** ******** ********* *** ** ***** **** ******** (1) Weigh 10 grains of sample seeds (fresh weight) (2) Add the seeds into a 1.5-mL eppendorf tube (PP) and then add one tangsten bead (Qiagen) (3) Add 0.002% BHT and 0.1 mM C15:0-OMe /MeOH (500 pl each) (4) Crush (mixer mill MM300, Qiagen) under conditions of Freq: 1/20 seconds for 1 minute. (5) Add 500 pl of 10% HCl/MeOH (Tokyo Chemical Industry, stored at 4*C) (6) Maintain at 80*C for 1 hour (Heat block, Iwaki) (7) Add 1 ml of n-hexane (for phthalate analysis, Tokyo Chemical Industry) (8) Vortex (mixing) for 5 seconds (9) Transfer the upper layer (hexane phase, 800 pl) into a 1.5-ml eppendolf tube (7) Add again 800 pl of n-hexane (for phthalate analysis, Tokyo Chemical Industry) (8) Vortex (mixing) for 5 seconds (9) Transfer the upper layer (hexane phase, 800 p.l) into a 1.5-ml eppendolf tube (10) Dry and solidify under reduced pressure (Concentrator 5301, Eppendolf) (11) Add 100 pl of n-hexane (for phthalate analysis, Tokyo Chemical Industry) (12) Transfer the resultant into a GC glass tube vial and then seal the vial (13) GC/MS analysis (HP) The thus obtained spectra obtained by treatment performed according to the above protocols were analyzed under conditions in Table 5. Under the conditions, regioisomers, oleic acid (C18:1, A9) and petroselinic acid (C18:1, A6), which differ in the positions of double bonds, can be separated by GC and then analyzed. - 36 - Table 5 Analytical instrument: HEWLETT PACKARD GC/MS HP6890 series GC system, 5973 Mass Selective Detector Column: SUPELCO SP-2380 (internal diameter: 0.25 mm, 100 m) Separation program: 80*C (at the start), temperature rise at 3*C/minute -+ 180*C (keep the temperature for 35 minutes), Temperature rise at 30*C/minute -+ 240*C (keep the temperature for 10 minutes) Total: approximately 80 minutes Analysis mode: Split (split ratio of 20 : 1) Carrier gas: Helium at a flow rate of 1 mL/min, pressure of 29.9 psi, and average velocity of 20 cm/sec The integration value at each peak in the Total Ion Chromatogram obtained with a GC-MS system mass detector was divided by the internal standard and the relevant molecular weight. The sum of the thus calculated values was determined to be the total amount of fatty acid. The percentage of each fatty acid in the total amount of fatty acid is represented by molar fraction (mol-%). In addition, fragment intensity detected with a mass spectrometer differs depending on substances and is known not to simply reflect the molecular weight. However, the reproducibility and the quantitative reliability are very high when analysis is performed under the same conditions. Hence, relative comparison is sufficiently possible. Analysis result I To examine the effect of the Dc4DES gene obtained according to the present invention on petroselinic acid production, continuous-systemic-expression-type transformed plants were produced using a pB-4DES construct expressing the Dc4DES gene under the control of a CaMV35S promoter. Leaves were collected from the transformed plants, fat and oil components were extracted, and then fatty acid composition analysis was performed by the above method. In addition, the fatty acid components analyzed herein were petroselinic acid and cis-4-hexadecenoic acid (16:1A4) that is a precursor of petroselinic acid. Table 6 shows the results of analyzing these monoene unsaturated fatty acids. - 37 - Table 6 Plant tissue analyzed Amount of fatty acid (wt%/total amount of fatty acid) C16:1A4 ±SE C18:1A6 ±SE WTleaf 0±0 0± 0 pB-4DES/WT T2 leaf 4.60 ± 0.1 7.67 ± 0.17 As a result, 7.67 wt% petroselinic acid (not synthesized in wild-type Arabidopsis thaliana) was accumulated in the leaves of the transformants into which pB-Dc4DES had been introduced, with respect to the total amount of fatty acid. Specifically, it was demonstrated that petroselinic acid can be efficiently produced in plant cells with the use of the carrot-derived Dc4DES gene (Table 6). As reported previously (Patent document 1), petroselinic acid content was 2.7 wt% when the coriander-derived Cs4DES gene was introduced into cultured tobacco cells. Hence, it was revealed that in terms of petroselinic acid synthesis and accumulation, the functions of the carrot-derived Dc4DES gene is superior to that of the coriander-derived Cs4DES gene. Analysis result 2 Transformed plants expressing the Dc4DES gene, Cs4DES gene, or DcPTE gene in a seed-specific manner were produced using a rapeseed napin promoter expressing seed-specifically. Fat and oil components were extracted from these seeds and then fatty acid composition analysis was performed by the above method. Table 7 shows the results of analyzing monoene unsaturated fatty acid. In addition, "NT" in Table 7 is an abbreviation of "not tested." Table 7 -38- Genotype Fatty acid (mol%) C16:lA4 SE C18:1A6 SE C20:IA8 SE Total SE Experimental value WT 0.00 0.00 0.00 0.00 pNCs4DES/WT T2(n=3) 0.38 ±0.04 0.81 ±0.11 0.35 ±0.05 1.54 ±0.20 pNDc4DES/WT T2(n=3) 0.40 ±0.03 0.89 ±0.09 0.41 ±0.03 1.70 ±0.15 pNDc4DESPTE/WT T2(n=6) 0.37 ±0.01 1.37 ±0.07 0.53 ±0.03 2.28 ±0.15 pNDc4DESPTE/WT T3(n=3) 0.42 ±0.02 1.83 ±0.09 0.68 ±0.05 2.93 ±0.15 Value in document WT 0.00 0.00 0.00 0.00 pN-Cs4DES/WT T2 (n=10) NT NT NT 0.6 ±0.04 pN-Cs4DES/fabl T2 (n=6) NT NT NT 2.4 ±0.2 As a result, 0.81 mol% petroselinic acid was accumulated in the seeds of transformants into which pN-Cs4DES had been introduced. In contrast, 0.89 mol% petroselinic acid was accumulated in the seeds of transformants into which pN-Dc4DES had been introduced. Regarding petroselinic acid synthesis and accumulation in seeds, the functions of the carrot-derived Dc4DES gene was again superior to that of the coriander-derived Cs4DES gene. In the transformants into which pN-Dc4DES-DcPTE had been introduced, 1.83 mol% petroselinic acid was accumulated. Specifically, compared with the case in which the Cs4DES gene alone had been introduced, the amount of petroselinic acid accumulated was increased to 226%. Petroselinic acid production was promoted via introduction of DcPTE having high substrate specificity to petroselinoyl-ACP. Further specifically, it was revealed that co-expression of the Dc4DES gene and the DcPTE gene can have an effect of increasing the amount of petroselinic acid accumulated to a level that is significantly higher than that in the case of expression of the Dc4DES gene alone. Moreover, a value obtained via such co-expression was nearly twice or more than that obtained according to such conventional technology that involves using the coriander-derived Cs4DES gene alone. Such a petroselinic acid biosynthesis system gene that enables an increased amount of petroselinic acid accumulated through coexpression with the 4DES gene has never been known. The petroselinic acid biosynthesis system gene was discovered for the first time in the world. -39- Furthermore, as shown in Table 6, it was discovered that through expression of the Dc4DES gene alone or through co-expression of the Dc4DES gene and the DcPTE gene, production of not only petroselinic acid, but also cis-8-icosenoic acid is possible. Moreover, it could be confirmed that the production amount of cis-8-icosenoic acid can be more increased via co-expression of the Dc4DES gene and the DcPTE gene than the case in which the Dc4DES gene is expressed alone. In addition, there are no successful cases concerning production of cis-8-icosenoic acid in plants. Analysis result 3 - Furthermore, each fatty acid composition (including saturated fatty acid) in transformed plants in which the Dc4DES gene or DcPTE gene had been expressed alone or the transformed plants in which the Dc4DES gene and DcPTE gene had been co-expressed was analyzed. As a result, it was revealed that saturated fatty acid contents were significantly increased in the seeds of transformants in which the DcPTE gene had been expressed alone or transformants in which the DcPTE gene and the Dc4DES gene had been co-expressed, compared with the same in a wild type strain, transformed plants in which the Cs4DES gene had been introduced, and transformed plants in which the Dc4DES gene had been introduced (Fig. 9). Stearic acid (C18:0) content, icosanoic acid (C20:0) content, or docosanoic acid (C22:0) content in the seeds of plants in which the DcPTE gene had been introduced reached a level that was nearly twice the relevant content in the seeds of plants in which no DcPTE gene had been introduced. The DcPTE gene is classified as a thioesterase gene in a group referred to as Fat A (members of which have specificity to unsaturated fatty acid-ACP) as a result of molecular phylogenetic tree analysis based on the predicted amino acid sequence. Moreover, an effect of increasing unsaturated fatty acid content can be expected according to the results shown in Example 2 such that the DcPTE gene shows substrate specificity to petroselinoyl ACP. However, it is difficult to predict an effect of increasing saturated fatty acid production. Hence, it was revealed that the DcPTE gene has a special significant effect that is unpredictable for persons skilled in the art. - 40 - The DcPTE gene has very advantageous properties for production of a nylon raw material (dicarboxylic acid) via oxidative decomposition of plant-derived fatty acid. Specifically, the properties are to exert an effect of increasing the contents of saturated fatty acids with C 18 (or greater number of carbons) in addition to its effect of promoting petroselinic acid accumulation. More specifically, the DcPTE gene can increase saturated fatty acid contents, so as to lower unsaturated fatty acid contents (that cause the generation of impurities because of oxidative decomposition) other than petroselinic acid. Furthermore, such effects can be obtained not only by the use of another gene (e.g., Dc4DES gene) in combination, but also by the use of the DcPTE gene alone. Therefore, the DcPTE gene can also be used for applications (e.g., saturated fatty acid production) other than petroselinic acid accumulation for the purpose of producing a resin raw material. Industrial Applicability According to the present invention, novel genes that are used for increasing the amount of petroselinic acid synthesized upon production of petroselinic acid can be provided. Furthermore, a novel method for producing petroselinic acid using the genes can be provided. With the use of genes involved in production of petroselinic acid according to the present invention, petroselinic acid can be accumulated in large amounts in plant seeds, for example. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety. -41-

Claims (11)

1. An isolated gene encoding a protein that comprises the amino acid sequence shown in SEQ ID NO: 4 or 6. 5

2, An isolated gene comprising the sequence shown in SEQ ID NO:3 or 5.

3. An expression vector comprising the gene according to claim I or 2. 10

4. A plant transformant, into which the gene according to claim I or 2 is introduced, or the expression vector according to claim 3 is introduced.

5. The transformant according to claim 4, into which a A4-palmitoyl-ACP desaturase gene derived from Daucus carota shown in SEQ ID NO: 1, or encoding a 15 protein that comprises the amino acid sequence shown in SEQ ID NO: 2 is introduced.

6. A transformed plant cell or transformed plant, into which a gene according to claim 1 or 2 and a A4-palmitoyl-ACP desaturase gene derived from Daucus carota shown in SEQ ID NO: 1, or encoding a protein that comprises the amino acid sequence 20 shown in SEQ ID NO: 2 are introduced.

7. A transformed plant cell or a transformed plant having an improved productivity of saturated fatty acid, into which a gene according to claim I or 2 is introduced. 25

8. A method for producing saturated fatty acid, comprising steps of: introducing a gene according to claim I or 2 into a plant cell to produce a transformed plant; and extracting saturated fatty acid from tissues collected from the transformed plant. 30

9. The method according to claim 8, wherein a A4-palmitoyl-ACP desaturase gene derived from Daucus carota shown in SEQ ID NO: 1, or encoding a protein that comprises the amino acid sequence shown in SEQ ID NO: 2 is further introduced into the transformed plant. 42

10. An isolated gene encoding a protein having petroselinoyl-ACP thioesterase activity substantially as hereinbefore described with reference to the Figures and/or Examples, excluding comparative Examples. 5

11. A transformed plant cell or transformed plant according to claim 6 substantially as hereinbefore described with reference to the Figures and/or Examples, excluding comparative Examples. 43