KR101854961B1 - Use of novel genes for increasing malonyl-ginsenoside Rb2 syntheis in Ginseng - Google Patents

Abstract

The present invention relates to the use of a novel gene derived from ginseng. The novel glycosyltransferase inducing ginseng saponin identified in the present invention significantly increases the synthesis of Malonyl-ginsenoside Rb2 of ginseng As a result, it is possible to develop a transgenic plant capable of mass-producing a useful substance by transforming the above-mentioned glycosyltransferase into ginseng and other plants, and it is also possible to mass-produce useful substances by in vitro reaction, And can be usefully used for the development of high value-added products such as health functional foods and cosmetics containing the useful substances derived therefrom.

Description

Use of novel genes that increase the synthesis of malonyl-ginsenoside Rb2 {Use of novel genes for increasing malonyl-ginsenoside Rb2 syntheis in Ginseng}

The present invention relates to the use of novel ginseng-derived genes, and more particularly to the use of two novel ginsenoside-derived glycosyltransferases to increase the synthesis of malonyl-ginsenoside Rb2.

Ginseng has been widely known in the world as a unique product of Korea and has been used as an important medicinal material for oriental countries for many years. The efficacy of ginseng as a medicinal product was first described in detail in the "Chinese Divine Herald (BC 100)," a Chinese treaty about 2000 years ago. The main effects of ginseng are 1) central nervous system, memory, improvement of learning efficacy and antistress action, 2) 2) anti-cancer activity and immune function control, 3) anti-diabetic effect, 4) hyperfunction of liver function and toxic substance detoxifying action, 5) improvement of cardiovascular disorder, antiarteriosclerosis and cholesterol metabolism improvement, 6) menopausal disorder and osteoporosis prevention Ginseng has characteristics that its quality and efficacy are changed according to natural conditions. For this reason, ginseng produced in Korea is known as "Korean Ginseng It is may be referred to as bulryeojigo synonymous with ginseng.

Worldwide, ginseng is cultivated commercially in Korea, China, Japan, the United States, Canada and Europe. By the end of 1980, Korea had produced about 46% of ginseng in the world. In the 1990s, its market share decreased to 39%, China accounted for more than 50%, USA and Canada gained 10% The market share is declining further. It is known that the ginseng of Korea is very excellent in the pharmacological effect, but it is very weak in price competitiveness. Therefore, the characteristics and merits of Korean ginseng are very many, but it is the time when there is an urgent need for efforts to improve the international competitiveness of ginseng products, in line with the rapidly changing world conditions and the WTO, major investment in bio-industry between countries and economic crisis .

To date, about 30 different kinds of ginsenosides have been isolated from ginseng and ginseng processed products (Shibata, 2001), and antidiabetic activity, immune inflammation Antioxidant activity, anticancer activity, and so on. In addition, phenolic components, polyacetylenes, alkaloids, and polysaccharides are known as other physiologically active ingredients besides ginsenosides. More than 10 kinds of antioxidative and phenolic substances have been identified as phenolic compounds as effective ingredients for inhibiting aging, , Anticancer, antioxidant, and whitening activity. Recent research has shown that anti-stress effects are maintained nonspecifically for various stresses, both physically and mentally (Lee et al., 2008). The results of pharmacological studies of these ginsengs have increased the interest in ginsenoside, ginsenoside, and their necessity for mass production has been raised. However, mass production of ginseng useful materials through general cultivation methods has been limited to 4 ~ Development of a new alternative production method is urgently required because it includes problems such as a long period of 6 years of cultivation, difficulty in controlling pests caused by shading cultivation, and cultivation of rotogens. Recently, ginseng saponin-related genes have been discovered on the basis of biotechnology, but there has been no development of ginseng plant having specific saponin-reinforced genes using these genes. Looking at the ginsenoside biosynthetic pathway, squalene is synthesized by fusion of two farnesyl diphosphates by the enzyme squalene synthase. Squalene synthesizes 2,3-oxidosqualene by a squalene epoxidase enzyme reaction. At this stage, the synthesis of the ginsenoside precursors dammarenediol, beta-amyrin and the plant sterol precursor cycloartenol is determined by oxidosqualene cyclase. Dammarenediol is converted to ginsenoside by hydroxylation and glycosylation, and the function of these genes has not been elucidated even though a large number of genes involved in this step have been discovered.

Meanwhile, gene discovery and functional analysis related to saponin biosynthesis of ginseng is one of the most important technologies in the development of new ginseng. The reason for applying genetic engineering and metabolism engineering is that ginseng and intragastric ginseng As a result. Recently, we have developed saponin-enhanced ginseng through overexpression of squalene synthase gene involved in saponin biosynthesis. We also identified DDS gene function using a transformant that has knocked out the dammarenediol synthase (DDS) gene. However, the gene located under this gene has not been reported yet. In order to identify these genes, Japan, China and Korea have been actively pursuing researches. China is trying to find genes to secure excellence of Chinese ancestor (P anax notoginseng) ) Are being studied. In Korea, we are promoting the Ginseng Genome Project starting from 2012 to improve the status of Korean Ginseng as a successor country, and are trying to secure a large amount of genes related to ginseng saponin biosynthesis.

Korean Patent Publication No. 10-2004-0086918 Korean Patent No. 10-1303686

It is an object of the present invention to search for a novel gene for developing a technique capable of enhancing a useful substance in ginseng and to establish an optimum condition for mass production of the useful substance derived from ginseng.

In order to accomplish the above object, the present invention provides a recombinant vector comprising a glycosyltransferase of SEQ ID NO: 1 or SEQ ID NO: 2.

The present invention also provides a plant transformed with said vector.

The present invention also relates to a method for transforming plant cells, And regenerating the transformed plant from the transformed plant cell.

In addition, the present invention provides a method for producing a transgenic plant that over-expresses malonyl-ginsenoside Rb2 comprising the step of over-expressing a glycosyltransferase gene by transforming a plant cell with the vector.

In one embodiment of the present invention, the plant may be ginseng.

The present invention also provides a method for increasing the synthesis of malonyl-ginsenoside Rb2 by reacting an enzyme protein synthesized by translation of the gene of SEQ ID NO: 1 or SEQ ID NO: 2 with a ginseng extract.

In addition, the present invention provides a method for increasing the synthesis of malonyl-ginsenoside Rb2 by treating MJ (methyl jasmonate) with ginseng root.

In one embodiment of the present invention, the increase in the synthesis of malonyl-ginsenoside Rb2 is characterized in that the expression of the gene of SEQ ID NO: 1 or SEQ ID NO: 2 is increased.

The novel glycosyltransferase that induces ginsenoside saponin identified in the present invention significantly enhances the synthesis of malonyl-ginsenoside Rb2 of ginseng. As a result, the glycosyltransferase, which is a glycosyltransferase, By transforming ginseng and other plants, it is possible to develop transgenic plants capable of mass production of useful substances, and it is possible to mass-produce useful substances even in in vitro reaction, and to provide health functional foods and cosmetics containing useful substances derived from ginseng It will be useful for the development of high value-added products.

FIG. 1 shows the amino acid sequences of four isoforms of glycosyltransferase with increased expression level after treatment with MJ (methyl jasmonate) in the ginseng root.
FIG. 2 is a graph showing the relationship between the two kinds of glycosyltransferase genes of SEQ ID NO: 1 (gene encoding PgG6 1-6 of FIG. 1) and SEQ ID NO: 2 (A gene encoding PgGT 1-10 in Fig. 1) was over-expressed in E. coli and then subjected to column purification and SDS-PAGE analysis.
FIG. 3 shows the result of HPLC analysis of the final extract after reacting ginseng extract with two kinds of glycosyltransferase enzymes which were increased in expression level after treatment with MJ (methyl jasmonate) in the ginseng cultured muscle (A: B: an enzyme-treated ginseng extract expressed by SEQ ID NO: 1, and C: an enzyme-treated ginseng extract expressed by SEQ ID NO: 2).
FIG. 4 is a graph showing the relationship between the amount of glycosyltransferase and the amount of glycosyltransferase added to ginseng saponin treated with MJ (methyl jasmonate) spectra.

Hereinafter, the present invention will be described in detail.

The present invention provides a recombinant vector comprising a glycosyltransferase of SEQ ID NO: 1 or SEQ ID NO: 2. The vector is preferably a recombinant plant expression vector.

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, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. In addition, the recombinant cell can express a gene found in a cell in its natural state, but the gene has been modified and reintroduced intracellularly by an artificial means.

The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "carrier" is often used interchangeably with "vector ". The term "expression vector" means a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism.

A preferred example of a plant expression vector is a Ti-plasmid vector that is capable of transferring a so-called T-region into a plant cell when it is present in a suitable host such as Agrobacterium tumefaciens. Other types of Ti-plasmid vectors (see EP 0 116 718 B1) are currently used to transfer hybrid DNA sequences to plant cells or protoplasts in which new plants capable of properly inserting hybrid DNA into the plant's genome can be produced have. A particularly preferred form of the Ti-plasmid vector is a so-called binary vector as claimed in EP 0 120 516 B1 and U.S. Patent No. 4,940,838. Other suitable vectors that may be used to introduce CaHBl according to the present invention into a plant host include a double-stranded plant virus (e. G., CaMV) and a uracil vector such as may be derived from single- For example, from non -complete plant virus vectors. The use of such vectors may be particularly advantageous when it is difficult to transform the plant host properly.

The expression vector preferably comprises one or more selectable markers. The marker is typically a nucleic acid sequence having a property that can be selected by a chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. Examples include antibiotic resistance genes such as herbicide resistance genes such as glyphosate or phosphinotricin, kanamycin, G418, Bleomycin, hygromycin, chloramphenicol, It is not. The expression vector preferably comprises one or more selectable markers. The marker is typically a nucleic acid sequence having a property that can be selected by a chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. Examples include antibiotic resistance genes such as herbicide resistance genes such as glyphosate or phosphinotricin, kanamycin, G418, Bleomycin, hygromycin, chloramphenicol, It is not.

In a 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 the region of DNA upstream from the 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. Constructive promoters may be preferred in the present invention because the choice of transformants can be made by various tissues at various stages. Thus, constitutive promoters do not limit selectivity.

The plant expression vector of the present invention can be a conventional terminator, for example, nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaciens (Agrobacterium tumefaciens ) Octopine gene terminator, but the present invention is not limited thereto. Regarding 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 a terminator is highly desirable in the context of the present invention.

The present invention provides a plant transformed with said vector.

Methods for introducing the recombinant vectors of the present invention into Agrobacterium can be carried out through various methods known to those skilled in the art and include, for example, particle bombardment, electroporation, transfection A lithium acetate method, a heat shock method, and a freeze-thaw method. Most preferably, the freeze-thaw method is used.

The present invention relates to a method for transforming plant cells, And regenerating the transformed plant from the transformed plant cell.

Since the method for producing a transgenic plant of the present invention is produced using the composition of the present invention described above, the description common to both is omitted in order to avoid the excessive complexity of the present specification.

(Methods of Enzymology, Vol. 153, (1987)) to produce transformed plant cells and transgenic plants of the invention. An exogenous polynucleotide may be inserted into a carrier such as a plasmid, a virus, or the like to transform the plant, and Agrobacterium bacteria may be used as an agent (Chilton, et al., Cell, 11: 263: 271 ), Direct extrinsic polynucleotides can be introduced into plant cells to transform plants (Lorz et al., MoI Genet., 199: 178-182; (1985)). For example, when a vector not containing a T-DNA region is used, electroporation, particle impact method (microparticle

bombardment, and polyethylene glycol-mediated uptake.

In general, a commonly used method for transforming plants is to infect plant cells or seeds with Agrobacterium tumefaciens transformed with an exogenous polynucleotide (see, for example, U.S. Patent No. 5,004,863 5,349,124 and 5,416,011). One of ordinary skill in the art can cultivate or plant transformed plant cells or seeds under suitable known conditions to develop into plants.

As used herein, the term "plants" is understood to mean not only mature plants but also plant cells, plant tissues and plant seeds that develop into mature plants.

The plants to which the method of the present invention can be applied are not particularly limited. As the plants to which the method of the present invention can be applied, most of the dicotyledonous plants including the lettuce, cabbage, potato and radish, or monocotyledonous plants such as rice, barley and banana can be used, The method of the present invention is applicable to food crops including rice, wheat, barley, corn, soybean, potato, wheat, red bean, oats and millet; Vegetable crops including Arabidopsis, cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, squash, onions, onions and carrots; Special crops including ginseng, wild ginseng, tobacco, cotton, sesame, sugar cane, beet, perilla, peanut and rapeseed; Apple trees, pears, jujubes, peaches, sheep, grapes, citrus, persimmon, plums, apricots and banana; Roses, gladiolus, gerberas, carnations, chrysanthemums, lilies and tulips; And feed crops including rice grass, red clover, orchardgrass, alpha-alpha, tall fescue and perennial rice.

The explant used in the present invention is a plant cell or a plant tissue, and when plant tissue is used, a callus is preferably used.

In the method of the present invention, transformation of plant cells can be carried out according to conventional methods known in the art, which can be carried out by electroporation (Neumann, E., et al., EMBO J., 1: 841 ), Particle bombardment (Yang, et al., Proc. Natl. Acad. Sci., 87: 9568-9572 (1990)) and Agrobacterium-mediated transformation (US Pat. No. 5,004,863 5,349,124 and 5,416,011). Of these, Agrobacterium-mediated transformation is most preferred.

Selection of transformed plant cells can be carried out by exposing the transformed culture to a selection agent such as a metabolic inhibitor, an antibiotic and a herbicide. Plant cells that stably contain a marker gene that is transformed and conferring selectative resistance are grown and divided in the above cultures. Exemplary labels include, but are not limited to, hygromycin phosphotransferase gene, glycophosphate tolerance gene and neomycin phosphotransferase (nptII) system.

The development or regeneration of a plant comprising a foreign gene introduced by Agrobacterium is accomplished according to methods known in the art, including, but not limited to, (U.S. Patent Nos. 5,004,863, 5,349,124, and 5,416,011). In the present invention, a preferred transformation method is carried out using an Agrobacterium system, more preferably, an Agrobacterium tumefaciens-binary vector system.

In a method of using an Agrobacterium system, a specific embodiment includes the steps of: (a ') introducing Agrobacterium tumefaciens, which can be inserted into the genomic DNA of a plant cell and have the following sequence, (I) a nucleotide sequence encoding a protein of the present invention; (ii) a promoter operably linked to the nucleotide sequence of (i) and acting on plant cells to form RNA molecules; (iii) a 3'-non-detoxified site that acts in plant cells to cause polyadenylation of the 3'-end of the RNA molecule; (b ') Regenerating the infected explant in a regeneration medium to obtain a transformed plant.

Transformation of plant cells is carried out with Agrobacterium tumefaciens containing Ti plasmids (Depicker, A., et al., Plant cell transformation by Agrobacterium plasmids. In Genetic Engineering of Plants, Plenum Press, New York 1983). More preferably, binary vector systems such as pBin19, pRD400, pRD320, pGA1611 and pGA1991 are used (An, G., et al., Binary vectors "In Plant Gene Res. Manual, Martinus Nijhoff Publisher, New York An et al., 1988; and Lee et al., 1999).

Binary vectors suitable for the present invention include (i) a promoter that is operative in plants; (ii) a structural gene operably linked to the promoter; And (iii) a polyadenylation signal sequence. Alternatively, the vector additionally carries a gene encoding a reporter molecule (e.g., luciferase and glucuronidase). Examples of the promoter used for the binary vector include, but are not limited to, maize ubiquitin promoter, CaMV 35S promoter, 1 promoter, 2 promoter and nopaline synthase (nos) promoter.

Infection of the explant by Agrobacterium tumefaciens includes methods known in the art. Most preferably, the infection process comprises co-culturing Agrobacterium tumefaciens culture with an explant. Through this, Agrobacterium tumefaciens is infected into plants.

The explant transformed by Agrobacterium tumefaciens regulates in the regeneration medium, which ultimately forms the transgenic plant.

The transformed plants according to the present invention are confirmed to be transformed by methods known in the art. For example, PCR using DNA samples from tissues of transformed plants can identify foreign genes inserted into the genome of the transgenic plant. Alternatively, Northern or Southern blotting can be performed to confirm the transformation (Maniatis, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. (1989)).

The present invention also provides a method for increasing the synthesis of malonyl-ginsenoside Rb2 by reacting an enzyme protein synthesized by translation of the gene of SEQ ID NO: 1 or SEQ ID NO: 2 with a ginseng extract. This may be utilized for the large-scale synthesis of malonyl-ginsenoside Rb2 by reacting the gene of SEQ ID NO: 1 or SEQ ID NO: 2 with an encoded protein (enzyme) in the ginseng extract in vitro.

The present invention also provides a method for increasing the synthesis of malonyl-ginsenoside Rb2 by treating MJ (methyl jasmonate) with a ginseng root, wherein the increase in the synthesis of malonyl-ginsenoside Rb2 is represented by SEQ ID NO: 1 or SEQ ID NO: 2 gene expression is increased and induced.

Hereinafter, the present invention will be described in detail with reference to Examples. However, the following examples are intended to illustrate the present invention and the scope of the present invention is not limited by the following examples.

≪ Example 1 >

MJ  The expression is increased by treatment Transglycosylase ( glycosyltransferase ) Gene cloning

Glycine EST sequence data 10,355 genes were analyzed by microarray analysis to identify glycosyltransferases useful for saponin biosynthesis. The microarray conditions were as shown in Table 1 below.

Sample  Ginseng cultured root total RNA platform  CambiMatrix Eukaryotic 12K Marker  Biotin Data processing  Microarray imager v5.9.3 software  ArrayAssist 5.51 (Stratagene)

The ginseng cultured muscle was treated with MJ for 3 days. Specifically, ginseng adventitious roots were induced and cultured for 4 weeks on the MS medium. Ginseng adventitious roots were cultured in liquid MS medium for 5 weeks. 0.1 mM MeJA dissolved in ethanol was added to the MS liquid medium for 3 days and then collected and used as a sample. After culturing, total RNA was extracted from the cultured muscle and the amount of expression was analyzed using these as probes. The glycosyltransferase gene, which increases the expression level after MJ treatment, was investigated. The expression of 87 genes of glycosyltransferase gene was found to be up to 17 times higher than that of the control (MJ untreated), except for the overlapping genes. RACE cloning was performed to obtain full-length cDNAs of these genes. As a result, one of these genes was successfully obtained a complete base sequence, and the total length of the gene was composed of 1,480 bp, consisting of 476 amino acids, and its molecular weight was 53.548 kDa. The gene was amplified using a bi-directional primer including the initiation codon and stop codon of the gene and analyzed for the nucleotide sequence. As a result, four isoforms were found (see FIG. 1).

≪ Example 2 >

Production of E. coli transformants expressing proteins

To identify the function of the four genes, each full-length cDNA was ligated using a protein expression system (Invitrogen, pET100-TOPO vector). Sequencing was performed to confirm that two isoforms of four isoforms were successfully ligated (SEQ ID NO: 1, SEQ ID NO: 2), and the constructed vector was inserted into E. coli BL21star (DE) Respectively. Each transformed Escherichia coli was induced by IPTG induction until OD ₆₀₀ = 0.8, and cultured at 37 ° C and precipitated by centrifugation. Collected precipitated E. coli was reconstituted in buffer and extracted with ultrasonic disruption. The extract was purified using a His-tag column. The protein extract was subjected to SDS-PAGE analysis after being denatured by treating at 100 ° C for 5 minutes in SDS buffer. Analysis of the extract of the elution buffer revealed that the band was clearly observed at about 56 kDa, which is a His-tag 3 kDa binding (FIG. 2).

≪ Example 3 >

Identification of Substance after Recombinant Protein Reaction with Ginseng Extract as Substrate

These synthesized enzyme proteins and ginseng extracts were used as substrates to perform enzyme reaction and the final extracts were analyzed by HPLC. The enzyme reaction was performed by incubating the purified enzyme protein, UDP-glucose and ginseng extract at 30 ° C for 30 minutes. The butanol corresponding to twice the amount of the cultured mixture was mixed and sufficiently mixed. This mixture was concentrated in a vacuum concentrator. The concentrated mixture was diluted in 1 ml of 80% methanol. HPLC analysis was carried out at a column oven temperature of 45 < 0 > C. The mobile phase solutions were A (0.1% formic acid in water, v / v) and B (0.1% formic acid in acetonitrile, v / v) The mobile phase condition is B: 0-2 min, 28% B; 2-15 min, 35% B; 15-18 min, 90% B; 18-19 min, 100% B; 19-20 min, 100% B, and 20-23 min and 80% B, respectively. The flow rate was 0.50 ml / min. The injection volume was analyzed by 2 쨉 l. The absorbance of the UV detector was measured at a wavelength of 230 nm.

As a result, the amount of a specific component (peak 1, peak 2) was greatly increased in the final extract chromatogram of SEQ ID NO: 1 compared to the control, and in the final extract chromatogram of SEQ ID NO: 2, It was confirmed that the synthesized amount of the components (peak 1, peak 2) increased much more (see FIG. 3).

The synthesized peak 1 was analyzed by Q-TOF / MS to find the exact component of the increased peak 1. The desolvation temperature was analyzed at 350 ° C at a flow rate of 700 L / h and the basic temperature was set at 100 ° C. The capillary and the voltage were set at 2,800 V and 75 V, respectively. Data analysis was performed using Agilent software Masshunter. Data collection was performed between 100 and 1,500 m / z, and ion precursor generation was analyzed at 5 eV and fragment generation at 35 eV.

As a result, the peak 1 was identified as malonyl-ginsenoside Rb2 (see Fig. 4). Therefore, it was found that the above-mentioned SEQ ID NO: 1 and SEQ ID NO: 2 are effective for mass production of malonyl-ginsenoside Rb2.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

<110> Chungbuk National University Industry-Academic Cooperation Foundation <120> Use of novel genes for increasing malonyl-ginsenoside Rb2          syntheis in Ginseng <130> PN1507-202 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 1425 <212> DNA <213> a glycosyltransferase from ginseng <400> 1 atgaattcag aattgatatt cttgcccgcc ccggccatcg gacacctcgt gggaatggtg 60 gagatggcta aactcttcat cagtcgacat gaaaacctct cggtcaccgt cttcatctcg 120 aaattctaca tggatacggg ggtagacaac tacaataaat cactcttaac aaaccctacc 180 ccgcgtctca caattgtaaa tctcccggga accgaccccc aaaactatat gctcaaacca 240 cgccacgcca tccttcctag cgtcatcgag actcagaaga cacacgtgcg agacataata 300 tccggcatga ctcagtccga gtcgactcgg gtcgttggtt tgctggctga ccttttgttc 360 atcaacatca tggacattgc caatgagttc aatgttccaa tttatgtata ctcccctgcc 420 ggagccggtt atcttggtct cgcgttccat ctccagacac tttacgacaa aaagcaagat 480 gtgaccgagt tcagagactc ggacactgag ttattggtac cgggttttgc aaaccccgtt 540 cccgccgagg tcttgccgtc gatgtatgtg gataaagaag gtgggtatga ttatttgttt 600 tcattgttcc ggaggtgcag agagtcaaag gcaattatta ttaacacgtt tgaggagctg 660 gaaccctatg cgatcaattc cctccggatg gatagtatga tccctccgat ctacccggtg 720 ggacccatac taaatctcaa cggtgatggc caaaactctg atgaggctgc tgtgatcctt 780 ggttggttag atgatcaacc cctttcatct gtggtgtttt tgtgctttgg tagctatgga 840 acctttcaag aaaaccaggt gaaggagatt gcaatgggtc tagagcggag tgggcatcgc 900 ttcttgtggg ccttgcgtcc gtctatccct aaaggtgaga caaagcttca gcttaaatac 960 tcaaatttgg aagaaattct cccagtcgga ttcttggaca ggacatcatg cgtcggaaaa 1020 gttattggat gggccccgca agtggccgtg ctcggacacg aggcagtcgc agggttcatg 1080 tctcattgcg gttggaattc gacattagag ggtgtgtggt ttggcgtgcc cgtcgcaaca 1140 tggccaatgt acggtgagca acacctcaat gcttttgaga tggttaagga gttgggtcta 1200 gcggtggaaa ttgaggtgga ctataagaat gaatatttta acacgaagaa tgattttatt 1260 gttagggcag aagaaattga gacgaaaata aagaagttga tgatggatga aaagaatagt 1320 gaaataagga agaaggtaaa ggaaatgaaa gaaaagagta gagttgccat gtcggagaat 1380 ggatcatctt ataattcatt ggcgaagcta tttgaggaaa ttatg 1425 <210> 2 <211> 1424 <212> DNA <213> a glycosyltransferase from ginseng <400> 2 tgaattcaga attgatattc ttgcccgccc cggccatcgg acacctcgtg ggaatggtgg 60 agatggctaa actcttcatc agtcgacatg aaaatctctc ggtcaccgtc ctcatcgcga 120 aattctacat ggatacgggg gtagacaact acaataaatc actcttaaca aaccctaccc 180 cgcgtctcac aattgtaaat ctcccggaaa ccgaccccca aaactatatg ctcaaaccac 240 gccacgccat ctttcctagc gtcatcgaga ctcagaagac acacgtgcga gacataatat 300 ccggtatgac tcagtccgag tcgactcagg tcgttggttt gctggctgac cttttgttca 360 tcaacatcat ggacattgcc aatgagttca atgttccaac ttatgtatac tcccctgccg 420 gagccggtca tcttggcctc gcgttccatc tccagacact caacgacaaa aaacaagatg 480 tgaccgagtt caggaactcg gatactgagt tattggtacc gagttttgca aacccggttc 540 ccgccgaggt cttgccgtcg atgtatgtgg ataaagaagg tgggtatgat tatctgtttt 600 cattgttccg gaggtgcaga gagtcaaagg caattattat taacacgttt gaggagctgg 660 aaccctatgc gatcaattcc ctccggatgg atagtatgat ccctccgatc tacccggtgg 720 gacccatact aaatctcaac ggtgatggcc aaaactccga tgaggctgct gtgatccttg 780 gttggttaga tgatcaacca ctttcatctg tggtgttttt gtgctttggt agctatggaa 840 gctttcaaga aaaccaggtg aaggagattg caatgggtct agagcgcagt gggcatcgct 900 tcttgtggtc cttgcgtccg tctatcccta aaggcgagac aaagcttcag cttaaatact 960 caaatttgaa agaaattctc ccagtaggat tcttggacag gacatcatgc gtcggaaaag 1020 tgattggatg ggccccgcaa gtggccgtgc tcggacatga gtcagtcgga gggttcctgt 1080 ctcattgcgg ttggaattcg acattggaga gtgtttggtg tggggtgccc gttgcaacat 1140 ggccaatgta tggtgagcaa caactcaatg cttttgagat ggttaaggag ttaggtattg 1200 cggtggaaat tgaggtggac tataagaaag attattttaa catgaagaat gattttattg 1260 ttagggcaga agaaatcgag acaaaaataa agaagttgat gatggatgaa aataatagtg 1320 aaataagaaa gaaggtaaag gaaatgaaag aaaagagtag ggctgcaatg tctgagaatg 1380 gatcatctta taattcatgg gcgaagctat ttgaggaaat tatg 1424

Claims (8)

delete delete delete Synthesis of malonyl-ginsenoside Rb2 comprising the step of over-expressing a glycosyltransferase gene by transforming a plant cell with a recombinant vector comprising a glycosyltransferase gene consisting of the nucleotide sequence of SEQ ID NO: 2 &Lt; / RTI &gt; 5. The method of claim 4,
Wherein the plant is ginseng. A method for increasing the synthesis of malonyl-ginsenoside Rb2 by reacting a glycosyltransferase gene comprising the nucleotide sequence of SEQ ID NO: 2 with an enzyme protein synthesized and synthesized with ginseng extract.
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