KR100931845B1 - Ginseng-derived PPS genes involved in protofa narcotic diol biosynthesis - Google Patents

Abstract

The present invention relates to a ginseng-derived PPDS gene involved in protopanaxanalyse diol biosynthesis. ) protein; A gene encoding the PPDS protein; A recombinant vector comprising the gene; The vector relates to a transformed yeast and a transformed dicotyledonous plant.

Description

 Ginseng PPDS gene involved in protopanaxadiol biosynthetic pathway

The present invention relates to a ginseng-derived PPDS gene that is involved in protofaanacodiol biosynthesis.

Saponin is the name given to the microbubble of soap in aqueous solution. In general, saponin lowers the surface tension of water, so it foams easily and shows hemolytic action. Ginseng saponins, however, were found to be mildly toxic and nontoxic, with little hemolytic action. In addition, ginseng saponin component has a completely different chemical structure from saponins found in other plants. Therefore, ginseng saponin is called "ginsenoside" in the sense of ginseng glycoside to distinguish it from other plant-based saponins.

Of the 30 ginsenosides in Korean ginseng, the pharmacological action of more than 10 individual ginsenosides has been revealed. These ginsenosides may act similarly or vice versa. Therefore, Korean ginseng contains similar or opposite functional ingredients. For example, the representative gnsenoside-Rb1 of PD-based saponins has an inhibitory effect on the central nervous system, and the representative ginsenoside-Rg1 of PT-based saponins has a stimulating effect. However, these components are known to not antagonize each other.

Simply defined, the chemical structure is called the sugar part "sugar" (glycone), the non-sugar part "nonsugar" (also known as aglycone or genin), and especially the aglycone of saponin is called sapogenin. Saponins are classified into two types, dammarane and oleanane, depending on the skeleton structure of the aglycone.

Ginseng saponin (ginsenoside) is divided into two types, mainly tetracyclic saponins and pentacyclic olean-based saponins, depending on the non-sugar portion. Dmarmarane saponin is divided into protopanaxadiol-based saponins (PPD) in two cases and protopanaxatriol-based saponins (PPT) in three cases depending on the number of hydroxyl groups (-OH) attached to the non-sugar portion. There are 19 types of protopanaxadiol-based saponins: G-Ra1, G-Ra2, G-Ra3, G-Rb1, G-Rb2, G-Rb3, G-Rc, G-Rd, 20 (S) G-Ra3, 20 ( R) G-Rg3, G-Rh2, G-Rs1, G-Rs2, Quinquenoside-R1, Notoginsenoside-R4, Malonyl-G-Rb1, Malonyl-G-Rb2, Malonyl-G-Rc, Malonyl-G-Rd Protopanaxatriol-based saponins are 10 species, G-Rc, G-Rf, G-Rg2, G-Rh1, 20-Glc-G-Rf, Notoginsenoside-R1, 20 (R) G-Rg2, 20 (R) G-Rh1, Rh4, etc., and Oleanane-type saponin is G-Ro.

However, nothing is known about the ProtoPanaxadiol Synthetase (PPDS) gene involved in ProtoPanaxadiol biosynthesis.

The present invention has been made in accordance with the above requirements, and the present invention is intended to provide the usefulness of the PPDS gene involved in protopananaxadiol-based ginseng saponin biosynthesis as well as the biosynthesis of prophylactic acid diol, which is itself physiologically active. .

In order to solve the above problems, the present invention provides a ginseng-derived PPDS (Protopanaxadiol synthase) protein, which is involved in protoparanaxadiol biosynthesis, consisting of the amino acid sequence represented by SEQ ID NO: 2.

The present invention also provides a gene encoding the PPDS protein.

The present invention also provides a recombinant vector comprising the gene.

The present invention also provides a transformed yeast and a transformed dicotyledonous plant transformed with the vector.

The ginseng-derived PPDS gene involved in the protopanaxanadiol biosynthesis according to the present invention can be usefully used in a method for mass-producing protopanaxanadiol or increasing the proteophaxadiol-based ginseng saponin biosynthesis.

In order to achieve the object of the present invention, the present invention provides a PPDS (Protopanaxadiol synthase) protein derived from ginseng involved in protopananaxadiol biosynthesis, consisting of the amino acid sequence represented by SEQ ID NO: 2

The scope of the PPDS protein according to the present invention includes a protein having an amino acid sequence represented by SEQ ID NO: 2 isolated from ginseng and a functional equivalent of the protein. "Functional equivalent" means at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 70% of the amino acid sequence represented by SEQ ID NO: 2 as a result of the addition, substitution, or deletion of the amino acid Is 95% or more of sequence homology, and refers to a protein that exhibits substantially homogeneous physiological activity with the protein represented by SEQ ID NO: 2. By "substantially homogeneous physiological activity" is meant the activity involved in protopanaxadiol biosynthesis in plants.

The present invention provides a gene encoding the PPDS protein. Genes of the invention include both genomic DNA and cDNA encoding PPDS proteins. Preferably, the gene of the present invention may include the nucleotide sequence represented by SEQ ID NO: 1.

In addition, variants of the above nucleotide sequences are included within the scope of the present invention. Specifically, the gene has a base sequence having a sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% with the nucleotide sequence of SEQ ID NO: 1, respectively. It may include. The "% sequence homology" for a polynucleotide is identified by comparing two optimally arranged sequences with a comparison region, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include the addition or deletion (ie, gap) compared to).

The present invention also provides a recombinant vector comprising the PPDS gene according to the present invention. The recombinant vector is preferably a recombinant yeast expression vector or a recombinant plant expression vector.

The recombinant yeast expression vector according to an embodiment of the present invention is characterized in that pYeDP60_PPDS described in FIG. 2.

The recombinant plant expression vector according to one embodiment of the present invention is characterized in that the pPZP211_PPDS vector described in FIG. 3.

The term “recombinant” refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, a heterologous peptide, or a heterologous nucleic acid. Recombinant cells can express genes or gene fragments that are not found in their natural form in either the sense or antisense form. Recombinant cells can also express genes found in natural cells, but the genes have been modified and reintroduced into cells by artificial means.

The term “vector” is used to refer to a DNA fragment (s), a nucleic acid molecule, that is delivered into a cell. Vectors can replicate DNA and be reproduced independently in host cells. The term "carrier" is often used interchangeably with "vector". The term “expression vector” refers to a recombinant DNA molecule comprising a coding sequence of interest and a suitable nucleic acid sequence necessary to express a coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

Yeast expression vectors include promoter genes, genes encoding the target protein from which translational initiation and termination codons have been removed, and terminators, wherein the promoter genes are genes selected from the group consisting of GAPDH, PGK, ADH, PHO5, GAL1 and GAL10. Preferably, but not limited to.

The PPDS gene of the present invention comprises a nucleic acid sequence encoding a signal peptide, which allows for the export of the expressed protein. Here, the nucleic acid sequence encoding the signal peptide is preferably bound directly to 5 'of the heterologous gene to be expressed. In the secretion and modification of many eukaryotic cell proteins, fusion with a protein sequence having a signal sequence at the N-terminus is required to steer the polypeptide into the secretion apparatus.

The vector may be both an integrative yeast plasmid (YIp) and an extrachromosomal plasmid vector (YP). The extrachromosomal plasmid vector is divided into an episomal yeast plasmid (YEp), a replicative yeast plasmid (YRp) and a yeast centromeric plasmid (YCp). Furthermore, artificial yeast chromosomes (YACs) are also possible as expression vectors according to the present invention.

In addition, particularly preferred yeast vectors are yeast replication plasmids that can be propagated and selected in E. coli containing the origin of replication ori and an antibiotic resistance cassette. Furthermore, they have ARS sequences capable of independent chromosome replication in yeast cells, such as HARS1 from H. polymorpha , and metabolic yeast selection markers such as URA3 or HLEU2.

Preferred examples of plant expression vectors are Ti-plasmid vectors which, when present in a suitable host such as Agrobacterium tumerfaciens, can transfer part of themselves, the so-called T-region, into plant cells. Another type of Ti-plasmid vector (see EP 0 116 718 B1) is used to transfer hybrid DNA sequences to protoplasts from which current plant cells or new plants can be produced that properly insert hybrid DNA into the plant's genome. have. A particularly preferred form of the Ti-plasmid vector is the so-called binary vector as claimed in EP 0 120 516 B1 and US Pat. No. 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the invention into a plant host are viral vectors, such as those which can be derived from double stranded plant viruses (eg CaMV) and single stranded viruses, gemini viruses, etc. For example, it may be selected from an incomplete plant viral vector. The use of such vectors can be advantageous especially when it is difficult to properly transform a plant host.

The expression vector will preferably comprise one or more selectable markers. The marker is typically a nucleic acid sequence having properties that can be selected by chemical methods, and all genes that can distinguish transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate or phosphinothricin (phosphinothricin), kanamycin, G418, bleomycin, hygromycin, chloramphenicol There are antibiotic resistance genes such as, but not limited to.

In the plant expression vector according to an embodiment of the present invention, the promoter may be CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter, but is not limited thereto. The term "promoter" refers to a region of DNA upstream from a structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. A "constitutive promoter" is a promoter that is active under most environmental conditions and developmental conditions or cell differentiation. Constitutive promoters may be preferred in the present invention because selection of the transformants may be made by various tissues at various stages. Thus, the constitutive promoter does not limit the selection possibilities.

The terminator may be a conventional terminator, and examples thereof include nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, agrobacterium tumefaciens (ocrobacterium tumefaciens) Terminator of the Fine (Octopine) gene, etc., but is not limited thereto. With regard to the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of terminators is highly desirable in the context of the present invention.

The present invention provides a transformed yeast transformed with a recombinant yeast vector comprising the PPDS gene of the present invention, preferably the transformed yeast transformed with the pYeDP60_PPDS vector described in FIG. 2.

Preferably, the yeast belongs to the genus selected from Pichia, Hansenula, Candida, Torulopsis, Saccharomyces, Schizosaccharomyces, Kluyveromyces and Yarrowia . Especially preferably, the microorganism belongs to a species selected from Hansenula polymorpha, Saccharomyces Cervisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, and Yarrowia lipolytica .

Transformation of yeast allows the nucleic acid molecule or vector to be introduced into the cell by standard methods known to those skilled in the art, preferably by electroporation, chemical transformation, transformation by plasma fusion, or particle bombardment ( See: Current Protocols in Molecular Biology, John Wiley & Sons, Edited by: Fred M. Ausubel et al .; Molecular Cloning: A Laboratory Manual (Third Edition), J. Sambrook and D. Russell, 2001, Cold Spring Harbor Laboratory Press ).

The present invention provides a plant transformed with a recombinant plant vector comprising the PPDS gene of the present invention, and preferably provides a transformed dicotyledonous plant transformed with the pPZP211_PPDS vector described in FIG. 3.

The plant is tobacco, eggplant, tobacco, pepper, tomato, burdock, garland chrysanthemum, lettuce, bellflower, spinach, chard, sweet potato, celery, carrots, buttercups, parsley, cabbage, cabbage, mustard, watermelon, melon, cucumber pumpkin, gourd, It may be a dicotyledonous plant, characterized in that selected from strawberries, soybeans, mung beans, kidney beans and peas, but is preferably Arabidopsis.

Plant transformation refers to any method of transferring DNA to a plant. Such transformation methods do not necessarily have a period of regeneration and / or tissue culture. Transformation of plant species is now common for plant species, including both dicotyledonous plants as well as monocotyledonous plants. In principle, any transformation method can be used to introduce hybrid DNA according to the invention into suitable progenitor cells. Method is calcium / polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373), protoplasts Electroporation (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102), microscopic injection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185 ), (DNA or RNA-coated) particle bombardment of various plant elements (Klein TM et al., 1987, Nature 327, 70), Agrobacterium tumulopasis by plant infiltration or transformation of mature pollen or vesicles And infection with (incomplete) virus (EP 0 301 316) in en mediated gene transfer. Preferred methods according to the invention include Agrobacterium mediated DNA delivery. Especially preferred is the use of the so-called binary vector technology as described in EP A 120 516 and US Pat. No. 4,940,838.

The "plant cells" used for plant transformation may be any plant cells. The plant cells may be cultured cells, cultured tissues, cultured organs or whole plants, preferably cultured cells, cultured tissues or cultured organs and more preferably any form of cultured cells.

"Plant tissue" refers to the tissues of differentiated or undifferentiated plants, such as, but not limited to, roots, stems, leaves, pollen, seeds, cancer tissues and various types of cells used in culture, ie single cells, protoplasts. (protoplast), shoots and callus tissue. The plant tissue may be in planta or in an organ culture, tissue culture or cell culture.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

<Example 1; PPDS Cloning Method>

As shown in FIG. 1, an addition reaction of an OH group is required for the synthesis of protoparnaxadiol from dammarenediol, and therefore, it is assumed that the enzyme that biosynthesizes the protoparnaxadiol as a substrate is P450 series. . About 20,000 ESTs were produced from Panax Ginseng for PPDS gene cloning, and a total of 69 Ginseng P450 genes were selected from the produced ESTs. Among the total ginseng P450 genes selected, P450 genes with P450 genes with high amino acid sequence similarity and increased expression of ginsenoside synthesis and PDS genes using triterpene family structurally similar to dammarenediol were PPDS genes. Candidates were selected. The function of the selected gene is to screen the P450 gene, which expresses the Ginseng P450 gene in yeast, treats the substrate dammarendiol, and then analyzes the metabolite by LC / MS to convert the dammarendiol substrate into protopanaxadiol. Protoparnaxadiol synthase (PPDS) gene was cloned.

<Example 2; Vector manufacturing

As shown in FIG. 2, only the ORF of the Ginseng P450 gene selected to express the ginseng PPDS gene in yeast was PCR amplified and inserted into the EcoRI and BamHI sites of the yeast expression vector pYeDP60 vector. Primers used for PPDS amplification are as follows; 5'-ATGGTTGTTTTCATATTTGTGCT-3 '(SEQ ID NO: 3), 5'-GTAAGCTGAAACCCTAAGGTGTA-3' (SEQ ID NO: 4). Recombinant DNA with PPDS was transformed into E. coli to select E. coli transformed with the recombinant DNA. The recombinant DNA into which the PPDS gene was introduced was introduced into the yeast, WAT21, after the mutation was confirmed through sequencing.

As shown in FIG. 3, only the ORF of the Ginseng P450 gene (PPDS) was PCR amplified and inserted into the KpnI and SalI sites of the plant expression vector pZPP211 to express the ginseng PPDS gene in plants. Primers used for PPDS amplification are as follows; 5'-ATGGCTGTTTTCATAGTTGTG-3 '(SEQ ID NO: 5) 5'-TTAGTAAGCTGAAACCCTAAG 3 '(SEQ ID NO: 6). PCR products were cloned into the pGEMT-Easy vector, and then examined for mutation by sequencing. The PCR products were digested with restriction enzymes KpnI and SalI to insert only the PPDS gene into the plant expression vector pPZP211 to complete pPZP211_PPDS.

Recombinant DNA (pPZP211_PPDS) into which ginseng PPDS was inserted was introduced into Agrobacterium tumefaciens GV3101 using electroporation. Finally, the ginseng PPDS gene was introduced into Arabidopsis thaliana (ecotype Col_0) using the Agrobacterium transformation method.

<Example 3; Transformant Production Method>

Transformation of yeast by recombinant DNA inserted with the PPDS gene was carried out according to the method provided using "Yeastmaker Yeast Transformation System2 kit" by "Clontech". Yeast transformants with the recombinant pYeDP60 vector inserted with the PPDS gene were selected in SD / Ura-, Ade- medium. Introduction of recombinant DNA into the transformed yeast was confirmed by survival in the selection medium and PCR using PPDS specific primers.

Arabidopsis transformants in which the ginseng PPDS gene was inserted were selected from a medium containing kanamycin. The introduction and expression of the ginseng PPDS gene in the transformed and grown germination in the kanamycin-added medium was confirmed using PCR and RT-PCR methods.

<Example 4; PPDS Isolation and Activity Assessment from Transformants>

Yeast transformants were harvested by centrifugation after incubation in a medium containing the substrate, dammarenediol. The harvested yeast was extracted intracellular metabolites using 20% KOH / 50% EtOH solution. The cell extract was again extracted with hexane and used as a sample for TLC. As a solvent for TLC, water and acetonitrile were mixed in a 5:95 ratio. TLC results of the cell extracts were able to detect protopanaxanadiol, and proropanaxadiol expected substances on the TLC of the transgenic enzyme extract through the introduction of PPDS gene were extracted from the TLC plate and subjected to LC / MS analysis. .

As a result of LC / MS analysis, it was confirmed that there was a substance which was not synthesized in the control yeast in which the PPDS gene was not introduced at the same position as the protopanaxadiol from the yeast transformant into which the ginseng PPDS gene was inserted. (Figure 4). As a result of mass spectrometry of this substance, it was judged that it was the same substance as the protoparanaxadiol used as a standard. (Figure 5). From these results, the ginseng gene PPDS is considered to encode an enzyme that biosynthesizes protoparnaxadiol as a substrate.

1 is a diagram showing the ginsenoside biosynthetic pathway.

Figure 2 is a recombinant yeast expression vector pYePD60_PPDS with the PPDS gene inserted.

3 is a recombinant plant expression vector pPZP211_PPDS inserted with the PPDS gene.

FIG. 4 is a liquid chromatogram of material extracted from control yeast (A) and transformed yeast (B) with PPDS gene (pYePD60_PPDS) inserted. 4C is an LC chromatogram of ProtoPanaxadiol standard.

5 is a mass spectrum result obtained by LC / APCIMS of a material isolated by TLC of the extract from the transformed yeast inserted with the PPDS gene (pYePD60_PPDS) (provided above) and a protoparanaxadiol standard. The extract from the transformed yeast with the PPDS gene (pYePD60_PPDS) inserted into the protoparnaxadiol gene shows that the PPDS is the protoparanaxadiol biosynthesis gene.

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Ginseng PPDS gene involved in protopanaxadiol biosynthetic          pathway <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 1590 <212> DNA <213> Panax ginseng <400> 1 ggtctctctc tctctctgtg aagtgtattg gagcttttag gaagaatcaa tggttgtttt 60 catatttgtg cttggtgtgg ttttggaact cagtctcgta agtacagctt tgttgagatg 120 gaatgaggtg aggtacagga agaagggttt gcctccaggt accatgggtt ggccagtttt 180 tggtgagacc actgagttcc tcaaacaagg cccaagcttc atgaaaaacc agagagccag 240 gttcggaagt tttttcaaat cccacatatt gggttgtcct acaattgttt ccatggatcc 300 agagctcaac aaatatatcc ttatgaatga agcaaaaggg cttgtacctg gctatccaca 360 atccatgctt gacattttag ggaaatgcaa cattgccgct gtccatggtt ctactcacaa 420 gaacatgaga ggggcattgc ttgcacttgt tagccacacc atgatcaaag accaactttt 480 gccaaaaatc gatcatttca tgagatctca cctgagcaat tgggataaca aagtcattga 540 tattcaagaa aaaaccaaag agatggcact actgtcctca cttaagcaaa ttgctggtat 600 agaatctggc tcaatgtgcc aggaattcat gcctgaattt ttcaagttag ttttaggaac 660 tctttcattg ccaatcaacc ttccgggtac agattatcat catggctttc aggcaaggca 720 gaacattgtc aggatgttga gacaacttat tgaggacaga agatcttccc aaaaaaccca 780 tcatgacatg cttggttatc ttatgaacag tgaaggaaac agatataaga taactgatga 840 agagataatt gatcaaatta tcacaattct ctactctgga tatgaaactg tttcaacaac 900 ttcaatgatg gctgtcaagt atcttcatga tcatccaagg gttcttgaag aactcagaaa 960 agaacatatg gcaattagag agaagaaaag gccagaggat ccaatcaact ggaatgacta 1020 taagtccatg cgcttcactc gagctgtgat ctttgagacc tcaagattag ctacaattgt 1080 taatggggtt ttgaggaaaa ctactcgaga gatggaactg aatggttata ttattcccga 1140 aggatggaga atatatgttt atacaagaga aattaattat gacccacggc tatatccaga 1200 accacaaacc tttaatccct ggagatggct ggataagagc atagatgccc aaaactactt 1260 cttcatattt ggaggaggaa ctaggcaatg tcctggaaag gaactaggga tagcagaaat 1320 ctcaactttt ctgcattact tcgtcactag atatagatgg aatgaagttg ggggagataa 1380 actgatgaaa tttccaagag ttgaagcacc aaatgggtta caccttaggg tttcagctta 1440 ctaaccaacc tggtatacat atgtacagaa gagatctagg atataagcat aacaaggatg 1500 taaaataatg ttctaacaaa gttttagaaa ttgattgaaa ttaaggttct gcgttcaaaa 1560 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1590 <210> 2 <211> 464 <212> PRT <213> Panax ginseng <400> 2 Met Val Val Phe Ile Phe Val Leu Gly Val Val Leu Glu Leu Ser Leu   1 5 10 15 Val Ser Thr Ala Leu Leu Arg Trp Asn Glu Val Arg Tyr Arg Lys Lys              20 25 30 Gly Leu Pro Pro Gly Thr Met Gly Trp Pro Val Phe Gly Glu Thr Thr          35 40 45 Glu Phe Leu Lys Gln Gly Pro Ser Phe Met Lys Asn Gln Arg Ala Arg      50 55 60 Phe Gly Ser Phe Phe Lys Ser His Ile Leu Gly Cys Pro Thr Ile Val  65 70 75 80 Ser Met Asp Pro Glu Leu Asn Lys Tyr Ile Leu Met Asn Glu Ala Lys                  85 90 95 Gly Leu Val Pro Gly Tyr Pro Gln Ser Met Leu Asp Ile Leu Gly Lys             100 105 110 Cys Asn Ile Ala Ala Val His Gly Ser Thr His Lys Asn Met Arg Gly         115 120 125 Ala Leu Leu Ala Leu Val Ser His Thr Met Ile Lys Asp Gln Leu Leu     130 135 140 Pro Lys Ile Asp His Phe Met Arg Ser His Leu Ser Asn Trp Asp Asn 145 150 155 160 Lys Val Ile Asp Ile Gln Glu Lys Thr Lys Glu Met Ala Leu Leu Ser                 165 170 175 Ser Leu Lys Gln Ile Ala Gly Ile Glu Ser Gly Ser Met Cys Gln Glu             180 185 190 Phe Met Pro Glu Phe Phe Lys Leu Val Leu Gly Thr Leu Ser Leu Pro         195 200 205 Ile Asn Leu Pro Gly Thr Asp Tyr His His Gly Phe Gln Ala Arg Gln     210 215 220 Asn Ile Val Arg Met Leu Arg Gln Leu Ile Glu Asp Arg Arg Ser Ser 225 230 235 240 Gln Lys Thr His His Asp Met Leu Gly Tyr Leu Met Asn Ser Glu Gly                 245 250 255 Asn Arg Tyr Lys Ile Thr Asp Glu Glu Ile Ile Asp Gln Ile Ile Thr             260 265 270 Ile Leu Tyr Ser Gly Tyr Glu Thr Val Ser Thr Thr Ser Met Met Ala         275 280 285 Val Lys Tyr Leu His Asp His Pro Arg Val Leu Glu Glu Leu Arg Lys     290 295 300 Glu His Met Ala Ile Arg Glu Lys Lys Arg Pro Glu Asp Pro Ile Asn 305 310 315 320 Trp Asn Asp Tyr Lys Ser Met Arg Phe Thr Arg Ala Val Ile Phe Glu                 325 330 335 Thr Ser Arg Leu Ala Thr Ile Val Asn Gly Val Leu Arg Lys Thr Thr             340 345 350 Arg Glu Met Glu Leu Asn Gly Tyr Ile Ile Pro Glu Gly Trp Arg Ile         355 360 365 Tyr Val Tyr Thr Arg Glu Ile Asn Tyr Asp Pro Arg Leu Tyr Pro Glu     370 375 380 Pro Gln Thr Phe Asn Pro Trp Arg Trp Leu Asp Lys Ser Ile Asp Ala 385 390 395 400 Gln Asn Tyr Phe Phe Ile Phe Gly Gly Gly Thr Arg Gln Cys Pro Gly                 405 410 415 Lys Glu Leu Gly Ile Ala Glu Ile Ser Thr Phe Leu His Tyr Phe Val             420 425 430 Thr Arg Tyr Arg Trp Asn Glu Val Gly Gly Asp Lys Leu Met Lys Phe         435 440 445 Pro Arg Val Glu Ala Pro Asn Gly Leu His Leu Arg Val Ser Ala Tyr     450 455 460 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 atggttgttt tcatatttgt gct 23 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gtaagctgaa accctaaggt gta 23 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 atggctgttt tcatagttgt g 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 ttagtaagct gaaaccctaa g 21  

Claims (10)

A ginseng-derived PPDS (Protopanaxadiol synthase) protein, which is involved in protoparnaxadiol biosynthesis, consisting of the amino acid sequence represented by SEQ ID NO: 2. The gene encoding the PPDS protein of claim 1. The gene according to claim 2, wherein the gene consists of a nucleotide sequence represented by SEQ ID NO: 1. A recombinant vector comprising the gene of claim 2 or 3. 5. The recombinant vector of claim 4, wherein said vector is a recombinant yeast vector. The recombinant vector according to claim 5, wherein the vector is a pYeDP60_PPDS vector described in FIG. Transformed yeast transformed with the vector of claim 5. 5. The recombinant vector of claim 4, wherein said vector is a recombinant plant vector. The recombinant vector of claim 8, wherein the vector is a pPZP211_PPDS vector described in FIG. 3. A transformed dicotyledonous plant transformed with the vector of claim 8.

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