CN115094046A

CN115094046A - Group of glycosyltransferases and application thereof

Info

Publication number: CN115094046A
Application number: CN202111642975.0A
Authority: CN
Inventors: 周志华; 严兴; 王平平; 魏勇军; 魏维; 许云鹏; 李晓东; 杨成帅
Original assignee: Individual
Current assignee: Shenghe Everything Suzhou Biotechnology Co ltd
Priority date: 2014-06-09
Filing date: 2015-06-09
Publication date: 2022-09-23
Also published as: CN115341008A; WO2015188742A3; CN105177100A; WO2015188742A2

Abstract

The invention relates to a group of glycosyltransferases and their use. Specifically, the application of glycosyltransferase gGT29-7 and its derivative polypeptide in terpenoid glycosylation catalysis and new saponin synthesis is provided, wherein the glycosyltransferase can specifically and efficiently transfer glycosyl from glycosyl donor to the first glycosyl at C-3 position and/or C-6 position of tetracyclic triterpenoid to extend sugar chain. The glycosyltransferase can also be applied to the construction of artificially synthesized rare ginsenoside and a plurality of new ginsenoside and derivatives thereof.

Description

Group of glycosyltransferases and application thereof

The application is a divisional application of an invention patent application with the application date of 2015, 6 and 9, and the application number of 201510313727.X, and the name of the invention is 'a group of glycosyltransferases and application thereof'.

Technical Field

The present invention relates to the fields of biotechnology and plant biology, and in particular, the present invention relates to a group of novel glycosyltransferases and their uses.

Background

Ginsenoside is a general term for saponins separated from ginseng and other congeneric plants (such as panax notoginseng, American ginseng, etc.), belongs to triterpenoid saponin, and is a main effective component in ginseng. At present, at least 60 saponins have been isolated from ginseng, some of which have been shown to have a wide range of physiological functions and medicinal value: including the functions of resisting tumor, regulating immunity, resisting fatigue, protecting heart, protecting liver, etc.

The ginsenosides Rg3, Rf and Rg2 are all rare ginsenosides, and have very strong physiological activities. For example, ginsenoside Rg3 has good antitumor activity, and can induce apoptosis of tumor cells and inhibit metastasis of tumor cells. It is used in combination with radiotherapy and chemotherapy to enhance the effect of radiotherapy and chemotherapy; ginsenoside Rf has anti-tumor and anti-fatigue effects, can reduce uterine contraction, has analgesic effect associated with cranial nerve cells, and has physiological function of regulating phospholipid metabolism; ginsenoside Rg2 has effects of protecting brain of rat with Alzheimer disease, enhancing learning and memory ability of rat, and repairing myocardial injury, and ginsenoside Rg2 also has effects of protecting cell from ultraviolet injury.

However, since these rare ginsenosides having low glycosylation are very easily modified by glycosyltransferase in natural ginseng and produce ginsenosides having complex sugar chains, these ginsenosides having high biological activity are contained in ginseng in an extremely low amount.

At present, the method for producing the rare ginsenoside is to convert a large amount of ginsenoside in ginseng by a method of selectively hydrolyzing glycosyl, and then extract and purify the ginsenoside. Takes total saponins or protopanaxadiol saponins of panax plants as raw materials, and carries out conversion, separation and extraction by chemical and enzymatic methods. The chemical preparation method has the disadvantages of large raw material loss, complex operation and more byproducts, so that the cost is increased and the yield is difficult to improve. In addition, since the obtaining of total ginsenosides depends on the planting of ginseng, the market price of rare ginsenoside monomers produced by the traditional method is high.

Currently, an effective method for producing rare ginsenosides Rg3, Rf and Rg2 is lacked in the field, so that the development of various specific and efficient glycosyltransferases is urgently needed.

Disclosure of Invention

The invention aims to provide a group of glycosyltransferases and application thereof.

In a first aspect of the invention, there is provided an in vitro glycosylation method, comprising the steps of:

transferring the glycosyl group of the glycosyl donor to the following sites of the tetracyclic triterpenoid in the presence of a glycosyltransferase:

on the first glycosyl at position C-3 and/or C-6;

thereby forming a glycosylated tetracyclic triterpene compound;

wherein the glycosyltransferase is the glycosyltransferase shown in SEQ ID No. 61 or a derivative polypeptide thereof.

In another preferred embodiment, the sugar chain extension comprises direct extension or substitution extension.

In another preferred embodiment, the direct extension is to add a sugar group to the first sugar group at the C-3 and/or C-6 position to extend the sugar chain.

In another preferred embodiment, the substitution extension is to substitute the terminal sugar group of the sugar chain at the C-3 and/or C-6 position with a different sugar group, and to extend the sugar chain from the first sugar group at the C-3 and/or C-6 position.

In another preferred embodiment, said derivative polypeptide is selected from the group consisting of:

a derivative polypeptide which is formed by substituting, deleting or adding one or more amino acid residues of the polypeptide of the amino acid sequence shown in SEQ ID NO. 61 or adding a signal peptide sequence and has glycosyltransferase activity; or

A derivative polypeptide having glycosyltransferase activity, wherein the homology of the amino acid sequence with the amino acid sequence of SEQ ID No. 61 is more than or equal to 85% (preferably more than or equal to 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%);

wherein the glycosyltransferase activity refers to an activity capable of transferring a glycosyl group of a glycosyl donor to a first glycosyl group at C-3 and/or C-6 of the tetracyclic triterpenoid to extend a sugar chain.

In another preferred embodiment, the derivative polypeptide comprises any one of the sequences shown in SEQ ID No. 26, 28, 55, 57, 59, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 93,94 or 95.

In a second aspect of the invention, there is provided an isolated polypeptide selected from the group consisting of:

(a) a polypeptide having an amino acid sequence as set forth in SEQ ID No. 61;

(b) a derivative polypeptide which is formed by substituting, deleting or adding one or more amino acid residues of the polypeptide of the amino acid sequence shown in SEQ ID NO. 61 or adding a signal peptide sequence and has glycosyltransferase activity;

(c) a derivative polypeptide having the sequence of the polypeptide of (a) or (b);

(d) a derivative polypeptide having an amino acid sequence homology of 85% or more (preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) to the amino acid sequence of SEQ ID No. 61 and having glycosyltransferase activity;

In another preferred embodiment, the sequence (c) is a fusion protein formed by adding a tag sequence, a signal sequence or a secretion signal sequence to (a) or (b).

In another preferred embodiment, the derivative polypeptide comprises a polypeptide selected from the amino acid sequences shown in SEQ ID No. 26, 28, 55, 57, 59, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 93,94 or 95.

In a third aspect of the invention, there is provided an isolated polynucleotide, said polynucleotide being a sequence selected from the group consisting of:

(A) a nucleotide sequence encoding the polypeptide of claim 3;

(B) a nucleotide sequence encoding a polypeptide as set forth in SEQ ID No. 61 or a polypeptide derived therefrom;

(C) a nucleotide sequence set forth as SEQ ID No. 60;

(D) a nucleotide sequence having a homology of 90% or more (preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) to the sequence shown in SEQ ID No. 60;

(E) 60 nucleotide sequence formed by truncating or adding 1-60 (preferably 1-30, more preferably 1-10) nucleotides at the 5 'end and/or the 3' end of the nucleotide sequence shown in SEQ ID NO;

(F) a nucleotide sequence complementary to the nucleotide sequence of any one of (A) to (E).

In another preferred embodiment, the nucleotide sequence is as set forth in SEQ ID No. 25, 27, 54, 56, 58, 60, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, or 91.

In another preferred embodiment, the polynucleotide having a sequence as set forth in SEQ ID No. 25, 27, 54, 56, 58, 60, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, or 91 encodes a polypeptide having an amino acid sequence as set forth in SEQ ID No. 26, 28, 55, 57, 59, 61, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 93,94, or 95, respectively.

In a fourth aspect of the invention, there is provided a vector comprising the polynucleotide of the third aspect. Preferably, the vector comprises an expression vector, a shuttle vector and an integration vector.

In a fifth aspect of the invention there is provided the use of an isolated polypeptide of the first or second aspects of the invention for catalysing one or more of the following reactions, or for the preparation of a catalytic formulation for catalysing one or more of the following reactions: transferring a glycosyl group from a glycosyl donor to a first glycosyl group at position C-3 or position C6 of the tetracyclic triterpenoid to extend a sugar chain; preferably, the first and second electrodes are formed of a metal,

it is used to catalyze one or more of the following in vitro reactions, or is used to prepare a catalytic formulation that catalyzes one or more of the following reactions:

(i) transferring the glycosyl from glycosyl donor to the first glycosyl at C-3 position of the tetracyclic triterpene compound to extend the sugar chain;

(ii) transferring the glycosyl from glycosyl donor to the first glycosyl at C-6 position of the tetracyclic triterpene compound to extend the sugar chain;

(iii) the glycosyl group of the glycosyl donor is substituted with the terminal glycosyl group of the C-6 position sugar chain of the tetracyclic triterpene compound, and the sugar chain is extended from the first glycosyl group at the C-3 and/or C-6 position.

In another preferred embodiment, the glycosyl donor comprises a nucleoside diphosphate sugar selected from the group consisting of: UDP-glucose, ADP-glucose, TDP-glucose, CDP-glucose, GDP-glucose, UDP-acetylglucose, ADP-acetylglucose, TDP-acetylglucose, CDP-acetylglucose, GDP-acetylglucose, UDP-xylose, ADP-xylose, TDP-xylose, CDP-xylose, GDP-xylose, UDP-galacturonic acid, ADP-galacturonic acid, TDP-galacturonic acid, CDP-galacturonic acid, GDP-galacturonic acid, UDP-galactose, ADP-galactose, TDP-galactose, CDP-galactose, GDP-galactose, UDP-arabinose, ADP-arabinose, TDP-arabinose, CDP-arabinose, GDP-arabinose, UDP-rhamnose, ADP-rhamnose, TDP-rhamnose, CDP-rhamnose, GDP-rhamnose, or other nucleoside diphosphate hexoses or nucleoside diphosphate pentoses, or a combination thereof.

In another preferred embodiment, said glycosyl donor comprises a Uridine Diphosphate (UDP) sugar selected from the group consisting of: UDP-glucose, UDP-xylose, UDP-galacturonic acid, UDP-galactose, UDP-arabinose, UDP-rhamnose, or other uridine diphosphate hexoses or uridine diphosphate pentoses, or a combination thereof.

In another preferred embodiment, the isolated polypeptide is used to catalyze one or more of the following reactions or is used to prepare a catalytic formulation that catalyzes one or more of the following reactions:

(A)

wherein R1 is a glycosyl group; r2 and R3 are OH or H; r4 is a glycosyl group or H; r5 is glycosyl, R5-R1-O is glycosyl derived from the first glycosyl of C3, and the polypeptide is selected from the polypeptide shown in SEQ ID NO. 61 or derived polypeptide thereof; preferably, it is selected from 26, 28, 55, 57, 59, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 93,94 or 95 or a polypeptide derived thereof.

The compounds substituted with R1-R4 are shown in the following table:

substrate	R1	R2	R3	R4
					Rh2	Sugar radical	H	OH	H
F2	Sugar radical	H	OH	Sugar radical

When R1 is glucosyl; r2 is H, R3 is OH, R4 is H, and the compound of formula (I) is Rh 2.

R1 is glucosyl; r2 is H, R3 is OH, R4 is glucosyl, and the compound of formula (I) is F2.

When UDP-glucose is used as glycosyl donor, the substrate (I) compound is Rh2, and the product (II) compound is Rg 3; the substrate (I) compound is F2, then the product compound of formula (II) is Rd;

when UDP-xylose is used as glycosyl donor, the substrate compound (I) is Rh2, and the product compound shown as the formula (II) is 3-O-beta- (D-xylopyranosyl) -beta- (D-glucopyranosyl) -PPD; the substrate (I) compound is F2, and the product compound of formula (II) is 3-O-beta- (D-xylopyranosyl) -beta- (D-glucopyranosyl) -CK.

(B)

Wherein R1 and R2 are H or a glycosyl, and R3 and R4 are glycosyl. R3-R4-O is glycosyl derived from the first glycosyl of C6, and the polypeptide is selected from the polypeptide shown in SEQ ID NO. 61 or a derivative polypeptide thereof, preferably, the polypeptide is SEQ ID NO. 55, 57, 59, 78, 82, 92, 94 or 95 or a derivative polypeptide thereof.

When R1 and R2 are H, and R3 is glucosyl, the compound of formula (III) is Rh 1.

When UDP-glucose is used as the glycosyl donor, the substrate compound (III) is Rh1, and the product compound of formula (IV) is Rf;

when UDP-rhamnose is used as glycosyl donor, the substrate (III) compound is Rh1, and the product (IV) compound is Rg 2.

(C)

Wherein R1 is a glycosyl group; r2 and R3 are OH or H; r4 is a glycosyl group or H; r5 is a glycosyl, R5-R1-O is a glycosyl derived from the first glycosyl of C3; r6 is glycosyl, R6-R1-O is glycosyl derived from the first glycosyl of C3, and the polypeptide is selected from the polypeptide shown in SEQ ID NO. 61 or derived polypeptide thereof, preferably the polypeptide shown in SEQ ID NO. 26, 28, 59, 76, 84, 86 or 88.

R1 is two glucosyl groups, R2 is H, R3 is OH, R4 is H, and the compound of formula (V) is Rg 3.

R1 is two glucosyl groups, R2 is H, R3 is OH, R4 is glucosyl group, and the compound of formula (V) is Rd

When UDP-xylose is used as glycosyl donor, the substrate (V) compound is Rg3, and the product compound shown as the formula (VI) is 3-O-beta- (D-xylopyranosyl) -beta- (D-glucopyranosyl) -PPD; when the substrate (V) is Rd, the product compound of formula (VI) is 3-O-beta- (D-xylopyranosyl) -beta- (D-glucopyranosyl) -CK.

In another preferred embodiment, said glycosyl is selected from the group consisting of: glucosyl, galacturonic acid, xylosyl, galactosyl, arabinosyl, rhamnosyl, and other hexose or pentosyl groups.

In another preferred embodiment, the compounds of the formula (I), or (III) include, but are not limited to: an S-configuration or R-configuration dammarane-type tetracyclic triterpene compound, a lanoline-type tetracyclic triterpene compound, an apotorucane-type tetracyclic triterpene compound, a euphorbiane-type tetracyclic triterpene compound, a cycloartenane (cycloartane) -type tetracyclic triterpene compound, a cucurbitane tetracyclic triterpene compound, or a meliane-type tetracyclic triterpene compound.

In another preferred embodiment, the polypeptide is selected from the group consisting of:

(a) a polypeptide having an amino acid sequence as set forth in any one of SEQ ID nos. 26, 28, 55, 57, 59, 61, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 93,94, or 95;

(b) 26, 28, 55, 57, 59, 61, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 93,94 or 95, or a derivative polypeptide which is formed by adding a signal peptide sequence and has glycosyltransferase activity;

(d) the amino acid sequence is similar to SEQ ID NO: 26. 28, 55, 57, 59, 61, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 93,94 or 95, and having glycosyltransferase activity.

In another preferred embodiment, the polynucleotide encoding the nucleotide sequence of the polypeptide is a sequence selected from the group consisting of seq id no:

(A) a nucleotide sequence encoding a polypeptide according to the second aspect of the invention;

(B) a nucleotide sequence encoding a polypeptide as set forth in SEQ ID No. 26, 28, 55, 57, 59, 61, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 93,94, or 95;

(C) 25, 27, 54, 56, 58, 60, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, or 91;

(D) a nucleotide sequence having a homology of 85% or more (preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) to a sequence represented by SEQ ID NO. 25, 27, 54, 56, 58, 60, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89 or 91;

(E) a nucleotide sequence formed by truncating or adding 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides at the 5 'end and/or the 3' end of the nucleotide sequence shown in SEQ ID No. 25, 27, 54, 56, 58, 60, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89 or 91;

(F) a nucleotide sequence complementary (preferably fully complementary) to a nucleotide sequence described in any one of (A) to (E).

In a sixth aspect of the present invention, there is provided a method of performing a glycosyltransfer catalytic reaction, comprising the steps of: the glycosyltransfer catalytic reaction is carried out in the presence of a polypeptide according to the second aspect of the invention or a polypeptide derived therefrom.

In another preferred embodiment, the method further comprises the steps of:

(II) in the presence of a glycosyl donor and a polypeptide and polypeptide derived therefrom according to the second aspect of the invention, or a compound of formula (I) into said compound of formula (II), or a compound of formula (III) into said compound of formula (IV), or a compound of formula (V) into said compound of formula (VI);

in another preferred embodiment, the method further comprises adding the polypeptide and its derivative polypeptide to a catalytic reaction respectively; and/or

The polypeptide and its derivative polypeptide are added into catalytic reaction at the same time.

In another preferred embodiment, the method further comprises co-expressing a nucleotide sequence encoding a glycosyltransferase with a key gene in the anabolic pathway of dammarediol and/or protopanaxadiol and/or protopanaxatriol and/or other glycosyltransferase genes in a host cell, thereby obtaining the compound of formula (II), (IV), or (VI).

In another preferred embodiment, the host cell is yeast or E.coli.

In another preferred embodiment, the polypeptide is a polypeptide having an amino acid sequence as set forth in SEQ ID No. 26, 28, 55, 57, 59, 61, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 93,94 or 95, and derivatives thereof.

In another preferred embodiment, the nucleotide sequence encoding the polypeptide is as set forth in SEQ ID NO. 25, 27, 54, 56, 58, 60, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, or 91.

In another preferred example, the method further comprises: an additive for regulating the activity of the enzyme is provided to the reaction system.

In another preferred embodiment, the additive for regulating the enzyme activity is: additives for increasing or inhibiting the activity of an enzyme.

In another preferred embodiment, the additive for regulating the enzymatic activity is selected from the group consisting of: ca ²⁺ 、Co ²⁺ 、Mn ²⁺ 、Ba ²⁺ 、Al ³⁺ 、Ni ²⁺ 、Zn ²⁺ Or Fe ²⁺ 。

In another preferred embodiment, the additive for regulating the enzyme activity is: can generate Ca ²⁺ 、Co ²⁺ 、Mn ²⁺ 、Ba ²⁺ 、Al ³⁺ 、Ni ²⁺ 、Zn ²⁺ Or Fe ²⁺ The substance of (1).

In another preferred embodiment, the glycosyl donor is a nucleoside diphosphate sugar selected from the group consisting of: UDP-glucose, ADP-glucose, TDP-glucose, CDP-glucose, GDP-glucose, UDP-acetylglucose, ADP-acetylglucose, TDP-acetylglucose, CDP-acetylglucose, GDP-acetylglucose, UDP-xylose, ADP-xylose, TDP-xylose, CDP-xylose, GDP-xylose, UDP-galacturonic acid, ADP-galacturonic acid, TDP-galacturonic acid, CDP-galacturonic acid, GDP-galacturonic acid, UDP-galactose, ADP-galactose, TDP-galactose, CDP-galactose, GDP-galactose, UDP-arabinose, ADP-arabinose, TDP-arabinose, CDP-arabinose, GDP-arabinose, UDP-rhamnose, ADP-rhamnose, TDP-rhamnose, CDP-rhamnose, GDP-rhamnose, or other nucleoside diphosphate hexoses or nucleoside diphosphate pentoses, or a combination thereof.

In another preferred embodiment, said glycosyl donor is a uridine diphosphate sugar selected from the group consisting of: UDP-glucose, UDP-xylose, UDP-galacturonic acid, UDP-galactose, UDP-arabinose, UDP-rhamnose, or other uridine diphosphogliose or uridine diphosphogliose, or a combination thereof.

In another preferred embodiment, the pH of the reaction system is: pH4.0-10.0, preferably pH 5.5-9.0.

In another preferred embodiment, the temperature of the reaction system is: 10 ℃ to 105 ℃, preferably 20 ℃ to 50 ℃.

In another preferred embodiment, the key genes in the dammarenediol anabolic pathway include (but are not limited to): dammarenediol synthetase gene.

In another preferred embodiment, the key genes in the protopanaxadiol anabolic pathway include (but are not limited to): dammarenediol synthetase gene, cytochrome P450CYP716A47 gene and reductase gene thereof, or a combination thereof. .

In another preferred embodiment, the key genes in the protopanaxatriol anabolic pathway include (but are not limited to): dammarenediol synthetase gene, cytochrome P450CYP716A47 gene and reductase gene thereof, cytochrome P450CYP716A53V2 gene and reductase gene thereof, or a combination thereof.

In another preferred embodiment, the key genes in the anabolic pathway of ginsenoside Rh2 include (but are not limited to): dammarenediol synthetase gene, cytochrome P450CYP716A47 gene and reductase gene thereof, tetracyclic triterpene C-3 glycosyl transferase gene UGTPg45, or combination thereof.

In another preferred embodiment, the key genes in the anabolic pathway of ginsenoside Rh1 include (but are not limited to): dammarenediol synthetase gene, cytochrome P450CYP716A47 gene and reductase gene thereof, cytochrome P450CYP716A53V2 gene and reductase gene thereof, and glycosyl transferase gene UGTPg100 at C-6 position of tetracyclic triterpene, or combination thereof.

In another preferred embodiment, the substrate of the glycosyl-catalyzed reaction is a compound of formula (I), or (III), respectively, and the product is a compound of formula (II), or (IV), respectively;

in another preferred embodiment, the compound of formula (I) is ginsenoside Rh2, and the compound of formula (II) is ginsenoside Rg 3;

or, the compound of formula (I) is ginsenoside Rh2, and the compound of formula (II) is 3-O-beta- (D-xylopyranosyl) -beta- (D-glucopyranosyl) -PPD;

or, the compound of formula (I) is ginsenoside F2, and the compound of formula (II) is ginsenoside Rd;

or the compound shown in the formula (I) is ginsenoside F2, and the compound shown in the formula (II) is 3-O-beta- (D-xylopyranosyl) -beta- (D-glucopyranosyl) -CK;

or, the compound of formula (III) is ginsenoside Rh1, and the compound of formula (IV) is ginsenoside Rf;

or, the compound of formula (III) is ginsenoside Rh1, and the compound of formula (IV) is ginsenoside Rg 2;

or, the compound of formula (V) is ginsenoside Rg3, and the compound of formula (VI) is 3-O-beta- (D-xylopyranosyl) -beta- (D-glucopyranosyl) -PPD;

or, the compound of formula (V) is ginsenoside Rd, and the compound of formula (IV) is 3-O-beta- (D-xylopyranosyl) -beta- (D-glucopyranosyl) -CK. In a seventh aspect of the invention, there is provided a genetically engineered host cell comprising a vector according to the fourth aspect of the invention, or a genome thereof into which has been integrated a polynucleotide according to the third aspect of the invention.

In another preferred embodiment, the glycosyltransferase is a polypeptide as described in the second aspect of the invention or a polypeptide derived therefrom.

In another preferred embodiment, the nucleotide sequence encoding said glycosyltransferase is as described in the third aspect of the invention.

In another preferred embodiment, the cell is a prokaryotic cell or a eukaryotic cell.

In another preferred embodiment, the host cell is a eukaryotic cell, such as a yeast cell or a plant cell.

In another preferred embodiment, the host cell is a Saccharomyces cerevisiae cell.

In another preferred embodiment, the host cell is a prokaryotic cell, such as E.coli.

In another preferred embodiment, the host cell is a ginseng cell.

In another preferred embodiment, the host cell is not a cell which naturally produces a compound of formula (II), (IV), (VI), (VIII), (II), (IIII).

In another preferred example, the host cell is not a cell naturally producing rare ginsenoside Rg3 and/or rare ginsenoside Rf and/or rare ginsenoside Rg2, and/or novel ginsenosides 3-O-beta- (D-xylopyranosyl) -beta- (D-glucopyranosyl) -PPD and 3-O-beta- (D-xylopyranosyl) -beta- (D-glucopyranosyl) -CK, etc.

In another preferred embodiment, the host cell contains key genes in the protopanaxadiol anabolic pathway including (but not limited to): dammarenediol synthetase gene, cytochrome P450CYP716A47 gene and reductase gene thereof, or combination thereof.

In another preferred embodiment, the host cell contains key genes in the protopanaxatriol anabolic pathway including (but not limited to): dammarenediol synthetase gene, cytochrome P450CYP716A47 gene and reductase thereof, reductase gene of P450CYP716A47 and gene thereof, or combination thereof. .

In an eighth aspect of the invention there is provided the use of a host cell as described in the eighth aspect for the preparation of an enzyme-catalysed reagent, or for the production of a glycosyltransferase, or as a catalytic cell, or for the production of a compound of formula (II), (IV) or (VI).

In another preferred example, the host cell is used for producing the new saponins 3-O-beta- (D-xylopyranosyl) -beta- (D-glucopyranosyl) -PPD and 3-O-beta- (D-xylopyranosyl) -beta- (D-glucopyranosyl) -CKII and/or the rare ginsenosides Rg3 and/or the rare ginsenosides Rf and/or the rare ginsenosides Rg2 by glycosylation reaction of the ginsenosides Rh2, F2, Rg3, Rd and/or the ginsenosides Rh1, Rg 1.

In a ninth aspect of the present invention, there is provided a method of producing a transgenic plant comprising the steps of: regenerating the genetically engineered host cell of the eighth aspect into a plant, and the genetically engineered host cell is a plant cell.

In another preferred embodiment, the genetically engineered host cell is a ginseng cell.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. For reasons of space, they will not be discussed in detail herein.

Drawings

The following drawings are included to illustrate specific embodiments of the invention and are not intended to limit the scope of the invention as defined by the claims.

FIG. 1 shows agarose gel electrophoresis of PCR products of (a) gGT29/gGT29-3 gene and (b) gGT29-4/gGT29-5/gGT29-6 and gGT29-7 gene. (b) Lane 1, nucleic acid Marker; lane 2, gGT29/gGT29-3 gene PCR product; (b) lane 1, PCR product of gGT29-4/gGT29-5/gGT29-6 gene; lane 2, gGT29-7 PCR product of the gene; lane 3, nucleic acid Marker.

FIG. 2 shows SDS-PAGE detecting expression of gGT29 and gGT29-3 in Saccharomyces cerevisiae; lane 1, lysate supernatant of the empty vector pYES2 recombinant; lane 2, supernatant of lysate of gGT29-pYES2 yeast recombinant; lane 3, gGT29-3-pYES2 yeast recombinant supernatant.

FIG. 3 shows Western Blot detection of gGT29 and gGT29-3 expression in Saccharomyces cerevisiae; lane 1, supernatant of lysate of empty vector pYES2 recombinant; lane 2, supernatant of lysate of gGT29-pYES2 yeast recombinant; lane 3, gGT29-3-pYES2 yeast recombinant supernatant.

FIG. 4 shows TLC detection patterns of products of glycosyltransferases gGT29 and gGT29-3 catalyzing ginsenosides Rh2 and F2; lane 1, mixed standard sample of PPD and PPD type saponins, Lane 2, gGT29 crude enzyme (supernatant of gGT29-pYES2 yeast recombinant lysate) catalyzes Rh2 to generate Rg3, Lane 3, gGT29 crude enzyme catalyzes Rh2 contrast, adds pYES2 empty plasmid yeast recombinant lysate to replace enzyme solution; lane 4, gGT29 catalyzed F2 to generate Rd, lane 5, gGT29 catalyzed F2 control, and pYES2 empty plasmid yeast recombinant lysate was added instead of enzyme solution; lane 6, gGT29-3 crude enzyme (supernatant of gGT29-3-pYES2 yeast recombinant) catalyzed Rh2 to produce Rg 3; lane 7, gGT29-3 crude enzyme catalyzed the formation of Rd from F2.

FIG. 5 shows that glycosyltransferase gGT29 and BvUGT73C10 or gGT29 and UGTPg45 in combination catalyze TLC detection of the PPD product; (a) gGT29 and BvUGT73C10 in combination catalyzed PPD, lane 1, PPD and mixed standard PPD-type saponins; lane 2, BvUGT73C10 catalyzed the formation of Rh2 by PPD; lane 3, gGT29 catalyzes Rh2 to Rg 3; lane 4, BvUGT73C10 and gGT29 in combination catalyze the production of Rg3 from PPD; (b) gGT29 and UGTPg45 in combination catalyzing PPD, lane 1, PPD and mixed standard of PPD-type saponins; lane 2, UGTPg45 catalyzes PPD to Rh 2; lane 3, PPD; lane 4, UGTPg45 and gGT29 in combination catalyze the production of Rg3 from PPD.

FIG. 6 shows TLC detection patterns of products of glycosyltransferases BvUGT73C10 and gGT29 catalyzing 20(R) -PPD and 20(R) -PPD, respectively, and 20(R) -PPD in combination; lane 1, BvUGT73C10 catalyzed the formation of 20(R) -Rh2 from 20(R) -PPD; lane 2, gGT29 catalyzes the production of 20(R) -Rg3 from 20(R) -Rh 2; lane 3, BvUGT73C10 and gGT29, in combination catalyzed the formation of 20(R) -Rg3 from 20(R) -PPD.

FIG. 7 shows the results of HPLC detection of glycosyltransferase gGT29 and BvUGT73C10 or gGT29 in combination with UGTPg45 to catalyze the PPD product. First row, Rg3, Rh2 and PPD mixed standard samples; the second row, gGT29 and BvUGT73C10 jointly catalyze PPD, and the third row, gGT29 and UGTPg45 jointly catalyze PPD.

FIG. 8 shows the product LC/MS detection results of the combination of glycosyltransferase gGT29 and BvUGT73C10 or gGT29 and UGTPg45 catalyzing PPD. The mass spectrum of the standard sample Rg3 and the mass spectra of the P1 peak (gGT29 and BvUGT73C10 in combination catalyzing the product of PPD) and the P2 peak (gGT29 and UGTPg45 in combination catalyzing the product of PPD) in figure 7 are shown.

Fig. 9 shows the HPLC detection results of the lysate extract of Rg 3-producing engineered yeast a2 cell, the first row of samples: mixed standard samples of protopanaxadiol (PPD), Dammarenediol (DM), ginsenosides Rh2 and Rg 3; second row of samples: producing Rg3 yeast engineering bacteria A2 cell lysate extract.

FIG. 10 shows that expression of gGT29-4, gGT29-5, gGT29-6, gGT29-7 in recombinant E.coli was detected by SDS-PAGE. Lane 1, gGT29-4-pET28a recombinant E.coli lysate total protein; lane 2, gGT29-4-pET28a recombinant E.coli lysate supernatant; lane 3, gGT29-5-pET28a recombinant E.coli lysate total protein; lane 4, gGT29-5-pET28a recombinant E.coli lysate supernatant; lane 5, gGT29-6-pET28a recombinant E.coli lysate total protein; lane 6, gGT29-6-pET28a recombinant E.coli lysate supernatant; lane 7, gGT29-7-pET28a recombinant E.coli lysate total protein; lane 8, gGT29-7-pET28a recombinant E.coli lysate supernatant; lane 9, protein molecular weight Marker.

FIG. 11 shows the expression of gGT29-4, gGT29-5, gGT29-6, gGT29-7 in recombinant E.coli by Western Blot assay. Lane 1, gGT29-4-pET28a recombinant E.coli lysate total protein; lane 2, gGT29-4-pET28a recombinant E.coli lysed supernatant; lane 3, gGT29-5-pET28a recombinant E.coli lysate total protein; lane 4, gGT29-5-pET28a recombinant E.coli lysate supernatant; lane 5, gGT29-6-pET28a recombinant E.coli lysate total protein; lane 6, gGT29-6-pET28a recombinant E.coli lysate supernatant; lane 7, gGT29-7-pET28a recombinant E.coli lysates in total protein; lane 8, gGT29-7-pET28a recombinant E.coli lysate supernatant.

FIG. 12 shows the TLC detection profiles of products of glycosyltransferases gGT29-4, gGT29-5, gGT29-6, gGT29-7 catalyzing Rh2 and F2, respectively. Lane Rh2 shows the use of saponin Rh2 as substrate; lane F2 shows the use of saponin F2 as substrate. gGT29-4, gGT29-5, gGT29-6 and gGT29-7 show that different enzyme solutions are used for catalytic reaction.

FIG. 13 shows TLC detection profiles of products catalyzing Rh1 by glycosyltransferases gGT29-4, gGT29-5, gGT29-6, gGT29-7, respectively. (a)

Lanes

1, 2 and 3 represent the products of glycosyltransferases gGT29-4, gGT29-5 and gGT29-6, respectively, catalyzing Rh1, and lane 4 represents the protopanaxatriol-type saponin mixture standard; (b) lane 1 represents the product of catalysis of Rh1 by glycosyltransferase gGT29-7, and lane 2 represents the protopanaxatriol-type saponin cocktail standard.

FIG. 14 shows the results of a test showing that glycosyltransferase gGT29-7 and its muteins gGT29-7-N343G, gGT29-7-A359P and gGT29-7-N343G/A359P catalyze Rh1 (UDP-glucose and UDP-rhamnose were used simultaneously as glycosyl donors). First row, Rg1, Rf, Rg2 and Rh1 mixed standard samples; second row, gGT29-7 catalyzes Rh 1; third line, gGT29-7-N343G catalyzes Rh 1; fourth, gGT29-7-A359P catalyzes Rh 1; the fifth row, gGT29-7-N343G/A359P catalyzes Rh 1.

FIG. 15 shows TLC detection patterns of catalytic ginsenoside Rh2, Rg3 and Rd products by glycosyltransferases gGT29-3 and gGT29-14 with UDP-xylose as glycosyl donor; (A) catalyzing by using the supernatant of the enterobacter lysate for expressing an empty vector pET28a as an enzyme solution; (B) catalyzing by using an enterobacter lysate supernatant which expresses pET28a-gGT29-3 as an enzyme solution; (C) the supernatant of the enterobacter lysate expressing pET28a-gGT29-14 was used as an enzyme solution for catalysis. Lanes Rh2, Rg3 and Rd show the use of saponins Rh2, Rg3 and Rd as substrates, respectively, and lane M represents protopanaxadiol type saponin mix standards.

Detailed Description

The present inventors have conducted extensive and intensive studies and have for the first time provided glycosyltransferases gGT29-7(SEQ ID NO: 61) and polypeptides derived therefrom, such as gGT29(SEQ ID NO: 26), gGT29-3(SEQ ID NO: 28), gGT29-4(SEQ ID NO: 55), gGT29-5(SEQ ID NO: 57), gGT29-6(SEQ ID NO: 59), gGT29-8(SEQ ID NO: 72), gGT29-9(SEQ ID NO: 74), gGT29-10(SEQ ID NO: 76), gGT29-11(SEQ ID NO: 78), gGT29-12(SEQ ID NO: 80), gGT29-13(SEQ ID NO: 82), gGT29-14(SEQ ID NO: 84), gGT29-15(SEQ ID NO: 86), gGT29-16(SEQ ID NO: 80), gGT29-17(SEQ ID NO: 90), and polypeptides derived therefrom, gGT29-18(SEQ ID NO.:92), gGT29-7-N343G (SEQ ID NO.:93), gGT29-7-A359P (SEQ ID NO.:94), gGT29-7-N343G/A359P (SEQ ID NO.:95) in terpenoid glycosylation catalysis and new saponin synthesis. Specifically, the glycosyltransferase of the present invention is capable of specifically and efficiently catalyzing a tetracyclic triterpene compound substrate and/or transferring a sugar group from a sugar group donor to the first sugar group at C-3 or C-6 of a tetracyclic triterpene compound to extend a sugar chain. In particular, the ginsenoside Rh2 can be converted into rare ginsenoside Rg3 with anticancer activity, the ginsenoside F2 is converted into ginsenoside Rd, the ginsenoside Rh1 is converted into rare ginsenoside Rf with anti-tumor and anti-fatigue effects, and the ginsenoside Rh1 is converted into rare ginsenoside Rg2 with neuroprotective effect and ultraviolet protection effect. The invention also provides conversion and catalytic processes. The glycosyl transferase can be co-expressed with key enzyme in the anabolism pathway of dammarenediol and/or protopanoxadiol or protopanaxatriol and glycosyl transferase at C-3 or C-6 position of tetracyclic triterpene in host cells, or applied to genetic engineering cells for preparing ginsenoside Rh2 and ginsenoside Rh1, and applied to construction of artificially synthesized rare ginsenoside Rg3 and Rf. In addition, the glycosyltransferase can be co-expressed with key enzymes in the anabolism pathway of dammarenediol and/or protopanaxadiol or protopanaxatriol, C-6 glycosyltransferase and key enzymes for synthesizing UDP-rhamnose in host cells, and is applied to constructing strains for artificially synthesizing rare ginsenoside Rg 2. The present invention has been completed based on this finding.

Definition of

As used herein, the terms "active polypeptide", "polypeptide of the invention and its derived polypeptide", "enzyme of the invention", "glycosyltransferase", "gGT 29 of the invention or its derived polypeptide" or "glycosyltransferase of the invention", all refer to glycosyltransferase gGT29-7(SEQ ID No.:61) or its derived polypeptide. Wherein preferred derivative polypeptides include gGT29(SEQ ID NO: 26), gGT29-3(SEQ ID NO: 28), gGT29-4(SEQ ID NO: 55), gGT29-5(SEQ ID NO: 57), gGT29-6(SEQ ID NO: 59), gGT29-8(SEQ ID NO: 72), gGT29-9(SEQ ID NO: 74), gGT29-10(SEQ ID NO: 76), gGT29-11(SEQ ID NO: 78), gGT29-12(SEQ ID NO: 80), gGT29-13(SEQ ID NO: 82) gGT29-14(SEQ ID NO: 84), gGT29-15(SEQ ID NO: 86), gGT29-16(SEQ ID NO: 88), 48-17 (SEQ ID NO: 90), gGT29-18(SEQ ID NO: 92), and gGT 29-7-343N (SEQ ID NO: 93), gGT29-7-A359P (SEQ ID No.:94), gGT29-7-N343G/A359P (SEQ ID No.: 95).

Unless otherwise specified, ginsenosides and sapogenins referred to herein are ginsenosides and sapogenins having S and/or R configuration at position C20.

As used herein, "isolated polypeptide" means that the polypeptide is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. One skilled in the art can purify the polypeptide using standard protein purification techniques. Substantially pure polypeptides are capable of generating a single major band on a non-reducing polyacrylamide gel. The purity of the polypeptide can be further analyzed by amino acid sequence.

The active polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide. The polypeptides of the invention can be naturally purified products, or chemically synthesized products, or using recombinant technology from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, plants). Depending on the host used in the recombinant production scheme, the polypeptides of the invention may be glycosylated or may be non-glycosylated. The polypeptides of the invention may or may not also include an initial methionine residue.

The invention also includes fragments, derivatives and analogues of the polypeptides. As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that retains substantially the same biological function or activity as the polypeptide.

A fragment, derivative or analogue of a polypeptide of the invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which the mature polypeptide is fused to another compound, such as a compound that increases the half-life of the polypeptide, e.g. polyethylene glycol, or (iv) a polypeptide in which an additional amino acid sequence is fused to the sequence of the polypeptide (e.g. a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with an antigenic IgG fragment). Such fragments, derivatives and analogs are well within the skill of those in the art in light of the teachings herein.

The active polypeptide of the invention has glycosyltransferase activity and is capable of catalyzing one or more of the following reactions:

(A)

wherein R1 is a glycosyl group; r2 and R3 are OH or H; r4 is a glycosyl group or H; r5 is a glycosyl group, and the polypeptide is selected from the group consisting of SEQ ID NO. 26, 28, 55, 57, 59, 61, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 93,94 or 95 or a derivative polypeptide thereof.

The compounds substituted with R1-R4 are shown in the following table:

That is, when R1 is glucosyl; r2 is H, R3 is OH, R4 is H, and the compound of formula (I) is Rh 2.

when UDP-xylose is used as glycosyl donor, the substrate (I) compound is Rh2, and the product (II) compound is 3-O-beta- (D-xylopyranosyl) -beta- (D-glucopyranosyl) -PPD; when the compound of the substrate (I) is F2, the product of the compound of the formula (II) is 3-O-beta- (D-xylopyranosyl) -beta- (D-glucopyranosyl) -CK.

(B)

Wherein R1 and R2 are H or a glycosyl group, and R3 and R4 are glycosyl groups. R3-R4-O is a glycosyl derived from the first glycosyl of C6, and the polypeptide is selected from SEQ ID NO. 55, 57, 59, 61, 78, 82, 92, 94 or 95 or a polypeptide derived from the polypeptide.

When UDP-glucose is used as the glycosyl donor, the substrate (IIII) compound is Rh1, and the product compound of formula (IV) is Rf;

when UDP-rhamnose is taken as glycosyl donor, the substrate (IIII) compound is Rh1, and the product compound shown in formula (IV) is Rg 2;

(C)

wherein R1 is a glycosyl group; r2 and R3 are OH or H; r4 is a glycosyl group or H; r5 is a glycosyl, R5-R1-O is a glycosyl derived from the first glycosyl of C3; r6 is glycosyl, R6-R1-O is glycosyl derived from the first glycosyl of C3, and the polypeptide is selected from SEQ ID NO. 26, 28, 59, 76, 84, 86 or 88 or derived polypeptide thereof. R1 is two glucosyl groups, R2 is H, R3 is OH, R4 is H, and the compound of formula (V) is Rg 3.

R1 is two glucosyl groups, R2 is H, R3 is OH, R4 is glucosyl group, the compound of formula (V) is Rd, when UDP-xylose is used as a glycosyl donor, the substrate (V) compound is Rg3, and the product of the compound of formula (VI) is 3-O-beta- (D-xylopyranosyl) -beta- (D-glucopyranosyl) -PPD; when the substrate (V) compound is Rd, the product compound of formula (VI) is 3-O-beta- (D-xylopyranosyl) -beta- (D-glucopyranosyl) -CK.

The polypeptide sequence is SEQ ID No. 61 or a polypeptide derived therefrom, preferably a polypeptide of SEQ ID No. 26, 28, 55, 57, 59, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 93,94 or 95, and the term also includes variants of SEQ ID No. 26, 28, 55, 57, 59, 61, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 93,94 or 95 sequences having the same function as the polypeptide. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. The term also includes active fragments and active derivatives of the proteins of the invention. The invention also provides analogs of the polypeptides. These analogs may differ from the native polypeptide by amino acid sequence differences, by modifications that do not affect the sequence, or by both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by irradiation or exposure to mutagens, site-directed mutagenesis, or other known molecular biological techniques. Analogs also include analogs having residues other than the natural L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It is to be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

Modified (generally without altering primary structure) forms include: chemically derivatized forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those that result from glycosylation modifications during synthesis and processing of the polypeptide or during further processing steps. Such modification may be accomplished by exposing the polypeptide to an enzyme that effects glycosylation, such as a mammalian glycosylase or deglycosylase. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides modified to increase their resistance to proteolysis or to optimize solubility.

The gGT29-7 polypeptide or derivative polypeptide thereof of the invention, such as preferably gGT29, gGT29-3, gGT29-4, gGT29-5, gGT29-6, gGT29-8, gGT29-9, gGT29-10, gGT29-11, gGT29-12, gGT29-13, gGT29-14, gGT29-15, gGT29-16, gGT29-17, gGT29-18, gGT29-7-N343G, gGT29-7-A359P or gGT29-7-N343G/A359P protein may also contain one or more polypeptide fragments at the amino-terminus or carboxy-terminus as protein tags. Any suitable label may be used with the present invention. For example, the tag can be FLAG, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ε, B, gE, and Ty 1. These tags can be used to purify proteins. Some of these tags and their sequences are listed in table 1.

TABLE 1

In order to make the translated protein be secreted and expressed (for example, secreted to the outside of cells), a signal peptide sequence, such as pelB signal peptide and the like, can be added to the amino terminal of the amino acid of the gGT29-7 polypeptide or a derivative polypeptide thereof, preferably gGT29, gGT29-3, gGT29-4, gGT29-5, gGT29-6, gGT29-8, gGT29-9, gGT29-10, gGT29-11, gGT29-12, gGT29-13, gGT29-14, gGT29-15, gGT29-16, gGT29-17, gGT29-18, gGT29-7-N343G, gGT29-7-A359P or gGT 29-7-N343G/A359P. The signal peptide may be cleaved off during secretion of the polypeptide from the cell.

The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The sequence of the coding region encoding the mature polypeptide may be identical to the sequence of the coding region as shown in SEQ ID No. 60 or may be a degenerate variant. As used herein, "degenerate variant" refers in the present invention to a nucleic acid sequence that encodes a protein having SEQ ID No. 61 or a polypeptide derived therefrom, preferably SEQ ID No. 26, 28, 55, 57, 59, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 93,94 or 95, but differs from the coding sequence of SEQ ID No. 60 or a protein derived therefrom, preferably the sequence set forth in SEQ ID No. 25, 27, 54, 56, 58, 60, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89 or 91.

Polynucleotides encoding the polypeptide of SEQ ID No. 61 or a polypeptide derived therefrom, preferably the mature polypeptide of SEQ ID No. 26, 28, 55, 57, 59, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 93,94 or 95, include: a coding sequence encoding only the mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature polypeptide.

The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, and may also include additional coding and/or non-coding sequences.

The present invention also relates to variants of the above polynucleotides which encode polypeptides having the same amino acid sequence as the present invention or fragments, analogs and derivatives of the polypeptides. The variant of the polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the polypeptide encoded thereby.

The present invention also relates to polynucleotides which hybridize to the above-described sequences and which have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides hybridizable under stringent conditions (or stringent conditions) with the polynucleotides of the present invention. In the present invention, "stringent conditions" mean: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 XSSC, 0.1% SDS, 60 ℃; or (2) adding denaturant during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 deg.C, etc.; or (3) hybridization occurs only when the identity between two sequences is at least 90% or more, preferably 95% or more. And, the hybridizable polynucleotide encodes a polypeptide having the same biological function and activity as the mature polypeptide of SEQ ID No. 26, 28, 55, 57, 59, 61, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 93,94, or 95.

The invention also relates to nucleic acid fragments which hybridize to the sequences described above. As used herein, a "nucleic acid fragment" is at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides in length. The nucleic acid fragment can be used in amplification technology (such as PCR) of nucleic acid to determine and/or isolate gGT29-7 polypeptide or its derivative polypeptide, preferably gGT29, gGT29-3, gGT29-4, gGT29-5, gGT29-6, gGT29-8, gGT29-9, gGT29-10, gGT29-11, gGT29-12, gGT29-13, gGT29-14, gGT29-15, gGT29-16, gGT29-17, gGT29-18, gGT29-7-N343G, gGT29-7-A359P, or gGT29-7-N343G/A359P protein polynucleotide.

The polypeptides and polynucleotides of the present invention are preferably provided in isolated form, more preferably purified to homogeneity.

The gGT29-7 polypeptide or derivative polypeptide thereof of the invention, preferably gGT29, gGT29-3, gGT29-4, gGT29-5, gGT29-6, gGT29-8, gGT29-9, gGT29-10, gGT29-11, gGT29-12, gGT29-13, gGT29-14, gGT29-15, gGT29-16, gGT29-17, gGT29-18, gGT29-7-N343G, gGT29-7-A359P or gGT29-7-N343G/A359P nucleotide full-length sequence or its fragment can be obtained by PCR amplification method, recombinant method or artificial synthesis method. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, particularly open reading frame sequences, and amplified using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates to obtain the sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice together the amplified fragments in the correct order.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Typically, long fragments are obtained by first synthesizing a plurality of small fragments and then ligating them together.

At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

A method of amplifying DNA/RNA using PCR technology is preferably used to obtain the gene of the present invention. Particularly, when it is difficult to obtain a full-length cDNA from a library, it is preferable to use the RACE method (RACE-cDNA terminal rapid amplification method), and primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein and synthesized by a conventional method. The amplified DNA/RNA fragments can be isolated and purified by conventional methods, such as by gel electrophoresis.

The present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell genetically engineered with the vector of the present invention or gGT29-7 polypeptide or a polypeptide derived therefrom, preferably gGT29, gGT29-3, gGT29-4, gGT29-5, gGT29-6, gGT29-8, gGT29-9, gGT29-10, gGT29-11, gGT29-12, gGT29-13, gGT29-14, gGT29-15, gGT29-16, gGT29-17, gGT29-18, gGT29-7-N343G, gGT29-7-A359P or gGT29-7-N343G/A359P protein coding sequence, and a method for producing the polypeptide of the present invention by recombinant technology.

The polynucleotide sequence of the present invention can be used to express or produce recombinant gGT29-7 polypeptide or its derivative polypeptide by conventional recombinant DNA techniques, preferably gGT29, gGT29-3, gGT29-4, gGT29-5, gGT29-6, gGT29-8, gGT29-9, gGT29-10, gGT29-11, gGT29-12, gGT29-13, gGT29-14, gGT29-15, gGT29-16, gGT29-17, gGT29-18, gGT29-7-N343G, gGT29-7-A359P or gGT29-7-N343G/A359P polypeptide. Generally, the following steps are performed:

(1) transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding an gGT29-7 polypeptide or a polypeptide derived therefrom of the invention, preferably a gGT29, gGT29-3, gGT29-4, gGT29-5, gGT29-6, gGT29-8, gGT29-9, gGT29-10, gGT29-11, gGT29-12, gGT29-13, gGT29-14, gGT29-15, gGT29-16, gGT29-17, gGT29-18, gGT29-7-N343G, gGT29-7-A359P or gGT29-7-N343G/A359P polypeptide, or with a recombinant expression vector containing the polynucleotide;

(2) a host cell cultured in a suitable medium;

(3) isolating and purifying the protein from the culture medium or the cells.

In the invention, gGT29-7 polypeptide or derivative polypeptide thereof, preferably gGT29, gGT29-3, gGT29-4, gGT29-5, gGT29-6, gGT29-8, gGT29-9, gGT29-10, gGT29-11, gGT29-12, gGT29-13, gGT29-14, gGT29-15, gGT29-16, gGT29-17, gGT29-18, gGT29-7-N343G, gGT29-7-A359P or gGT29-7-N343G/A359P polynucleotide sequence can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors well known in the art. Any plasmid or vector may be used as long as it can replicate and is stable in the host. An important feature of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translation control elements.

Methods well known to those skilled in the art can be used to construct expression vectors comprising gGT29-7 polypeptide or derived polypeptides thereof, preferably gGT29, gGT29-3, gGT29-4, gGT29-5, gGT29-6, gGT29-8, gGT29-9, gGT29-10, gGT29-11, gGT29-12, gGT29-13, gGT29-14, gGT29-15, gGT29-16, gGT29-17, gGT29-18, gGT29-7-N343G, gGT29-7-A359P or gGT29-7-N343G/A359P encoding DNA sequences and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. Representative examples of such promoters are: lac or trp promoter of E.coli; a lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, LTRs of retrovirus, and other known promoters which can control the expression of genes in prokaryotic or eukaryotic cells or viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli.

Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: escherichia coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; a plant cell; insect cells of Drosophila S2 or Sf 9; CHO, COS, 293 cells, or Bowes melanoma cells.

When the polynucleotide of the present invention is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs, that act on a promoter to increase transcription of a gene. Examples include the SV40 enhancer at the late side of the replication origin at 100 to 270 bp, the polyoma enhancer at the late side of the replication origin, and adenovirus enhancers.

It will be clear to one of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl ₂ Methods, the steps used are well known in the art. Another method is to use MgCl ₂ . If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

Applications of

The active polypeptide or the peptide transferase gGT29-7 polypeptide or the derivative polypeptide thereof, preferably gGT29, gGT29-3, gGT29-4, gGT29-5, gGT29-6, gGT29-8, gGT29-9, gGT29-10, gGT29-11, gGT29-12, gGT29-13, gGT29-14, gGT29-15, gGT29-16, gGT29-17, gGT29-18, gGT29-7-N343G, gGT29-7-A359P or gGT29-7-N343G/A359P, includes (but is not limited to): the glycosyl group from the glycosyl donor is transferred to the first glycosyl group at the C-3 position and the C-6 position of the tetracyclic triterpenoid to extend the sugar chain specifically and efficiently. In particular, the ginsenoside Rh2 can be converted into rare ginsenoside Rg3 with better anticancer activity; converting ginsenoside F2 into ginsenoside Rd; converting ginsenoside Rh1 into rare ginsenoside Rf with antitumor and antifatigue activities; converting ginsenoside Rh1 into rare ginsenoside Rg2 with neuroprotective effect and ultraviolet protection effect; the glycosyltransferase can also synthesize novel saponins, namely 3-O-beta- (D-xylopyranosyl) -beta- (D-glucopyranosyl) -PPD and (3-O-beta- (D-xylopyranosyl) -beta- (D-glucopyranosyl) -CK which are not reported before by ginsenoside Rh2, Rg3 and Rd.

The tetracyclic triterpene compounds comprise (but are not limited to): tetracyclic triterpenoids such as dammarane type, lanoline type, kansuine type, cycloartane (cycloartane) type, apotorucallane type, cucurbitane type, and meliane type in S or R configuration.

The invention provides an industrial catalysis method, which comprises the following steps: compounds of formula (II), (IV) or formula (VI) are obtained using an gGT29-7 polypeptide or a polypeptide derived therefrom of the invention, preferably gGT29, gGT29-3, gGT29-4, gGT29-5, gGT29-6, gGT29-8, gGT29-9, gGT29-10, gGT29-11, gGT29-12, gGT29-13, gGT29-14, gGT29-15, gGT29-16, gGT29-17, gGT29-18, gGT29-7-N343G, gGT29-7-A359P or gGT29-7-N343G/A359P active polypeptide or a peptidyl transferase under conditions that provide a glycosyl donor. Specifically, the polypeptide used in the reaction (A) is selected from the polypeptide shown in SEQ ID No. 61 or a derivative polypeptide thereof, preferably an active polypeptide of an amino acid sequence shown in SEQ ID No. 26, 28, 55, 57, 59, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 93,94 or 95; the polypeptide used in the reaction of (B) is selected from the group consisting of SEQ ID No. 55, 57, 59, 61, 78, 82, 92, 94 or 95; the polypeptide used in the reaction (C) is selected from the polypeptide shown in SEQ ID No. 61 or a derivative polypeptide thereof, and preferably is selected from the polypeptide shown in SEQ ID No. 26, 28, 59, 76, 84, 86 or 88.

The glycosyl donor is nucleoside diphosphate sugar, and is selected from the following group: UDP-glucose, ADP-glucose, TDP-glucose, CDP-glucose, GDP-glucose, UDP-acetylglucose, ADP-acetylglucose, TDP-acetylglucose, CDP-acetylglucose, GDP-acetylglucose, UDP-xylose, ADP-xylose, TDP-xylose, CDP-xylose, UDP-xylose, GDP-xylose, UDP-galacturonic acid, ADP-galacturonic acid, TDP-galacturonic acid, CDP-galacturonic acid, GDP-galacturonic acid, UDP-galactose, ADP-galactose, TDP-galactose, CDP-galactose, GDP-galactose, UDP-arabinose, ADP-arabinose, TDP-arabinose, CDP-arabinose, GDP-arabinose, UDP-rhamnose, ADP-rhamnose, TDP-rhamnose, CDP-rhamnose, GDP-rhamnose, or other nucleoside diphosphate hexoses or nucleoside diphosphate pentoses, or a combination thereof.

Said glycosyl donor is preferably a uridine diphosphate sugar selected from the group consisting of: UDP-glucose, UDP-xylose, UDP-rhamnose, UDP-galacturonic acid, UDP-galactose, UDP-arabinose, or other uridine diphosphogliose or uridine diphosphogliose, or combinations thereof.

In the method, an enzyme activity supplement (a supplement for increasing the enzyme activity or inhibiting the enzyme activity) may be added. The enzymatic activity additive may be selected from the group consisting of: ca ²⁺ 、Co ²⁺ 、Mn ²⁺ 、Ba ²⁺ 、Al ³⁺ 、Ni ²⁺ 、Zn ²⁺ Or Fe ²⁺ (ii) a Or can generate Ca ²⁺ 、Co ²⁺ 、Mn ²⁺ 、Ba ²⁺ 、Al ³⁺ 、Ni ²⁺ 、Zn ²⁺ Or Fe ²⁺ The substance of (1).

The pH conditions of the method are as follows: pH4.0-10.0, preferably pH6.0-pH8.5, more preferably 8.5.

The temperature conditions of the method are as follows: 10 ℃ to 105 ℃, preferably 25 ℃ to 35 ℃, more preferably 35 ℃.

The present invention also provides a composition comprising an effective amount of an active polypeptide or a peptidyl transferase gGT29-7 polypeptide or a polypeptide derived therefrom of the present invention, preferably gGT29, gGT29-3, gGT29-4, gGT29-5, gGT29-6, gGT29-8, gGT29-9, gGT29-10, gGT29-11, gGT29-12, gGT29-13, gGT29-14, gGT29-15, gGT29-16, gGT29-17, gGT29-18, gGT29-7-N343G, gGT29-7-A359P or gGT29-7-N343G/A359P, and a dietetically or industrially acceptable carrier or excipient. Such vectors include (but are not limited to): water, buffer, glucose, water, glycerol, ethanol, and combinations thereof.

The composition may further comprise a substance which regulates the glycosyltransferase activity of the present invention. Any of those having the function of enhancing the enzymatic activityAll substances are usable. Preferably, the substance for improving the glycosyltransferase activity of the present invention is selected from mercaptoethanol. In addition, many substances may reduce enzymatic activity, including but not limited to: ca ²⁺ 、Co ²⁺ 、Mn ²⁺ 、Ba ²⁺ 、Al ³⁺ 、Ni ²⁺ 、Zn ²⁺ And Fe ²⁺ (ii) a Or can be hydrolyzed to form Ca upon addition to the substrate ²⁺ 、Co ²⁺ 、Mn ²⁺ 、Ba ²⁺ 、Al ³⁺ 、Ni ²⁺ 、Zn ²⁺ And Fe ²⁺ The substance of (1).

After the gGT29-7 polypeptide or its derivative of the present invention is obtained, preferably gGT29, gGT29-3, gGT29-4, gGT29-5, gGT29-6, gGT29-8, gGT29-9, gGT29-10, gGT29-11, gGT29-12, gGT29-13, gGT29-14, gGT29-15, gGT29-16, gGT29-17, gGT29-18, gGT29-7-N343G, gGT29-7-A359P or gGT29-7-N343G/A359P, one skilled in the art can conveniently use the enzyme to exert the transglycosylation effect, particularly on dammarane glycol, protopanaxadiol and protopanaxatriol. As a preferred embodiment of the present invention, there are also provided two methods for forming rare ginsenosides, one of the methods comprising: the gGT29-7 polypeptide or derivative polypeptide thereof is preferably gGT29, gGT29-3, gGT29-4, gGT29-5, gGT29-6, gGT29-8, gGT29-9, gGT29-10, gGT29-11, gGT29-12, gGT29-13, gGT29-14, gGT29-15, gGT29-16, gGT29-17, gGT29-18, gGT29-7-N343G, gGT29-7-A359P or gGT29-7-N343G/A359P to treat the substrate to be transglycosylated, wherein the substrate comprises tetracyclic triterpenoids such as dammarylene glycol, protopanaxadiol, protopanaxatriol and derivatives thereof. Preferably, a substrate to be transglycosylated is enzymatically treated with said gGT29-7 polypeptide or a polypeptide derived therefrom, preferably gGT29, gGT29-3, gGT29-4, gGT29-5, gGT29-6, gGT29-8, gGT29-9, gGT29-10, gGT29-11, gGT29-12, gGT29-13, gGT29-14, gGT29-15, gGT29-16, gGT29-17, gGT29-18, gGT29-7-N343G or gGT29-7-A359P or gGT29-7-N343G/A359P, at pH 3.5-10. Preferably, the substrate to be transglycosylated is treated by the gGT29-7 polypeptide or the derived polypeptide thereof, preferably gGT29, gGT29-3, gGT29-4, gGT29-5, gGT29-6, gGT29-8, gGT29-9, gGT29-10, gGT29-11, gGT29-12, gGT29-13, gGT29-14, gGT29-15, gGT29-16, gGT29-17, gGT29-18, gGT29-7-N343G, gGT29-7-A359P, gGT29-7-N343G/A359P and gGT29-3 enzyme at the temperature of 30-105 ℃.

The second method comprises: gGT29-7 polypeptide or derivative polypeptide thereof, preferably gGT29, gGT29-3, gGT29-4, gGT29-5, gGT29-6, gGT29-8, gGT29-9, gGT29-10, gGT29-11, gGT29-12, gGT29-13, gGT29-14, gGT29-15, gGT29-16, gGT29-17, gGT29-18, gGT29-7-N343G, gGT29-7-A359P or gGT29-7-N343G/A359P gene is transferred into an engineering bacterium (for example, yeast or Escherichia coli engineering bacterium) capable of synthesizing ginsenoside Rh2 or Rh1, or gGT29-7 polypeptide or derivative polypeptide thereof, preferably gGT29, gGT29-3, gGT29-4, gGT29-5, gGT29-6, gGT29-8, gGT29-9, gGT29-10, gGT29-11, gGT29-12, gGT29-13, gGT29-14, gGT29-15, gGT29-16, gGT29-17, gGT29-18, gGT29-7-N343G, gGT29-7-A359P or gGT29-7-N343G/A359P gene is co-expressed with dammar-diol, key gene in anabolic pathway of protopanaxadiol and protopanaxatriol and glycosyl transferase at C-3 and/or C-6 position of tetracyclic triterpene in host cell (such as yeast cell or Escherichia coli) to obtain recombinant bacteria for directly producing rare ginsenoside Rg3 or Rf. Alternatively, gGT29-7 polypeptide or its derivative polypeptide, preferably gGT29, gGT29-3, gGT29-4, gGT29-5, gGT29-6, gGT29-8, gGT29-9, gGT29-10, gGT29-11, gGT29-12, gGT29-13, gGT29-14, gGT29-15, gGT29-16, gGT29-17, gGT29-18, gGT29-7-N343G, or gGT29-7-A359P, or gGT29-7-N343G/A359P gene is co-expressed with dammarenediol and/or protopanaxadiol or protopanaxatriol anabolic pathway key enzyme and tetracyclic triterpene C-6 glycosyltransferase and UDP-rhamnose key enzyme in host cell, and applied to constructing recombinant strain for artificially synthesizing rare ginsenoside 2.

The key genes in the dammarenediol anabolic pathway include (but are not limited to): dammarenediol synthetase gene.

In another preferred embodiment, the key genes in the anabolic pathway of protopanaxadiol include (but are not limited to): dammarenediol synthetase gene, cytochrome P450CYP716A47 gene and its reductase gene, or their combination. Or isozymes of the above enzymes and combinations thereof. Wherein the dammarenediol synthetase converts oxidosqualene (synthesized by saccharomyces cerevisiae itself) into dammarenediol, and the cytochrome P450CYP716A47 and the reductase thereof convert the dammarenediol into protopanoxadiol. (Han et al, plant & cell physiology, 2011, 52.2062-73)

In another preferred embodiment, the key genes in the anabolic pathway of protopanaxatriol include (but are not limited to): dammarenediol synthetase gene, cytochrome P450CYP716A47 gene and reductase gene thereof, cytochrome P450CYP716A53V2 gene and reductase gene thereof, or a combination thereof. Or isozymes of the above enzymes and combinations thereof. Wherein the dammarenediol synthetase converts oxidosqualene (synthesized by saccharomyces cerevisiae) into dammarenediol, the cytochrome P450CYP716A47 and the reductase thereof convert the dammarenediol into protopanaxadiol, and the cytochrome P450CYP716A53v2(JII036031) and the reductase thereof further convert the protopanaxadiol into protopanaxatriol. (Han et al plant & cell physiology, 2012, 53.1535-45)

In another preferred embodiment, the key genes in the anabolic pathway of ginsenoside Rh2 include (but are not limited to): dammarenediol synthetase gene, cytochrome P450CYP716A47 gene and reductase gene thereof, tetracyclic triterpene C-3 position glycosyl transferase UGTPg45 or combination thereof. Or isozymes of the above enzymes and combinations thereof. Among them, dammarenediol synthetase converts oxidosqualene (synthesized by saccharomyces cerevisiae itself) into dammarenediol, cytochrome P450CYP716a47 and its reductase further convert dammarenediol into protopanoxadiol, and glycosyltransferase UGTPg45 may further convert protopanaxadiol into Rh2(Wang et al, Metabolic Engineering,2015, 29.97-105).

In another preferred embodiment, the key genes in the anabolic pathway of ginsenoside Rh1 include (but are not limited to): dammarenediol synthetase gene, cytochrome P450CYP716A47 gene and reductase gene thereof, cytochrome P450CYP716A53V2 gene and reductase gene thereof, and C-6 glycosyltransferase UGTPg100 or combination thereof. Or isozymes of the above enzymes and combinations thereof. Among them, dammarenediol synthetase converts oxidosqualene (synthesized by saccharomyces cerevisiae itself) into dammarenediol, cytochrome P450CYP716a47 and its reductase further convert damenediol into protopanaxadiol, cytochrome P450CYP716a53v2(JII036031) and its reductase further convert protopanaxadiol into protopanaxatriol, and glycosyltransferase UGTPg100 may further convert protopanaxatriol into Rh1(Wei et al, Molecular Plant,2015,15.doi:10.1016/j. molp.2015.05.010).

The invention has the main advantages that:

(1) the glycosyltransferase of the present invention can specifically and efficiently transfer a glycosyl group from a glycosyl donor to the first glycosyl group at C-3 and/or C-6 of a tetracyclic triterpene compound to extend a sugar chain

(2) The glycosyltransferase can particularly respectively convert ginsenoside Rh2 and Rh1 into rare ginsenoside Rg3 with better anticancer activity, rare ginsenoside Rf with the efficacies of tumor resistance and fatigue resistance, and rare ginsenoside Rg2 with the functions of neuroprotection and ultraviolet protection

(3) A synthetic pathway of ginsenoside (dammar diol, protopanaxadiol and protopanaxatriol) or a synthetic pathway of rare ginsenoside Rh2 or Rh1 is constructed in yeast, so that the rare ginsenosides Rg3 and Rf are produced by fermenting the yeast with monosaccharide such as glucose as a substrate. A synthesis way of rare ginsenoside Rh1 and a way of synthesizing UDP-rhamnose are constructed in yeast, and the yeast is used for fermenting to produce the rare ginsenoside Rg 2. The method not only can solve the problem of raw material sources in saponin production, but also can greatly reduce the production cost of the rare saponins Rg3, Rg2 and Rf.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations.

Example 1 isolation of glycosyltransferase and Gene encoding the same

100 cDNA sequences predicted to be glycosyltransferase are extracted from published expression profile data of the ginseng plants, 60 cDNA full-length sequences are cloned from the cDNA sequences and are subjected to expression and transglycosylation reaction analysis, and 17 expression products have transglycosylation activity on the ginsengenin and the saponin.

Extracting RNA of ginseng and carrying out reverse transcription to obtain cDNA of ginseng. PCR amplification was performed using the cDNA as a template, using primer set 1(SEQ ID NO.:7, 8); primer pair 2(SEQ ID NO: 9, 10); primer pair 3(SEQ ID NO: 11, 12); primer pair 5(SEQ ID NO: 34, 35); primer pair 7(SEQ ID NO: 46, 47); primer pair 8(SEQ ID No.:62, 63); amplification products were obtained for all of the primer pairs 9(SEQ ID NO: 64, 65). The DNA polymerase is KOD DNA polymerase with high fidelity from BAO bioengineering GmbH. The PCR products were detected by agarose gel electrophoresis (FIG. 1). The target DNA band is cut off by irradiating under ultraviolet. Then, the amplified DNA fragment was recovered from the agarose Gel using the AIIygen Gel EIItraction Kit (AEYGEN). The DNA fragment was ligated to a commercially available cloning Vector pMD18-T Vector after adding A to the end of rTaq DNA polymerase from Takara Bio Inc., the ligation product was used to transform commercially available E.coli EPI300 competent cells, the transformed E.coli solution was spread on LB plates supplemented with ampicillin 50ug/mL, IPTG 0.5mM, II-Gal 25. mu.g/mL, and recombinant cloning was further verified by PCR and digestion. One of the clones is selected to extract recombinant plasmids and then sequencing is carried out. Open Reading Frames (ORFs) were searched using the BESTORF software. By sequence alignment, ORF encodes the conserved functional domain PSPG box of family 1 of glycosyltransferase, indicating that it is a glycosyltransferase gene.

The gene obtained by using the primer pair 5(SEQ ID NO. 34, 35) has the nucleotide sequence shown in SEQ ID NO. 25, 27 and is respectively named gGT29 and gGT 29-3. Wherein, the corresponding gene information is shown in Table 2.

TABLE 2

SEQ ID NO.:	From the 5' start site	Name of Gene	Action	Sequence of
					25	1-1329	gGT29	Open reading frame
25	1-3	gGT29	Initiation codon	ATG
					25	1327-1329	gGT29	Stop codon	TAG
27	1-1329	gGT29-3	Open reading frame
					27	1-3	gGT29-3	Initiation codon	ATG
27	1327-1329	gGT29-3	Stop codon	TAG

The nucleotide sequence of the gene having gGT 29-7-derived polypeptide shown in Table 3 was obtained using primer set 7(SEQ ID NO.:62, 63), and named gGT29-4, gGT29-5, gGT29-6, gGT29-8, gGT29-9, gGT29-10, gGT29-11, gGT29-12, gGT29-13, gGT29-14, gGT29-17 and gGT 29-18. The corresponding gene information is shown in Table 3.

TABLE 3

The genes obtained with primer pair 8(SEQ ID NO.:64, 65) had the nucleotide sequences shown in Table 4, and were designated gGT29-7, gGT29-15 and gGT 29-16. The corresponding gene information is shown in Table 4.

TABLE 4

SEQ ID NO.:	From the 5' start site	Name of Gene	Function of	Sequence of
					60	1-1341	gGT29-7	Open reading frame
60	1-3	gGT29-7	Initiation codon	ATG
					60	1339-1341	gGT29-7	Stop codon	TAG
86	1-1329	gGT29-15	Open reading frame
					86	1-3	gGT29-15	Initiation codon	ATG
86	1327-1329	gGT29-15	Stop codon	TAG
					88	1-1329	gGT29-16	Open reading frame
88	1-3	gGT29-16	Initiation codon	ATG
					88	1327-1329	gGT29-16	Stop codon	TAG

The proteins involved and their properties are shown in table 5:

TABLE 5

Example 2 construction of Yeast recombinant expression vectors for glycosyltransferase genes gGT29 and gGT29-3

The plasmids gGT29-pMD18T and gGT29-3-pMD18T containing gGT29 and gGT29-3 genes constructed in example 1 were used as templates to amplify the target genes, respectively.

gGT29(SEQ ID NO: 36), with a Kpn I recognition site added to the 5' end: GGATCC; the reverse primers used were all (SEQ ID No.:37) with an IIhoI recognition site added to the 5' end: CTCGAG, reverse primer introduction of 6-His Tag for Western Blot detection of expression and purification.

gGT29-3, wherein the 5' end of the forward primer is added with a Kpn I recognition site: GGATCC; all reverse primers used were (SEQ ID No.:39) with an IIhoI recognition site added to the 5' end: CTCGAG, reverse primer introduction of 6-His Tag for Western Blot detection of expression and purification.

The genes of gGT29 and gGT29-3 were amplified by PCR using the above primers using plasmids gGT29-pMD18T and gGT29-3-pMD18T as templates. The high-fidelity DNA polymerase kod from Toyobo was used as the DNA polymerase, and the PCR procedure was set up by referring to the specification: 2min at 94 ℃; 15s at 94 ℃, 30s at 58 ℃ and 1.5min at 68 ℃ for 30 cycles; 10min at 68 ℃; keeping the temperature at 10 ℃. And detecting the PCR product through agarose gel electrophoresis, and cutting off a band with the size consistent with that of the target DNA under ultraviolet light. Then, the DNA fragment was recovered from the agarose Gel using the AIIyPrep DNA Gel EIItraction Kit from AIIYGEN. The recovered DNA fragment was digested with Quickcut restriction enzymes Kpn I and IIba I of Takara for 30min, and the digested product was collected by cleaning with AIIyPrep PCR clean Kit of AIIYGEN. The cleavage products were ligated with the Saccharomyces cerevisiae expression plasmid pYES2 (likewise cleaved with Kpn I and IIba I and recovered by tapping) using T4 DNA ligase from NEB for 2h at 25 ℃. Coli TOP10 competent cells were transformed and plated on LB plates supplemented with 100. mu.g/mL ampicillin. Positive transformants were verified by colony PCR and sequencing further verified the successful construction of expression plasmids gGT29-pYES2 and gGT29-3-pYES 2.

Example 3 expression of glycosyltransferase genes gGT29 and gGT29-3 in Saccharomyces cerevisiae

The constructed expression plasmids gGT29-pYES2 and gGT29-3-pYES2 were transformed into Saccharomyces cerevisiae (Saccharomyces cerevisiae) by an electrotransformation method, and spread on a screening plate SC-Ura (0.67% yeast has no basic nitrogen source of amino acids, 2% glucose). Yeast recombinants were verified by colony PCR. Yeast recombinant colonies were picked and cultured in 10mL of SC-Ura (2% glucose) medium at 30 ℃ and 200rpm for 20 hours. 3500g of thallus is collected by centrifugation at 4 ℃, the thallus is washed twice by sterile deionized water, the thallus is re-suspended by an induction culture medium SC-Ura (2% galactose), and the thallus is inoculated into 50mL of induction culture medium to ensure OD ₆₀₀ The induction of expression started at around 0.4, 200rpm at 30 ℃. 3500g of thalli for inducing expression for 12h is collected by centrifugation at 4 ℃, the thalli is washed twice by sterile deionized water and is suspended in yeast lysis buffer solution to ensure OD ₆₀₀ Between 50 and 100. The yeast cells were disrupted by shaking with a Fastprep cell disrupter, centrifuged at 12000g at 4 ℃ for 10min to remove cell debris, and the supernatant of the cell lysate was collected. The appropriate amount of lysate supernatant was subjected to SDS-PAGE, and compared with the recombinants of pYES2 empty vector, the gGT29-pYES2 and gGT29-3-pYES2 recombinants had no obvious band representation (FIG. 2). When the expression condition is detected by using anti-6-His Tag Western Blot, Saccharomyces cerevisiae expressing gGT29 and gGT29-3 shows strong Western Blot signals, which indicates that gGT29 and gGT29-3 can be expressed in the yeast in a soluble way, while a recombinant transferred with pYES2 empty vector does not have the anti-6-His Tag Western Blot signal (figure 3).

Example 4 Yeast expression products gGT29 and gGT29-3 transglycosylation reactions and product identification

The recombinant yeast cracking supernatant for expressing gGT29 and gGT29-3 is used as enzyme liquid to catalyze the transglycosylation reaction of ginsenoside Rh2 and F2, and the recombinant yeast cracking supernatant for expressing an empty vector is used as a control. The reaction system of 100. mu.L is shown in Table 3. The reaction was carried out at 35 ℃ for 12h, then 100. mu.L of butanol was added to terminate the reaction and the product was extracted. The product was dried under vacuum and dissolved in methanol.

The reaction product is detected by Thin Layer Chromatography (TLC), and the yeast host lysis supernatant enzyme solution expressing gGT29 and gGT29-3 can extend a glycosyl group at the 3-position of ginsenoside Rh2 and F2 to be converted into ginsenoside Rg3 and Rd (figure 4). gGT29 and gGT29-3 are not affected by the configuration of 20-glycosyl or hydroxyl of ginsenoside, and can convert 20(R) -Rh2 into 20(R) -Rg3 (figure 6).

Example 5 Combined transglycosylation reaction and product identification of the glycosyltransferases BvUGT73C10/UGTPg45 and gGT29

Coli host lysis supernatant expressing BvUGT73C10(JQ291613) or UGTPg45(KM401918) and yeast host lysis supernatant expressing gGT29 were used as enzyme solutions to co-catalyze protopanaxadiol (PPD). The reaction system of 100. mu.L is shown in Table 3. 40. mu.L of the large intestine host lysis supernatant was BvUGT73C10 in 73.4. mu.L of the enzyme solution, and the remaining 33.4. mu.L was gGT 29-expressing yeast host lysis supernatant. The reaction was carried out at 35 ℃ for 12h, then 100. mu.L of butanol was added to terminate the reaction and the product was extracted. The product was dried under vacuum and dissolved in methanol. The reaction product was first detected by Thin Layer Chromatography (TLC) (FIG. 5), and it can be seen that the glycosyltransferases BvUGT73C10 and gGT29 or UGTPg45 and gGT29 in combination can convert PPD to Rg 3.

The glycosyltransferases BvUGT73C10 and gGT29 or 3GT2 and gGT29 in combination catalyze 20(R) -PPD to produce 20(R) -Rg3 (FIG. 6).

Example 6 construction and product identification of Rg 3-producing engineered yeast

6.1 on pESC-HIS plasmid ((Stratagene, Agilent), Dammarenediol synthase) (ACZ71036.1) (GAL1/GAL10 GAL10 side promoter, ADH1 terminator), cytochrome P450CYP716A47 (AEY75213.1) (FBA1 promoter, CYC1 terminator) and glycosyltransferase UGTPg45 and gGT29(GAL1/GAL10 GAL1 side promoter, TDH2 terminator) were assembled together to construct a free plasmid, Saccharomyces cerevisiae BY4742 was transformed, and ATR2-1 (NP-849472.2) derived from Arabidopsis thaliana was integrated into the trp1 gene site (GAL1 promoter, using trp1 original terminator) in Saccharomyces cerevisiae BY4742 chromosome, and recombinant yeast A2 was constructed to supplement the corresponding amino acids of yeast A2 (0.01% tryptophan, 0.01% lysine).

The recombinant yeast A2 lysate was transferred to 2mL EP tubes, each containing 1mL, extracted for about 30min with an equal volume (1mL) of n-butanol and centrifuged at 12000g for 10 min. The supernatant was aspirated into a new EP tube. The n-butanol was evaporated to dryness at 45 ℃ and under vacuum. Dissolved in 100. mu.L of methanol and used for HPLC detection.

Cell lysates of recombinant yeast a2 contained dammarenediol, protopanaxadiol (PPD), and ginsenoside active metabolite Rg3 (fig. 8 and 9) by HPLC and LC-MS analysis.

6.2 the method is the same as 6.1, except that the glycosyl transferase BvUGT73C10 is used for replacing UGTPg45, and the recombinant yeast A6 is obtained. By HPLC analysis, the cell lysate of recombinant yeast A6 also contains dammarenediol, protopanaxadiol (PPD) and ginsenoside active metabolite Rg 3.

Example 7 construction of recombinant expression vectors for glycosyltransferase genes gGT29-4, gGT29-5, gGT29-6 and gGT29-7 in E.coli

The plasmids gGT29-4-pMD18T, gGT29-5-pMD18T, gGT29-6-pMD18T and gGT29-7-pMD18T containing gGT29-4, gGT29-5, gGT29-6 and gGT29-7 genes, which are constructed in example 1, are used as templates to amplify target genes.

gGT29-5 and gGT29-6 genes, and the 5' end of the forward primer is shown in SEQ ID NO. 66, and a sequence which is homologous with a vector pET28a is added: CTGGTGCCGCGCGGCAGC, respectively; the reverse primer used is shown as SEQ ID NO. 68, and a sequence homologous to the vector pET28a is added at the 5' end: TGCGGCCGCAAGCTTGTC are provided.

gGT29-4 and gGT29-7 genes adopt a forward primer of SEQ ID NO. 67, and a sequence which is homologous with a vector pET28a is added at the 5' end of the forward primer: CTGGTGCCGCGCGGCAGC; the reverse primer used was SEQ ID No. 68, to the 5' end of which was added an 18 base fragment homologous to vector pET28 a: TGCGGCCGCAAGCTTGTC are provided.

gGT29-4, gGT29-5, gGT29-6 and gGT29-7 genes were amplified by PCR using the above primers. The Q5 high-fidelity DNA polymerase of NEB company is selected as the amplification gene, and the PCR program is set according to the specification: 30s at 98 ℃; 35 cycles of 98 ℃ for 15s, 58 ℃ for 30s and 72 ℃ for 1 min; 2min at 72 ℃; keeping the temperature at 10 ℃.

Meanwhile, the vector pET28a was amplified using SEQ ID No. 69 and SEQ ID No. 70 as forward and reverse primers, respectively, to obtain the linearized vector pET28 a. The linear vector for pET28a amplification was also obtained using Q5 high fidelity DNA polymerase from NEB, and the PCR program was set up with reference to the description: 30s at 98 ℃; 35 cycles of 98 ℃ for 15s, 58 ℃ for 30s and 72 ℃ for 3 min; 2min at 72 ℃; keeping the temperature at 10 ℃.

The PCR products of the gGT29-4, gGT29-5, gGT29-6 and gGT29-7 genes and the linearized vector pET28a are detected by agarose gel electrophoresis, and bands with the same size as the target DNA are cut under ultraviolet light. Then, the DNA fragment was recovered from the agarose Gel using AxyPrep DNA Gel Extraction Kit from AXYGEN. Referring to the BGclonart seamless cloning kit of NocGreenwich Biotech, 20. mu.l of the recovered linearized pET28a vector fragment, the recovered gGT29-4, gGT29-5, gGT29-6 and gGT29-7 gene fragments and the BGclonart seamless cloning reaction solution of NocGreenwich Biotech are mixed in a proper ratio. After mixing, the mixture was incubated at 50 ℃ for 30 minutes, and then the mixed reaction solution was transferred to ice. Coli EPI300 competent cells were transformed with 5. mu.l of the reaction solution and plated on LB plates supplemented with 50. mu.g/mL kanamycin. Positive transformants were verified by colony PCR and sequenced to further verify the successful construction of expression plasmids gGT29-4-pET28a, gGT29-5-pET28a, gGT29-6-pET28a and gGT29-7-pET28 a.

Example 8 expression of glycosyltransferase genes gGT29-4, gGT29-5, gGT29-6 and gGT29-7 in E.coli

Coli expression vectors gGT29-4-pET28a, gGT29-5-pET28a, gGT29-6-pET28a and gGT29-7-pET28a constructed in example 19 were transformed into E.coli BL21 available on the market. Inoculating a recombinant into LB medium, culturing at 30 deg.C and 200rpm to OD ₆₀₀ About 0.6-0.8, cooling the bacterial liquid to 4 ℃, adding IPTG with the final concentration of 50 mu M, and inducing expression for 15h at 18 ℃ and 200 rpm. The cells were collected by centrifugation at 4 ℃ and disrupted by sonication, and the supernatant of cell lysate was collected by centrifugation at 12000g at 4 ℃ and subjected to SDS-PAGE on a sample (FIG. 10). gGT29-4-pET28a, gGT29-5-pET28a, gGT29-6-pET28a and gGT29-7-pET28a recombinant lysates and distinct bands of the protein of interest (approximately 50kD) in the total protein and supernatant were obtained, characterizing glycosyltransferases gGT29-4, gGT29-5, gGT29-6 and gG, respectivelyT29-7. From the results of Western Blot (FIG. 11), it was also confirmed that the target proteins gGT29-4, gGT29-5, gGT29-6 and gGT29-7 were expressed in a soluble form in the host.

Example 9 E.coli expression products gGT29-4, gGT29-5, gGT29-6 and gGT29-7 transglycosylation reactions and identification of the products

The recombinant yeast cracked supernatant expressed by gGT29-4, gGT29-5, gGT29-6 and gGT29-7 is used as an enzyme solution to catalyze the transglycosylation reaction of ginsenoside Rh2 and F2. The reaction system of 100. mu.L is shown in Table 6. The reaction was carried out at 35 ℃ for 12h, then 100. mu.L of butanol was added to terminate the reaction and the product was extracted. The product was dried under vacuum and dissolved in methanol.

TABLE 6

9% Tween 20	11.1μL
		50mM UDP-glucose	10μL
1M Tris-HCl pH8.5	5μL
		100mM substrate (ethanol dissolved)	0.5μL
Enzyme solution	73.4μL

Detecting the reaction product by Thin Layer Chromatography (TLC), wherein the crude enzyme solution of gGT29-6 can be obtained by extending a glycosyl on 3-position glycosyl of ginsenoside Rh2 and F2 to obtain ginsenoside Rg3 and Rd (FIG. 12); gGT29-4, gGT29-5 and gGT29-7 can extend a glycosyl group at position 3 of ginsenoside F2 to generate saponin Rd, but they can not catalyze saponin Rh2 (FIG. 12). gGT29-4, gGT29-5, gGT29-6 and gGT29-7 crude enzyme solutions can further extend a glycosyl on C-6 position of protopanaxatriol type saponin Rh1 to form ginsenoside Rf (FIG. 13), wherein gGT29-4, gGT29-5 and gGT29-6 have weaker activity, and gGT29-7 has stronger activity (Table 7).

Example 10 construction of recombinant expression vectors for glycosyltransferase genes gGT29-8, gGT29-9, gGT29-10, gGT29-11, gGT29-12, gGT29-13, gGT29-14, gGT29-15, gGT29-16, gGT29-17, gGT29-18 E.coli, expression in E.coli and product identification

Methods as in examples 7 to 8, expression vectors for E.coli (gGT29-8-pET28 gGT29, gGT29-9, gGT29-12, gGT29-13, gGT29-14, gGT29-15, gGT29-16, gGT29-17, gGT 29-18) were constructed for gGT29-8, gGT29-9-pET28 gGT29, gGT29-10-pET28 gGT29, gGT29-11-pET28 gGT29, gGT29-12-pET28 gGT29, gGT29-13-pET28 gGT29, gGT29-14-pET28 gGT29, gGT29-15-pET28 gGT29, gGT29-16-pET28 gGT29, gGT29-17-pET28 gGT29, gGT29-18-pET28 gGT 29) and soluble expression was achieved in E.coli. The elongation activity of the first glycosyl residue at a different position (C3 or C6) on the substrate from the other gGT29 series glycosyltransferases identified according to the method described in example 9 is shown in Table 7, where "+" indicates activity at that position and "+" indicates a relatively strong activity at that position.

TABLE 7

Example 11 Change of specificity of gGT29-7 for glycosyl Donor by amino acid site-directed mutagenesis

The glycosyl transferase gGT29-7 can take UDP-glucose as glycosyl donor to catalyze C6-O-Glc of ginsenoside Rh1 to extend one glucose to generate ginsenoside Rf. The specificity of glycosyltransferase gGT29-7 for its glycosyl donor was altered by site-directed mutagenesis of the glycosyl donor-binding region (PSPG). The amino acid sequence of rhamnosyl transferase which is reported to be capable of catalyzing other glycosyl acceptors in the prior research is compared with gGT29-7, and two sites on a gGT29-7 PSPG box are screened and finally selected for site-directed mutagenesis (N343G and A359P).

Two primers were designed and synthesized by the conventional method using Hieff Mut from Yeasen as a template and plasmid pET28a-gGT29-7 as a template ^TM site-directed mutagenesis kit carries out site-directed mutagenesis at position gGT 29-7N 343, respectively.

The PCR amplification procedure was: 30s at 95 ℃; 5s at 95 ℃, 10s at 56 ℃ and 1.5min at 72 ℃ for 30 cycles; the temperature is reduced to 10 ℃. The PCR product was digested with DpnI and reacted in 37 ℃ water bath for 2 h. Then 3 μ L of the recombinant enzyme EIInase in the kit is used for recombination at 37 ℃ for 30min, and the TOP10 competence of the escherichia coli is transformed. A single clone is picked and extracted, the sequence is carried out to verify the mutation site, and the obtained plasmid is named as pET28a-gGT 29-7-N343G.

Plasmid pET28a-gGT29-7-N343G was transformed into E.coli BL21(DE3), and recombinant pET28a-gGT29-7-N343G-BL21 was constructed, and the procedure for inducing expression was the same as in example 8.

The A359 site of gGT29-7 was point-mutated by conventionally designing and synthesizing two primer pairs and constructing mutant plasmid pET28a-gGT29-7-A359P and recombinant strain gGT29-7-N343G-BL21, as above. The procedure for inducing expression was the same as in example 8. A plasmid pET28a-gGT29-7-N343G is used as a template, 29-7-A359P-F and 29-7-A359P-R are used as primers, and A359 is subjected to point mutation to construct a double-mutation plasmid pET28a-gGT29-7-N343G/A359P and a recombinant strain gGT29-7-N343G/A359P-BL21, and the steps are the same as above. The procedure for induction of expression was the same as in example 8, transglycosylation reaction and identification of the product were the same as in example 9, and the reaction system was as shown in Table 6, except that UDP-glucose/UDP-rhamnose mixed in a ratio of 1/1 was used instead of UDP-glucose as the glycosyl donor.

The result is shown in FIG. 14, the enzyme activity of the single mutein gGT29-7-N343G for catalyzing the C6-O-Glc of Rh1 to extend one glucose to generate Rf is not detected basically, and the enzyme activity of gGT29-7-A359P for catalyzing the C6-O-Glc of Rh1 to extend one glucose is still remained. The double mutant protein gGT29-7-N343G/A359P not only retains the enzyme activity of wild protein for catalyzing C6-O-Glc of Rh1 to extend one glucose to generate Rf, but also obtains the enzyme activity of catalyzing C6-O-Glc of Rh1 to extend one rhamnose to generate Rg 2.

Example 12 transglycosylation reaction of E.coli expression product of glycosyltransferase with UDP-xylose as glycosyl Donor and identification of the product

Recombinant Escherichia coli supernatants expressing gGT29-10 and gGT29-14 are used as enzyme liquid to catalyze the transglycosylation reaction of the ginsenosides Rh2 and F2. The 100. mu.L reaction system is shown in Table 6, but UDP-xylose was used instead of UDP-glucose as the glycosyl donor. The reaction was carried out at 35 ℃ for 12h, then 100. mu.L of butanol was added to terminate the reaction and the product was extracted. The product was dried under vacuum and dissolved in methanol.

The reaction product is detected by Thin Layer Chromatography (TLC), gGT29-10 and gGT29-14 can extend one xylose in C-3 glycosyl of ginsenoside Rh2 to generate a new triterpene saponin, (3-O-beta- (D-xylopyranosyl) -beta- (D-glucopyranosyl) -PPD; gGT29-10 and gGT29-14 can replace the second glucose at C-3 position of ginsenoside Rg3 with xylose to generate a new triterpene saponin (3-O-beta- (D-xylopyranosyl) -beta- (D-glucopyranosyl) -PPD); gGT29-10 and gGT29-14 can also replace the second glucose at C-3 position of ginsenoside Rd with xylose to generate a new triterpene saponin (3-O-beta- (D-xylopyranosyl) -beta- (D-glucopyranosyl) -CK) (FIG. 15).

Other glycosyltransferases gGT29, gGT29-4, gGT29-5, gGT29-6 and, gGT29-7 gGT29-8, gGT29-9, gGT29-10, gGT29-11, gGT29-12, gGT29-13, gGT29-15, gGT29-16, gGT29-17 and gGT29-18, with UDP-xylose as the glycosyl donor, catalyze the activities of Rh2, Rg3 and Rd as shown in Table 8.

TABLE 8

Example 13 construction of engineering bacteria producing Rf Yeast and identification of the products

On pESC-HIS plasmid ((Stratagene, Agilent), Dammarenediol synthase (ACZ71036.1) (GAL1/GAL10 GAL10 side promoter, ADH1 terminator), cytochrome P450CYP716A47 (AEY75213.1) (FBA1 promoter, CYC1 terminator), cytochrome P450CYP716A53V2 gene (ENO2 promoter, CYC1 terminator) and glycosyltransferase gene UGTPg100(KP795113) (GAL1/GAL10 side promoter, GAL1 terminator) and glycosyltransferase gGT29-7(TEF1 promoter, FBA1 terminator) were simultaneously assembled to construct Arabidopsis thaliana episomal plasmid, Saccharomyces cerevisiae BY4742 was transformed, and cytochrome P450 reductase 9-1 (NP 6862) derived from Saccharomyces cerevisiae was integrated into Saccharomyces cerevisiae chromosomal gene site (GAL 8454, GAL 1) of Saccharomyces cerevisiae, recombinant DNA (GAL 1) using the original chromosomal gene promoter, GAL1, and chromosomal DNA fragment 865 was constructed, 0.01% leucine, 0.01% lysine).

The recombinant yeast A7 lysate was transferred to 2mL EP tubes, each containing 1mL, extracted for about 30min with an equal volume (1mL) of n-butanol and centrifuged at 12000g for 10 min. The supernatant was pipetted into a new EP tube. The n-butanol was evaporated to dryness at 45 ℃ and under vacuum. Dissolved in 100. mu.L of methanol and used for HPLC detection.

By HPLC analysis, the cell lysate of recombinant yeast A7 contains protopanaxatriol (PPT) and ginsenoside active metabolites Rh1 and Rf.

Example 14 construction and product identification of Rg 2-producing engineered yeast

On pESC-HIS plasmid ((Stratagene, Agilent), Dammarenediol synthase (Dammarenediol synthase) (ACZ71036.1) (GAL1/GAL10 GAL10 side promoter, ADH1 terminator), cytochrome P450CYP716A47 (AEY75213.1) (FBA1 promoter, CYC1 terminator), cytochrome P450CYP716A53V2 gene (ENO2 promoter, CYC 2 terminator) and two glycosyltransferase genes TPg100(KP795113) (GAL 2/GAL 2 side promoter, GAL 2 terminator) and 2-7-N343 2/A2 (TEF 2 promoter, FBA 2 terminator) were simultaneously assembled, a episomal plasmid was constructed, Saccharomyces cerevisiae BY4742 was transformed, and cytochrome P450-reductase-1 (ATR 2-2) (GAL promoter derived from Saccharomyces cerevisiae, GAL3672 terminator was synthesized BY using the chromosomal DNA of the RHA 2, GAL3672 terminator of the RG2 gene (RG 2) and the chromosomal DNA 2, GAL3672 terminator were integrated in the chromosomal DNA 2, a recombinant yeast A8 was constructed. The recombinant yeast needs to be supplemented with the corresponding amino acids (0.01% tryptophan, 0.01% leucine, 0.01% lysine).

The recombinant yeast A8 lysate was transferred to 2mL EP tubes, each containing 1mL, extracted for about 30min with an equal volume (1mL) of n-butanol and centrifuged at 12000g for 10 min. The supernatant was pipetted into a new EP tube. The n-butanol was evaporated to dryness at 45 ℃ and under vacuum. Dissolved in 100. mu.L of methanol and used for HPLC detection. Through HPLC analysis, the cell lysate of the recombinant yeast A8 contains protopanaxatriol (PPT) and ginsenoside active metabolites Rh1 and Rg 2.

All documents referred to herein are incorporated by reference into this application as if each had been individually incorporated by reference. Furthermore, it should be understood that various changes or modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the appended claims of the present application.

Sequence listing

<110> China academy of sciences molecular plant science remarkable innovation center

<120> a group of glycosyltransferases and uses thereof

<130> P2015-0697

<150> CN201410254021.6

<151> 2014-06-09

<160> 95

<170> PatentIn version 3.5

<210> 1

<211> 1425

<212> DNA

<213> Ginseng radix (Panax ginseng)

<220>

<221> misc_feature

<223> gGT25

<400> 1

atgaagtcag aattgatatt cttgcccgcc ccggccatcg gacacctcgt gggaatggtg 60

gagatggcta aactcttcat cagtcgacat gaaaacctct cggtcaccgt cctcatcgcg 120

aaattctaca tggatacggg ggtagacaac tacaataaat cactcttaac aaaccctacc 180

ccgcgtctca caattgtaaa tctcccggaa accgaccccc aaaactatat gctcaaacca 240

cgccatgcca tctttcctag cgtcatcgag actcagaaga cacacgtgcg agacataata 300

tcaggcatga ctcagtccga gtcgactcgg gtcgttggtt tgctggctga ccttttgttc 360

atcaacatta tggacattgc caatgagttc aatgttccaa cttatgtata ctcccctgcc 420

ggagccggtc atcttggcct cgcgttccat ctccagacac tcaacgacaa aaagcaagat 480

gtgaccgagt tcaggaactc ggacactgag ttattggtac cgagttttgc aaacccggtt 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 caaaactccg atgaggctgc tgtgatcctt 780

ggttggttag atgatcaacc accttcatct gtggtgtttt tgtgctttgg tagctatgga 840

agctttcaag aaaaccaggt gaaggagatt gcaatgggtc tagagcgcag tgggcatcgc 900

ttcttgtggt ccttgcgtcc gtctatccct aaaggcgaga caaagcttca gcttaaatac 960

tcaaatttga aagaaattct cccagtagga ttcttggaca ggacatcatg cgtcggaaaa 1020

gtgattggat gggccccgca agtggccgtg ctcggacatg agtcagtcgg agggttcctg 1080

tctcattgcg gttggaattc gacattggag agtgtttggt gtggggtgcc cgttgcaaca 1140

tggccaatgt atggtgagca acaactcaat gcttttgaga tggttaagga gttaggtatt 1200

gcggtggaaa ttgaggtgga ctataagaaa gattatttta acatgaagaa tgattttatt 1260

gttagggcag aagaaatcga gacaaaaata aagaagttga tgatggatga aaataatagt 1320

gaaataagaa agaaggtaaa ggaaatgaaa gaaaagagta gggctgcaat gtctgagaat 1380

ggatcatctt ataattcatt ggcgaagcta tttgaggaaa ttatg 1425

<210> 2

<211> 475

<212> PRT

<213> Ginseng radix (Panax ginseng)

<220>

<221> misc_feature

<223> gGT25

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Met Lys Ser Glu Leu Ile Phe Leu Pro Ala Pro Ala Ile Gly His Leu

1 5 10 15

Val Gly Met Val Glu Met Ala Lys Leu Phe Ile Ser Arg His Glu Asn

20 25 30

Leu Ser Val Thr Val Leu Ile Ala Lys Phe Tyr Met Asp Thr Gly Val

35 40 45

Asp Asn Tyr Asn Lys Ser Leu Leu Thr Asn Pro Thr Pro Arg Leu Thr

50 55 60

Ile Val Asn Leu Pro Glu Thr Asp Pro Gln Asn Tyr Met Leu Lys Pro

65 70 75 80

Arg His Ala Ile Phe Pro Ser Val Ile Glu Thr Gln Lys Thr His Val

85 90 95

Arg Asp Ile Ile Ser Gly Met Thr Gln Ser Glu Ser Thr Arg Val Val

100 105 110

Gly Leu Leu Ala Asp Leu Leu Phe Ile Asn Ile Met Asp Ile Ala Asn

115 120 125

Glu Phe Asn Val Pro Thr Tyr Val Tyr Ser Pro Ala Gly Ala Gly His

130 135 140

Leu Gly Leu Ala Phe His Leu Gln Thr Leu Asn Asp Lys Lys Gln Asp

145 150 155 160

Val Thr Glu Phe Arg Asn Ser Asp Thr Glu Leu Leu Val Pro Ser Phe

165 170 175

Ala Asn Pro Val Pro Ala Glu Val Leu Pro Ser Met Tyr Val Asp Lys

180 185 190

Glu Gly Gly Tyr Asp Tyr Leu Phe Ser Leu Phe Arg Arg Cys Arg Glu

195 200 205

Ser Lys Ala Ile Ile Ile Asn Thr Phe Glu Glu Leu Glu Pro Tyr Ala

210 215 220

Ile Asn Ser Leu Arg Met Asp Ser Met Ile Pro Pro Ile Tyr Pro Val

225 230 235 240

Gly Pro Ile Leu Asn Leu Asn Gly Asp Gly Gln Asn Ser Asp Glu Ala

245 250 255

Ala Val Ile Leu Gly Trp Leu Asp Asp Gln Pro Pro Ser Ser Val Val

260 265 270

Phe Leu Cys Phe Gly Ser Tyr Gly Ser Phe Gln Glu Asn Gln Val Lys

275 280 285

Glu Ile Ala Met Gly Leu Glu Arg Ser Gly His Arg Phe Leu Trp Ser

290 295 300

Leu Arg Pro Ser Ile Pro Lys Gly Glu Thr Lys Leu Gln Leu Lys Tyr

305 310 315 320

Ser Asn Leu Lys Glu Ile Leu Pro Val Gly Phe Leu Asp Arg Thr Ser

325 330 335

Cys Val Gly Lys Val Ile Gly Trp Ala Pro Gln Val Ala Val Leu Gly

340 345 350

His Glu Ser Val Gly Gly Phe Leu Ser His Cys Gly Trp Asn Ser Thr

355 360 365

Leu Glu Ser Val Trp Cys Gly Val Pro Val Ala Thr Trp Pro Met Tyr

370 375 380

Gly Glu Gln Gln Leu Asn Ala Phe Glu Met Val Lys Glu Leu Gly Ile

385 390 395 400

Ala Val Glu Ile Glu Val Asp Tyr Lys Lys Asp Tyr Phe Asn Met Lys

405 410 415

Asn Asp Phe Ile Val Arg Ala Glu Glu Ile Glu Thr Lys Ile Lys Lys

420 425 430

Leu Met Met Asp Glu Asn Asn Ser Glu Ile Arg Lys Lys Val Lys Glu

435 440 445

Met Lys Glu Lys Ser Arg Ala Ala Met Ser Glu Asn Gly Ser Ser Tyr

450 455 460

Asn Ser Leu Ala Lys Leu Phe Glu Glu Ile Met

465 470 475

<210> 3

<211> 1431

<212> DNA

<213> Ginseng (Panax ginseng)

<220>

<221> misc_feature

<223> gGT13

<400> 3

atgggagcgg agctcatctt aatcccttct ccgggagttg gtcatctggt atctactgtt 60

gagattgcaa agctcctcat cagtcgagat gaacggcttt ccatcacagt ccttgtaatg 120

aagttctcgc atgacactgg tgtgactgcc tacactcggt cattgcagaa agatgctcct 180

aaccgcatag tctttgtgga ccttcctcag aacgagtctc ttatctcatc gcccaagtct 240

ttctttacta gcttcatcga gagtcagacg agtccggtta gagattctgt cagacaaatt 300

gtgagtcggt ctgattctaa taagctcgct ggcttcgtca tcgacatgtt ctgcacccca 360

atgatagacg tggcaaatga atttggagtc ccaacctatg tgttcttcac ttcaggtgct 420

gcatttcttg gcctccagtt ttaccatctg agtctcagtg atgaacataa ccaggacctt 480

accgagtata aggacacgga tgttgagtta tctatcccga gtttcatcaa cccagtgccc 540

gctaaggttt tgccttcggt gattctgaac aaggaaggat cgaccatgct ccaatctatt 600

tcccgaaggt ttaaagaagc caaggccatt ctagtcaaca cgttcgcgga gctggaacca 660

catgccatta aggcccttgg tgataactgc aagatccctc ctatctatcc cgtgggaccc 720

ataatcaacc tcaagaacaa ggagggaaca acccaaaacc atagttctga agatggtatc 780

attagctggt tggacaatca gccaccatct tcggtagtgt ttttgtgctt tgggagcttt 840

gggagctttg atgaaggcca agtcagggag atagcaaacg gattggagca gagtggacag 900

cgattcttgt ggtctctacg ccggcggcca gaaaaaatgg aattgcctaa agactatgag 960

aatcccgaag aagtgttgcc agaaggattc atagaacgaa catcagggat ggggaaggtg 1020

atcggatggg cgccacaaac ggcgattctt tcccaccctg ctgtgggagg attcgtgtct 1080

cattgtggat ggaattctac attggagagt atatggtgtg gggttccaat ggctacttgg 1140

cctatatatg cagagcagca aatcaatgcg tttgagttgg tgaaggagtt gggaatggct 1200

gtggagatca aaatggatta cagagaagat tatatttttg cacctgaaaa taatttagtt 1260

gtgaccgcag accagataga gaaaggaatg cgatgtctga tgatggatgg agagagtgaa 1320

atgaggaaga aggtggaaga gatgaaagag aagagcagaa tggccatggt gaagggcggg 1380

tcttcttaca tttcactcgg gcattttatt gaggatgtca tgcgtaatta a 1431

<210> 4

<211> 476

<212> PRT

<213> Ginseng radix (Panax ginseng)

<220>

<221> misc_feature

<223> gGT13

<400> 4

Met Gly Ala Glu Leu Ile Leu Ile Pro Ser Pro Gly Val Gly His Leu

1 5 10 15

Val Ser Thr Val Glu Ile Ala Lys Leu Leu Ile Ser Arg Asp Glu Arg

20 25 30

Leu Ser Ile Thr Val Leu Val Met Lys Phe Ser His Asp Thr Gly Val

35 40 45

Thr Ala Tyr Thr Arg Ser Leu Gln Lys Asp Ala Pro Asn Arg Ile Val

50 55 60

Phe Val Asp Leu Pro Gln Asn Glu Ser Leu Ile Ser Ser Pro Lys Ser

65 70 75 80

Phe Phe Thr Ser Phe Ile Glu Ser Gln Thr Ser Pro Val Arg Asp Ser

85 90 95

Val Arg Gln Ile Val Ser Arg Ser Asp Ser Asn Lys Leu Ala Gly Phe

100 105 110

Val Ile Asp Met Phe Cys Thr Pro Met Ile Asp Val Ala Asn Glu Phe

115 120 125

Gly Val Pro Thr Tyr Val Phe Phe Thr Ser Gly Ala Ala Phe Leu Gly

130 135 140

Leu Gln Phe Tyr His Leu Ser Leu Ser Asp Glu His Asn Gln Asp Leu

145 150 155 160

Thr Glu Tyr Lys Asp Thr Asp Val Glu Leu Ser Ile Pro Ser Phe Ile

165 170 175

Asn Pro Val Pro Ala Lys Val Leu Pro Ser Val Ile Leu Asn Lys Glu

180 185 190

Gly Ser Thr Met Leu Gln Ser Ile Ser Arg Arg Phe Lys Glu Ala Lys

195 200 205

Ala Ile Leu Val Asn Thr Phe Ala Glu Leu Glu Pro His Ala Ile Lys

210 215 220

Ala Leu Gly Asp Asn Cys Lys Ile Pro Pro Ile Tyr Pro Val Gly Pro

225 230 235 240

Ile Ile Asn Leu Lys Asn Lys Glu Gly Thr Thr Gln Asn His Ser Ser

245 250 255

Glu Asp Gly Ile Ile Ser Trp Leu Asp Asn Gln Pro Pro Ser Ser Val

260 265 270

Val Phe Leu Cys Phe Gly Ser Phe Gly Ser Phe Asp Glu Gly Gln Val

275 280 285

Arg Glu Ile Ala Asn Gly Leu Glu Gln Ser Gly Gln Arg Phe Leu Trp

290 295 300

Ser Leu Arg Arg Arg Pro Glu Lys Met Glu Leu Pro Lys Asp Tyr Glu

305 310 315 320

Asn Pro Glu Glu Val Leu Pro Glu Gly Phe Ile Glu Arg Thr Ser Gly

325 330 335

Met Gly Lys Val Ile Gly Trp Ala Pro Gln Thr Ala Ile Leu Ser His

340 345 350

Pro Ala Val Gly Gly Phe Val Ser His Cys Gly Trp Asn Ser Thr Leu

355 360 365

Glu Ser Ile Trp Cys Gly Val Pro Met Ala Thr Trp Pro Ile Tyr Ala

370 375 380

Glu Gln Gln Ile Asn Ala Phe Glu Leu Val Lys Glu Leu Gly Met Ala

385 390 395 400

Val Glu Ile Lys Met Asp Tyr Arg Glu Asp Tyr Ile Phe Ala Pro Glu

405 410 415

Asn Asn Leu Val Val Thr Ala Asp Gln Ile Glu Lys Gly Met Arg Cys

420 425 430

Leu Met Met Asp Gly Glu Ser Glu Met Arg Lys Lys Val Glu Glu Met

435 440 445

Lys Glu Lys Ser Arg Met Ala Met Val Lys Gly Gly Ser Ser Tyr Ile

450 455 460

Ser Leu Gly His Phe Ile Glu Asp Val Met Arg Asn

465 470 475

<210> 5

<211> 1353

<212> DNA

<213> Ginseng radix (Panax ginseng)

<220>

<221> misc_feature

<223> gGT30

<400> 5

atggcaaccc aaaaatgttt ccgtgtcctc gtgttcccat ggttagctca tggtcacatc 60

tcccccttcc tagaactagc cagaaaactt atagaaaaag gaaacttttt catttacttc 120

tgctccactc ctataaatct catctccata aagaaaaaac ttagtggtga tgatcatcaa 180

aattacacca agtcaataca actagtggaa cacaatctac cgaccttacc tcaacttcct 240

cctcactacc acaccaccga cggcctccca ccaaatctca atcctaccct tcgaaaggca 300

tttgaaatgt caaaactatc cttccccaac accctaaaca ctttgaaacc agatctactc 360

atttgtgatg acttatttca atggccagaa atagtagctt catcacatgg tgttccggtt 420

gttcggttcc aaacatgcag cgtgacagcc gctagttttc tagctcatat ttttacgaac 480

ccagacgtta catacccttt tccagccatt tatcttcatg agtatgaaac tgatcagatc 540

aggcgttgcg ttgacgcggt ttttgaaagc ggcagagaag aatctcgcaa cctgttggtt 600

gtcaacacgt ctaaagcgat cgaggaaaaa tatttttatt attattctct acttcggggt 660

aacaccaaaa ttatgccagt tggtccgctt attcagcaag ccccaaatgg cgacgaggat 720

atgaaggtca tcgaatggct tgacaagaag gatccgtgtt caaccgtgtt cgtgtccttt 780

gggagcgagt attttatgca aaaagaagag gttgaagaga tggctcatgg tttagagctt 840

agcaacgtca atttcatctg ggtttttagg gctccggtgg gagcggaaaa ggttaagctg 900

ccgttagggt ttgttgagag ggttgggggg aggggaattg ttatggaggg gtgggcccca 960

caggcaagga ttttgggaca ttcaagtatt ggtgggtttg tgagtcattg tgggtggaat 1020

tctgtgttgg aaactataaa ttttggtgtt ccaataatag ggatgccaat gaaatttgag 1080

cagcctatga atgctaggct tctatctgaa cttggtgttt gtgttgaaat tgtgggggac 1140

gaaactagga ggtttggaag agaagaggta ggaaatgtaa taagagaagt ggttggtggg 1200

aagatagggg acgatttgag aaggaaagtg aaagaacttg gagcaaagat aaaggaaaaa 1260

caagaggaag agatggatga tgtgcttgat gaattagtac aaatttgtaa caagaaaaaa 1320

cggactgttg tacatcatca tcatcatcat tga 1353

<210> 6

<211> 450

<212> PRT

<213> Ginseng (Panax ginseng)

<220>

<221> misc_feature

<223> gGT30

<400> 6

Met Ala Thr Gln Lys Cys Phe Arg Val Leu Val Phe Pro Trp Leu Ala

1 5 10 15

His Gly His Ile Ser Pro Phe Leu Glu Leu Ala Arg Lys Leu Ile Glu

20 25 30

Lys Gly Asn Phe Phe Ile Tyr Phe Cys Ser Thr Pro Ile Asn Leu Ile

35 40 45

Ser Ile Lys Lys Lys Leu Ser Gly Asp Asp His Gln Asn Tyr Thr Lys

50 55 60

Ser Ile Gln Leu Val Glu His Asn Leu Pro Thr Leu Pro Gln Leu Pro

65 70 75 80

Pro His Tyr His Thr Thr Asp Gly Leu Pro Pro Asn Leu Asn Pro Thr

85 90 95

Leu Arg Lys Ala Phe Glu Met Ser Lys Leu Ser Phe Pro Asn Thr Leu

100 105 110

Asn Thr Leu Lys Pro Asp Leu Leu Ile Cys Asp Asp Leu Phe Gln Trp

115 120 125

Pro Glu Ile Val Ala Ser Ser His Gly Val Pro Val Val Arg Phe Gln

130 135 140

Thr Cys Ser Val Thr Ala Ala Ser Phe Leu Ala His Ile Phe Thr Asn

145 150 155 160

Pro Asp Val Thr Tyr Pro Phe Pro Ala Ile Tyr Leu His Glu Tyr Glu

165 170 175

Thr Asp Gln Ile Arg Arg Cys Val Asp Ala Val Phe Glu Ser Gly Arg

180 185 190

Glu Glu Ser Arg Asn Leu Leu Val Val Asn Thr Ser Lys Ala Ile Glu

195 200 205

Glu Lys Tyr Phe Tyr Tyr Tyr Ser Leu Leu Arg Gly Asn Thr Lys Ile

210 215 220

Met Pro Val Gly Pro Leu Ile Gln Gln Ala Pro Asn Gly Asp Glu Asp

225 230 235 240

Met Lys Val Ile Glu Trp Leu Asp Lys Lys Asp Pro Cys Ser Thr Val

245 250 255

Phe Val Ser Phe Gly Ser Glu Tyr Phe Met Gln Lys Glu Glu Val Glu

260 265 270

Glu Met Ala His Gly Leu Glu Leu Ser Asn Val Asn Phe Ile Trp Val

275 280 285

Phe Arg Ala Pro Val Gly Ala Glu Lys Val Lys Leu Pro Leu Gly Phe

290 295 300

Val Glu Arg Val Gly Gly Arg Gly Ile Val Met Glu Gly Trp Ala Pro

305 310 315 320

Gln Ala Arg Ile Leu Gly His Ser Ser Ile Gly Gly Phe Val Ser His

325 330 335

Cys Gly Trp Asn Ser Val Leu Glu Thr Ile Asn Phe Gly Val Pro Ile

340 345 350

Ile Gly Met Pro Met Lys Phe Glu Gln Pro Met Asn Ala Arg Leu Leu

355 360 365

Ser Glu Leu Gly Val Cys Val Glu Ile Val Gly Asp Glu Thr Arg Arg

370 375 380

Phe Gly Arg Glu Glu Val Gly Asn Val Ile Arg Glu Val Val Gly Gly

385 390 395 400

Lys Ile Gly Asp Asp Leu Arg Arg Lys Val Lys Glu Leu Gly Ala Lys

405 410 415

Ile Lys Glu Lys Gln Glu Glu Glu Met Asp Asp Val Leu Asp Glu Leu

420 425 430

Val Gln Ile Cys Asn Lys Lys Lys Arg Thr Val Val His His His His

435 440 445

His His

450

<210> 7

<211> 26

<212> DNA

<213> Ginseng radix (Panax ginseng)

<400> 7

ccaagttcat tcaagatgaa gtcaga 26

<210> 8

<211> 28

<212> DNA

<213> oligonucleotides

<400> 8

cataatttcc tcaaatagct tcgccaat 28

<210> 9

<211> 24

<212> DNA

<213> oligonucleotides

<400> 9

actggagcag ttgcaacata caaa 24

<210> 10

<211> 24

<212> DNA

<213> oligonucleotides

<400> 10

tcatttatgc gctttcctgg atgt 24

<210> 11

<211> 21

<212> DNA

<213> oligonucleotides

<400> 11

acgagggtgg ttttaatggc a 21

<210> 12

<211> 27

<212> DNA

<213> oligonucleotides

<400> 12

acaccattca taatcataca acagtcc 27

<210> 13

<211> 29

<212> DNA

<213> oligonucleotides

<400> 13

atatggtacc atggcagtag ccggcgctg 29

<210> 14

<211> 57

<212> DNA

<213> oligonucleotides

<400> 14

gcgctctaga ttaatgatga tgatgatgat gttttattat atgttttttg gggtcgc 57

<210> 15

<211> 1428

<212> DNA

<213> Ginseng radix (Panax ginseng)

<220>

<221> misc_feature

<223> gGT25-1

<400> 15

atgaagtcag aattgatatt cttgcccgcc ccggccatcg gacacctcgt gggaatggtg 60

gagatggcta aactcttcat cagtcgacat gaaaacctct cggtcaccgt cctcatcgcg 120

aaattctaca tggatacggg ggtagacaac tacaataaat cactcttaac aaaccctacc 180

ccgcgtctca caattgtaaa tctcccggaa accgaccccc aaaactatat gctcaaacca 240

cgccatgcca tctttcctag cgtcatcgag actcagaaga cacacgtgcg agacataata 300

tcaggcatga ctcagtccga gtcgactcgg gtcgttggtt tgctggctga ccttttgttc 360

atcaacatta tggacattgc caatgagttc aatgttccaa cttatgtata ctcccctgcc 420

ggagccggtc atcttggcct cgcgttccat ctccagacac tcaacgacaa aaagcaagat 480

gtgaccgagt tcaggaactc ggacactgag ttattggtac cgagttttgc aaacccggtt 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 caaaactccg atgaggctgc tgtgatcctt 780

ggttggttag atgatcaacc accttcatct gtggtgtttt tgtgctttgg tagctatgga 840

acctttcaag aaaaccaggt gaaggagatt gcaatgggtc tagagcgcag tgggcatcgc 900

ttcttgtggt ccttgcgtcc gtctatccct aaaggcgaga caaagcttca gcttaaatac 960

tcaaatttgg aagaaattct cccagtcgga ttcttggaca ggacatcatg cgtcggaaaa 1020

gttattggat gggccccgca agtggcggtg ctcggacacg aggcagtcgg agggttcctg 1080

tctcattgtg gttggaattc gacattagag agtgtgtggt gtggcgtgcc cgtcgcaaca 1140

tggccaatgt acggcgagca acaactcaat gcttttgaga tggttaagga gttaggtatt 1200

gcggtggaaa ttgaggtgga ctataagaat gaatatttta acatgaataa tgattttatt 1260

gttagggcag aagaaatcga gacgaaaata aagaagttga tgatggatga aaagaatagt 1320

gaaataagga agaaggtaaa ggaaatgaaa gaaaagagta ggcttgcaat gtctgagaat 1380

ggatcatctt ataattcatt ggcgaagcta tttgaggaaa tcatgtaa 1428

<210> 16

<211> 475

<212> PRT

<213> Ginseng radix (Panax ginseng)

<220>

<221> misc_feature

<223> gGT25-1

<400> 16

Met Lys Ser Glu Leu Ile Phe Leu Pro Ala Pro Ala Ile Gly His Leu

1 5 10 15

Val Gly Met Val Glu Met Ala Lys Leu Phe Ile Ser Arg His Glu Asn

20 25 30

Leu Ser Val Thr Val Leu Ile Ala Lys Phe Tyr Met Asp Thr Gly Val

35 40 45

Asp Asn Tyr Asn Lys Ser Leu Leu Thr Asn Pro Thr Pro Arg Leu Thr

50 55 60

Ile Val Asn Leu Pro Glu Thr Asp Pro Gln Asn Tyr Met Leu Lys Pro

65 70 75 80

Arg His Ala Ile Phe Pro Ser Val Ile Glu Thr Gln Lys Thr His Val

85 90 95

Arg Asp Ile Ile Ser Gly Met Thr Gln Ser Glu Ser Thr Arg Val Val

100 105 110

Gly Leu Leu Ala Asp Leu Leu Phe Ile Asn Ile Met Asp Ile Ala Asn

115 120 125

Glu Phe Asn Val Pro Thr Tyr Val Tyr Ser Pro Ala Gly Ala Gly His

130 135 140

Leu Gly Leu Ala Phe His Leu Gln Thr Leu Asn Asp Lys Lys Gln Asp

145 150 155 160

Val Thr Glu Phe Arg Asn Ser Asp Thr Glu Leu Leu Val Pro Ser Phe

165 170 175

Ala Asn Pro Val Pro Ala Glu Val Leu Pro Ser Met Tyr Val Asp Lys

180 185 190

Glu Gly Gly Tyr Asp Tyr Leu Phe Ser Leu Phe Arg Arg Cys Arg Glu

195 200 205

Ser Lys Ala Ile Ile Ile Asn Thr Phe Glu Glu Leu Glu Pro Tyr Ala

210 215 220

Ile Asn Ser Leu Arg Met Asp Ser Met Ile Pro Pro Ile Tyr Pro Val

225 230 235 240

Gly Pro Ile Leu Asn Leu Asn Gly Asp Gly Gln Asn Ser Asp Glu Ala

245 250 255

Ala Val Ile Leu Gly Trp Leu Asp Asp Gln Pro Pro Ser Ser Val Val

260 265 270

Phe Leu Cys Phe Gly Ser Tyr Gly Thr Phe Gln Glu Asn Gln Val Lys

275 280 285

Glu Ile Ala Met Gly Leu Glu Arg Ser Gly His Arg Phe Leu Trp Ser

290 295 300

Leu Arg Pro Ser Ile Pro Lys Gly Glu Thr Lys Leu Gln Leu Lys Tyr

305 310 315 320

Ser Asn Leu Glu Glu Ile Leu Pro Val Gly Phe Leu Asp Arg Thr Ser

325 330 335

Cys Val Gly Lys Val Ile Gly Trp Ala Pro Gln Val Ala Val Leu Gly

340 345 350

His Glu Ala Val Gly Gly Phe Leu Ser His Cys Gly Trp Asn Ser Thr

355 360 365

Leu Glu Ser Val Trp Cys Gly Val Pro Val Ala Thr Trp Pro Met Tyr

370 375 380

Gly Glu Gln Gln Leu Asn Ala Phe Glu Met Val Lys Glu Leu Gly Ile

385 390 395 400

Ala Val Glu Ile Glu Val Asp Tyr Lys Asn Glu Tyr Phe Asn Met Asn

405 410 415

Asn Asp Phe Ile Val Arg Ala Glu Glu Ile Glu Thr Lys Ile Lys Lys

420 425 430

Leu Met Met Asp Glu Lys Asn Ser Glu Ile Arg Lys Lys Val Lys Glu

435 440 445

Met Lys Glu Lys Ser Arg Leu Ala Met Ser Glu Asn Gly Ser Ser Tyr

450 455 460

Asn Ser Leu Ala Lys Leu Phe Glu Glu Ile Met

465 470 475

<210> 17

<211> 1428

<212> DNA

<213> Ginseng radix (Panax ginseng)

<220>

<221> misc_feature

<223> gGT25-3

<400> 17

atgaagtcag aattgatatt cttgcccgcc ccggccatcg gacacctcgt gggaatggtg 60

gagatggcta aactcttcat cagtcgacat gaaaatctct cggtcaccgt cctcatcgcg 120

aaattctaca tggatacggg ggtagacaac tacaataaat cactcttaac aaaccctacc 180

ccgcgtctca caattgtaaa tctcccggaa accgaccccc aaaactatat gctcaaacca 240

cgccacgcca tctttcctag cgtcatcgag actcagaaga cacacgtgcg agacataata 300

tccggtatga ctcagtccga gtcgactcag gtcgttggtt tgctggctga ccttttgttc 360

atcaacatca tggacattgc caatgagttc aatgttccaa cttatgtata ctcccctgcc 420

ggagccggtc atcttggcct cgcgttccat ctccagacac tcaacgacaa aaaacaagat 480

gtgaccgagt tcaggaactc ggatactgag ttattggtac cgagttttgc aaacccggtt 540

cccgccgagg tcttgccgtc gatgtatgtg gataaagaag gtgggtatga ttatctgttt 600

tcattgttcc ggaggtgcag agagtcaaag gcaattatta ttaacacgtt tgaggagctg 660

gaaccctatg cgatcaattc cctccggatg gatagtatga tccctccgat ctacccggtg 720

ggacccatac taaatctcaa cggtgatggc caaaactccg atgaggctgc tgtgatcctt 780

ggttggttag atgatcaacc accttcatct gtggtgtttt tgtgctttgg tagctatgga 840

agctttcaag aaaaccaggt gaaggagatt gcaatgggtc tagagcgcag tgggcatcgc 900

ttcttgtggt ccttgcgtcc gtctatccct aaaggcgaga caaagcttca gcttaaatac 960

tcaaatttga aagaaattct cccagtagga ttcttggaca ggacatcatg cgtcggaaaa 1020

gtgattggat gggccccgca agtggccgtg ctcggacatg agtcagtcgg agggttcctg 1080

tctcattgcg gttggaattc gacattggag agtgtttggt gtggggtgcc cgttgcaaca 1140

tggccaatgt atggtgagca acaactcaat gcttttgaga tggttaagga gttaggtatt 1200

gcggtggaaa ttgaggtgga ctataagaaa gattatttta acatgaagaa tgattttatt 1260

gttagggcag aagaaatcga gacaaaaata aagaagttga tgatggatga aaataatagt 1320

gaaataagaa agaaggtaaa ggaaatgaaa gaaaagagta gggctgcaat gtctgagaat 1380

ggatcatctt ataattcatt ggcgaagcta tttgaggaaa ttatgtaa 1428

<210> 18

<211> 475

<212> PRT

<213> Ginseng radix (Panax ginseng)

<220>

<221> misc_feature

<223> gGT25-3

<400> 18

Met Lys Ser Glu Leu Ile Phe Leu Pro Ala Pro Ala Ile Gly His Leu

1 5 10 15

Val Gly Met Val Glu Met Ala Lys Leu Phe Ile Ser Arg His Glu Asn

20 25 30

Leu Ser Val Thr Val Leu Ile Ala Lys Phe Tyr Met Asp Thr Gly Val

35 40 45

Asp Asn Tyr Asn Lys Ser Leu Leu Thr Asn Pro Thr Pro Arg Leu Thr

50 55 60

Ile Val Asn Leu Pro Glu Thr Asp Pro Gln Asn Tyr Met Leu Lys Pro

65 70 75 80

Arg His Ala Ile Phe Pro Ser Val Ile Glu Thr Gln Lys Thr His Val

85 90 95

Arg Asp Ile Ile Ser Gly Met Thr Gln Ser Glu Ser Thr Gln Val Val

100 105 110

Gly Leu Leu Ala Asp Leu Leu Phe Ile Asn Ile Met Asp Ile Ala Asn

115 120 125

Glu Phe Asn Val Pro Thr Tyr Val Tyr Ser Pro Ala Gly Ala Gly His

130 135 140

Leu Gly Leu Ala Phe His Leu Gln Thr Leu Asn Asp Lys Lys Gln Asp

145 150 155 160

Val Thr Glu Phe Arg Asn Ser Asp Thr Glu Leu Leu Val Pro Ser Phe

165 170 175

Ala Asn Pro Val Pro Ala Glu Val Leu Pro Ser Met Tyr Val Asp Lys

180 185 190

Glu Gly Gly Tyr Asp Tyr Leu Phe Ser Leu Phe Arg Arg Cys Arg Glu

195 200 205

Ser Lys Ala Ile Ile Ile Asn Thr Phe Glu Glu Leu Glu Pro Tyr Ala

210 215 220

Ile Asn Ser Leu Arg Met Asp Ser Met Ile Pro Pro Ile Tyr Pro Val

225 230 235 240

Gly Pro Ile Leu Asn Leu Asn Gly Asp Gly Gln Asn Ser Asp Glu Ala

245 250 255

Ala Val Ile Leu Gly Trp Leu Asp Asp Gln Pro Pro Ser Ser Val Val

260 265 270

Phe Leu Cys Phe Gly Ser Tyr Gly Ser Phe Gln Glu Asn Gln Val Lys

275 280 285

Glu Ile Ala Met Gly Leu Glu Arg Ser Gly His Arg Phe Leu Trp Ser

290 295 300

Leu Arg Pro Ser Ile Pro Lys Gly Glu Thr Lys Leu Gln Leu Lys Tyr

305 310 315 320

Ser Asn Leu Lys Glu Ile Leu Pro Val Gly Phe Leu Asp Arg Thr Ser

325 330 335

Cys Val Gly Lys Val Ile Gly Trp Ala Pro Gln Val Ala Val Leu Gly

340 345 350

His Glu Ser Val Gly Gly Phe Leu Ser His Cys Gly Trp Asn Ser Thr

355 360 365

Leu Glu Ser Val Trp Cys Gly Val Pro Val Ala Thr Trp Pro Met Tyr

370 375 380

Gly Glu Gln Gln Leu Asn Ala Phe Glu Met Val Lys Glu Leu Gly Ile

385 390 395 400

Ala Val Glu Ile Glu Val Asp Tyr Lys Lys Asp Tyr Phe Asn Met Lys

405 410 415

Asn Asp Phe Ile Val Arg Ala Glu Glu Ile Glu Thr Lys Ile Lys Lys

420 425 430

Leu Met Met Asp Glu Asn Asn Ser Glu Ile Arg Lys Lys Val Lys Glu

435 440 445

Met Lys Glu Lys Ser Arg Ala Ala Met Ser Glu Asn Gly Ser Ser Tyr

450 455 460

Asn Ser Leu Ala Lys Leu Phe Glu Glu Ile Met

465 470 475

<210> 19

<211> 1419

<212> DNA

<213> Ginseng radix (Panax ginseng)

<220>

<221> misc_feature

<223> gGT25-5

<400> 19

atgaagtcag aattgatatt cgtgcccgtc ccggccttcg ggcacctcgt ggggatggtg 60

gagatggcta aacttttcat cagtcgacac gaaaacctat cagtcaccgt cctcatttcg 120

aaatttttca ttgatacggg gatagacaac tacaataaat cactcttagc gaaacctacc 180

ccgcgtctca caattataaa tctcccggaa atcgatcccc aaaaatattt gctcaaacca 240

cgttgcgcca tctttccttc cctcatcgag aatcagaaga cacacgtgcg agacgtaatg 300

tcccgcatga ctcagtccga gtcgactcgg gtcgttggtt tgctggcaga cattttgttc 360

gtcgacatct tcgacattgc cgatgagttc aatgttccaa cttatgtata ctcccctgcc 420

ggagccggtt ttcttggcct cgcgttccat ctccagacac tcaacgacga caaaaagcaa 480

gatgtgaccg agttcaggaa ttcggacact gagttattgg taccgagttt tgcaaacccg 540

gtccccgccg agttcttgcc gtcgatattt ttggaaaaag atggtaggca tgatgttttg 600

ttatcattgt actggaggtg cagggaggca aagggaatta ttgttaacac gtttgaggag 660

ctggaaccct atgcgatcaa ttccctccgg atggatagta tgatccctcc gatctacccg 720

gtgggaccca tactaaatct caacggtgag ggacaaaact ccgatgaggc tgctgtgatc 780

cttggttggt tagatgatca accaccttca tctgtggtgt ttttgtgctt tggtagcttt 840

ggaagctttc cagaaaacca ggtgaaggag attgcaatgg gtttagagcg cagcgggcat 900

cgcttcttgt ggtccttgcg tccgtgtatc tctgaaggtg agacaacgct tcaacttaaa 960

tactcaaatt tggaacttcc ggccggattc ttggatagga catcatgcgt cggaaaagtg 1020

attggatggg ccccacaaat ggccatccta gcacacgagg cagtcggagg gttcgtgtct 1080

cattgtggtt ggaattcggt actagagagt gtgtggtatg gcatgcctgt cgcaacatgg 1140

ccaatgtacg gtgagcaaca actcaacgct tttgagatgg ttaaggagtt gggtcttgcg 1200

gtggaaattg aggtggacta taggaatgaa tataacaagt ctgattttat tgttaaggct 1260

gacgaaattg agacaaaaat aaagaagttg atgatggatg gaaagaatag taaaataagg 1320

aagaaggtaa aggaaatgaa agaaaagagt agggttgcca tgtcggagaa tgggtcatct 1380

tatacttcat tggcgaagct atttgaggaa attatgtaa 1419

<210> 20

<211> 472

<212> PRT

<213> Ginseng radix (Panax ginseng)

<220>

<221> misc_feature

<223> gGT25-5

<400> 20

Met Lys Ser Glu Leu Ile Phe Val Pro Val Pro Ala Phe Gly His Leu

1 5 10 15

Val Gly Met Val Glu Met Ala Lys Leu Phe Ile Ser Arg His Glu Asn

20 25 30

Leu Ser Val Thr Val Leu Ile Ser Lys Phe Phe Ile Asp Thr Gly Ile

35 40 45

Asp Asn Tyr Asn Lys Ser Leu Leu Ala Lys Pro Thr Pro Arg Leu Thr

50 55 60

Ile Ile Asn Leu Pro Glu Ile Asp Pro Gln Lys Tyr Leu Leu Lys Pro

65 70 75 80

Arg Cys Ala Ile Phe Pro Ser Leu Ile Glu Asn Gln Lys Thr His Val

85 90 95

Arg Asp Val Met Ser Arg Met Thr Gln Ser Glu Ser Thr Arg Val Val

100 105 110

Gly Leu Leu Ala Asp Ile Leu Phe Val Asp Ile Phe Asp Ile Ala Asp

115 120 125

Glu Phe Asn Val Pro Thr Tyr Val Tyr Ser Pro Ala Gly Ala Gly Phe

130 135 140

Leu Gly Leu Ala Phe His Leu Gln Thr Leu Asn Asp Asp Lys Lys Gln

145 150 155 160

Asp Val Thr Glu Phe Arg Asn Ser Asp Thr Glu Leu Leu Val Pro Ser

165 170 175

Phe Ala Asn Pro Val Pro Ala Glu Phe Leu Pro Ser Ile Phe Leu Glu

180 185 190

Lys Asp Gly Arg His Asp Val Leu Leu Ser Leu Tyr Trp Arg Cys Arg

195 200 205

Glu Ala Lys Gly Ile Ile Val Asn Thr Phe Glu Glu Leu Glu Pro Tyr

210 215 220

Ala Ile Asn Ser Leu Arg Met Asp Ser Met Ile Pro Pro Ile Tyr Pro

225 230 235 240

Val Gly Pro Ile Leu Asn Leu Asn Gly Glu Gly Gln Asn Ser Asp Glu

245 250 255

Ala Ala Val Ile Leu Gly Trp Leu Asp Asp Gln Pro Pro Ser Ser Val

260 265 270

Val Phe Leu Cys Phe Gly Ser Phe Gly Ser Phe Pro Glu Asn Gln Val

275 280 285

Lys Glu Ile Ala Met Gly Leu Glu Arg Ser Gly His Arg Phe Leu Trp

290 295 300

Ser Leu Arg Pro Cys Ile Ser Glu Gly Glu Thr Thr Leu Gln Leu Lys

305 310 315 320

Tyr Ser Asn Leu Glu Leu Pro Ala Gly Phe Leu Asp Arg Thr Ser Cys

325 330 335

Val Gly Lys Val Ile Gly Trp Ala Pro Gln Met Ala Ile Leu Ala His

340 345 350

Glu Ala Val Gly Gly Phe Val Ser His Cys Gly Trp Asn Ser Val Leu

355 360 365

Glu Ser Val Trp Tyr Gly Met Pro Val Ala Thr Trp Pro Met Tyr Gly

370 375 380

Glu Gln Gln Leu Asn Ala Phe Glu Met Val Lys Glu Leu Gly Leu Ala

385 390 395 400

Val Glu Ile Glu Val Asp Tyr Arg Asn Glu Tyr Asn Lys Ser Asp Phe

405 410 415

Ile Val Lys Ala Asp Glu Ile Glu Thr Lys Ile Lys Lys Leu Met Met

420 425 430

Asp Gly Lys Asn Ser Lys Ile Arg Lys Lys Val Lys Glu Met Lys Glu

435 440 445

Lys Ser Arg Val Ala Met Ser Glu Asn Gly Ser Ser Tyr Thr Ser Leu

450 455 460

Ala Lys Leu Phe Glu Glu Ile Met

465 470

<210> 21

<211> 1488

<212> DNA

<213> European wasabi (Barbarea vulgaris)

<220>

<221> misc_feature

<223> 3GT1

<400> 21

atggtttccg aaatcaccca taaatcttat cctcttcact ttgttctctt ccctttcatg 60

gctcaaggcc acatgattcc catggttgat attgcaaggc tcttggctca gcgcggtgtg 120

aaaataacaa ttgtcacaac gccgcacaat gcagcgaggt tcgagaatgt cctaagccgt 180

gccattgagt ctggcttgcc catcagcata gtgcaagtca agcttccatc tcaagaagct 240

ggcttaccag aaggaaatga gactttcgat tcacttgtct caacaaagtt gctggtacct 300

ttctttaaag cggttaacat gcttgaagaa ccggtccaga agctctttga agagatgagc 360

cctcaaccaa gctgtataat ttctgatttt tgtttgcctt atacaagcaa aatcgccaag 420

aagttcaata tcccaaagat cctcttccat ggcatgtgtt gcttttgtct tctgtgtatg 480

catgttttac gcaagaaccg tgagatcttg gaaaacttaa agtctgacaa ggagcatttc 540

gttgttcctt attttcctga tcgagttgaa ttcacaagac ctcaagttcc attggcaaca 600

tatgttcctg gggaatggca cgagatcaag gaggatatgg tagaagcgga taagacttcc 660

tatggtgtga tagtcaacac atatcaagag ctcgagcctg cttatgccaa cggctacaag 720

gaggcaaggt ctggtaaagc atggaccatt ggacctgttt ccttgtgcaa caaggtggga 780

gccgacaaag cagagagggg aaacaaagca gacattgatc aagatgagtg tcttaaatgg 840

cttgattcta aagaagaagg ttcggttcta tatgtttgcc ttggaagtat ctgcagtctt 900

cctctgtctc agctcaagga gctggggcta ggccttgagg aatcccaaag acctttcatt 960

tgggtcgtaa gaggttggga gaagaacaaa gagttacttg agtggttctc ggagagcgga 1020

tttgaagaaa gagtaaaaga cagagggctt ctcatcaaag gatggtcacc tcaaatgctt 1080

atccttgcac atcattccgt tggagggttc ttaacacact gtggatggaa ctcgaccctc 1140

gaaggaatca cttcaggcgt tccattgctc acttggccac tgtttggaga ccaattctgc 1200

aaccaaaaac ttgtcgtgca ggtgctaaaa gtgggtgtaa gtgccggggt tgaagaggtt 1260

acgaattggg gagaagagga gaaaatagga gtattagtgg ataaagaggg agtgaagaag 1320

gcagtggaag aattaatggg tgagagtgat gatgctaaag aaataagaaa aagagtcaaa 1380

gagcttggac aattagctca caaggctgtg gaggaaggag gctcatctca ttctaatatc 1440

acatccttgc tagaagacat aatgcaacta gcacaaccta ataattaa 1488

<210> 22

<211> 495

<212> PRT

<213> European wasabi (Barbarea vulgaris)

<220>

<221> misc_feature

<223> 3GT1

<400> 22

Met Val Ser Glu Ile Thr His Lys Ser Tyr Pro Leu His Phe Val Leu

1 5 10 15

Phe Pro Phe Met Ala Gln Gly His Met Ile Pro Met Val Asp Ile Ala

20 25 30

Arg Leu Leu Ala Gln Arg Gly Val Lys Ile Thr Ile Val Thr Thr Pro

35 40 45

His Asn Ala Ala Arg Phe Glu Asn Val Leu Ser Arg Ala Ile Glu Ser

50 55 60

Gly Leu Pro Ile Ser Ile Val Gln Val Lys Leu Pro Ser Gln Glu Ala

65 70 75 80

Gly Leu Pro Glu Gly Asn Glu Thr Phe Asp Ser Leu Val Ser Thr Lys

85 90 95

Leu Leu Val Pro Phe Phe Lys Ala Val Asn Met Leu Glu Glu Pro Val

100 105 110

Gln Lys Leu Phe Glu Glu Met Ser Pro Gln Pro Ser Cys Ile Ile Ser

115 120 125

Asp Phe Cys Leu Pro Tyr Thr Ser Lys Ile Ala Lys Lys Phe Asn Ile

130 135 140

Pro Lys Ile Leu Phe His Gly Met Cys Cys Phe Cys Leu Leu Cys Met

145 150 155 160

His Val Leu Arg Lys Asn Arg Glu Ile Leu Glu Asn Leu Lys Ser Asp

165 170 175

Lys Glu His Phe Val Val Pro Tyr Phe Pro Asp Arg Val Glu Phe Thr

180 185 190

Arg Pro Gln Val Pro Leu Ala Thr Tyr Val Pro Gly Glu Trp His Glu

195 200 205

Ile Lys Glu Asp Met Val Glu Ala Asp Lys Thr Ser Tyr Gly Val Ile

210 215 220

Val Asn Thr Tyr Gln Glu Leu Glu Pro Ala Tyr Ala Asn Gly Tyr Lys

225 230 235 240

Glu Ala Arg Ser Gly Lys Ala Trp Thr Ile Gly Pro Val Ser Leu Cys

245 250 255

Asn Lys Val Gly Ala Asp Lys Ala Glu Arg Gly Asn Lys Ala Asp Ile

260 265 270

Asp Gln Asp Glu Cys Leu Lys Trp Leu Asp Ser Lys Glu Glu Gly Ser

275 280 285

Val Leu Tyr Val Cys Leu Gly Ser Ile Cys Ser Leu Pro Leu Ser Gln

290 295 300

Leu Lys Glu Leu Gly Leu Gly Leu Glu Glu Ser Gln Arg Pro Phe Ile

305 310 315 320

Trp Val Val Arg Gly Trp Glu Lys Asn Lys Glu Leu Leu Glu Trp Phe

325 330 335

Ser Glu Ser Gly Phe Glu Glu Arg Val Lys Asp Arg Gly Leu Leu Ile

340 345 350

Lys Gly Trp Ser Pro Gln Met Leu Ile Leu Ala His His Ser Val Gly

355 360 365

Gly Phe Leu Thr His Cys Gly Trp Asn Ser Thr Leu Glu Gly Ile Thr

370 375 380

Ser Gly Val Pro Leu Leu Thr Trp Pro Leu Phe Gly Asp Gln Phe Cys

385 390 395 400

Asn Gln Lys Leu Val Val Gln Val Leu Lys Val Gly Val Ser Ala Gly

405 410 415

Val Glu Glu Val Thr Asn Trp Gly Glu Glu Glu Lys Ile Gly Val Leu

420 425 430

Val Asp Lys Glu Gly Val Lys Lys Ala Val Glu Glu Leu Met Gly Glu

435 440 445

Ser Asp Asp Ala Lys Glu Ile Arg Lys Arg Val Lys Glu Leu Gly Gln

450 455 460

Leu Ala His Lys Ala Val Glu Glu Gly Gly Ser Ser His Ser Asn Ile

465 470 475 480

Thr Ser Leu Leu Glu Asp Ile Met Gln Leu Ala Gln Pro Asn Asn

485 490 495

<210> 23

<211> 1488

<212> DNA

<213> European wasabi (Barbarea vulgaris)

<220>

<221> misc_feature

<223> 3GT2

<400> 23

atggtttccg aaatcaccca taaatcttat cctcttcact ttgttctctt ccctttcatg 60

gctcaaggcc acatgattcc catggttgat attgcaaggc tcttggccca gcgcggtgtg 120

aaaataacaa ttgtcacaac cccgcacaat gcagcgaggt tcaagaatgt cctaagtcgt 180

gccattgagt ctggcttgcc catcagcata gtgcaagtca agcttccatc tcaagaagct 240

ggcttaccag aaggaaatga gactctcgat tcacttgtct cgatggagtt gatgatacat 300

ttcttaaaag cggttaacat gctggaagaa ccggtccaga agctctttga agagatgagc 360

cctcaaccaa gctgtataat ttctgatttt tgtttgcctt atacaagcaa aatcgccaag 420

aagttcaata tcccaaagat cctcttccat ggcatgtgct gcttttgtct tctgtgtatg 480

catattttac gcaagaaccg tgagatcgtg gaaaacttaa agtctgacaa ggagcatttc 540

gttgttcctt attttcctga tcgagttgaa ttcacaagac ctcaagttcc agtggcaaca 600

tatgttcctg gagactggca cgagatcacg gaggatatgg tagaagcgga taagacttcc 660

tatggtgtga tagtcaacac atatcaagag ctcgagcctg cttatgccaa cgactacaag 720

gaggcaaggt ctggtaaagc atggaccatt ggacctgttt ccttgtgcaa caaggtggga 780

gcggacaaag cagagagggg aaacaaagca gacattgatc aagatgagtg tcttaaatgg 840

cttaattcta aagaagaagg ttcggttcta tatgtttgcc ttggaagtat ctgcaatctt 900

cctctgtctc agctcaagga gctcgggcta ggccttgagg aatcccaaag acctttcatt 960

tgggtcataa gaggttggga gaagaacaaa gagttacatg agtggttctc ggagagcgga 1020

ttcgaagaaa gaatcaaaga cagaggactt ctcatcaaag gatgggctcc tcaaatgctt 1080

atactttcac atcattccgt tggagggttc ttaacacact gtggatggaa ctcgactctt 1140

gaggggctaa ccgctggtct accactgctg acatggccgc ttttcgcaga ccagttctgc 1200

aacgagaaac ttgccgtgca ggtattaaaa gccggtgtaa gcgccggggt tgaccagcct 1260

atgaaatggg gagaagagga gaaaatagga gtgttggtgg ataaagaagg agtgaagaag 1320

gcagtggaag aattaatggg tgagagtgat gatgctaaag agataagaag aagagccaaa 1380

gagcttggag aattagctca caaggctgtg gaggaaggag gctcatctca ttctaatatc 1440

acatcccttc tagaagacat aatgcaacta gcacaatcca ataattaa 1488

<210> 24

<211> 495

<212> PRT

<213> European wasp (Barbarea vulgaris)

<220>

<221> misc_feature

<223> 3GT2

<400> 24

Met Val Ser Glu Ile Thr His Lys Ser Tyr Pro Leu His Phe Val Leu

1 5 10 15

Phe Pro Phe Met Ala Gln Gly His Met Ile Pro Met Val Asp Ile Ala

20 25 30

Arg Leu Leu Ala Gln Arg Gly Val Lys Ile Thr Ile Val Thr Thr Pro

35 40 45

His Asn Ala Ala Arg Phe Lys Asn Val Leu Ser Arg Ala Ile Glu Ser

50 55 60

Gly Leu Pro Ile Ser Ile Val Gln Val Lys Leu Pro Ser Gln Glu Ala

65 70 75 80

Gly Leu Pro Glu Gly Asn Glu Thr Leu Asp Ser Leu Val Ser Met Glu

85 90 95

Leu Met Ile His Phe Leu Lys Ala Val Asn Met Leu Glu Glu Pro Val

100 105 110

Gln Lys Leu Phe Glu Glu Met Ser Pro Gln Pro Ser Cys Ile Ile Ser

115 120 125

Asp Phe Cys Leu Pro Tyr Thr Ser Lys Ile Ala Lys Lys Phe Asn Ile

130 135 140

Pro Lys Ile Leu Phe His Gly Met Cys Cys Phe Cys Leu Leu Cys Met

145 150 155 160

His Ile Leu Arg Lys Asn Arg Glu Ile Val Glu Asn Leu Lys Ser Asp

165 170 175

Lys Glu His Phe Val Val Pro Tyr Phe Pro Asp Arg Val Glu Phe Thr

180 185 190

Arg Pro Gln Val Pro Val Ala Thr Tyr Val Pro Gly Asp Trp His Glu

195 200 205

Ile Thr Glu Asp Met Val Glu Ala Asp Lys Thr Ser Tyr Gly Val Ile

210 215 220

Val Asn Thr Tyr Gln Glu Leu Glu Pro Ala Tyr Ala Asn Asp Tyr Lys

225 230 235 240

Glu Ala Arg Ser Gly Lys Ala Trp Thr Ile Gly Pro Val Ser Leu Cys

245 250 255

Asn Lys Val Gly Ala Asp Lys Ala Glu Arg Gly Asn Lys Ala Asp Ile

260 265 270

Asp Gln Asp Glu Cys Leu Lys Trp Leu Asn Ser Lys Glu Glu Gly Ser

275 280 285

Val Leu Tyr Val Cys Leu Gly Ser Ile Cys Asn Leu Pro Leu Ser Gln

290 295 300

Leu Lys Glu Leu Gly Leu Gly Leu Glu Glu Ser Gln Arg Pro Phe Ile

305 310 315 320

Trp Val Ile Arg Gly Trp Glu Lys Asn Lys Glu Leu His Glu Trp Phe

325 330 335

Ser Glu Ser Gly Phe Glu Glu Arg Ile Lys Asp Arg Gly Leu Leu Ile

340 345 350

Lys Gly Trp Ala Pro Gln Met Leu Ile Leu Ser His His Ser Val Gly

355 360 365

Gly Phe Leu Thr His Cys Gly Trp Asn Ser Thr Leu Glu Gly Leu Thr

370 375 380

Ala Gly Leu Pro Leu Leu Thr Trp Pro Leu Phe Ala Asp Gln Phe Cys

385 390 395 400

Asn Glu Lys Leu Ala Val Gln Val Leu Lys Ala Gly Val Ser Ala Gly

405 410 415

Val Asp Gln Pro Met Lys Trp Gly Glu Glu Glu Lys Ile Gly Val Leu

420 425 430

Val Asp Lys Glu Gly Val Lys Lys Ala Val Glu Glu Leu Met Gly Glu

435 440 445

Ser Asp Asp Ala Lys Glu Ile Arg Arg Arg Ala Lys Glu Leu Gly Glu

450 455 460

Leu Ala His Lys Ala Val Glu Glu Gly Gly Ser Ser His Ser Asn Ile

465 470 475 480

Thr Ser Leu Leu Glu Asp Ile Met Gln Leu Ala Gln Ser Asn Asn

485 490 495

<210> 25

<211> 1329

<212> DNA

<213> Ginseng (Panax ginseng)

<220>

<221> misc_feature

<223> gGT29

<400> 25

atggataacc aaaaaggtag aatcagtata gcgttgctac catttttagc ccatggtcac 60

atatctccct tctttgagct agccaaacaa ctcgcgaaaa gaaattgcaa tgttttcctc 120

tgttctaccc caatcaatct tagctccatc aaggataagg attcctctgc ttctataaaa 180

ctagttgagc ttcatcttcc atcttcccct gatcttcctc ctcactatca caccacaaat 240

ggcctccctt cccatctcat gctcccactc agaaacgcct ttgaaactgc aggccccacc 300

ttctctgaaa tccttaaaac cttaaacccc gatttgctta tttatgattt caatccctca 360

tgggcaccgg agatcgcttc gtctcacaat attccggcag tttatttcct aaccacggca 420

gcagccagct cttccattgg cctacatgct ttcaaaaacc caggtgaaaa atacccattt 480

ccagattttt atgataacag taatattacc cctgaaccac cttctgcaga taacatgaag 540

ctacttcatg attttatcgc ttgtttcgaa cgatcttgcg acattatttt gattaagagt 600

tttagagaac tagaagggaa atatattgat ttgctttcca ctttatctga taaaactttg 660

gttcctgttg gtccactcgt tcaagatcct atgggccata atgaagatcc aaaaacagag 720

cagattataa actggcttga caaaagggct gaatctacag tggtgtttgt ctgctttgga 780

agtgagtatt ttctctccaa tgaggaattg gaagaagtag caattgggct agagattagc 840

acggttaatt tcatatgggc tgtgagatta attgaaggag agaaaaaagg gattttacca 900

gaggggtttg ttcaaagggt aggagacaga ggattggttg tggaggggtg ggctccacag 960

gcaagaattt taggacattc aagcaccggt gggtttgtga gccattgtgg gtggagttct 1020

attgcggaga gtatgaagtt tggggttcca gtaattgcca tggccaggca tcttgatcag 1080

cctttgaatg gtaagctggc ggcggaggtt ggtgtgggca tggaggttgt gagagatgag 1140

aatgggaagt ataagagaga agggattgca gaggtaataa gaaaagtggt tgtggagaaa 1200

agtggggagg ttatcaggag gaaagcaagg gagttgagtg agaaaatgaa agagaaagga 1260

gagcaagaga ttgatagggc attggaggag ctagtacaaa tttgtaagaa gaagaaagat 1320

gaacaatag 1329

<210> 26

<211> 442

<212> PRT

<213> Ginseng radix (Panax ginseng)

<220>

<221> misc_feature

<223> gGT29

<400> 26

Met Asp Asn Gln Lys Gly Arg Ile Ser Ile Ala Leu Leu Pro Phe Leu

1 5 10 15

Ala His Gly His Ile Ser Pro Phe Phe Glu Leu Ala Lys Gln Leu Ala

20 25 30

Lys Arg Asn Cys Asn Val Phe Leu Cys Ser Thr Pro Ile Asn Leu Ser

35 40 45

Ser Ile Lys Asp Lys Asp Ser Ser Ala Ser Ile Lys Leu Val Glu Leu

50 55 60

His Leu Pro Ser Ser Pro Asp Leu Pro Pro His Tyr His Thr Thr Asn

65 70 75 80

Gly Leu Pro Ser His Leu Met Leu Pro Leu Arg Asn Ala Phe Glu Thr

85 90 95

Ala Gly Pro Thr Phe Ser Glu Ile Leu Lys Thr Leu Asn Pro Asp Leu

100 105 110

Leu Ile Tyr Asp Phe Asn Pro Ser Trp Ala Pro Glu Ile Ala Ser Ser

115 120 125

His Asn Ile Pro Ala Val Tyr Phe Leu Thr Thr Ala Ala Ala Ser Ser

130 135 140

Ser Ile Gly Leu His Ala Phe Lys Asn Pro Gly Glu Lys Tyr Pro Phe

145 150 155 160

Pro Asp Phe Tyr Asp Asn Ser Asn Ile Thr Pro Glu Pro Pro Ser Ala

165 170 175

Asp Asn Met Lys Leu Leu His Asp Phe Ile Ala Cys Phe Glu Arg Ser

180 185 190

Cys Asp Ile Ile Leu Ile Lys Ser Phe Arg Glu Leu Glu Gly Lys Tyr

195 200 205

Ile Asp Leu Leu Ser Thr Leu Ser Asp Lys Thr Leu Val Pro Val Gly

210 215 220

Pro Leu Val Gln Asp Pro Met Gly His Asn Glu Asp Pro Lys Thr Glu

225 230 235 240

Gln Ile Ile Asn Trp Leu Asp Lys Arg Ala Glu Ser Thr Val Val Phe

245 250 255

Val Cys Phe Gly Ser Glu Tyr Phe Leu Ser Asn Glu Glu Leu Glu Glu

260 265 270

Val Ala Ile Gly Leu Glu Ile Ser Thr Val Asn Phe Ile Trp Ala Val

275 280 285

Arg Leu Ile Glu Gly Glu Lys Lys Gly Ile Leu Pro Glu Gly Phe Val

290 295 300

Gln Arg Val Gly Asp Arg Gly Leu Val Val Glu Gly Trp Ala Pro Gln

305 310 315 320

Ala Arg Ile Leu Gly His Ser Ser Thr Gly Gly Phe Val Ser His Cys

325 330 335

Gly Trp Ser Ser Ile Ala Glu Ser Met Lys Phe Gly Val Pro Val Ile

340 345 350

Ala Met Ala Arg His Leu Asp Gln Pro Leu Asn Gly Lys Leu Ala Ala

355 360 365

Glu Val Gly Val Gly Met Glu Val Val Arg Asp Glu Asn Gly Lys Tyr

370 375 380

Lys Arg Glu Gly Ile Ala Glu Val Ile Arg Lys Val Val Val Glu Lys

385 390 395 400

Ser Gly Glu Val Ile Arg Arg Lys Ala Arg Glu Leu Ser Glu Lys Met

405 410 415

Lys Glu Lys Gly Glu Gln Glu Ile Asp Arg Ala Leu Glu Glu Leu Val

420 425 430

Gln Ile Cys Lys Lys Lys Lys Asp Glu Gln

435 440

<210> 27

<211> 1329

<212> DNA

<213> Ginseng radix (Panax ginseng)

<220>

<221> misc_feature

<223> gGT29-3

<400> 27

atggataacc aagaaggtag aatcagtata gcgttgctac catttttagc ccatggtcac 60

atatctccct tctttgagct agccaaacaa ctcgcaaaaa gaaattgcaa tgttttcctc 120

tgttctaccc caatcaatct tagctccatc aagaataagg attcctctgc ttctataaaa 180

ctagttgagc ttcatcttcc atcttcccct gatcttcctc ctcactatca caccacaaat 240

ggcctccctt cccatctcat ggtcccactc ataaacgcct ttgaaacagc aggccccacc 300

ttctctgaaa tccttaaaac cttaaacccc gatttgctta tttatgattt caatccctca 360

tgggcaccgg agatcgcttc gtctcacaat attccggcag tttatttcct aaccacggca 420

gcagccagct cttccattgg cctacatgct ttcaaaaacc caggtgaaaa atacccattt 480

ccagattttt atgataacag taataatacc cctgaaccac cttctgcaga taacatgaag 540

ctacttcatg attttatcgc ttgtttcgaa cgatcttgcg acattatttt gattaagagt 600

tttatagaac tagaagggaa atatatcgat ttgctttcca ctttatctga taaaactttg 660

gttcctgttg gtccactcgt tcaagatcct atgggccata atgaagatcc aaaaacagag 720

cagattataa actggcttga caaaagggct gaatctacag tggtgtttgt ctgctttgga 780

agtgagtatt ttctctccaa tgaggaattg gaagaagtag caattgggct agagattagc 840

atggttaatt tcatatgggc tgtgagatta attgaaggag agaaaaaagg ggttttacca 900

gagggatttg ttcaaagggt aggagacaga ggattggttg tggaggggtg ggctccacag 960

gcaagaattt taggacattc aagcaccggt gggtttgtga gccattgtgg gtggagttct 1020

attgcggaga gtatgaagtt tggggttcca gtaattgcca tggccaggca tcttgatcag 1080

cctttgaatg gtaagctggc ggcggaggtt ggtgtgggca tggaggttgt gagagatgaa 1140

aatgggaagt ataagagaga agggattgca gaggtaataa gaaaagtcgt tgtggagaaa 1200

agtggggagg ttatgaggag gaaagcaagg gaattgagtg agaaaatgaa agagaaagga 1260

gaggaagaga ttgatagggc agtggaggag ctagtacaaa tttgtaagaa gaagaaagat 1320

gcacaatag 1329

<210> 28

<211> 442

<212> PRT

<213> Ginseng radix (Panax ginseng)

<220>

<221> misc_feature

<223> gGT29-3

<400> 28

Met Asp Asn Gln Glu Gly Arg Ile Ser Ile Ala Leu Leu Pro Phe Leu

1 5 10 15

Ala His Gly His Ile Ser Pro Phe Phe Glu Leu Ala Lys Gln Leu Ala

20 25 30

Lys Arg Asn Cys Asn Val Phe Leu Cys Ser Thr Pro Ile Asn Leu Ser

35 40 45

Ser Ile Lys Asn Lys Asp Ser Ser Ala Ser Ile Lys Leu Val Glu Leu

50 55 60

His Leu Pro Ser Ser Pro Asp Leu Pro Pro His Tyr His Thr Thr Asn

65 70 75 80

Gly Leu Pro Ser His Leu Met Val Pro Leu Ile Asn Ala Phe Glu Thr

85 90 95

Ala Gly Pro Thr Phe Ser Glu Ile Leu Lys Thr Leu Asn Pro Asp Leu

100 105 110

Leu Ile Tyr Asp Phe Asn Pro Ser Trp Ala Pro Glu Ile Ala Ser Ser

115 120 125

His Asn Ile Pro Ala Val Tyr Phe Leu Thr Thr Ala Ala Ala Ser Ser

130 135 140

Ser Ile Gly Leu His Ala Phe Lys Asn Pro Gly Glu Lys Tyr Pro Phe

145 150 155 160

Pro Asp Phe Tyr Asp Asn Ser Asn Asn Thr Pro Glu Pro Pro Ser Ala

165 170 175

Asp Asn Met Lys Leu Leu His Asp Phe Ile Ala Cys Phe Glu Arg Ser

180 185 190

Cys Asp Ile Ile Leu Ile Lys Ser Phe Ile Glu Leu Glu Gly Lys Tyr

195 200 205

Ile Asp Leu Leu Ser Thr Leu Ser Asp Lys Thr Leu Val Pro Val Gly

210 215 220

Pro Leu Val Gln Asp Pro Met Gly His Asn Glu Asp Pro Lys Thr Glu

225 230 235 240

Gln Ile Ile Asn Trp Leu Asp Lys Arg Ala Glu Ser Thr Val Val Phe

245 250 255

Val Cys Phe Gly Ser Glu Tyr Phe Leu Ser Asn Glu Glu Leu Glu Glu

260 265 270

Val Ala Ile Gly Leu Glu Ile Ser Met Val Asn Phe Ile Trp Ala Val

275 280 285

Arg Leu Ile Glu Gly Glu Lys Lys Gly Val Leu Pro Glu Gly Phe Val

290 295 300

Gln Arg Val Gly Asp Arg Gly Leu Val Val Glu Gly Trp Ala Pro Gln

305 310 315 320

Ala Arg Ile Leu Gly His Ser Ser Thr Gly Gly Phe Val Ser His Cys

325 330 335

Gly Trp Ser Ser Ile Ala Glu Ser Met Lys Phe Gly Val Pro Val Ile

340 345 350

Ala Met Ala Arg His Leu Asp Gln Pro Leu Asn Gly Lys Leu Ala Ala

355 360 365

Glu Val Gly Val Gly Met Glu Val Val Arg Asp Glu Asn Gly Lys Tyr

370 375 380

Lys Arg Glu Gly Ile Ala Glu Val Ile Arg Lys Val Val Val Glu Lys

385 390 395 400

Ser Gly Glu Val Met Arg Arg Lys Ala Arg Glu Leu Ser Glu Lys Met

405 410 415

Lys Glu Lys Gly Glu Glu Glu Ile Asp Arg Ala Val Glu Glu Leu Val

420 425 430

Gln Ile Cys Lys Lys Lys Lys Asp Ala Gln

435 440

<210> 29

<211> 20

<212> DNA

<213> oligonucleotides

<400> 29

atggtttccg aaatcaccca 20

<210> 30

<211> 24

<212> DNA

<213> oligonucleotides

<400> 30

ttaattattr grttgtgcta gttg 24

<210> 31

<211> 35

<212> DNA

<213> oligonucleotides

<400> 31

gactggatcc atggtttccg aaatcaccca taaat 35

<210> 32

<211> 30

<212> DNA

<213> oligonucleotides

<400> 32

gcgcgtcgac attattaggt tgtgctagtt 30

<210> 33

<211> 30

<212> DNA

<213> oligonucleotides

<400> 33

gcgcgtcgac attattggat tgtgctagtt 30

<210> 34

<211> 26

<212> DNA

<213> oligonucleotides

<400> 34

agtaagaaaa acagagttca tcatgg 26

<210> 35

<211> 20

<212> DNA

<213> oligonucleotides

<400> 35

gcctcggtta ggctagctgt 20

<210> 36

<211> 35

<212> DNA

<213> oligonucleotides

<400> 36

ggcgggtacc atggataacc aaaaaggtag aatca 35

<210> 37

<211> 56

<212> DNA

<213> oligonucleotides

<400> 37

gccgctcgag tcaatgatga tgatgatgat gttgttcatc tttcttcttc ttacaa 56

<210> 38

<211> 35

<212> DNA

<213> oligonucleotides

<400> 38

ggcgggtacc atggataacc aagaaggtag aatca 35

<210> 39

<211> 56

<212> DNA

<213> oligonucleotides

<400> 39

gccgctcgag tcaatgatga tgatgatgat gttgtgcatc tttcttcttc ttacaa 56

<210> 40

<211> 1494

<212> DNA

<213> Medicago truncatula (Medicago truncatula)

<220>

<221> misc_feature

<223> 3GT3

<400> 40

atggaaggtg ttgaagttga acaaccattg aaagtttact tcattccatt tcttgcatct 60

ggacatatga tccctctttt tgacatagca actatgtttg catcccgtgg ccagcaagta 120

acagtcatca ccactcccgc caacgcaaaa tcccttacca aatctctctc atccgacgct 180

ccttcattcc ttcgtcttca caccgttgac ttcccctccc aacaagtcgg cctccctgaa 240

ggtattgaat ctatgtcttc aactacggac cccaccacca cttggaagat ccatactggc 300

gcgatgctcc ttaaagaacc tattggggat ttcattgaga atgatccacc ggattgtatc 360

atctccgact ccacgtaccc atgggttaat gacttggccg ataagtttca gatcccaaac 420

atcacattca atggattgtg cctttttgct gtctccctcg tggaaaccct caaaacaaac 480

aatttactta agtctcagac agattctgat tcggattcaa gttcctttgt tgttccaaat 540

tttcctcacc atatcacctt gtgtggaaaa ccgccaaagg taatcggtat attcatggga 600

atgatgcttg agacggtgct taaaagtaaa gcactaatca tcaacaactt cagtgaactt 660

gatggagaag agtgcataca acactacgag aaagccacgg gtcacaaggt ttggcatctt 720

ggtccaactt ctcttattcg caaaactgct caagagaaat cagagagggg aaatgagggt 780

gctgtgaatg tgcacgagag cctgagttgg ctcgattcag agagagttaa ctcagtgttg 840

tacatatgtt ttggaagcat caactatttt tctgataaac aactatacga gatggcatgt 900

gcgatagaag catccggtca cccattcata tgggttgttc ctgagaagaa agggaaagaa 960

gatgagagcg aagaagagaa agaaaagtgg ttaccgaagg gatttgaaga gagaaatatc 1020

gggaagaagg gtttgatcat taggggttgg gccccacagg ttaagatatt gagccaccct 1080

gcagtggggg gatttatgac gcattgcggg gggaactcaa ccgtagaggc tgttagtgcg 1140

ggagttccaa tgataacgtg gccggttcat ggagatcaat tctacaatga gaaactgata 1200

acacaattcc gaggaattgg agttgaagtc ggtgcaacag aatggtgtac gagtggtgtc 1260

gcggagagaa agaagttagt gagcagagat agcatagaga aggctgtaag gagattgatg 1320

gacggtggtg atgaagctga aaatatcagg ctacgtgctc gagagtttgg agaaaaagct 1380

atacaagcta ttcaagaagg tggctcgtct tataataatt tgttggcttt gattgacgaa 1440

cttaaaagat cgagagacct taaaagattg agagacctca agctggatga ttaa 1494

<210> 41

<211> 497

<212> PRT

<213> Medicago truncatula (Medicago truncatula)

<220>

<221> misc_feature

<223> 3GT3

<400> 41

Met Glu Gly Val Glu Val Glu Gln Pro Leu Lys Val Tyr Phe Ile Pro

1 5 10 15

Phe Leu Ala Ser Gly His Met Ile Pro Leu Phe Asp Ile Ala Thr Met

20 25 30

Phe Ala Ser Arg Gly Gln Gln Val Thr Val Ile Thr Thr Pro Ala Asn

35 40 45

Ala Lys Ser Leu Thr Lys Ser Leu Ser Ser Asp Ala Pro Ser Phe Leu

50 55 60

Arg Leu His Thr Val Asp Phe Pro Ser Gln Gln Val Gly Leu Pro Glu

65 70 75 80

Gly Ile Glu Ser Met Ser Ser Thr Thr Asp Pro Thr Thr Thr Trp Lys

85 90 95

Ile His Thr Gly Ala Met Leu Leu Lys Glu Pro Ile Gly Asp Phe Ile

100 105 110

Glu Asn Asp Pro Pro Asp Cys Ile Ile Ser Asp Ser Thr Tyr Pro Trp

115 120 125

Val Asn Asp Leu Ala Asp Lys Phe Gln Ile Pro Asn Ile Thr Phe Asn

130 135 140

Gly Leu Cys Leu Phe Ala Val Ser Leu Val Glu Thr Leu Lys Thr Asn

145 150 155 160

Asn Leu Leu Lys Ser Gln Thr Asp Ser Asp Ser Asp Ser Ser Ser Phe

165 170 175

Val Val Pro Asn Phe Pro His His Ile Thr Leu Cys Gly Lys Pro Pro

180 185 190

Lys Val Ile Gly Ile Phe Met Gly Met Met Leu Glu Thr Val Leu Lys

195 200 205

Ser Lys Ala Leu Ile Ile Asn Asn Phe Ser Glu Leu Asp Gly Glu Glu

210 215 220

Cys Ile Gln His Tyr Glu Lys Ala Thr Gly His Lys Val Trp His Leu

225 230 235 240

Gly Pro Thr Ser Leu Ile Arg Lys Thr Ala Gln Glu Lys Ser Glu Arg

245 250 255

Gly Asn Glu Gly Ala Val Asn Val His Glu Ser Leu Ser Trp Leu Asp

260 265 270

Ser Glu Arg Val Asn Ser Val Leu Tyr Ile Cys Phe Gly Ser Ile Asn

275 280 285

Tyr Phe Ser Asp Lys Gln Leu Tyr Glu Met Ala Cys Ala Ile Glu Ala

290 295 300

Ser Gly His Pro Phe Ile Trp Val Val Pro Glu Lys Lys Gly Lys Glu

305 310 315 320

Asp Glu Ser Glu Glu Glu Lys Glu Lys Trp Leu Pro Lys Gly Phe Glu

325 330 335

Glu Arg Asn Ile Gly Lys Lys Gly Leu Ile Ile Arg Gly Trp Ala Pro

340 345 350

Gln Val Lys Ile Leu Ser His Pro Ala Val Gly Gly Phe Met Thr His

355 360 365

Cys Gly Gly Asn Ser Thr Val Glu Ala Val Ser Ala Gly Val Pro Met

370 375 380

Ile Thr Trp Pro Val His Gly Asp Gln Phe Tyr Asn Glu Lys Leu Ile

385 390 395 400

Thr Gln Phe Arg Gly Ile Gly Val Glu Val Gly Ala Thr Glu Trp Cys

405 410 415

Thr Ser Gly Val Ala Glu Arg Lys Lys Leu Val Ser Arg Asp Ser Ile

420 425 430

Glu Lys Ala Val Arg Arg Leu Met Asp Gly Gly Asp Glu Ala Glu Asn

435 440 445

Ile Arg Leu Arg Ala Arg Glu Phe Gly Glu Lys Ala Ile Gln Ala Ile

450 455 460

Gln Glu Gly Gly Ser Ser Tyr Asn Asn Leu Leu Ala Leu Ile Asp Glu

465 470 475 480

Leu Lys Arg Ser Arg Asp Leu Lys Arg Leu Arg Asp Leu Lys Leu Asp

485 490 495

Asp

<210> 42

<211> 1374

<212> DNA

<213> Ginseng (Panax ginseng)

<220>

<221> misc_feature

<223> 3GT4

<400> 42

atggagagag aaatgttgag caaaactcac attatgttca tcccattccc agctcaaggc 60

cacatgagcc caatgatgca attcgccaag cgtttagcct ggaaaggcct gcgaatcacg 120

atagttcttc cggctcaaat tcgagatttc atgcaaataa ccaacccatt gatcaacact 180

gagtgcatct cctttgattt tgataaagac gatgggatgc catacagcat gcaggcttat 240

atgggagttg taaaactcaa ggtcacaaat aaactgagtg acctactcga gaagcaaaga 300

acaaatggct accctgttaa tttgctagtg gttgattcat tatatccatc tcgggtagaa 360

atgtgccacc aacttggggt aaaaggagct ccatttttca ctcactcttg tgctgttggt 420

gccatttatt ataatgctcg cttagggaaa ttgaagatac ctcctgagga agggttgact 480

tctgtttcat tgccttcaat tccattgttg gggagagatg atttgccaat tattaggact 540

ggcacctttc ctgatctctt tgagcatttg gggaatcagt tttcagatct tgataaagcg 600

gattggatct ttttcaatac ttttgataag cttgaaaatg aggaagcaaa atggctatct 660

agccaatggc caattacatc catcggacca ttaatccctt caatgtactt agacaaacaa 720

ttaccaaatg acaaagacaa tggcattaat ttctacaagg cagacgtcgg atcgtgcatc 780

aagtggctag acgccaaaga ccctggctcg gtagtctacg cctcattcgg gagcgtgaag 840

cacaacctcg gcgatgacta catggacgaa gtagcatggg gcttgttaca tagcaaatat 900

cacttcatat gggttgttat agaatccgaa cgtacaaagc tctctagcga tttcttggca 960

gaggcagagg cagaggaaaa aggcctaata gtgagttggt gccctcaact ccaagttttg 1020

tcacataaat ctatagggag ttttatgact cattgtggtt ggaactcgac ggttgaggca 1080

ttgagtttgg gcgtgccaat ggtggcactg ccacaacagt ttgatcagcc tgctaatgcc 1140

aagtatatcg tggatgtatg gcaaattggg gttcgggttc cgattggtga agagggggtt 1200

gttttgaggg gagaagttgc taactgtata aaggatgtta tggaggggga aataggggat 1260

gagcttagag ggaatgcttt gaaatggaag gggttggctg tggaggcaat ggagaaaggg 1320

ggtagctctg ataagaatat tgatgagttc atttcaaagc ttgtttcctc ctga 1374

<210> 43

<211> 457

<212> PRT

<213> Ginseng radix (Panax ginseng)

<220>

<221> misc_feature

<223> 3GT4

<400> 43

Met Glu Arg Glu Met Leu Ser Lys Thr His Ile Met Phe Ile Pro Phe

1 5 10 15

Pro Ala Gln Gly His Met Ser Pro Met Met Gln Phe Ala Lys Arg Leu

20 25 30

Ala Trp Lys Gly Leu Arg Ile Thr Ile Val Leu Pro Ala Gln Ile Arg

35 40 45

Asp Phe Met Gln Ile Thr Asn Pro Leu Ile Asn Thr Glu Cys Ile Ser

50 55 60

Phe Asp Phe Asp Lys Asp Asp Gly Met Pro Tyr Ser Met Gln Ala Tyr

65 70 75 80

Met Gly Val Val Lys Leu Lys Val Thr Asn Lys Leu Ser Asp Leu Leu

85 90 95

Glu Lys Gln Arg Thr Asn Gly Tyr Pro Val Asn Leu Leu Val Val Asp

100 105 110

Ser Leu Tyr Pro Ser Arg Val Glu Met Cys His Gln Leu Gly Val Lys

115 120 125

Gly Ala Pro Phe Phe Thr His Ser Cys Ala Val Gly Ala Ile Tyr Tyr

130 135 140

Asn Ala Arg Leu Gly Lys Leu Lys Ile Pro Pro Glu Glu Gly Leu Thr

145 150 155 160

Ser Val Ser Leu Pro Ser Ile Pro Leu Leu Gly Arg Asp Asp Leu Pro

165 170 175

Ile Ile Arg Thr Gly Thr Phe Pro Asp Leu Phe Glu His Leu Gly Asn

180 185 190

Gln Phe Ser Asp Leu Asp Lys Ala Asp Trp Ile Phe Phe Asn Thr Phe

195 200 205

Asp Lys Leu Glu Asn Glu Glu Ala Lys Trp Leu Ser Ser Gln Trp Pro

210 215 220

Ile Thr Ser Ile Gly Pro Leu Ile Pro Ser Met Tyr Leu Asp Lys Gln

225 230 235 240

Leu Pro Asn Asp Lys Asp Asn Gly Ile Asn Phe Tyr Lys Ala Asp Val

245 250 255

Gly Ser Cys Ile Lys Trp Leu Asp Ala Lys Asp Pro Gly Ser Val Val

260 265 270

Tyr Ala Ser Phe Gly Ser Val Lys His Asn Leu Gly Asp Asp Tyr Met

275 280 285

Asp Glu Val Ala Trp Gly Leu Leu His Ser Lys Tyr His Phe Ile Trp

290 295 300

Val Val Ile Glu Ser Glu Arg Thr Lys Leu Ser Ser Asp Phe Leu Ala

305 310 315 320

Glu Ala Glu Ala Glu Glu Lys Gly Leu Ile Val Ser Trp Cys Pro Gln

325 330 335

Leu Gln Val Leu Ser His Lys Ser Ile Gly Ser Phe Met Thr His Cys

340 345 350

Gly Trp Asn Ser Thr Val Glu Ala Leu Ser Leu Gly Val Pro Met Val

355 360 365

Ala Leu Pro Gln Gln Phe Asp Gln Pro Ala Asn Ala Lys Tyr Ile Val

370 375 380

Asp Val Trp Gln Ile Gly Val Arg Val Pro Ile Gly Glu Glu Gly Val

385 390 395 400

Val Leu Arg Gly Glu Val Ala Asn Cys Ile Lys Asp Val Met Glu Gly

405 410 415

Glu Ile Gly Asp Glu Leu Arg Gly Asn Ala Leu Lys Trp Lys Gly Leu

420 425 430

Ala Val Glu Ala Met Glu Lys Gly Gly Ser Ser Asp Lys Asn Ile Asp

435 440 445

Glu Phe Ile Ser Lys Leu Val Ser Ser

450 455

<210> 44

<211> 24

<212> DNA

<213> oligonucleotides

<400> 44

atggaaggtg ttgaagttga acaa 24

<210> 45

<211> 24

<212> DNA

<213> oligonucleotides

<400> 45

ttaatcatcc agcttgaggt ctct 24

<210> 46

<211> 25

<212> DNA

<213> oligonucleotides

<400> 46

tacataaata tataatacat aggca 25

<210> 47

<211> 25

<212> DNA

<213> oligonucleotides

<400> 47

aaaatacata caaaatcttg aaata 25

<210> 48

<211> 45

<212> DNA

<213> oligonucleotides

<400> 48

actttaagaa ggagatatac catggaaggt gttgaagttg aacaa 45

<210> 49

<211> 45

<212> DNA

<213> Artificial sequence

<400> 49

ctcgagtgcg gccgcaagct tatcatccag cttgaggtct ctcaa 45

<210> 50

<211> 46

<212> DNA

<213> oligonucleotides

<400> 50

actttaagaa ggagatatac catggagaga gaaatgttga gcaaaa 46

<210> 51

<211> 46

<212> DNA

<213> oligonucleotides

<400> 51

ctcgagtgcg gccgcaagct tggaggaaac aagctttgaa atgaac 46

<210> 52

<211> 27

<212> DNA

<213> oligonucleotides

<400> 52

ggtatatctc cttcttaaag ttaaaca 27

<210> 53

<211> 27

<212> DNA

<213> oligonucleotides

<400> 53

aagcttgcgg ccgcactcga gcaccac 27

<210> 54

<211> 1341

<212> DNA

<213> Ginseng (Panax ginseng)

<220>

<221> misc_feature

<223> gGT29-4

<400> 54

atggataacc aaaaaggtag aatcagtata gcgttgctac catttttagc ccatggccac 60

atttctccat tctttgagct agccaagcat ctctcaaaaa gaaattgtaa tatattcctc 120

tgttctaccc caatcaatct tagctccatc aagaacagaa tatctgataa ggattcctct 180

gcttctataa aactagtaga gcttcatctt ccatcttccc ctgatcttcc tcctcactac 240

cacaccacaa atggcctccc ttcccatctc atggtcccac tcagaaacgc ctttgaaaca 300

gcagccccca ccttctctga aatccttaaa accttaaacc ctgatttgct tatttatgat 360

ttcaatccct catgggcacc ggagatcgct tcgtctcaca atattccggc agtttgtttc 420

ataattgggg gagcagcctc cttttccatg agcctacata gtttcaaaaa cccaggtgaa 480

aaatacccat ttctagattt tgatgataac agtaatatta cccctgaacc accttcagca 540

gataacatga agttattact tgattttatg acttgtttcg aacgatcttg cgacattatt 600

ttgattaaga gttttagaga actagaaggg aaatattttg atttttattc tactttatct 660

gataaaactt tggttcctgt tggtccactc gttcaagatc ctatgggcca taatgaagat 720

ccaaaaacag agcagtttat aaactggctt gacaaaaggg ctgaatctac agtggtgttt 780

gtctgctttg gaagtgagta ttttctctcc aatgaggaat tggaagaagt agcaattggg 840

ctagagatta gcatggttaa tttcatatgg gctgtgagat taattgaagg agagaaaaaa 900

ggggttttac cagaggggtt tgttcaaagg gtaggagaca gaggattggt tgtggagggg 960

tgggctccac aggcaagaat tttaggacat tcaagcaccg gtgggtttgt gagccattgt 1020

gggtggagtt ctattacgga gagtatgaag tttggggttc cagtaattgc catggccagg 1080

catcttgatc agcctttgaa tggtaagctg gcggcggagg ttggtgtggg catggaggtt 1140

gtgagagatg aaaatgggaa gtataagaga gaagggattg cagaggtaat aagaaaagtc 1200

gttgtggaga aaagtgggga ggttatgagg aggaaagcaa gggaattgag tgagaaaatg 1260

aaagagaaag gagaggaaga gattgatagg gcagtggagg agctagtaca aatttgtaag 1320

aagaagaaag atgaacaata g 1341

<210> 55

<211> 446

<212> PRT

<213> Ginseng (Panax ginseng)

<220>

<221> misc_feature

<223> gGT29-4

<400> 55

Met Asp Asn Gln Lys Gly Arg Ile Ser Ile Ala Leu Leu Pro Phe Leu

1 5 10 15

Ala His Gly His Ile Ser Pro Phe Phe Glu Leu Ala Lys His Leu Ser

20 25 30

Lys Arg Asn Cys Asn Ile Phe Leu Cys Ser Thr Pro Ile Asn Leu Ser

35 40 45

Ser Ile Lys Asn Arg Ile Ser Asp Lys Asp Ser Ser Ala Ser Ile Lys

50 55 60

Leu Val Glu Leu His Leu Pro Ser Ser Pro Asp Leu Pro Pro His Tyr

65 70 75 80

His Thr Thr Asn Gly Leu Pro Ser His Leu Met Val Pro Leu Arg Asn

85 90 95

Ala Phe Glu Thr Ala Ala Pro Thr Phe Ser Glu Ile Leu Lys Thr Leu

100 105 110

Asn Pro Asp Leu Leu Ile Tyr Asp Phe Asn Pro Ser Trp Ala Pro Glu

115 120 125

Ile Ala Ser Ser His Asn Ile Pro Ala Val Cys Phe Ile Ile Gly Gly

130 135 140

Ala Ala Ser Phe Ser Met Ser Leu His Ser Phe Lys Asn Pro Gly Glu

145 150 155 160

Lys Tyr Pro Phe Leu Asp Phe Asp Asp Asn Ser Asn Ile Thr Pro Glu

165 170 175

Pro Pro Ser Ala Asp Asn Met Lys Leu Leu Leu Asp Phe Met Thr Cys

180 185 190

Phe Glu Arg Ser Cys Asp Ile Ile Leu Ile Lys Ser Phe Arg Glu Leu

195 200 205

Glu Gly Lys Tyr Phe Asp Phe Tyr Ser Thr Leu Ser Asp Lys Thr Leu

210 215 220

Val Pro Val Gly Pro Leu Val Gln Asp Pro Met Gly His Asn Glu Asp

225 230 235 240

Pro Lys Thr Glu Gln Phe Ile Asn Trp Leu Asp Lys Arg Ala Glu Ser

245 250 255

Thr Val Val Phe Val Cys Phe Gly Ser Glu Tyr Phe Leu Ser Asn Glu

260 265 270

Glu Leu Glu Glu Val Ala Ile Gly Leu Glu Ile Ser Met Val Asn Phe

275 280 285

Ile Trp Ala Val Arg Leu Ile Glu Gly Glu Lys Lys Gly Val Leu Pro

290 295 300

Glu Gly Phe Val Gln Arg Val Gly Asp Arg Gly Leu Val Val Glu Gly

305 310 315 320

Trp Ala Pro Gln Ala Arg Ile Leu Gly His Ser Ser Thr Gly Gly Phe

325 330 335

Val Ser His Cys Gly Trp Ser Ser Ile Thr Glu Ser Met Lys Phe Gly

340 345 350

Val Pro Val Ile Ala Met Ala Arg His Leu Asp Gln Pro Leu Asn Gly

355 360 365

Lys Leu Ala Ala Glu Val Gly Val Gly Met Glu Val Val Arg Asp Glu

370 375 380

Asn Gly Lys Tyr Lys Arg Glu Gly Ile Ala Glu Val Ile Arg Lys Val

385 390 395 400

Val Val Glu Lys Ser Gly Glu Val Met Arg Arg Lys Ala Arg Glu Leu

405 410 415

Ser Glu Lys Met Lys Glu Lys Gly Glu Glu Glu Ile Asp Arg Ala Val

420 425 430

Glu Glu Leu Val Gln Ile Cys Lys Lys Lys Lys Asp Glu Gln

435 440 445

<210> 56

<211> 1341

<212> DNA

<213> Ginseng radix (Panax ginseng)

<220>

<221> misc_feature

<223> gGT29-5

<400> 56

atggataacc aaaagggtag aatcagtata gttatgctgc catttttagc ccatggccac 60

atttctccat tctttgagct agccaagcat ctctcaaaaa gaaattgtaa tatattcctc 120

tgttctaccc caatcaatct tagctccatc aagaacagaa tatctgataa ggattcctct 180

gcttctataa aactagtaga gcttcatctt ccatcttccc ctgatcttcc tcctcactac 240

cacaccacaa atggcctccc ttcccatctc atggtcccac tcagaaacgc ctttgaaaca 300

gcagccccca ccttctctga aatccttaaa accttaaacc ctgatttgct tatttatgat 360

ttcaatccct catgggcacc ggagatcgct tcgtctcaca atattccggc agtttgtttc 420

ataattgggg gagcagcctc cttttccatg agcctacata gtttcaaaaa cccaggtgaa 480

aaatacccat ttctagattt tgatgataac agtaatatta cccctgaacc accttcagca 540

gataacatga agttattact tgattttatg acttgtttcg aacgatcttg cgacattatt 600

ttgattaaga gttttagaga actagaaggg aaatatatcg atttgctttc cactttatct 660

gataaaactt tggttcctgt tggtccactc gttcaagatc ctatgggcca taatgaagat 720

ccaaaaacag agcagattat aaactggctt gacaaaaggg ctgaatctac agtggtgttt 780

gtctgctttg gaagtgagta ttttctctcc aatgaggaat tggaagaagt agcaattggg 840

ctagagatta gcatggttaa tttcatatgg gctgtgagat taattgaagg agagaaaaaa 900

ggggttttac cagagggatt tgttcaaagg gtaggagaca gaggattggt tgtggagggg 960

tgggctccac aggcaagaat tttaggacat tcaagcaccg gtgggtttgt gagccattgt 1020

gggtggagtt ctattgcgga gagtatgaag tttggggttc cagtaattgc catggccagg 1080

catcttgatc agcctttgaa tggtaagctg gcggcggagg ttggtgtggg catggaggtt 1140

gtgagagatg aaaatgggaa gtataagaga gaagggattg cagaggtaat aagaaaagtc 1200

gttgtggaga aaagtgggga ggttatcagg aggaaagcaa gggaattgag tgagaaaatg 1260

aaagagatag gagagcaatt gattgatagg gcagtggagg agctagtaca aatttgtaag 1320

aagaagaaag atgaacaata g 1341

<210> 57

<211> 446

<212> PRT

<213> Ginseng (Panax ginseng)

<220>

<221> misc_feature

<223> gGT29-5

<400> 57

Met Asp Asn Gln Lys Gly Arg Ile Ser Ile Val Met Leu Pro Phe Leu

1 5 10 15

Ala His Gly His Ile Ser Pro Phe Phe Glu Leu Ala Lys His Leu Ser

20 25 30

Lys Arg Asn Cys Asn Ile Phe Leu Cys Ser Thr Pro Ile Asn Leu Ser

35 40 45

Ser Ile Lys Asn Arg Ile Ser Asp Lys Asp Ser Ser Ala Ser Ile Lys

50 55 60

Leu Val Glu Leu His Leu Pro Ser Ser Pro Asp Leu Pro Pro His Tyr

65 70 75 80

His Thr Thr Asn Gly Leu Pro Ser His Leu Met Val Pro Leu Arg Asn

85 90 95

Ala Phe Glu Thr Ala Ala Pro Thr Phe Ser Glu Ile Leu Lys Thr Leu

100 105 110

Asn Pro Asp Leu Leu Ile Tyr Asp Phe Asn Pro Ser Trp Ala Pro Glu

115 120 125

Ile Ala Ser Ser His Asn Ile Pro Ala Val Cys Phe Ile Ile Gly Gly

130 135 140

Ala Ala Ser Phe Ser Met Ser Leu His Ser Phe Lys Asn Pro Gly Glu

145 150 155 160

Lys Tyr Pro Phe Leu Asp Phe Asp Asp Asn Ser Asn Ile Thr Pro Glu

165 170 175

Pro Pro Ser Ala Asp Asn Met Lys Leu Leu Leu Asp Phe Met Thr Cys

180 185 190

Phe Glu Arg Ser Cys Asp Ile Ile Leu Ile Lys Ser Phe Arg Glu Leu

195 200 205

Glu Gly Lys Tyr Ile Asp Leu Leu Ser Thr Leu Ser Asp Lys Thr Leu

210 215 220

Val Pro Val Gly Pro Leu Val Gln Asp Pro Met Gly His Asn Glu Asp

225 230 235 240

Pro Lys Thr Glu Gln Ile Ile Asn Trp Leu Asp Lys Arg Ala Glu Ser

245 250 255

Thr Val Val Phe Val Cys Phe Gly Ser Glu Tyr Phe Leu Ser Asn Glu

260 265 270

Glu Leu Glu Glu Val Ala Ile Gly Leu Glu Ile Ser Met Val Asn Phe

275 280 285

Ile Trp Ala Val Arg Leu Ile Glu Gly Glu Lys Lys Gly Val Leu Pro

290 295 300

Glu Gly Phe Val Gln Arg Val Gly Asp Arg Gly Leu Val Val Glu Gly

305 310 315 320

Trp Ala Pro Gln Ala Arg Ile Leu Gly His Ser Ser Thr Gly Gly Phe

325 330 335

Val Ser His Cys Gly Trp Ser Ser Ile Ala Glu Ser Met Lys Phe Gly

340 345 350

Val Pro Val Ile Ala Met Ala Arg His Leu Asp Gln Pro Leu Asn Gly

355 360 365

Lys Leu Ala Ala Glu Val Gly Val Gly Met Glu Val Val Arg Asp Glu

370 375 380

Asn Gly Lys Tyr Lys Arg Glu Gly Ile Ala Glu Val Ile Arg Lys Val

385 390 395 400

Val Val Glu Lys Ser Gly Glu Val Ile Arg Arg Lys Ala Arg Glu Leu

405 410 415

Ser Glu Lys Met Lys Glu Ile Gly Glu Gln Leu Ile Asp Arg Ala Val

420 425 430

Glu Glu Leu Val Gln Ile Cys Lys Lys Lys Lys Asp Glu Gln

435 440 445

<210> 58

<211> 1341

<212> DNA

<213> Ginseng radix (Panax ginseng)

<220>

<221> misc_feature

<223> gGT29-6

<400> 58

atggataacc aaaagggtag aatcagtata gttatgctgc catttttagc ccatggccac 60

atttctccat tctttgagct agccaagcat ctctcaaaaa gaaattgtaa tatattcctc 120

tgttctaccc caatcaatct tagctccatc aagaacagaa tatctgataa ggattcctct 180

gcttctataa aactagtaga gcttcatctt ccatcttccc ctgatcttcc tcctcactac 240

cacaccacaa atggcctccc ttcccatctc atggtcccac tcagaaacgc ctttgaaaca 300

gcagccccca ccttctctga aatccttaaa accttaaacc ctgatttgct tatttatgat 360

ttcaatccct catgggcacc ggagatcgct tcgtctcaca atattccggc agtttatttc 420

ctaaccacgg cagcagccag ctcttccatt ggcctacatg ctttcaaaaa cccaggtgaa 480

aaatacccat ttccagattt ttatgataac agtaataata cccctgaacc accttctgca 540

gataacatga agctacttca tgattttatc gcttgtttcg aacgatcttg cgacattatt 600

ttgattaaga gttttataga actagaaggg aaatatatcg atttgctttc cactttatct 660

gataaaactt tggttcctgt tggtccactc gttcaagatc ctatgggcca taatgaagat 720

ccaaaaacag agcagattat aaactggctt gacaaaaggg ctgaatctac agtggtgttt 780

gtctgctttg gaagtgagta ttttctctcc aatgaggaat tggaagaagt agcaattggg 840

ctagagatta gcatggttaa tttcatatgg gctgtgagat taattgaagg agagaaaaaa 900

ggggttttac cagagggatt tgttcaaagg gtaggagaca gaggattggt tgtggagggg 960

tgggctccac aggcaagaat tttaggacat tcaagcaccg gtgggtttgt gagccattgt 1020

gggtggagtt ctattgcgga gagtatgaag tttggggttc cagtaattgc catggccagg 1080

catcttgatc agcctttgaa tggtaagctg gcggcggagg ttggtgtggg catggaggtt 1140

gtgagagatg aaaatgggaa gtataagaga gaagatattg caggggtaat aagaaaagtc 1200

gtggtggaga aaagtgggga ggttatcagg aggaaagcaa gggaattgag tgagaaaatg 1260

aaagagatag gagagcaatt gattgatagg gcagtggagg agctagtaca aatttgtaag 1320

aagaagaaag atgaacaata g 1341

<210> 59

<211> 446

<212> PRT

<213> Ginseng radix (Panax ginseng)

<220>

<221> misc_feature

<223> gGT29-6

<400> 59

Met Asp Asn Gln Lys Gly Arg Ile Ser Ile Val Met Leu Pro Phe Leu

1 5 10 15

Ala His Gly His Ile Ser Pro Phe Phe Glu Leu Ala Lys His Leu Ser

20 25 30

Lys Arg Asn Cys Asn Ile Phe Leu Cys Ser Thr Pro Ile Asn Leu Ser

35 40 45

Ser Ile Lys Asn Arg Ile Ser Asp Lys Asp Ser Ser Ala Ser Ile Lys

50 55 60

Leu Val Glu Leu His Leu Pro Ser Ser Pro Asp Leu Pro Pro His Tyr

65 70 75 80

His Thr Thr Asn Gly Leu Pro Ser His Leu Met Val Pro Leu Arg Asn

85 90 95

Ala Phe Glu Thr Ala Ala Pro Thr Phe Ser Glu Ile Leu Lys Thr Leu

100 105 110

Asn Pro Asp Leu Leu Ile Tyr Asp Phe Asn Pro Ser Trp Ala Pro Glu

115 120 125

Ile Ala Ser Ser His Asn Ile Pro Ala Val Tyr Phe Leu Thr Thr Ala

130 135 140

Ala Ala Ser Ser Ser Ile Gly Leu His Ala Phe Lys Asn Pro Gly Glu

145 150 155 160

Lys Tyr Pro Phe Pro Asp Phe Tyr Asp Asn Ser Asn Asn Thr Pro Glu

165 170 175

Pro Pro Ser Ala Asp Asn Met Lys Leu Leu His Asp Phe Ile Ala Cys

180 185 190

Phe Glu Arg Ser Cys Asp Ile Ile Leu Ile Lys Ser Phe Ile Glu Leu

195 200 205

Glu Gly Lys Tyr Ile Asp Leu Leu Ser Thr Leu Ser Asp Lys Thr Leu

210 215 220

Val Pro Val Gly Pro Leu Val Gln Asp Pro Met Gly His Asn Glu Asp

225 230 235 240

Pro Lys Thr Glu Gln Ile Ile Asn Trp Leu Asp Lys Arg Ala Glu Ser

245 250 255

Thr Val Val Phe Val Cys Phe Gly Ser Glu Tyr Phe Leu Ser Asn Glu

260 265 270

Glu Leu Glu Glu Val Ala Ile Gly Leu Glu Ile Ser Met Val Asn Phe

275 280 285

Ile Trp Ala Val Arg Leu Ile Glu Gly Glu Lys Lys Gly Val Leu Pro

290 295 300

Glu Gly Phe Val Gln Arg Val Gly Asp Arg Gly Leu Val Val Glu Gly

305 310 315 320

Trp Ala Pro Gln Ala Arg Ile Leu Gly His Ser Ser Thr Gly Gly Phe

325 330 335

Val Ser His Cys Gly Trp Ser Ser Ile Ala Glu Ser Met Lys Phe Gly

340 345 350

Val Pro Val Ile Ala Met Ala Arg His Leu Asp Gln Pro Leu Asn Gly

355 360 365

Lys Leu Ala Ala Glu Val Gly Val Gly Met Glu Val Val Arg Asp Glu

370 375 380

Asn Gly Lys Tyr Lys Arg Glu Asp Ile Ala Gly Val Ile Arg Lys Val

385 390 395 400

Val Val Glu Lys Ser Gly Glu Val Ile Arg Arg Lys Ala Arg Glu Leu

405 410 415

Ser Glu Lys Met Lys Glu Ile Gly Glu Gln Leu Ile Asp Arg Ala Val

420 425 430

Glu Glu Leu Val Gln Ile Cys Lys Lys Lys Lys Asp Glu Gln

435 440 445

<210> 60

<211> 1341

<212> DNA

<213> Ginseng radix (Panax ginseng)

<220>

<221> misc_feature

<223> gGT29-7

<400> 60

atggataacc aaaaaggtag aatcagtata gcgttgctac catttttagc ccatggtcac 60

atatctccct tctttgagct agccaaacaa ctcgcaaaaa gaaattgcaa tgttttcctc 120

tgttctaccc caatcaatct tagctccatc aagaacagag tatctgataa ggattcctct 180

gcttctataa aactagtaga gcttcatctt ccatcttccc ctgatcttcc tcctcactac 240

cacaccacaa atggcctccc ttcccatctc atgatcccac tcagaaacgc ctttgataca 300

gcaggcccca ccttctctga aatccttaaa accttaaacc ctgatttgct tatttatgat 360

ttcaatccct catgggcacc ggagatcgct tcgtctcaca atattccggc agtttgtttc 420

ataattggtg gagcagcctc ctcttccatg agcctacata gtttcaaaaa cccaggtgaa 480

aaatacccat ttctagattt tgatgataac agtaatatta cccctgaacc accttcagca 540

gataacatga agctattaat taattttatg acttgtttcg aacgatcttg cgacattatt 600

ttgattaaga gttttagaga actagaaggg aaatattttg attttttttc cactttatct 660

gataaaactt tggttcctgt tggtccactc gttcaagatc ctatgggcca taatgaagat 720

ccaaaaacag agcagtttat aaactggctt gacaaaaggg ctgaatctac agtggtgttt 780

gtctgctttg gaagtgagtg ttttctctcc aatgaggaat tggaagaagt agcgattggg 840

ctagagatta gcatggttaa tttcatatgg gctgtgagat taattgaagg agagaaaaaa 900

ggggttttac cagaggggtt tgttcaaagg gtaggagaca gaggattggt tgtggaggag 960

tgggctccac aggcaagaat tttaggacat tcaagcaccg gtgggtttgt gagccattgt 1020

gggtggaatt ctattacgga gagtatgaag tttggggttc cagtaattgc catggccagg 1080

cattttgatc agcctttgaa tggtaagctg gcggcggagg ttggtgtggg catggaggtt 1140

gtgagagatg aaaatgggaa gtataagaga gaagggattg cagaggtaat aagaaaagtc 1200

gttgtggaga aaagtgggga ggttatcagg aggaaagcaa gggaattgag tgagaaaatg 1260

aaagagaaag gagagcaaga gattgatagg gtagtggagg agctagtaca aatttgtaag 1320

aagaagaaag atgaacaata g 1341

<210> 61

<211> 446

<212> PRT

<213> Ginseng (Panax ginseng)

<220>

<221> misc_feature

<223> gGT29-7

<400> 61

Met Asp Asn Gln Lys Gly Arg Ile Ser Ile Ala Leu Leu Pro Phe Leu

1 5 10 15

Ala His Gly His Ile Ser Pro Phe Phe Glu Leu Ala Lys Gln Leu Ala

20 25 30

Lys Arg Asn Cys Asn Val Phe Leu Cys Ser Thr Pro Ile Asn Leu Ser

35 40 45

Ser Ile Lys Asn Arg Val Ser Asp Lys Asp Ser Ser Ala Ser Ile Lys

50 55 60

Leu Val Glu Leu His Leu Pro Ser Ser Pro Asp Leu Pro Pro His Tyr

65 70 75 80

His Thr Thr Asn Gly Leu Pro Ser His Leu Met Ile Pro Leu Arg Asn

85 90 95

Ala Phe Asp Thr Ala Gly Pro Thr Phe Ser Glu Ile Leu Lys Thr Leu

100 105 110

Asn Pro Asp Leu Leu Ile Tyr Asp Phe Asn Pro Ser Trp Ala Pro Glu

115 120 125

Ile Ala Ser Ser His Asn Ile Pro Ala Val Cys Phe Ile Ile Gly Gly

130 135 140

Ala Ala Ser Ser Ser Met Ser Leu His Ser Phe Lys Asn Pro Gly Glu

145 150 155 160

Lys Tyr Pro Phe Leu Asp Phe Asp Asp Asn Ser Asn Ile Thr Pro Glu

165 170 175

Pro Pro Ser Ala Asp Asn Met Lys Leu Leu Ile Asn Phe Met Thr Cys

180 185 190

Phe Glu Arg Ser Cys Asp Ile Ile Leu Ile Lys Ser Phe Arg Glu Leu

195 200 205

Glu Gly Lys Tyr Phe Asp Phe Phe Ser Thr Leu Ser Asp Lys Thr Leu

210 215 220

Val Pro Val Gly Pro Leu Val Gln Asp Pro Met Gly His Asn Glu Asp

225 230 235 240

Pro Lys Thr Glu Gln Phe Ile Asn Trp Leu Asp Lys Arg Ala Glu Ser

245 250 255

Thr Val Val Phe Val Cys Phe Gly Ser Glu Cys Phe Leu Ser Asn Glu

260 265 270

Glu Leu Glu Glu Val Ala Ile Gly Leu Glu Ile Ser Met Val Asn Phe

275 280 285

Ile Trp Ala Val Arg Leu Ile Glu Gly Glu Lys Lys Gly Val Leu Pro

290 295 300

Glu Gly Phe Val Gln Arg Val Gly Asp Arg Gly Leu Val Val Glu Glu

305 310 315 320

Trp Ala Pro Gln Ala Arg Ile Leu Gly His Ser Ser Thr Gly Gly Phe

325 330 335

Val Ser His Cys Gly Trp Asn Ser Ile Thr Glu Ser Met Lys Phe Gly

340 345 350

Val Pro Val Ile Ala Met Ala Arg His Phe Asp Gln Pro Leu Asn Gly

355 360 365

Lys Leu Ala Ala Glu Val Gly Val Gly Met Glu Val Val Arg Asp Glu

370 375 380

Asn Gly Lys Tyr Lys Arg Glu Gly Ile Ala Glu Val Ile Arg Lys Val

385 390 395 400

Val Val Glu Lys Ser Gly Glu Val Ile Arg Arg Lys Ala Arg Glu Leu

405 410 415

Ser Glu Lys Met Lys Glu Lys Gly Glu Gln Glu Ile Asp Arg Val Val

420 425 430

Glu Glu Leu Val Gln Ile Cys Lys Lys Lys Lys Asp Glu Gln

435 440 445

<210> 62

<211> 26

<212> DNA

<213> Artificial sequence

<400> 62

acagcaagag agagacacag agttca 26

<210> 63

<211> 23

<212> DNA

<213> oligonucleotides

<400> 63

gttcaaagcc caacctaagc gca 23

<210> 64

<211> 25

<212> DNA

<213> oligonucleotides

<400> 64

atgaaattat acagagaggg agaga 25

<210> 65

<211> 25

<212> DNA

<213> oligonucleotides

<400> 65

tggttttctt cacaacacaa agtac 25

<210> 66

<211> 43

<212> DNA

<213> oligonucleotides

<400> 66

ctggtgccgc gcggcagcat ggataaccaa aagggtagaa tca 43

<210> 67

<211> 43

<212> DNA

<213> oligonucleotides

<400> 67

ctggtgccgc gcggcagcat ggataaccaa aaaggtagaa tca 43

<210> 68

<211> 43

<212> DNA

<213> oligonucleotides

<400> 68

tgcggccgca agcttgtctt gttcatcttt cttcttctta caa 43

<210> 69

<211> 30

<212> DNA

<213> oligonucleotides

<400> 69

gctgccgcgc ggcaccaggc cgctgctgtg 30

<210> 70

<211> 30

<212> DNA

<213> oligonucleotides

<400> 70

tccgtcgaca agcttgcggc cgcactcgag 30

<210> 71

<211> 1329

<212> DNA

<213> Ginseng radix (Panax ginseng)

<400> 71

atggataacc aagaaggtag aatcagtata gcgttgctac catttttagc ccatggtcac 60

atatctccct tctttgagct agccaaacaa ctcgcaaaaa gaaattgcaa tgttttcctc 120

tgttctaccc caatcaatct tagctccatc aagaataagg attcctctgc ttctataaaa 180

ctagtagagc ttcatcttcc atcttcccct gatcttcctc ctcactacca caccacaaat 240

ggcctccctt cccatctcat ggtcccactc agaaacgcct ttgaaacagc agcccccacc 300

ttctctgaaa tccttaaaac cttaaaccct gatttgctta tttatgattt caatccctca 360

tgggcaccgg agatcgcttc gtctcacaat attccggcag tttgtttcat aattggggga 420

gcagcctcct tttccatgag cctacatagt ttcaaaaacc caggtgaaaa atacccattt 480

ctagattttg atgataacag taatattacc cctgaaccac cttcagcaga taacatgaag 540

ttattacttg attttatgac ttgtttcgaa cgatcttgcg acattatttt gattaagagt 600

tttagagaac tagaagggaa atattttgat ttttattcta ctttatctga taaaactttg 660

gttcctgttg gtccactcgt tcaagatcct atgggccata atgaagatcc aaaaacagag 720

cagtttataa actggcttga caaaagggct gaatctacag tggtgtttgt ctgctttgga 780

agtgagtatt ttctctccaa tgaggaattg gaagaagtag caattgggct agagattagc 840

atggttaatt tcatatgggc tgtgagatta attgaaggag agaaaaaagg ggttttacca 900

gaggggtttg ttcaaagggt aggagacaga ggattggttg tggaggggtg ggctccacag 960

gcaagaattt taggacattc aagcaccggt gggtttgtga gccattgtgg gtggagttct 1020

attacggaga gtatgaagtt tggggttcca gtatttgcca tggccaggca ttttgatcag 1080

cctttgaatg ctaagctggc ggcggaggtt ggtgtgggca tggaggttgt gagagatgaa 1140

aatgggaagt ataagagaga agatattgca ggggtaataa gaaaagtcgt ggtggagaaa 1200

agtggggagg ttatcaggag gaaagcaagg gaattgagtg agaaaatgaa agagatagga 1260

gagcaattga ttgatagggc agtggaggag ctagtacaaa tttgtaagaa gaagaaagat 1320

gaacaatag 1329

<210> 72

<211> 442

<212> PRT

<213> Ginseng radix (Panax ginseng)

<400> 72

Met Asp Asn Gln Glu Gly Arg Ile Ser Ile Ala Leu Leu Pro Phe Leu

1 5 10 15

Ala His Gly His Ile Ser Pro Phe Phe Glu Leu Ala Lys Gln Leu Ala

20 25 30

Lys Arg Asn Cys Asn Val Phe Leu Cys Ser Thr Pro Ile Asn Leu Ser

35 40 45

Ser Ile Lys Asn Lys Asp Ser Ser Ala Ser Ile Lys Leu Val Glu Leu

50 55 60

His Leu Pro Ser Ser Pro Asp Leu Pro Pro His Tyr His Thr Thr Asn

65 70 75 80

Gly Leu Pro Ser His Leu Met Val Pro Leu Arg Asn Ala Phe Glu Thr

85 90 95

Ala Ala Pro Thr Phe Ser Glu Ile Leu Lys Thr Leu Asn Pro Asp Leu

100 105 110

Leu Ile Tyr Asp Phe Asn Pro Ser Trp Ala Pro Glu Ile Ala Ser Ser

115 120 125

His Asn Ile Pro Ala Val Cys Phe Ile Ile Gly Gly Ala Ala Ser Phe

130 135 140

Ser Met Ser Leu His Ser Phe Lys Asn Pro Gly Glu Lys Tyr Pro Phe

145 150 155 160

Leu Asp Phe Asp Asp Asn Ser Asn Ile Thr Pro Glu Pro Pro Ser Ala

165 170 175

Asp Asn Met Lys Leu Leu Leu Asp Phe Met Thr Cys Phe Glu Arg Ser

180 185 190

Cys Asp Ile Ile Leu Ile Lys Ser Phe Arg Glu Leu Glu Gly Lys Tyr

195 200 205

Phe Asp Phe Tyr Ser Thr Leu Ser Asp Lys Thr Leu Val Pro Val Gly

210 215 220

Pro Leu Val Gln Asp Pro Met Gly His Asn Glu Asp Pro Lys Thr Glu

225 230 235 240

Gln Phe Ile Asn Trp Leu Asp Lys Arg Ala Glu Ser Thr Val Val Phe

245 250 255

Val Cys Phe Gly Ser Glu Tyr Phe Leu Ser Asn Glu Glu Leu Glu Glu

260 265 270

Val Ala Ile Gly Leu Glu Ile Ser Met Val Asn Phe Ile Trp Ala Val

275 280 285

Arg Leu Ile Glu Gly Glu Lys Lys Gly Val Leu Pro Glu Gly Phe Val

290 295 300

Gln Arg Val Gly Asp Arg Gly Leu Val Val Glu Gly Trp Ala Pro Gln

305 310 315 320

Ala Arg Ile Leu Gly His Ser Ser Thr Gly Gly Phe Val Ser His Cys

325 330 335

Gly Trp Ser Ser Ile Thr Glu Ser Met Lys Phe Gly Val Pro Val Phe

340 345 350

Ala Met Ala Arg His Phe Asp Gln Pro Leu Asn Ala Lys Leu Ala Ala

355 360 365

Glu Val Gly Val Gly Met Glu Val Val Arg Asp Glu Asn Gly Lys Tyr

370 375 380

Lys Arg Glu Asp Ile Ala Gly Val Ile Arg Lys Val Val Val Glu Lys

385 390 395 400

Ser Gly Glu Val Ile Arg Arg Lys Ala Arg Glu Leu Ser Glu Lys Met

405 410 415

Lys Glu Ile Gly Glu Gln Leu Ile Asp Arg Ala Val Glu Glu Leu Val

420 425 430

Gln Ile Cys Lys Lys Lys Lys Asp Glu Gln

435 440

<210> 73

<211> 1341

<212> DNA

<213> Ginseng (Panax ginseng)

<400> 73

atggataacc aaaagggtag aatcagtata gttatgctgc catttttagc ccatggccac 60

atttctccat tctttgagct agccaagcat ctctcaaaaa gaaattgtaa tatattcctc 120

tgttctaccc caatcaatct tagctccatc aagaacagaa tatctgataa ggattcctct 180

gcttctataa aactagtaga gcttcatctt ccatcttccc ctgatcttcc tcctcactac 240

cacaccacaa atggcctccc ttcccatctc atggtcccac tcagaaacgc ctttgaaaca 300

gcagccccca ccttctctga aatccttaaa accttaaacc ctgatttgct tatttatgat 360

ttcaatccct catgggcacc ggagatcgct tcgtctcaca atattccggc agtttgtttc 420

ataattgggg gagcagcctc cttttccatg agcctacata gtttcaaaaa cccaggtgaa 480

aaatacccat ttctagattt tgatgataac agtaatatta cccctgaacc accttcagca 540

gataacatga agttattact tgattttatg acttgtttcg aacgatcttg cgacattatt 600

ttgattaaga gttttagaga actagaaggg aaatattttg atttttattc tactttatct 660

gataaaactt tggttcctgt tggtccactc gttcaagatc ctatgggcca taatgaagat 720

ccaaaaacag agcagtttat aaactggctt gacaaaaggg ctgaatctac agtggtgttt 780

gtctgctttg gaagtgagta ttttctctcc aatgaggaat tggaagaagt agcaattggg 840

ctagagatta gcatggttaa tttcatatgg gctgtgagat taattgaagg agagaaaaaa 900

ggggttttac cagagggatt tgttcaaagg gtaggagaca gaggattggt tgtggagggg 960

tgggctccac aggcaagaat tttaggacat tcaagcaccg gtgggtttgt gagccattgt 1020

gggtggagtt ctattgcgga gagtatgaag tttggggttc cagtaattgc catggccagg 1080

catcttgatc agcctttgaa tggtaagctg gcggcggagg ttggtgtggg catggaggtt 1140

gtgagagatg aaaatgggaa gtataagaga gaagatattg caggggtaat aagaaaagtc 1200

gtggtggaga aaagtgggga ggttatcagg aggaaagcaa gggaattgag tgagaaaatg 1260

aaagagatag gagagcaatt gattgatagg gcagtggagg agctagtaca aatttgtaag 1320

aagaagaaag atgaacaata g 1341

<210> 74

<211> 446

<212> PRT

<213> Ginseng (Panax ginseng)

<400> 74

Met Asp Asn Gln Lys Gly Arg Ile Ser Ile Val Met Leu Pro Phe Leu

1 5 10 15

Ala His Gly His Ile Ser Pro Phe Phe Glu Leu Ala Lys His Leu Ser

20 25 30

Lys Arg Asn Cys Asn Ile Phe Leu Cys Ser Thr Pro Ile Asn Leu Ser

35 40 45

Ser Ile Lys Asn Arg Ile Ser Asp Lys Asp Ser Ser Ala Ser Ile Lys

50 55 60

Leu Val Glu Leu His Leu Pro Ser Ser Pro Asp Leu Pro Pro His Tyr

65 70 75 80

His Thr Thr Asn Gly Leu Pro Ser His Leu Met Val Pro Leu Arg Asn

85 90 95

Ala Phe Glu Thr Ala Ala Pro Thr Phe Ser Glu Ile Leu Lys Thr Leu

100 105 110

Asn Pro Asp Leu Leu Ile Tyr Asp Phe Asn Pro Ser Trp Ala Pro Glu

115 120 125

Ile Ala Ser Ser His Asn Ile Pro Ala Val Cys Phe Ile Ile Gly Gly

130 135 140

Ala Ala Ser Phe Ser Met Ser Leu His Ser Phe Lys Asn Pro Gly Glu

145 150 155 160

Lys Tyr Pro Phe Leu Asp Phe Asp Asp Asn Ser Asn Ile Thr Pro Glu

165 170 175

Pro Pro Ser Ala Asp Asn Met Lys Leu Leu Leu Asp Phe Met Thr Cys

180 185 190

Phe Glu Arg Ser Cys Asp Ile Ile Leu Ile Lys Ser Phe Arg Glu Leu

195 200 205

Glu Gly Lys Tyr Phe Asp Phe Tyr Ser Thr Leu Ser Asp Lys Thr Leu

210 215 220

Val Pro Val Gly Pro Leu Val Gln Asp Pro Met Gly His Asn Glu Asp

225 230 235 240

Pro Lys Thr Glu Gln Phe Ile Asn Trp Leu Asp Lys Arg Ala Glu Ser

245 250 255

Thr Val Val Phe Val Cys Phe Gly Ser Glu Tyr Phe Leu Ser Asn Glu

260 265 270

Glu Leu Glu Glu Val Ala Ile Gly Leu Glu Ile Ser Met Val Asn Phe

275 280 285

Ile Trp Ala Val Arg Leu Ile Glu Gly Glu Lys Lys Gly Val Leu Pro

290 295 300

Glu Gly Phe Val Gln Arg Val Gly Asp Arg Gly Leu Val Val Glu Gly

305 310 315 320

Trp Ala Pro Gln Ala Arg Ile Leu Gly His Ser Ser Thr Gly Gly Phe

325 330 335

Val Ser His Cys Gly Trp Ser Ser Ile Ala Glu Ser Met Lys Phe Gly

340 345 350

Val Pro Val Ile Ala Met Ala Arg His Leu Asp Gln Pro Leu Asn Gly

355 360 365

Lys Leu Ala Ala Glu Val Gly Val Gly Met Glu Val Val Arg Asp Glu

370 375 380

Asn Gly Lys Tyr Lys Arg Glu Asp Ile Ala Gly Val Ile Arg Lys Val

385 390 395 400

Val Val Glu Lys Ser Gly Glu Val Ile Arg Arg Lys Ala Arg Glu Leu

405 410 415

Ser Glu Lys Met Lys Glu Ile Gly Glu Gln Leu Ile Asp Arg Ala Val

420 425 430

Glu Glu Leu Val Gln Ile Cys Lys Lys Lys Lys Asp Glu Gln

435 440 445

<210> 75

<211> 1341

<212> DNA

<213> Ginseng radix (Panax ginseng)

<400> 75

atggataacc aaaagggtag aatcagtata gttatgctgc catttttagc ccatggccac 60

atttctccat tctttgagct agccaagcat ctctcaaaaa gaaattgtaa tatattcctc 120

tgttctaccc caatcaatct tagctccatc aagaacagaa tatctgataa ggattcctct 180

gcttctataa aactagtaga gcttcatctt ccatcttccc ctgatcttcc tcctcactat 240

cacaccacaa atggcctccc ttcccatctc atggtcccac tcataaacgc ctttgaaaca 300

gcaggcccca ccttctctga aatccttaaa accttaaacc ccgatttgct tatttatgat 360

ttcaatccct catgggcacc ggagatcgct tcgtctcaca atattccggc agtttatttc 420

ctaaccacgg cagcagccag ctcttccatt ggcctacatg ctttcaaaaa cccaggtgaa 480

aaatacccat ttccagattt ttatgataac agtaataata cccctgaacc accttctgca 540

gataacatga agctacttca tgattttatc gcttgtttcg aacgatcttg cgacattatt 600

ttgattaaga gttttataga actagaaggg aaatatatcg atttgctttc cactttatct 660

gataaaactt tggttcctgt tggtccactc gttcaagatc ctatgggcca taatgaagat 720

ccaaaaacag agcagattat aaactggctt gacaaaaggg ctgaatctac agtggtgttt 780

gtctgctttg gaagtgagta ttttctctcc aatgaggaat tggaagaagt agcaattggg 840

ctagagatta gcatggttaa tttcatatgg gctgtgagat taattgaagg agagaaaaaa 900

ggggttttac cagagggatt tgttcaaagg gtaggagaca gaggattggt tgtggagggg 960

tgggctccac aggcaagaat tttaggacat tcaagcaccg gtgggtttgt gagccattgt 1020

gggtggagtt ctattgcgga gagtatgaag tttggggttc cagtaattgc catggccagg 1080

catcttgatc agcctttgaa tggtaagctg gcggcggagg ttggtgtggg catggaggtt 1140

gtgagagatg aaaatgggaa gtataagaga gaagggattg cagaggtaat aagaaaagtc 1200

gttgtggaga aaagtgggga ggttatgagg aggaaagcaa gggaattgag tgagaaaatg 1260

aaagagaaag gagaggaaga gattgatagg gcagtggagg agctagtaca aatttgtaag 1320

aagaagaaag atgcacaata g 1341

<210> 76

<211> 446

<212> PRT

<213> Ginseng radix (Panax ginseng)

<400> 76

Met Asp Asn Gln Lys Gly Arg Ile Ser Ile Val Met Leu Pro Phe Leu

1 5 10 15

Ala His Gly His Ile Ser Pro Phe Phe Glu Leu Ala Lys His Leu Ser

20 25 30

Lys Arg Asn Cys Asn Ile Phe Leu Cys Ser Thr Pro Ile Asn Leu Ser

35 40 45

Ser Ile Lys Asn Arg Ile Ser Asp Lys Asp Ser Ser Ala Ser Ile Lys

50 55 60

Leu Val Glu Leu His Leu Pro Ser Ser Pro Asp Leu Pro Pro His Tyr

65 70 75 80

His Thr Thr Asn Gly Leu Pro Ser His Leu Met Val Pro Leu Ile Asn

85 90 95

Ala Phe Glu Thr Ala Gly Pro Thr Phe Ser Glu Ile Leu Lys Thr Leu

100 105 110

Asn Pro Asp Leu Leu Ile Tyr Asp Phe Asn Pro Ser Trp Ala Pro Glu

115 120 125

Ile Ala Ser Ser His Asn Ile Pro Ala Val Tyr Phe Leu Thr Thr Ala

130 135 140

Ala Ala Ser Ser Ser Ile Gly Leu His Ala Phe Lys Asn Pro Gly Glu

145 150 155 160

Lys Tyr Pro Phe Pro Asp Phe Tyr Asp Asn Ser Asn Asn Thr Pro Glu

165 170 175

Pro Pro Ser Ala Asp Asn Met Lys Leu Leu His Asp Phe Ile Ala Cys

180 185 190

Phe Glu Arg Ser Cys Asp Ile Ile Leu Ile Lys Ser Phe Ile Glu Leu

195 200 205

Glu Gly Lys Tyr Ile Asp Leu Leu Ser Thr Leu Ser Asp Lys Thr Leu

210 215 220

Val Pro Val Gly Pro Leu Val Gln Asp Pro Met Gly His Asn Glu Asp

225 230 235 240

Pro Lys Thr Glu Gln Ile Ile Asn Trp Leu Asp Lys Arg Ala Glu Ser

245 250 255

Thr Val Val Phe Val Cys Phe Gly Ser Glu Tyr Phe Leu Ser Asn Glu

260 265 270

Glu Leu Glu Glu Val Ala Ile Gly Leu Glu Ile Ser Met Val Asn Phe

275 280 285

Ile Trp Ala Val Arg Leu Ile Glu Gly Glu Lys Lys Gly Val Leu Pro

290 295 300

Glu Gly Phe Val Gln Arg Val Gly Asp Arg Gly Leu Val Val Glu Gly

305 310 315 320

Trp Ala Pro Gln Ala Arg Ile Leu Gly His Ser Ser Thr Gly Gly Phe

325 330 335

Val Ser His Cys Gly Trp Ser Ser Ile Ala Glu Ser Met Lys Phe Gly

340 345 350

Val Pro Val Ile Ala Met Ala Arg His Leu Asp Gln Pro Leu Asn Gly

355 360 365

Lys Leu Ala Ala Glu Val Gly Val Gly Met Glu Val Val Arg Asp Glu

370 375 380

Asn Gly Lys Tyr Lys Arg Glu Gly Ile Ala Glu Val Ile Arg Lys Val

385 390 395 400

Val Val Glu Lys Ser Gly Glu Val Met Arg Arg Lys Ala Arg Glu Leu

405 410 415

Ser Glu Lys Met Lys Glu Lys Gly Glu Glu Glu Ile Asp Arg Ala Val

420 425 430

Glu Glu Leu Val Gln Ile Cys Lys Lys Lys Lys Asp Ala Gln

435 440 445

<210> 77

<211> 1341

<212> DNA

<213> Ginseng radix (Panax ginseng)

<400> 77

atggataacc aaaagggtag aatcagtata gttatgctgc catttttagc ccatggccac 60

atttctccat tctttgagct agccaagcat ctctcaaaaa gaaattgtaa tatattcctc 120

tgttctaccc caatcaatct tagctccatc aagaacagaa tatctgataa ggattcctct 180

gcttctataa aactagtaga gcttcatctt ccatcttccc ctgatcttcc tcctcactac 240

cacaccacaa atggcctccc ttcccatctc atggtcccac tcagaaacgc ctttgaaaca 300

gcagccccca ccttctctga aatccttaaa accttaaacc ctgatttgct tatttatgat 360

ttcaatccct catgggcacc ggagatcgct tcgtctcaca atattccggc agtttgtttc 420

ataattgggg gagcagcctc cttttccatg agcctacata gtttcaaaaa cccaggtgaa 480

aaatacccat ttctagattt tgatgataac agtaatatta cccctgaacc accttcagca 540

gataacatga agttattact tgattttatg acttgtttcg aacgatcttg cgacattatt 600

ttgattaaga gttttagaga actagaaggg aaatattttg atttttattc tactttatct 660

gataaaactt tggttcctgt tggtccactc gttcaagatc ctatgggcca taatgaagat 720

ccaaaaacag agcagtttat aaactggctt gacaaaaggg ctgaatctac agtggtgttt 780

gtctgctttg gaagtgagta ttttctctcc aatgaggaat tggaagaagt agcaattggg 840

ctagagatta gcatggttaa tttcatatgg gctgtgagat taattgaagg agagaaaaaa 900

ggggttttac cagaggggtt tgttcaaagg gtaggagaca gaggattggt tgtggagggg 960

tgggctccac aggcaagaat tttaggacat tcaagcaccg gtgggtttgt gagccattgt 1020

gggtggagtt ctattacgga gagtatgaag tttggggttc cagtaattgc catggccagg 1080

cattttgatc agcctttgaa tgctaagctg gcggcggagg ttggtgtggg catggaggtt 1140

gtgagagatg aaaatgggaa gtataagaga gaagatattg caggggtaat aagaaaagtc 1200

gtggtggaga aaagtgggga ggttatcagg aggaaagcaa gggaattgag tgagaaaatg 1260

aaagagatag gagagcaatt gattgatagg gcagtggagg agctagtaca aatttgtaag 1320

aagaagaaag atgaacaata g 1341

<210> 78

<211> 446

<212> PRT

<213> Ginseng radix (Panax ginseng)

<400> 78

Met Asp Asn Gln Lys Gly Arg Ile Ser Ile Val Met Leu Pro Phe Leu

1 5 10 15

Ala His Gly His Ile Ser Pro Phe Phe Glu Leu Ala Lys His Leu Ser

20 25 30

Lys Arg Asn Cys Asn Ile Phe Leu Cys Ser Thr Pro Ile Asn Leu Ser

35 40 45

Ser Ile Lys Asn Arg Ile Ser Asp Lys Asp Ser Ser Ala Ser Ile Lys

50 55 60

Leu Val Glu Leu His Leu Pro Ser Ser Pro Asp Leu Pro Pro His Tyr

65 70 75 80

His Thr Thr Asn Gly Leu Pro Ser His Leu Met Val Pro Leu Arg Asn

85 90 95

Ala Phe Glu Thr Ala Ala Pro Thr Phe Ser Glu Ile Leu Lys Thr Leu

100 105 110

Asn Pro Asp Leu Leu Ile Tyr Asp Phe Asn Pro Ser Trp Ala Pro Glu

115 120 125

Ile Ala Ser Ser His Asn Ile Pro Ala Val Cys Phe Ile Ile Gly Gly

130 135 140

Ala Ala Ser Phe Ser Met Ser Leu His Ser Phe Lys Asn Pro Gly Glu

145 150 155 160

Lys Tyr Pro Phe Leu Asp Phe Asp Asp Asn Ser Asn Ile Thr Pro Glu

165 170 175

Pro Pro Ser Ala Asp Asn Met Lys Leu Leu Leu Asp Phe Met Thr Cys

180 185 190

Phe Glu Arg Ser Cys Asp Ile Ile Leu Ile Lys Ser Phe Arg Glu Leu

195 200 205

Glu Gly Lys Tyr Phe Asp Phe Tyr Ser Thr Leu Ser Asp Lys Thr Leu

210 215 220

Val Pro Val Gly Pro Leu Val Gln Asp Pro Met Gly His Asn Glu Asp

225 230 235 240

Pro Lys Thr Glu Gln Phe Ile Asn Trp Leu Asp Lys Arg Ala Glu Ser

245 250 255

Thr Val Val Phe Val Cys Phe Gly Ser Glu Tyr Phe Leu Ser Asn Glu

260 265 270

Glu Leu Glu Glu Val Ala Ile Gly Leu Glu Ile Ser Met Val Asn Phe

275 280 285

Ile Trp Ala Val Arg Leu Ile Glu Gly Glu Lys Lys Gly Val Leu Pro

290 295 300

Glu Gly Phe Val Gln Arg Val Gly Asp Arg Gly Leu Val Val Glu Gly

305 310 315 320

Trp Ala Pro Gln Ala Arg Ile Leu Gly His Ser Ser Thr Gly Gly Phe

325 330 335

Val Ser His Cys Gly Trp Ser Ser Ile Thr Glu Ser Met Lys Phe Gly

340 345 350

Val Pro Val Ile Ala Met Ala Arg His Phe Asp Gln Pro Leu Asn Ala

355 360 365

Lys Leu Ala Ala Glu Val Gly Val Gly Met Glu Val Val Arg Asp Glu

370 375 380

Asn Gly Lys Tyr Lys Arg Glu Asp Ile Ala Gly Val Ile Arg Lys Val

385 390 395 400

Val Val Glu Lys Ser Gly Glu Val Ile Arg Arg Lys Ala Arg Glu Leu

405 410 415

Ser Glu Lys Met Lys Glu Ile Gly Glu Gln Leu Ile Asp Arg Ala Val

420 425 430

Glu Glu Leu Val Gln Ile Cys Lys Lys Lys Lys Asp Glu Gln

435 440 445

<210> 79

<211> 1329

<212> DNA

<213> Ginseng radix (Panax ginseng)

<400> 79

atggataacc aagaaggtag aatcagtata gcgttgctac catttttagc ccatggtcac 60

atatctccct tctttgagct agccaaacaa ctcgcaaaaa gaaattgcaa tgttttcctc 120

tgttctaccc caatcaatct tagctccatc aagaataagg attcctctgc ttctataaaa 180

ctagttgagc ttcatcttcc atcttcccct gatcttcctc ctcactatca caccacaaat 240

ggcctccctt cccatctcat ggtcccactc ataaacgcct ttgaaacagc aggccccacc 300

ttctctgaaa tccttaaaac cttaaacccc gatttgctta tttatgattt caatccctca 360

tgggcaccgg agatcgcttc gtctcacaat attccggcag tttatttcct aaccacggca 420

gcagccagct cttccattgg cctacatgct ttcaaaaacc caggtgaaaa atacccattt 480

ccagattttt atgataacag taataatacc cctgaaccac cttctgcaga taacatgaag 540

ctacttcatg attttatcgc ttgtttcgaa cgatcttgcg acattatttt gattaagagt 600

tttatagaac tagaagggaa atatatcgat ttgctttcca ctttatctga taaaactttg 660

gttccttttg gtccactcgt tcaagatcct atgggccata atgaagatcc aaaaacagag 720

cagattataa actggcttga caaaagggct gaatctacag tggtgtttgt ctgctttgga 780

agtgagtatt ttctctccaa tgaggaattg gaagaagtag caattgggct agagattagc 840

atggttaatt tcatatgggc tgtgagatta attgaaggag agaaaaaagg ggttttacca 900

gagggatttg ttcaaagggt aggagacaga ggattggttg tggaggggtg ggctccacag 960

gcaagaattt taggacattc aagcaccggt gggtttgtga gccattgtgg gtggagttct 1020

attgcggaga gtatgaagtt tggggttcca gtaattgcca tggccaggca tcttgatcag 1080

cctttgaatg gtaagctggc ggcggaggtt ggtgtgggca tggaggttgt gagagatgaa 1140

aatgggaagt ataagagaga agggattgca gaggtaataa gaaaagtcgt tgtggagaaa 1200

agtggggagg ttatgaggag gaaagcaagg gaattgagtg agaaaatgaa agagaaagga 1260

gaggaagaga ttgatagggc agtggaggag ctagtacaaa tttgtaagaa gaagaaagat 1320

gcacaatag 1329

<210> 80

<211> 442

<212> PRT

<213> Ginseng radix (Panax ginseng)

<400> 80

Met Asp Asn Gln Glu Gly Arg Ile Ser Ile Ala Leu Leu Pro Phe Leu

1 5 10 15

Ala His Gly His Ile Ser Pro Phe Phe Glu Leu Ala Lys Gln Leu Ala

20 25 30

Lys Arg Asn Cys Asn Val Phe Leu Cys Ser Thr Pro Ile Asn Leu Ser

35 40 45

Ser Ile Lys Asn Lys Asp Ser Ser Ala Ser Ile Lys Leu Val Glu Leu

50 55 60

His Leu Pro Ser Ser Pro Asp Leu Pro Pro His Tyr His Thr Thr Asn

65 70 75 80

Gly Leu Pro Ser His Leu Met Val Pro Leu Ile Asn Ala Phe Glu Thr

85 90 95

Ala Gly Pro Thr Phe Ser Glu Ile Leu Lys Thr Leu Asn Pro Asp Leu

100 105 110

Leu Ile Tyr Asp Phe Asn Pro Ser Trp Ala Pro Glu Ile Ala Ser Ser

115 120 125

His Asn Ile Pro Ala Val Tyr Phe Leu Thr Thr Ala Ala Ala Ser Ser

130 135 140

Ser Ile Gly Leu His Ala Phe Lys Asn Pro Gly Glu Lys Tyr Pro Phe

145 150 155 160

Pro Asp Phe Tyr Asp Asn Ser Asn Asn Thr Pro Glu Pro Pro Ser Ala

165 170 175

Asp Asn Met Lys Leu Leu His Asp Phe Ile Ala Cys Phe Glu Arg Ser

180 185 190

Cys Asp Ile Ile Leu Ile Lys Ser Phe Ile Glu Leu Glu Gly Lys Tyr

195 200 205

Ile Asp Leu Leu Ser Thr Leu Ser Asp Lys Thr Leu Val Pro Phe Gly

210 215 220

Pro Leu Val Gln Asp Pro Met Gly His Asn Glu Asp Pro Lys Thr Glu

225 230 235 240

Gln Ile Ile Asn Trp Leu Asp Lys Arg Ala Glu Ser Thr Val Val Phe

245 250 255

Val Cys Phe Gly Ser Glu Tyr Phe Leu Ser Asn Glu Glu Leu Glu Glu

260 265 270

Val Ala Ile Gly Leu Glu Ile Ser Met Val Asn Phe Ile Trp Ala Val

275 280 285

Arg Leu Ile Glu Gly Glu Lys Lys Gly Val Leu Pro Glu Gly Phe Val

290 295 300

Gln Arg Val Gly Asp Arg Gly Leu Val Val Glu Gly Trp Ala Pro Gln

305 310 315 320

Ala Arg Ile Leu Gly His Ser Ser Thr Gly Gly Phe Val Ser His Cys

325 330 335

Gly Trp Ser Ser Ile Ala Glu Ser Met Lys Phe Gly Val Pro Val Ile

340 345 350

Ala Met Ala Arg His Leu Asp Gln Pro Leu Asn Gly Lys Leu Ala Ala

355 360 365

Glu Val Gly Val Gly Met Glu Val Val Arg Asp Glu Asn Gly Lys Tyr

370 375 380

Lys Arg Glu Gly Ile Ala Glu Val Ile Arg Lys Val Val Val Glu Lys

385 390 395 400

Ser Gly Glu Val Met Arg Arg Lys Ala Arg Glu Leu Ser Glu Lys Met

405 410 415

Lys Glu Lys Gly Glu Glu Glu Ile Asp Arg Ala Val Glu Glu Leu Val

420 425 430

Gln Ile Cys Lys Lys Lys Lys Asp Ala Gln

435 440

<210> 81

<211> 1341

<212> DNA

<213> Ginseng radix (Panax ginseng)

<400> 81

atggataacc aaaagggtag aatcagtata gttatgctgc catttttagc ccatggccac 60

atttctccat tctttgagct agccaagcat ctctcaaaaa gaaattgtaa tatattcctc 120

tgttctaccc caatcaatct tagctccatc aagaacagaa tatctgataa ggattcctct 180

gcttctataa aactagtaga gcttcatctt ccatcttccc ctgatcttcc tcctcactac 240

cacaccacaa atggcctccc ttcccatctc atggtcccac tcagaaacgc ctttgaaaca 300

gcagccccca ccttctctga aatccttaaa accttaaacc ctgatttgct tatttatgat 360

ttcaatccct catgggcacc ggagatcgct tcgtctcaca atattccggc agtttgtttc 420

ataattgggg gagcagcctc cttttccatg agcctacata gtttcaaaaa cccaggtgaa 480

aaatacccat ttctagattt tgatgataac agtaatatta cccctgaacc accttcagca 540

gataacatga agttattact tgattttatg acttgtttcg aacgatcttg cgacattatt 600

ttgattaaga gttttagaga actagaaggg aaatattttg atttttattc tactttatct 660

gataaaactt tggttcctgt tggtccactc gttcaagatc ctatgggcca taatgaagat 720

ccaaaaacag agcagtttat aaactggctt gacaaaaggg ctgaatctac agtggtgttt 780

gtctgctttg gaagtgagta ttttctctcc aatgaggaat tggaagaagt agcaattggg 840

ctagagatta gcatggttaa tttcatatgg gctgtgagat taattgaagg agagaaaaaa 900

ggggttttac cagaggggtt tgttcaaagg gtaggagaca gaggattggt tgtggagggg 960

tgggctccac aggcaagaat tttaggacat tcaagcaccg gtgggtttgt gagccattgt 1020

gggtggagtt ctattacgga gagtatgaag tttggggttc cagtaattgc catggccagg 1080

cattttgatc agcctttgaa tgctaagctg gcggcggagg ttggtgtggt catggaggtt 1140

gtgagagatg aaaatgggaa gtataagaga gaagatattg caggggtaat aagaaaagtc 1200

gtggtggaga aaagtgggga ggttatcagg aggaaagcaa gggaattgag tgagaaaatg 1260

aaagagatag gagagcaatt gattgatagg gcagtggagg agctagtaca aatttgtaag 1320

aagaagaaag atgaacaata g 1341

<210> 82

<211> 446

<212> PRT

<213> Ginseng radix (Panax ginseng)

<400> 82

Met Asp Asn Gln Lys Gly Arg Ile Ser Ile Val Met Leu Pro Phe Leu

1 5 10 15

Ala His Gly His Ile Ser Pro Phe Phe Glu Leu Ala Lys His Leu Ser

20 25 30

Lys Arg Asn Cys Asn Ile Phe Leu Cys Ser Thr Pro Ile Asn Leu Ser

35 40 45

Ser Ile Lys Asn Arg Ile Ser Asp Lys Asp Ser Ser Ala Ser Ile Lys

50 55 60

Leu Val Glu Leu His Leu Pro Ser Ser Pro Asp Leu Pro Pro His Tyr

65 70 75 80

His Thr Thr Asn Gly Leu Pro Ser His Leu Met Val Pro Leu Arg Asn

85 90 95

Ala Phe Glu Thr Ala Ala Pro Thr Phe Ser Glu Ile Leu Lys Thr Leu

100 105 110

Asn Pro Asp Leu Leu Ile Tyr Asp Phe Asn Pro Ser Trp Ala Pro Glu

115 120 125

Ile Ala Ser Ser His Asn Ile Pro Ala Val Cys Phe Ile Ile Gly Gly

130 135 140

Ala Ala Ser Phe Ser Met Ser Leu His Ser Phe Lys Asn Pro Gly Glu

145 150 155 160

Lys Tyr Pro Phe Leu Asp Phe Asp Asp Asn Ser Asn Ile Thr Pro Glu

165 170 175

Pro Pro Ser Ala Asp Asn Met Lys Leu Leu Leu Asp Phe Met Thr Cys

180 185 190

Phe Glu Arg Ser Cys Asp Ile Ile Leu Ile Lys Ser Phe Arg Glu Leu

195 200 205

Glu Gly Lys Tyr Phe Asp Phe Tyr Ser Thr Leu Ser Asp Lys Thr Leu

210 215 220

Val Pro Val Gly Pro Leu Val Gln Asp Pro Met Gly His Asn Glu Asp

225 230 235 240

Pro Lys Thr Glu Gln Phe Ile Asn Trp Leu Asp Lys Arg Ala Glu Ser

245 250 255

Thr Val Val Phe Val Cys Phe Gly Ser Glu Tyr Phe Leu Ser Asn Glu

260 265 270

Glu Leu Glu Glu Val Ala Ile Gly Leu Glu Ile Ser Met Val Asn Phe

275 280 285

Ile Trp Ala Val Arg Leu Ile Glu Gly Glu Lys Lys Gly Val Leu Pro

290 295 300

Glu Gly Phe Val Gln Arg Val Gly Asp Arg Gly Leu Val Val Glu Gly

305 310 315 320

Trp Ala Pro Gln Ala Arg Ile Leu Gly His Ser Ser Thr Gly Gly Phe

325 330 335

Val Ser His Cys Gly Trp Ser Ser Ile Thr Glu Ser Met Lys Phe Gly

340 345 350

Val Pro Val Ile Ala Met Ala Arg His Phe Asp Gln Pro Leu Asn Ala

355 360 365

Lys Leu Ala Ala Glu Val Gly Val Val Met Glu Val Val Arg Asp Glu

370 375 380

Asn Gly Lys Tyr Lys Arg Glu Asp Ile Ala Gly Val Ile Arg Lys Val

385 390 395 400

Val Val Glu Lys Ser Gly Glu Val Ile Arg Arg Lys Ala Arg Glu Leu

405 410 415

Ser Glu Lys Met Lys Glu Ile Gly Glu Gln Leu Ile Asp Arg Ala Val

420 425 430

Glu Glu Leu Val Gln Ile Cys Lys Lys Lys Lys Asp Glu Gln

435 440 445

<210> 83

<211> 1341

<212> DNA

<213> Ginseng radix (Panax ginseng)

<400> 83

atggataacc aaaagggtag aatcagtata gttatgctgc catttttagc ccatggccac 60

atttctccat tctttgagct agccaagcat ctctcaaaaa gaaattgtaa tatattcctc 120

tgttctaccc caatcaatct tagctccatc aagaacagaa tatctgataa ggattcctct 180

gcttctataa aactagtaga gcttcatctt ccatcttccc ctgatcttcc tcctcactac 240

cacaccacaa atggcctccc ttcccatctc atggtcccac tcagaaacgc ctttgaaaca 300

gcaggcccca ccttctctga aatccttaaa accttaaacc ccgatttgct tatttatgat 360

ttcaatccct catgggcacc ggagatcgct tcgtctcaca atattccggc agtttatttc 420

ctaaccacgg cagcagccag ctcttccatt ggcctacatg ctttcaaaaa cccaggtgaa 480

aaatacccat ttccagattt ttatgataac agtaataata cccctgaacc accttctgca 540

gataacatga agctacttca tgattttatc gcttgtttcg aacgatcttg cgacattatt 600

ttgattaaga gttttataga actagaaggg aaatatatcg atttgctttc cactttatct 660

gataaaactt tggttcctgt tggtccactc gttcaagatc ctatgggcca taatgaagat 720

ccaaaaacag agcagattat aaactggctt gacaaaaggg ctgaatctac agtggtgttt 780

gtctgctttg gaagtgagta ttttctctcc aatgaggaat tggaagaagt agcaattggg 840

ctagagatta gcatggttaa tttcatatgg gctgtgagat taattgaagg agagaaaaaa 900

ggggttttac cagagggatt tgttcaaagg gtaggagaca gaggattggt tgtggagggg 960

tgggctccac aggcaagaat tttaggacat tcaagcaccg gtgggtttgt gagccattgt 1020

gggtggagtt ctattgcgga gagtatgaag tttggggttc cagtaattgc catggccagg 1080

catcttgatc agcctttgaa tggtaagctg gcggcggagg ttggtgtggg catggaggtt 1140

gtgagagatg aaaatgggaa gtataagaga gaagggattg cagaggtaat aagaaaagtc 1200

gttgtggaga aaagtgggga ggttatgagg aggaaagcaa gggaattgag tgagaaaatg 1260

aaagagaaag gagaggaaga gattgatagg gcagtggagg agctagtaca aatttgtaag 1320

aagaagaaag atgcacaata g 1341

<210> 84

<211> 446

<212> PRT

<213> Ginseng radix (Panax ginseng)

<400> 84

Met Asp Asn Gln Lys Gly Arg Ile Ser Ile Val Met Leu Pro Phe Leu

1 5 10 15

Ala His Gly His Ile Ser Pro Phe Phe Glu Leu Ala Lys His Leu Ser

20 25 30

Lys Arg Asn Cys Asn Ile Phe Leu Cys Ser Thr Pro Ile Asn Leu Ser

35 40 45

Ser Ile Lys Asn Arg Ile Ser Asp Lys Asp Ser Ser Ala Ser Ile Lys

50 55 60

Leu Val Glu Leu His Leu Pro Ser Ser Pro Asp Leu Pro Pro His Tyr

65 70 75 80

His Thr Thr Asn Gly Leu Pro Ser His Leu Met Val Pro Leu Arg Asn

85 90 95

Ala Phe Glu Thr Ala Gly Pro Thr Phe Ser Glu Ile Leu Lys Thr Leu

100 105 110

Asn Pro Asp Leu Leu Ile Tyr Asp Phe Asn Pro Ser Trp Ala Pro Glu

115 120 125

Ile Ala Ser Ser His Asn Ile Pro Ala Val Tyr Phe Leu Thr Thr Ala

130 135 140

Ala Ala Ser Ser Ser Ile Gly Leu His Ala Phe Lys Asn Pro Gly Glu

145 150 155 160

Lys Tyr Pro Phe Pro Asp Phe Tyr Asp Asn Ser Asn Asn Thr Pro Glu

165 170 175

Pro Pro Ser Ala Asp Asn Met Lys Leu Leu His Asp Phe Ile Ala Cys

180 185 190

Phe Glu Arg Ser Cys Asp Ile Ile Leu Ile Lys Ser Phe Ile Glu Leu

195 200 205

Glu Gly Lys Tyr Ile Asp Leu Leu Ser Thr Leu Ser Asp Lys Thr Leu

210 215 220

Val Pro Val Gly Pro Leu Val Gln Asp Pro Met Gly His Asn Glu Asp

225 230 235 240

Pro Lys Thr Glu Gln Ile Ile Asn Trp Leu Asp Lys Arg Ala Glu Ser

245 250 255

Thr Val Val Phe Val Cys Phe Gly Ser Glu Tyr Phe Leu Ser Asn Glu

260 265 270

Glu Leu Glu Glu Val Ala Ile Gly Leu Glu Ile Ser Met Val Asn Phe

275 280 285

Ile Trp Ala Val Arg Leu Ile Glu Gly Glu Lys Lys Gly Val Leu Pro

290 295 300

Glu Gly Phe Val Gln Arg Val Gly Asp Arg Gly Leu Val Val Glu Gly

305 310 315 320

Trp Ala Pro Gln Ala Arg Ile Leu Gly His Ser Ser Thr Gly Gly Phe

325 330 335

Val Ser His Cys Gly Trp Ser Ser Ile Ala Glu Ser Met Lys Phe Gly

340 345 350

Val Pro Val Ile Ala Met Ala Arg His Leu Asp Gln Pro Leu Asn Gly

355 360 365

Lys Leu Ala Ala Glu Val Gly Val Gly Met Glu Val Val Arg Asp Glu

370 375 380

Asn Gly Lys Tyr Lys Arg Glu Gly Ile Ala Glu Val Ile Arg Lys Val

385 390 395 400

Val Val Glu Lys Ser Gly Glu Val Met Arg Arg Lys Ala Arg Glu Leu

405 410 415

Ser Glu Lys Met Lys Glu Lys Gly Glu Glu Glu Ile Asp Arg Ala Val

420 425 430

Glu Glu Leu Val Gln Ile Cys Lys Lys Lys Lys Asp Ala Gln

435 440 445

<210> 85

<211> 1329

<212> DNA

<213> Ginseng radix (Panax ginseng)

<400> 85

atggataacc aaaaaggtag aatcagtata gcgttgctac catttttagc ccatggtcac 60

atatctccct tctttgagct agccaaacaa ctcgcgaaaa gaaattgcaa tgttttcctc 120

tgttctaccc caatcaatct tagctccatc aaggataagg attcctctgc ttctataaaa 180

ctagttgagc ttcatcttcc atcttcccct gatcttcctc ctcactatca caccacaaat 240

ggcctccctt cccatctcat gctcccactc agaaacgcct ttgaaactgc aggccccacc 300

ttctctgaaa tccttaaaac cttaaacccc gatttgctta tttatgattt caatccctca 360

tgggcaccgg agatcgcttc gtctcacaat attccggcag tttatttcct aaccacggca 420

gcagccagct cttccattgg cctacatgct ttcaaaaacc caggtgaaaa atacccattt 480

ccagattttt atgataacag taatattacc cctgaaccac cttctgcaga taacatgaag 540

ctacttcatg attttatcgc ttgtttcgaa cgatcttgcg acattatttt gattaagagt 600

tttagagaac tagaagggaa atatattgat ttgctttcca ctttatctga taaaactttg 660

gttcctgttg gtccactcgt tcaagatcct atgggccata atgaagatcc aaaaacagag 720

cagattataa actggcttga caaaagggct gaatctacag tggtgtttgt ctgctttgga 780

agtgagtatt ttctctccaa tgaggaattg gaagaagtag caattgggct agagattagc 840

acggttaatt tcatatgggc tgtgagatta attgaaggag agaaaaaagg gattttacca 900

gagcggtttg ttcaaagggt aggagacaga ggattggttg tggaggggtg ggctccacag 960

gcaagaattt taggacattc aagcaccggt gggtttgtga gccattgtgg gtggagttct 1020

attgcggaga gtatgaagtt tggggttcca gtaattgcca tggccaggca tcttgatcag 1080

cctttgaatg gtaagctggc ggcggaggtt ggtgtgggca tggaggttgt gagagatgag 1140

aatgggaagt ataagagaga agggattgca gaggtaataa gaaaagtggt tgtggagaaa 1200

agtggggagg ttatcaggag gaaagcaagg gagttgagtg agaaaatgaa agagaaagga 1260

gagcaagaga ttgatagggc attggaggag ctagtacaaa tttgtaagaa gaagaaagat 1320

gaacaatag 1329

<210> 86

<211> 442

<212> PRT

<213> Ginseng radix (Panax ginseng)

<400> 86

Met Asp Asn Gln Lys Gly Arg Ile Ser Ile Ala Leu Leu Pro Phe Leu

1 5 10 15

Ala His Gly His Ile Ser Pro Phe Phe Glu Leu Ala Lys Gln Leu Ala

20 25 30

Lys Arg Asn Cys Asn Val Phe Leu Cys Ser Thr Pro Ile Asn Leu Ser

35 40 45

Ser Ile Lys Asp Lys Asp Ser Ser Ala Ser Ile Lys Leu Val Glu Leu

50 55 60

His Leu Pro Ser Ser Pro Asp Leu Pro Pro His Tyr His Thr Thr Asn

65 70 75 80

Gly Leu Pro Ser His Leu Met Leu Pro Leu Arg Asn Ala Phe Glu Thr

85 90 95

Ala Gly Pro Thr Phe Ser Glu Ile Leu Lys Thr Leu Asn Pro Asp Leu

100 105 110

Leu Ile Tyr Asp Phe Asn Pro Ser Trp Ala Pro Glu Ile Ala Ser Ser

115 120 125

His Asn Ile Pro Ala Val Tyr Phe Leu Thr Thr Ala Ala Ala Ser Ser

130 135 140

Ser Ile Gly Leu His Ala Phe Lys Asn Pro Gly Glu Lys Tyr Pro Phe

145 150 155 160

Pro Asp Phe Tyr Asp Asn Ser Asn Ile Thr Pro Glu Pro Pro Ser Ala

165 170 175

Asp Asn Met Lys Leu Leu His Asp Phe Ile Ala Cys Phe Glu Arg Ser

180 185 190

Cys Asp Ile Ile Leu Ile Lys Ser Phe Arg Glu Leu Glu Gly Lys Tyr

195 200 205

Ile Asp Leu Leu Ser Thr Leu Ser Asp Lys Thr Leu Val Pro Val Gly

210 215 220

Pro Leu Val Gln Asp Pro Met Gly His Asn Glu Asp Pro Lys Thr Glu

225 230 235 240

Gln Ile Ile Asn Trp Leu Asp Lys Arg Ala Glu Ser Thr Val Val Phe

245 250 255

Val Cys Phe Gly Ser Glu Tyr Phe Leu Ser Asn Glu Glu Leu Glu Glu

260 265 270

Val Ala Ile Gly Leu Glu Ile Ser Thr Val Asn Phe Ile Trp Ala Val

275 280 285

Arg Leu Ile Glu Gly Glu Lys Lys Gly Ile Leu Pro Glu Arg Phe Val

290 295 300

Gln Arg Val Gly Asp Arg Gly Leu Val Val Glu Gly Trp Ala Pro Gln

305 310 315 320

Ala Arg Ile Leu Gly His Ser Ser Thr Gly Gly Phe Val Ser His Cys

325 330 335

Gly Trp Ser Ser Ile Ala Glu Ser Met Lys Phe Gly Val Pro Val Ile

340 345 350

Ala Met Ala Arg His Leu Asp Gln Pro Leu Asn Gly Lys Leu Ala Ala

355 360 365

Glu Val Gly Val Gly Met Glu Val Val Arg Asp Glu Asn Gly Lys Tyr

370 375 380

Lys Arg Glu Gly Ile Ala Glu Val Ile Arg Lys Val Val Val Glu Lys

385 390 395 400

Ser Gly Glu Val Ile Arg Arg Lys Ala Arg Glu Leu Ser Glu Lys Met

405 410 415

Lys Glu Lys Gly Glu Gln Glu Ile Asp Arg Ala Leu Glu Glu Leu Val

420 425 430

Gln Ile Cys Lys Lys Lys Lys Asp Glu Gln

435 440

<210> 87

<211> 1329

<212> DNA

<213> Ginseng radix (Panax ginseng)

<400> 87

atggataacc aaaaaggtag aatcagtata gcgttgctac catttttagc ccatggtcac 60

atatctccct tctttgagct agccaaacaa ctcgcgaaaa gaaattgcaa tgttttcctc 120

tgttctaccc caatcaatct tagctccatc aaggataagg attcctctgc ttctataaaa 180

ctagttgagc ttcatcttcc atcttcccct gatcttcctc ctcactatca caccacaaat 240

ggcctccctt cccatctcat gctcccactc agaaacgcct ttgaaactgc aggccccacc 300

ttctctgaaa tccttaaaac cttaaacccc gatttgctta tttatgattt caatccctca 360

tgggcaccgg agatcgcttc gtctcacaat attccggcag tttatttcct aaccacggca 420

gcagccagct cttccattgg cctacatgct ttcaaaaacc caggtgaaaa atacccattt 480

ccagattttt atgataacag taatattacc cctgaaccac cttctgcaga taacatgaag 540

ctacttcatg attttatcgc ttgtttcgaa cgatcttgcg acattatttt gattaagagt 600

tttagagaac tagaagggaa atatattgat ttgctttcca ctttatctga taaaactttg 660

gttcctgttg gtccactcgt tcaagatcct atgggccata atgaagatcc aaaaacagag 720

cagattataa actggcttga caaaagggct gaatctacag tggtgtttgt ctgctttgga 780

agtgagtatt ttctctccaa tgaggaattg gaagaagtag caattgggct agagattagc 840

acggttaatt tcatatgggc tgtgagatta attgaaggag agaaaaaagg gattttacca 900

gaggggtttg ttcaaagggt aggagacaga ggattggttg tggaggggtg ggctccacag 960

gcaagaattt taggacattc aagcaccggt gggtttgtga gccattgtgg gtggagttct 1020

attgcggaga gtatgaagtt tggggttcca gtaattgcca tggccaggca tcttgatcag 1080

cctttgaatg gtaagctggc ggcggaggtt ggtgtgggca tggaggttgt gagagatgag 1140

aatgggaagt ataagagaga agggattgca gaggtaataa gaaaagtggt tgtggagaaa 1200

agtggggagg ttatcaggag gaaagcaagg gagttgagtg agaaaatgaa agagaaagga 1260

gagcaagaga ttgatagggt agtggaggag ctagtacaaa tttgtaagaa gaagaaagat 1320

gaacaatag 1329

<210> 88

<211> 442

<212> PRT

<213> Ginseng radix (Panax ginseng)

<400> 88

Met Asp Asn Gln Lys Gly Arg Ile Ser Ile Ala Leu Leu Pro Phe Leu

1 5 10 15

Ala His Gly His Ile Ser Pro Phe Phe Glu Leu Ala Lys Gln Leu Ala

20 25 30

Lys Arg Asn Cys Asn Val Phe Leu Cys Ser Thr Pro Ile Asn Leu Ser

35 40 45

Ser Ile Lys Asp Lys Asp Ser Ser Ala Ser Ile Lys Leu Val Glu Leu

50 55 60

His Leu Pro Ser Ser Pro Asp Leu Pro Pro His Tyr His Thr Thr Asn

65 70 75 80

Gly Leu Pro Ser His Leu Met Leu Pro Leu Arg Asn Ala Phe Glu Thr

85 90 95

Ala Gly Pro Thr Phe Ser Glu Ile Leu Lys Thr Leu Asn Pro Asp Leu

100 105 110

Leu Ile Tyr Asp Phe Asn Pro Ser Trp Ala Pro Glu Ile Ala Ser Ser

115 120 125

His Asn Ile Pro Ala Val Tyr Phe Leu Thr Thr Ala Ala Ala Ser Ser

130 135 140

Ser Ile Gly Leu His Ala Phe Lys Asn Pro Gly Glu Lys Tyr Pro Phe

145 150 155 160

Pro Asp Phe Tyr Asp Asn Ser Asn Ile Thr Pro Glu Pro Pro Ser Ala

165 170 175

Asp Asn Met Lys Leu Leu His Asp Phe Ile Ala Cys Phe Glu Arg Ser

180 185 190

Cys Asp Ile Ile Leu Ile Lys Ser Phe Arg Glu Leu Glu Gly Lys Tyr

195 200 205

Ile Asp Leu Leu Ser Thr Leu Ser Asp Lys Thr Leu Val Pro Val Gly

210 215 220

Pro Leu Val Gln Asp Pro Met Gly His Asn Glu Asp Pro Lys Thr Glu

225 230 235 240

Gln Ile Ile Asn Trp Leu Asp Lys Arg Ala Glu Ser Thr Val Val Phe

245 250 255

Val Cys Phe Gly Ser Glu Tyr Phe Leu Ser Asn Glu Glu Leu Glu Glu

260 265 270

Val Ala Ile Gly Leu Glu Ile Ser Thr Val Asn Phe Ile Trp Ala Val

275 280 285

Arg Leu Ile Glu Gly Glu Lys Lys Gly Ile Leu Pro Glu Gly Phe Val

290 295 300

Gln Arg Val Gly Asp Arg Gly Leu Val Val Glu Gly Trp Ala Pro Gln

305 310 315 320

Ala Arg Ile Leu Gly His Ser Ser Thr Gly Gly Phe Val Ser His Cys

325 330 335

Gly Trp Ser Ser Ile Ala Glu Ser Met Lys Phe Gly Val Pro Val Ile

340 345 350

Ala Met Ala Arg His Leu Asp Gln Pro Leu Asn Gly Lys Leu Ala Ala

355 360 365

Glu Val Gly Val Gly Met Glu Val Val Arg Asp Glu Asn Gly Lys Tyr

370 375 380

Lys Arg Glu Gly Ile Ala Glu Val Ile Arg Lys Val Val Val Glu Lys

385 390 395 400

Ser Gly Glu Val Ile Arg Arg Lys Ala Arg Glu Leu Ser Glu Lys Met

405 410 415

Lys Glu Lys Gly Glu Gln Glu Ile Asp Arg Val Val Glu Glu Leu Val

420 425 430

Gln Ile Cys Lys Lys Lys Lys Asp Glu Gln

435 440

<210> 89

<211> 1329

<212> DNA

<213> Ginseng (Panax ginseng)

<400> 89

atggataacc aagaaggtag aatcagtata gcgttgctac catttttagc ccatggtcac 60

atatctccct tctttgagct agccaaacaa ctcgcaaaaa gaaattgcaa tgttttcctc 120

tgttctaccc caatcaatct tagctccatc aagaataagg attcctctgc ttctataaaa 180

ctagttgagc ttcatcttcc atcttcccct gatcttcctc ctcactatca caccacaaat 240

ggcctccctt cccatctcat ggtcccactc ataaacgcct ttgaaacagc aggccccacc 300

ttctctgaaa tccttaaaac cttaaaccct gatttgctta tttatgattt caatccctca 360

tgggcaccgg agatcgcttc gtctcacaat attccggcag tttgtttcat aattggggga 420

gcagcctcct tttccatgag cctacatagt ttcaaaaacc caggtgaaaa atacccattt 480

ctagattttg atgataacag taatattacc cctgaaccac cttcagcaga taacatgaag 540

ttattacttg attttatgac ttgtttcgaa cgatcttgcg acattatttt gattaagagt 600

tttagagaac tagaagggaa atattttgat ttttattcta ctttatctga taaaactttg 660

gttcctgttg gtccactcgt tcaagatcct atgggccata atgaagatcc aaaaacagag 720

cagtttataa actggcttga caaaagggct gaatctacag tggtgtttgt ctgctttgga 780

agtgagtatt ttctctccaa tgaggaattg gaagaagtag caattgggct agagattagc 840

atggttaatt tcatatgggc tgtgagatta attgaaggag agaaaaaagg ggttttacca 900

gaggggtttg ttcaaagggt aggagacaga ggattggttg tggaggggtg ggctccacag 960

gcaagaattt taggacattc aagcaccggt gggtttgtga gccattgtgg gtggagttct 1020

attacggaga gtatgaagtt tggggttcca gtaattgcca tggccaggca ttttgatcag 1080

cctttgaatg ctaagctggc ggcggaggtt ggtgtgggca tggaggttgt gagagatgaa 1140

aatgggaagt ataagagaga agatattgca ggggtaataa gaaaagtcgt ggtggagaaa 1200

agtggggagg ttatcaggag gaaagcaagg gaattgagtg agaaaatgaa agagatagga 1260

gagcaattga ttgatagggc agtggaggag ctagtacaaa tttgtaagaa gaagaaagat 1320

gaacaatag 1329

<210> 90

<211> 442

<212> PRT

<213> Ginseng radix (Panax ginseng)

<400> 90

Met Asp Asn Gln Glu Gly Arg Ile Ser Ile Ala Leu Leu Pro Phe Leu

1 5 10 15

Ala His Gly His Ile Ser Pro Phe Phe Glu Leu Ala Lys Gln Leu Ala

20 25 30

Lys Arg Asn Cys Asn Val Phe Leu Cys Ser Thr Pro Ile Asn Leu Ser

35 40 45

Ser Ile Lys Asn Lys Asp Ser Ser Ala Ser Ile Lys Leu Val Glu Leu

50 55 60

His Leu Pro Ser Ser Pro Asp Leu Pro Pro His Tyr His Thr Thr Asn

65 70 75 80

Gly Leu Pro Ser His Leu Met Val Pro Leu Ile Asn Ala Phe Glu Thr

85 90 95

Ala Gly Pro Thr Phe Ser Glu Ile Leu Lys Thr Leu Asn Pro Asp Leu

100 105 110

Leu Ile Tyr Asp Phe Asn Pro Ser Trp Ala Pro Glu Ile Ala Ser Ser

115 120 125

His Asn Ile Pro Ala Val Cys Phe Ile Ile Gly Gly Ala Ala Ser Phe

130 135 140

Ser Met Ser Leu His Ser Phe Lys Asn Pro Gly Glu Lys Tyr Pro Phe

145 150 155 160

Leu Asp Phe Asp Asp Asn Ser Asn Ile Thr Pro Glu Pro Pro Ser Ala

165 170 175

Asp Asn Met Lys Leu Leu Leu Asp Phe Met Thr Cys Phe Glu Arg Ser

180 185 190

Cys Asp Ile Ile Leu Ile Lys Ser Phe Arg Glu Leu Glu Gly Lys Tyr

195 200 205

Phe Asp Phe Tyr Ser Thr Leu Ser Asp Lys Thr Leu Val Pro Val Gly

210 215 220

Pro Leu Val Gln Asp Pro Met Gly His Asn Glu Asp Pro Lys Thr Glu

225 230 235 240

Gln Phe Ile Asn Trp Leu Asp Lys Arg Ala Glu Ser Thr Val Val Phe

245 250 255

Val Cys Phe Gly Ser Glu Tyr Phe Leu Ser Asn Glu Glu Leu Glu Glu

260 265 270

Val Ala Ile Gly Leu Glu Ile Ser Met Val Asn Phe Ile Trp Ala Val

275 280 285

Arg Leu Ile Glu Gly Glu Lys Lys Gly Val Leu Pro Glu Gly Phe Val

290 295 300

Gln Arg Val Gly Asp Arg Gly Leu Val Val Glu Gly Trp Ala Pro Gln

305 310 315 320

Ala Arg Ile Leu Gly His Ser Ser Thr Gly Gly Phe Val Ser His Cys

325 330 335

Gly Trp Ser Ser Ile Thr Glu Ser Met Lys Phe Gly Val Pro Val Ile

340 345 350

Ala Met Ala Arg His Phe Asp Gln Pro Leu Asn Ala Lys Leu Ala Ala

355 360 365

Glu Val Gly Val Gly Met Glu Val Val Arg Asp Glu Asn Gly Lys Tyr

370 375 380

Lys Arg Glu Asp Ile Ala Gly Val Ile Arg Lys Val Val Val Glu Lys

385 390 395 400

Ser Gly Glu Val Ile Arg Arg Lys Ala Arg Glu Leu Ser Glu Lys Met

405 410 415

Lys Glu Ile Gly Glu Gln Leu Ile Asp Arg Ala Val Glu Glu Leu Val

420 425 430

Gln Ile Cys Lys Lys Lys Lys Asp Glu Gln

435 440

<210> 91

<211> 1341

<212> DNA

<213> Ginseng radix (Panax ginseng)

<400> 91

atggataacc aaaagggtag aatcagtata gttatgctgc catttttagc ccatggccac 60

atttctccat tctttgagct agccaagcat ctctcaaaaa gaaattgtaa tatattcctc 120

tgttctaccc caatcaatct tagctccatc aagaacagaa tatctgataa ggattcctct 180

gcttctataa aactagtaga gcttcatctt ccatcttccc ctgatcttcc tcctcactac 240

cacaccacaa atggcctccc ttcccatctc atggtcccac tcagaaacgc ctttgaaaca 300

gcagccccca ccttctctga aatccttaaa accttaaacc ctgatttgct tatttatgat 360

ttcaatccct catgggcacc ggagatcgct tcgtctcaca atattccggc agtttgtttc 420

ataattgggg gagcagcctc cttttccatg agcctacata gtttcaaaaa cccaggtgaa 480

aaatacccat ttctagattt tgatgataac agtaatatta cccctgaacc accttcagca 540

gataacatga agttattact tgattttatg acttgtttcg aacgatcttg cgacattatt 600

ttgattaaga gttttagaga actagaaggg aaatattttg atttttattc tactttatct 660

gataaaactt tggttcctgt tggtccactc gttcaagatc ctatgggcca taatgaagat 720

ccaaaaacag agcagtttat aaactggctt gacaaaaggg ctgaatctac agtggtgttt 780

gtctgctttg gaagtgagta ttttctctcc aatgaggaat tggaagaagt agcaattggg 840

ctagagatta gcatggttaa tttcatatgg gctgtgagat taattgaagg agagaaaaaa 900

ggggttttac cagaggggtt tgttcaaagg gtaggagaca gaggattggt tgtggagggg 960

tgggctccac aggcaagaat tttaggacat tcaagcaccg gtgggtttgt gagccattgt 1020

gggtggagtt ctattacgga gagtatgaag tttggggttc cagtaattgc catggccagg 1080

cattttgatc agcctttgaa tgctaagctg gcggcggagg ttggtgtggg catggaggtt 1140

gtgagagatg aaaatgggaa gtataagaga gaagatattg caggggtaat aagaaaagtc 1200

gtggtggaga aaagtgggga ggttatgagg aggaaagcaa gggaattgag tgagaaaatg 1260

aaagagaaag gagaggaaga gattgatagg gcagtggagg agctagtaca aatttgtaag 1320

aagaagaaag atgcacaata g 1341

<210> 92

<211> 446

<212> PRT

<213> Ginseng radix (Panax ginseng)

<400> 92

Met Asp Asn Gln Lys Gly Arg Ile Ser Ile Val Met Leu Pro Phe Leu

1 5 10 15

Ala His Gly His Ile Ser Pro Phe Phe Glu Leu Ala Lys His Leu Ser

20 25 30

Lys Arg Asn Cys Asn Ile Phe Leu Cys Ser Thr Pro Ile Asn Leu Ser

35 40 45

Ser Ile Lys Asn Arg Ile Ser Asp Lys Asp Ser Ser Ala Ser Ile Lys

50 55 60

Leu Val Glu Leu His Leu Pro Ser Ser Pro Asp Leu Pro Pro His Tyr

65 70 75 80

His Thr Thr Asn Gly Leu Pro Ser His Leu Met Val Pro Leu Arg Asn

85 90 95

Ala Phe Glu Thr Ala Ala Pro Thr Phe Ser Glu Ile Leu Lys Thr Leu

100 105 110

Asn Pro Asp Leu Leu Ile Tyr Asp Phe Asn Pro Ser Trp Ala Pro Glu

115 120 125

Ile Ala Ser Ser His Asn Ile Pro Ala Val Cys Phe Ile Ile Gly Gly

130 135 140

Ala Ala Ser Phe Ser Met Ser Leu His Ser Phe Lys Asn Pro Gly Glu

145 150 155 160

Lys Tyr Pro Phe Leu Asp Phe Asp Asp Asn Ser Asn Ile Thr Pro Glu

165 170 175

Pro Pro Ser Ala Asp Asn Met Lys Leu Leu Leu Asp Phe Met Thr Cys

180 185 190

Phe Glu Arg Ser Cys Asp Ile Ile Leu Ile Lys Ser Phe Arg Glu Leu

195 200 205

Glu Gly Lys Tyr Phe Asp Phe Tyr Ser Thr Leu Ser Asp Lys Thr Leu

210 215 220

Val Pro Val Gly Pro Leu Val Gln Asp Pro Met Gly His Asn Glu Asp

225 230 235 240

Pro Lys Thr Glu Gln Phe Ile Asn Trp Leu Asp Lys Arg Ala Glu Ser

245 250 255

Thr Val Val Phe Val Cys Phe Gly Ser Glu Tyr Phe Leu Ser Asn Glu

260 265 270

Glu Leu Glu Glu Val Ala Ile Gly Leu Glu Ile Ser Met Val Asn Phe

275 280 285

Ile Trp Ala Val Arg Leu Ile Glu Gly Glu Lys Lys Gly Val Leu Pro

290 295 300

Glu Gly Phe Val Gln Arg Val Gly Asp Arg Gly Leu Val Val Glu Gly

305 310 315 320

Trp Ala Pro Gln Ala Arg Ile Leu Gly His Ser Ser Thr Gly Gly Phe

325 330 335

Val Ser His Cys Gly Trp Ser Ser Ile Thr Glu Ser Met Lys Phe Gly

340 345 350

Val Pro Val Ile Ala Met Ala Arg His Phe Asp Gln Pro Leu Asn Ala

355 360 365

Lys Leu Ala Ala Glu Val Gly Val Gly Met Glu Val Val Arg Asp Glu

370 375 380

Asn Gly Lys Tyr Lys Arg Glu Asp Ile Ala Gly Val Ile Arg Lys Val

385 390 395 400

Val Val Glu Lys Ser Gly Glu Val Met Arg Arg Lys Ala Arg Glu Leu

405 410 415

Ser Glu Lys Met Lys Glu Lys Gly Glu Glu Glu Ile Asp Arg Ala Val

420 425 430

Glu Glu Leu Val Gln Ile Cys Lys Lys Lys Lys Asp Ala Gln

435 440 445

<210> 93

<211> 446

<212> PRT

<213> mutant proteins

<400> 93

Met Asp Asn Gln Lys Gly Arg Ile Ser Ile Ala Leu Leu Pro Phe Leu

1 5 10 15

Ala His Gly His Ile Ser Pro Phe Phe Glu Leu Ala Lys Gln Leu Ala

20 25 30

Lys Arg Asn Cys Asn Val Phe Leu Cys Ser Thr Pro Ile Asn Leu Ser

35 40 45

Ser Ile Lys Asn Arg Val Ser Asp Lys Asp Ser Ser Ala Ser Ile Lys

50 55 60

Leu Val Glu Leu His Leu Pro Ser Ser Pro Asp Leu Pro Pro His Tyr

65 70 75 80

His Thr Thr Asn Gly Leu Pro Ser His Leu Met Ile Pro Leu Arg Asn

85 90 95

Ala Phe Asp Thr Ala Gly Pro Thr Phe Ser Glu Ile Leu Lys Thr Leu

100 105 110

Asn Pro Asp Leu Leu Ile Tyr Asp Phe Asn Pro Ser Trp Ala Pro Glu

115 120 125

Ile Ala Ser Ser His Asn Ile Pro Ala Val Cys Phe Ile Ile Gly Gly

130 135 140

Ala Ala Ser Ser Ser Met Ser Leu His Ser Phe Lys Asn Pro Gly Glu

145 150 155 160

Lys Tyr Pro Phe Leu Asp Phe Asp Asp Asn Ser Asn Ile Thr Pro Glu

165 170 175

Pro Pro Ser Ala Asp Asn Met Lys Leu Leu Ile Asn Phe Met Thr Cys

180 185 190

Phe Glu Arg Ser Cys Asp Ile Ile Leu Ile Lys Ser Phe Arg Glu Leu

195 200 205

Glu Gly Lys Tyr Phe Asp Phe Phe Ser Thr Leu Ser Asp Lys Thr Leu

210 215 220

Val Pro Val Gly Pro Leu Val Gln Asp Pro Met Gly His Asn Glu Asp

225 230 235 240

Pro Lys Thr Glu Gln Phe Ile Asn Trp Leu Asp Lys Arg Ala Glu Ser

245 250 255

Thr Val Val Phe Val Cys Phe Gly Ser Glu Cys Phe Leu Ser Asn Glu

260 265 270

Glu Leu Glu Glu Val Ala Ile Gly Leu Glu Ile Ser Met Val Asn Phe

275 280 285

Ile Trp Ala Val Arg Leu Ile Glu Gly Glu Lys Lys Gly Val Leu Pro

290 295 300

Glu Gly Phe Val Gln Arg Val Gly Asp Arg Gly Leu Val Val Glu Glu

305 310 315 320

Trp Ala Pro Gln Ala Arg Ile Leu Gly His Ser Ser Thr Gly Gly Phe

325 330 335

Val Ser His Cys Gly Trp Gly Ser Ile Thr Glu Ser Met Lys Phe Gly

340 345 350

Val Pro Val Ile Ala Met Ala Arg His Phe Asp Gln Pro Leu Asn Gly

355 360 365

Lys Leu Ala Ala Glu Val Gly Val Gly Met Glu Val Val Arg Asp Glu

370 375 380

Asn Gly Lys Tyr Lys Arg Glu Gly Ile Ala Glu Val Ile Arg Lys Val

385 390 395 400

Val Val Glu Lys Ser Gly Glu Val Ile Arg Arg Lys Ala Arg Glu Leu

405 410 415

Ser Glu Lys Met Lys Glu Lys Gly Glu Gln Glu Ile Asp Arg Val Val

420 425 430

Glu Glu Leu Val Gln Ile Cys Lys Lys Lys Lys Asp Glu Gln

435 440 445

<210> 94

<211> 446

<212> PRT

<213> mutant proteins

<400> 94

Met Asp Asn Gln Lys Gly Arg Ile Ser Ile Ala Leu Leu Pro Phe Leu

1 5 10 15

Ala His Gly His Ile Ser Pro Phe Phe Glu Leu Ala Lys Gln Leu Ala

20 25 30

Lys Arg Asn Cys Asn Val Phe Leu Cys Ser Thr Pro Ile Asn Leu Ser

35 40 45

Ser Ile Lys Asn Arg Val Ser Asp Lys Asp Ser Ser Ala Ser Ile Lys

50 55 60

Leu Val Glu Leu His Leu Pro Ser Ser Pro Asp Leu Pro Pro His Tyr

65 70 75 80

His Thr Thr Asn Gly Leu Pro Ser His Leu Met Ile Pro Leu Arg Asn

85 90 95

Ala Phe Asp Thr Ala Gly Pro Thr Phe Ser Glu Ile Leu Lys Thr Leu

100 105 110

Asn Pro Asp Leu Leu Ile Tyr Asp Phe Asn Pro Ser Trp Ala Pro Glu

115 120 125

Ile Ala Ser Ser His Asn Ile Pro Ala Val Cys Phe Ile Ile Gly Gly

130 135 140

Ala Ala Ser Ser Ser Met Ser Leu His Ser Phe Lys Asn Pro Gly Glu

145 150 155 160

Lys Tyr Pro Phe Leu Asp Phe Asp Asp Asn Ser Asn Ile Thr Pro Glu

165 170 175

Pro Pro Ser Ala Asp Asn Met Lys Leu Leu Ile Asn Phe Met Thr Cys

180 185 190

Phe Glu Arg Ser Cys Asp Ile Ile Leu Ile Lys Ser Phe Arg Glu Leu

195 200 205

Glu Gly Lys Tyr Phe Asp Phe Phe Ser Thr Leu Ser Asp Lys Thr Leu

210 215 220

Val Pro Val Gly Pro Leu Val Gln Asp Pro Met Gly His Asn Glu Asp

225 230 235 240

Pro Lys Thr Glu Gln Phe Ile Asn Trp Leu Asp Lys Arg Ala Glu Ser

245 250 255

Thr Val Val Phe Val Cys Phe Gly Ser Glu Cys Phe Leu Ser Asn Glu

260 265 270

Glu Leu Glu Glu Val Ala Ile Gly Leu Glu Ile Ser Met Val Asn Phe

275 280 285

Ile Trp Ala Val Arg Leu Ile Glu Gly Glu Lys Lys Gly Val Leu Pro

290 295 300

Glu Gly Phe Val Gln Arg Val Gly Asp Arg Gly Leu Val Val Glu Glu

305 310 315 320

Trp Ala Pro Gln Ala Arg Ile Leu Gly His Ser Ser Thr Gly Gly Phe

325 330 335

Val Ser His Cys Gly Trp Asn Ser Ile Thr Glu Ser Met Lys Phe Gly

340 345 350

Val Pro Val Ile Ala Met Pro Arg His Phe Asp Gln Pro Leu Asn Gly

355 360 365

Lys Leu Ala Ala Glu Val Gly Val Gly Met Glu Val Val Arg Asp Glu

370 375 380

Asn Gly Lys Tyr Lys Arg Glu Gly Ile Ala Glu Val Ile Arg Lys Val

385 390 395 400

Val Val Glu Lys Ser Gly Glu Val Ile Arg Arg Lys Ala Arg Glu Leu

405 410 415

Ser Glu Lys Met Lys Glu Lys Gly Glu Gln Glu Ile Asp Arg Val Val

420 425 430

Glu Glu Leu Val Gln Ile Cys Lys Lys Lys Lys Asp Glu Gln

435 440 445

<210> 95

<211> 446

<212> PRT

<213> mutant proteins

<400> 95

Met Asp Asn Gln Lys Gly Arg Ile Ser Ile Ala Leu Leu Pro Phe Leu

1 5 10 15

Ala His Gly His Ile Ser Pro Phe Phe Glu Leu Ala Lys Gln Leu Ala

20 25 30

Lys Arg Asn Cys Asn Val Phe Leu Cys Ser Thr Pro Ile Asn Leu Ser

35 40 45

Ser Ile Lys Asn Arg Val Ser Asp Lys Asp Ser Ser Ala Ser Ile Lys

50 55 60

Leu Val Glu Leu His Leu Pro Ser Ser Pro Asp Leu Pro Pro His Tyr

65 70 75 80

His Thr Thr Asn Gly Leu Pro Ser His Leu Met Ile Pro Leu Arg Asn

85 90 95

Ala Phe Asp Thr Ala Gly Pro Thr Phe Ser Glu Ile Leu Lys Thr Leu

100 105 110

Asn Pro Asp Leu Leu Ile Tyr Asp Phe Asn Pro Ser Trp Ala Pro Glu

115 120 125

Ile Ala Ser Ser His Asn Ile Pro Ala Val Cys Phe Ile Ile Gly Gly

130 135 140

Ala Ala Ser Ser Ser Met Ser Leu His Ser Phe Lys Asn Pro Gly Glu

145 150 155 160

Lys Tyr Pro Phe Leu Asp Phe Asp Asp Asn Ser Asn Ile Thr Pro Glu

165 170 175

Pro Pro Ser Ala Asp Asn Met Lys Leu Leu Ile Asn Phe Met Thr Cys

180 185 190

Phe Glu Arg Ser Cys Asp Ile Ile Leu Ile Lys Ser Phe Arg Glu Leu

195 200 205

Glu Gly Lys Tyr Phe Asp Phe Phe Ser Thr Leu Ser Asp Lys Thr Leu

210 215 220

Val Pro Val Gly Pro Leu Val Gln Asp Pro Met Gly His Asn Glu Asp

225 230 235 240

Pro Lys Thr Glu Gln Phe Ile Asn Trp Leu Asp Lys Arg Ala Glu Ser

245 250 255

Thr Val Val Phe Val Cys Phe Gly Ser Glu Cys Phe Leu Ser Asn Glu

260 265 270

Glu Leu Glu Glu Val Ala Ile Gly Leu Glu Ile Ser Met Val Asn Phe

275 280 285

Ile Trp Ala Val Arg Leu Ile Glu Gly Glu Lys Lys Gly Val Leu Pro

290 295 300

Glu Gly Phe Val Gln Arg Val Gly Asp Arg Gly Leu Val Val Glu Glu

305 310 315 320

Trp Ala Pro Gln Ala Arg Ile Leu Gly His Ser Ser Thr Gly Gly Phe

325 330 335

Val Ser His Cys Gly Trp Gly Ser Ile Thr Glu Ser Met Lys Phe Gly

340 345 350

Val Pro Val Ile Ala Met Pro Arg His Phe Asp Gln Pro Leu Asn Gly

355 360 365

Lys Leu Ala Ala Glu Val Gly Val Gly Met Glu Val Val Arg Asp Glu

370 375 380

Asn Gly Lys Tyr Lys Arg Glu Gly Ile Ala Glu Val Ile Arg Lys Val

385 390 395 400

Val Val Glu Lys Ser Gly Glu Val Ile Arg Arg Lys Ala Arg Glu Leu

405 410 415

Ser Glu Lys Met Lys Glu Lys Gly Glu Gln Glu Ile Asp Arg Val Val

420 425 430

Glu Glu Leu Val Gln Ile Cys Lys Lys Lys Lys Asp Glu Gln

435 440 445

Claims

1. An in vitro glycosylation method, comprising the steps of:

a sugar chain extended on the first sugar group at the C-6 position;

thereby forming a glycosylated tetracyclic triterpene compound;

wherein the glycosyltransferase is the glycosyltransferase shown in SEQ ID NO. 61 or a derivative polypeptide thereof, the derivative polypeptide is selected from the polypeptide of an amino acid sequence shown in SEQ ID NO. 55, 57, 59, 78, 82, 92 or 95,

the extended sugar chain includes a direct extension,

the tetracyclic triterpene compounds are protopanaxatriol type saponin, the protopanaxatriol type saponin is ginsenoside Rh1,

and said glycosyl donor is a nucleoside diphosphate sugar, and said glycosyl donor is selected from the group consisting of: UDP-glucose, UDP-xylose, or a combination thereof.

2. The method of claim 1, wherein said direct extension is the addition of a sugar residue to the first sugar residue at the C-6 position to extend the sugar chain.

3. The method according to claim 1, wherein the substitution extension is an extension of the sugar chain from the first sugar group at the C-6 position by replacing the terminal sugar group of the sugar chain at the C-6 position with a different sugar group.

4. An isolated polypeptide selected from the group consisting of the amino acid sequences shown in SEQ ID No. 78, 82, 92, or 95.

5. An isolated polynucleotide, wherein said polynucleotide is a sequence selected from the group consisting of:

(B) encoding a polypeptide selected from the amino acid sequences shown in SEQ ID No. 78, 82, 92 or 95;

(F) a nucleotide sequence complementary to the nucleotide sequence of (B).

6. The polynucleotide of claim 5, wherein the polynucleotide has the sequence shown in SEQ ID No. 77, 81 or 91.

7. A vector comprising the polynucleotide of claim 5.

8. Use of the isolated polypeptide of claim 4 for catalyzing one or more of the following in vitro reactions, or for preparing a catalytic formulation for catalyzing one or more of the following reactions:

(ii) transferring glycosyl from glycosyl donor to the first glycosyl at C-6 of the tetracyclic triterpenoid to extend sugar chain;

(iii) replacing glycosyl of glycosyl donor with terminal glycosyl of C-6 position sugar chain of tetracyclic triterpenoid, and extending sugar chain from the first glycosyl at C-6 position, wherein the tetracyclic triterpenoid is protopanaxatriol saponin, the protopanaxatriol saponin is ginsenoside Rh1, and the glycosyl donor is selected from the following group: UDP-glucose, UDP-xylose, or a combination thereof.

9. Use according to claim 8, wherein the isolated polypeptide is used for catalyzing or is used for preparing a catalytic preparation for catalyzing:

(B)

wherein R1 and R2 are H or a glycosyl group, R3 and R4 are glycosyl groups; the polypeptide is selected from SEQ ID No. 61 or derived polypeptide thereof, and the derived polypeptide is selected from the polypeptide shown in SEQ ID No. 55, 57, 59, 78, 82, 92 or 95.

10. The method of claim 1, wherein the method further comprises: in the presence of additives for regulating the enzymatic activity.

11. The method of claim 10, wherein the additive for modulating enzyme activity is selected from the group consisting of: ca ²⁺ 、Co ²⁺ 、Mn ²⁺ 、Ba ²⁺ 、Al ³⁺ 、Ni ²⁺ 、Zn ²⁺ Or Fe ²⁺ 。

12. The method of claim 10, wherein the additive for modulating enzyme activity is: can generate Ca ²⁺ 、Co ²⁺ 、Mn ²⁺ 、Ba ²⁺ 、Al ³⁺ 、Ni ²⁺ 、Zn ²⁺ Or Fe ²⁺ The substance of (1).

13. The method of claim 1, wherein the substrate of the glycosylation process is compound (III) and the product is compound (IV); or the like, or, alternatively,

the compound of formula (III) is ginsenoside Rh1, and the compound of formula (IV) is ginsenoside Rf.

14. A genetically engineered host cell comprising the vector of claim 7, or having the polynucleotide of claim 5 integrated into its genome.

15. Use of the host cell of claim 14 for the preparation of an enzyme-catalysed reagent, or for the production of a glycosyltransferase, which is a glycosyltransferase as set forth in SEQ ID No. 61 or a polypeptide derived therefrom selected from the group consisting of the polypeptides having the amino acid sequences as set forth in SEQ ID No. 55, 57, 59, 78, 82, 92 or 95, or as a catalysed cell, or for the production of a glycosylated tetracyclic triterpene compound which is a protopanaxatriol-type saponin which is ginsenoside Rh 1.

16. The use according to claim 15, wherein the host cell is used for the production of rare ginsenoside Rf by glycosylation of ginsenoside Rh 1.

17. A method of producing a transgenic plant comprising the steps of: regenerating the genetically engineered host cell of claim 14 into a plant, and the genetically engineered host cell is a plant cell.

18. The method of claim 17, wherein the genetically engineered host cell is a ginseng cell.