JP2010193880A - Flavonoid-3-site-galactose-glucose transferase, and polynucleotide encoding the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an enzyme (F3Gal/GlcT) transferring any of galactose and glucose onto the 3-site of flavonoid, to provide a polynucleotide encoding the enzyme, to provide a vector including the polynucleotide, and to provide e.g. a transformant obtained using the vector. <P>SOLUTION: The vector includes a polynucleotide comprising a specific base sequence or a polynucleotide encoding a protein comprising a specific amino acid sequence. The transformant or the like is obtained by using the vector. Further, a UGT enzyme (F3Gal/GlcT) has new activity of transferring glucose or galactose onto the 3-site of flavonoid from grape. It becomes possible to artificially glycosylate the 3-site of flavonoid with glucose or galactose, thus enabling contributing to the development of new functional food materials and the development of plants producing useful compounds. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a grape-derived flavonoid 3-position galactose-glucosyltransferase, a polynucleotide encoding the enzyme, a vector containing the polynucleotide, a transformant obtained using the vector, and the like.

The wine ingredient Vitis vinifera is the world's major agricultural crop, and red wine is known for its useful functionality such as reducing heart disease risk and anti-obesity effect. As resveratrol, which is a kind of stilbene, and procyanidin as a major component that causes French paradox as the main functional component of the life-prolonging effect, it has attracted attention as a functional food (Non-patent Document 1). ~ 7).
For vine leaves, fruits and wine, as a flavonol glycoside that is a kind of flavonoid, in addition to 3-position glucose glycoside, 3-position rhamnose glycoside and 3-position glucuronic acid glycoside, 3-position galactose glycoside Has been reported (see Non-Patent Documents 8 and 9). Quercetin glycosides have antioxidative properties and NO-producing ability, and are expected to have an effect of inhibiting arteriosclerosis (see Non-Patent Documents 10 and 11).
Quercetin's 3-position galactose glycoside (Q3Gal) is called hyperoside and is said to increase its physiological activity by increasing the solubility of hypericin, the main component of St. John's wort used as an antidepressant. (See Non-Patent Documents 12 and 13). In addition, Q3Gal and quercetin 3-position glucuronic acid glycoside (Q3GA: Michelinin) have been confirmed to be effective in a forced swimming test for the purpose of antidepressant effect (see Non-Patent Document 14). Furthermore, it has been shown that cell damage caused by hydrogen peroxide is protected by the antioxidant property of Q3Gal (see Non-Patent Document 15). In recent years, the antihypertensive effect of Q3Gal has also been reported (see Non-Patent Document 16). In this way, since various useful glycosides of flavonoids have been found to have useful activity, flavonoids, especially enzymes that transfer galactose to the 3-position of flavonols, are thought to be useful for the production of functional flavonoids. .

  In general, enzymes that transfer sugars to flavonoids are catalyzed by UDP sugar-dependent glycosyltransferases (UGTs), and UGTs have very high selectivity for sugar donors. Only one kind of UDP sugar can be used as a substrate. That is, an enzyme that transfers UDP glucose cannot basically catalyze the sugar transfer reaction using other UDP sugar donors. So far, glycosyltransferases at the 3-position of flavonoids are known which transfer glucose, galactose, arabinose and rhamnose as sugar donors (see Non-Patent Documents 17 to 20). PetFGalT and ACGaT have been identified in order from Petunia and Udo, respectively, as enzymes that transfer flavonoid 3-position galactose (see Non-Patent Document 18). These galactosyltransferases (Flavonoid 3-O-galactosyltransferase; F3GalT) are also similar in structure to enzymes that transfer rhamnose, glucose, and arabinose to the 3rd position of the flavonoids. It is extremely difficult to infer selectivity.

By the way, in recent years, the genome sequence of grapes (Pinot Noir varieties) was decoded by a joint consortium of Italy and France, and 240 glycoside enzymes (UGT) genes were identified (Non-patent Document 21). , 22).
However, even if it is presumed to be a glycosyltransferase gene from gene sequence information, it is not easy to infer the type of sugar that is a substrate or a substance that is a receptor, and as described above, candidate genes Since there are a large number of 240 genes, it was extremely difficult to identify UDP-galactose as a sugar donor (F3GalT) from these UGT genes.

Renaud, S. and De Lorgeril, M., Lancet, vol. 339, p. 1523-1526, 1992 Gehm, B. D. et al., Proc. Natl. Acad. Sci. USA, vol. 94, p. 14138-14143, 1997 Corder, R. et al., Nature, vol. 414, p. 863-864, 2001 Lamming, D. W. et al. Mol. Microbiol., Vol. 53, p. 1003-1009, 2004 Corder, R. et al., Nature, vol. 444, p. 566, 2006 Baur, J. A. and Sinclair, D. A., Nature Rev. Drug Discovery, vol. 5, p. 493-506, 2006 Baur, J. A. et al., Nature, vol. 444, p. 337-342, 2006 Hmamouchi, M. et al., Am. J. Enol. Vitic., Vol. 47, p. 186-192, 1996 Castillo-Munoz, N. et al., J. Agric. Food. Chem., Vol. 55, p. 992-1002, 2007 Murota, K. and Terao, J., Arch. Biochem. Biophys., Vol. 417, p. 12-17, 2003

Steffen, Y. et al., Arch. Biochem. Biophys., Vol. 469, p. 209-219, 2008 Tatsis, E. C. et al., Phytochemistry, vol. 68, p. 383-393, 2007 Butterweck, V. et al., Planta Med., Vol. 69, p. 189-192, 2003 Butterweck, V. et al., Planta Med., Vol. 66, p. 3-6, 2000 Piao, M. J. et al., Biochimica et Biophysica Acta, vol. 1780, p. 1448-1457, 2008 Kagawa, T. et al., 3rd International Conference on Polyphenols and Health, Supplement P089, 2007 Ford, C. M. et al., J. Biol. Chem., Vol. 273, p. 9224-9233, 1998 Miller, K. D. et al., J. Biol. Chem., Vol. 274, p. 34011-34019, 1999 Yonekura-Sakakibara, K. et al., J. Biol. Chem., Vol. 282, p. 14932-14941, 2007 Yonekura-Sakakibara, K. et al., Plant Cell, vol. 20, p. 2160-2176, 2008 The French-Italian Public Consortium for Grapevine Genome Characterization, Nature, vol. 449, p. 463-468, 2007 Velasco, T. et al., PLoS ONE, vol. 12, e1326, p. 1-18, 2007

  Therefore, the problem to be solved by the present invention is an enzyme capable of transferring galactose to the 3-position of a flavonoid, a polynucleotide encoding the enzyme, a vector containing the polynucleotide, and a transformation obtained using the vector The body is to provide.

  The present inventor has intensively studied to solve the above problems. As a result, the present inventor paid attention to the correlation between the gene sequence structure of the flavonoid UGT and the position specificity for the substrate which is a sugar receptor, and was homologous to the enzyme gene that transfers sugar to the flavonoid 3 position. 6 candidate grape-derived UGT genes (Vitis vinifera UDP-sugar: glycosyltransferase; VvGT) were obtained by extracting UGT genes derived from grapes with a high level from the genome sequence information. One of the six VvGTs (VvGT6) is an enzyme that has the activity of transferring galactose to the 3-position of flavonoids in a UDP-galactose-dependent manner, that is, flavonoid 3-O-galactosyltransferase (Flavonoid 3-O-galactosyltransferase; The gene was found to encode F3GalT). Moreover, the present inventor has found that this VvGT6 is an enzyme having the activity of transferring glucose to the 3-position of flavonoids using UDP-glucose as a sugar donor to the same extent as UDP-galactose, that is, -O-glucosyltransferase (F3GlcT) was found to be a gene encoding a glycosyltransferase having activity. That is, unlike the glycosyltransferases reported so far, this enzyme is a novel flavonoid 3-position galactose-glucosyltransferase (Flavonoid 3-O-galactosyl / glucosyltransferase; F3Gal / GlcT) that uses two types of UDP sugars as substrates. ). The present invention was thus completed.

That is, the present invention is as follows.
(1) The polynucleotide according to any one of the following (a) to (f):
(A) a polynucleotide comprising a polynucleotide comprising the base sequence represented by SEQ ID NO: 3;
(B) a polynucleotide containing a polynucleotide encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 4;
(C) consisting of an amino acid sequence in which 1 to 15 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 4, and UDP-galactose transferase activity and UDP-glucose transferase activity A polynucleotide comprising a polynucleotide encoding a protein having
(D) a polyprotein encoding a protein having an amino acid sequence having 80% or more homology to the amino acid sequence represented by SEQ ID NO: 4 and having UDP-galactose transferase activity and UDP-glucose transferase activity A polynucleotide containing nucleotides;
(E) a protein that hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 3 and has UDP-galactosyltransferase activity and UDP-glucose transferase activity Or (f) a polynucleotide comprising a base sequence complementary to the base sequence of a polynucleotide encoding a protein comprising the amino acid sequence represented by SEQ ID NO: 4 and stringent conditions A polynucleotide comprising a polynucleotide that hybridizes with and encodes a protein having UDP-galactosyltransferase activity and UDP-glucose transferase activity.

Examples of the polynucleotide (1) include any of the following polynucleotides (g) to (j).
(G) consisting of an amino acid sequence in which no more than 10 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 4, and exhibit UDP-galactose transferase activity and UDP-glucose transferase activity A polynucleotide containing a polynucleotide encoding the protein having;
(H) a polyprotein encoding a protein having an amino acid sequence having 90% or more homology with the amino acid sequence represented by SEQ ID NO: 4 and having UDP-galactose transferase activity and UDP-glucose transferase activity A polynucleotide containing nucleotides;
(I) hybridizes with a polynucleotide comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 3 under highly stringent conditions and has UDP-galactose transferase activity and UDP-glucose transferase activity A polynucleotide containing a polynucleotide encoding a protein; or (j) a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 4 and a highly stringent A polynucleotide comprising a polynucleotide that hybridizes under conditions and encodes a protein having UDP-galactose transferase activity and UDP-glucose transferase activity.

In the polynucleotide (1), for example, the polynucleotides described in (c) to (j) above are the polynucleotides described in (c ′) to (j ′) below, respectively. Can be mentioned.
(C ′) consisting of an amino acid sequence in which 1 to 15 amino acids excluding the 19th and / or 373rd amino acid in the amino acid sequence represented by SEQ ID NO: 4 have been deleted, substituted, inserted and / or added, And a polynucleotide comprising a polynucleotide encoding a protein having UDP-galactose transferase activity and UDP-glucose transferase activity;
(D ′) an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 4 and corresponding to the 19th amino acid in the amino acid sequence represented by SEQ ID NO: 4 Contains a polynucleotide encoding a protein that is proline and / or has an amino acid sequence in which the amino acid corresponding to the 373rd amino acid is glutamine and has UDP-galactosyltransferase activity and UDP-glucose transferase activity Polynucleotides to:
(E ′) a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 3 in the base sequence represented by SEQ ID NO: 3; The polynucleotide corresponding to the 57th base is a polynucleotide encoding proline and / or the base corresponding to the 1117th to 1119th bases is a base encoding glutamine, and UDP-galactosyltransferase A polynucleotide comprising a polynucleotide encoding a protein having activity and UDP-glucose transferase activity;
(F ′) a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to the base sequence of a polynucleotide encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 4; The base corresponding to the 55th to 57th bases in the base sequence of the polynucleotide encoding the protein consisting of the amino acid sequence represented by 4 is a base encoding proline and / or the 1117th to 1119th bases A polynucleotide containing a polynucleotide encoding a protein wherein the corresponding base is a base encoding glutamine and having UDP-galactosyltransferase activity and UDP-glucose transferase activity;
(G ′) consisting of an amino acid sequence in which no more than 10 amino acids excluding the 19th and / or 373rd amino acid are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 4; A polynucleotide comprising a polynucleotide encoding a protein having UDP-galactosyltransferase activity and UDP-glucose transferase activity;
(H ′) an amino acid sequence having 90% or more homology with the amino acid sequence represented by SEQ ID NO: 4 and corresponding to the 19th amino acid in the amino acid sequence represented by SEQ ID NO: 4 Contains a polynucleotide encoding a protein that is proline and / or has an amino acid sequence in which the amino acid corresponding to the 373rd amino acid is glutamine and has UDP-galactosyltransferase activity and UDP-glucose transferase activity A polynucleotide to:
(I ′) a polynucleotide that hybridizes with a polynucleotide comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 3 under highly stringent conditions, wherein the 55th in the base sequence represented by SEQ ID NO: 3 The base corresponding to the 57th base is a polynucleotide encoding proline and / or the base corresponding to the 1117th to 1119th base is a base encoding glutamine, and UDP-galactose transfer A polynucleotide comprising a polynucleotide encoding a protein having enzymatic activity and UDP-glucosyltransferase activity; or (j ′) complementary to the nucleotide sequence of a polynucleotide encoding a protein comprising the amino acid sequence represented by SEQ ID NO: 4 Under a highly stringent condition with a polynucleotide consisting of a simple base sequence Whether the base corresponding to the 55th to the 57th base in the base sequence of the polynucleotide encoding the protein comprising the amino acid sequence represented by SEQ ID NO: 4 is a base encoding proline, / Or a polynucleotide in which the base corresponding to the 1117th to 1119th bases is a base encoding glutamine, and a polynucleotide encoding a protein having UDP-galactosyltransferase activity and UDP-glucosyltransferase activity Polynucleotide.

Examples of the polynucleotide (1) include those in which the UDP-galactosyltransferase activity is flavonoid 3-position galactose transferase activity, and the UDP-glucosyltransferase activity is flavonoid 3-position glucose transferase activity. It is done.
Examples of the polynucleotide (1) include those containing a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 3 or a polynucleotide encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 4. .
Examples of the polynucleotide (1) include DNA.

(2) A protein encoded by the polynucleotide of (1) above.
(3) A vector containing the polynucleotide of (1) above.
(4) A transformant introduced with the polynucleotide of (1) above.
(5) A transformant introduced with the vector of (3) above.
(6) The method for producing a protein of (2) above, using the transformant of (4) or (5).
(7) Galactose glycoside and / or glucose glycoside which produces galactose glycoside and / or glucose glycoside from UDP-galactose and / or UDP-glucose and a sugar acceptor substrate using the protein of (2) above as a catalyst Or the manufacturing method of glucose glycoside.
Examples of the method (7) include a method in which the sugar receptor substrate is a flavonoid.

ADVANTAGE OF THE INVENTION According to this invention, the glycosyltransferase (glycosylase) which can transfer both galactose and glucose to 3rd-position of a flavonoid can be provided. Moreover, according to the present invention, a polynucleotide encoding the enzyme, a vector containing the polynucleotide, a transformant obtained using the vector, and a galactose glycoside and / or glucose using the enzyme A method for producing a glycoside can also be provided.
Conventionally, when producing a flavonoid glycoside by enzymatic reaction in vitro, one product was obtained using one enzyme and one sugar donor. If used, the addition of two sugar donors in vitro enables simultaneous transfer of glucose and galactose at the 3-position of the flavonoid, making it easier to produce useful flavonoid glycosides such as hyperoside. Therefore, the present invention is extremely useful.

It is a figure which shows the result of the systematic analysis of the grape origin flavonoid glycoside enzyme homologue gene. This is a molecular phylogenetic tree of cluster I of flavonoid UGT genes including 8 grape UGTs (VvGT) by NJ method. Out group is the glucose transferase gene (VlRSgt) of resveratrol of American grape (Concord species). It is a figure which shows the result of the chromosome mapping of the grape origin glucosylase homolog gene. The VvGT1 homologue gene is located in tandem on the LG11 and LG6 chromosomes. It is a figure which shows the result of refinement | purification (SDS-PAGE) of E. coli expression VvGT6 protein. The band (arrow in the figure) recognized in the fraction eluted with 500 mM imidazole is His-tagged VvGT6 protein (His-VvGT6). “M” indicates a molecular size marker, “coarse” indicates a crude enzyme solution, “element” indicates a normal solution, and “wash” indicates a solution after washing. It is a figure which shows the result of the enzyme activity measurement of VvGT6. Specifically, it is an HPLC chart (chromatogram) monitored at 360 nm. The top chart is for the standard quercetin, the second chart is for the reaction solution of quercetin and VvGT6 (UDP sugar donor: UDP-galactose), the third chart from the top. The chart is for the standard quercetin 3-O-galactoside, the fourth chart from the top is for the reaction solution of quercetin and VvGT6 (UDP sugar donor: UDP-glucose), the bottom chart Is for the standard quercetin 3-O-glucoside. “Q” indicates the peak of the substrate quercetin, and “A” and “B” indicate the peaks of the reaction product. It is a graph which shows the buffer system used for the optimal pH assay of VvGT6. It is a figure which shows the result of the reaction pH dependence analysis (optimum pH analysis) of VvGT6. The vertical axis shows the relative activity (%) when the highest activity is taken as 100%.

It is a figure which shows the result of the reaction temperature dependence analysis (optimum reaction temperature analysis) of VvGT6. The vertical axis shows the relative activity (%) when the highest activity is taken as 100%. It is a graph which shows the result of the sugar donor selectivity analysis of VvGT6. The relative activity (%) is shown when the selectivity to UDP-glucose is 100%. “N. D.” Indicates below the detection limit. It is a graph which shows the result of the sugar receptor selectivity analysis of VvGT6. The relative activity (%) is shown when the selectivity for quercetin is 100%. “N. D.” Indicates below the detection limit. It is a graph which shows the result of the kinetic analysis of VvGT6. The above two tables show the results for flavonoid 3-position galactosyltransferase activity (F3GalT activity) and flavonoid 3-position glucose transferase activity (F3GlcT activity), respectively. The following HPLC chart (chromatogram; monitored at 360 nm) shows the reaction mixture when quercetin is used as a sugar acceptor and VvGT6 is reacted simultaneously with two sugar donors (UDP-galactose and UDP-glucose). Is. “Q” indicates the peak of the substrate quercetin, “Q3Gal” indicates the peak of the reaction product when UDP-galactose is a sugar donor, and “Q3Glc” indicates that UDP-glucose is a sugar donor The peak of the reaction product is shown. It is a figure which shows the result of the VvGT gene expression analysis according to organ by quantitative RT-PCR. Specifically, it is the result of the gene expression analysis of VvGT in the Pinot Noir variety (left) and the Cabernet Sauvignon variety (right). In both varieties, the upper graph shows the analysis results for the VvGT1 gene, the middle graph for the VvGT5 gene, and the lower graph for the VvGT6. All graphs show the relative expression levels normalized with the internal standard gene (VvUBQ). VvGT1 (a grape-derived flavonoid 3-glucose transferase), VvGT5 (a grape-derived flavonoid 3-position glucuronosyltransferase), VvGT6 (a grape-derived flavonoid 3-position galactose-glucosyltransferase), ACGalT (Udo-derived flavonoid galactosyltransferase) FIG. 5 is a diagram showing the results of multiple alaminates by Clustal-W for PhF3GalT (Petunia-derived flavonoid 3-position galactosyltransferase).

  Hereinafter, the present invention will be described in detail. The scope of the present invention is not limited to these explanations, and modifications other than the following examples can be made as appropriate without departing from the spirit of the present invention. In addition, this specification includes the whole of Japanese Patent Application No. 2009-020105 specification (filed on January 30, 2009), which is the basis of the priority claim of the present application. In addition, all publications cited in the present specification, for example, prior art documents, and publications, patent publications and other patent documents are incorporated herein by reference.

1. First, the present invention provides (a) a polynucleotide containing a polynucleotide consisting of the base sequence of SEQ ID NO: 3 (specifically, DNA, hereinafter, also simply referred to as “DNA”); and (B) A polynucleotide containing a polynucleotide encoding a protein having the amino acid sequence represented by SEQ ID NO: 4 is provided. The DNA of interest in the present invention is not limited to DNA encoding the above galactose-glucosyltransferase (F3Gal / GlcT), and other DNA encoding a protein functionally equivalent to this protein Including.
Examples of functionally equivalent proteins include (c) an amino acid sequence in which 1 to 15 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 4, and UDP- Examples include proteins having galactose transferase activity and UDP-glucose transferase activity. As such a protein, in the amino acid sequence represented by SEQ ID NO: 4, for example, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1 -9, 1-8, 1-7, 1-6 (1 to several), 1-5, 1-4, 1-3, 1-2, 1 amino acid residue Examples include proteins having an amino acid sequence in which a group is deleted, substituted, inserted and / or added and having UDP-galactose transferase activity and UDP-glucose transferase activity. In general, the smaller the number of amino acid residue deletions, substitutions, insertions and / or additions, the better. In the present invention, the functionally equivalent protein of (c) above has (i) an amino acid residue corresponding to the 19th amino acid residue (proline) in the amino acid sequence represented by SEQ ID NO: 4. Similarly, an amino acid sequence consisting of an amino acid sequence that is proline, and (ii) an amino acid sequence in which the amino acid residue corresponding to the 373rd amino acid residue (glutamine) in the amino acid sequence represented by SEQ ID NO: 4 is also glutamine It is preferably composed of an amino acid sequence satisfying both the characteristics (i) and (ii). That is, the deletion, substitution, insertion and / or addition of the amino acid residue is performed for the amino acid other than the 19th amino acid and / or the 373rd amino acid in the amino acid sequence represented by SEQ ID NO: 4. Preferably there is. This preferred embodiment is the same for the polynucleotide (g).

  Examples of functionally equivalent proteins include (d) an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 4, and UDP-galactose transferase activity and A protein having UDP-glucose transferase activity may also be mentioned. As such a protein, about 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% with respect to the amino acid sequence represented by SEQ ID NO: 4 More than 88%, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, 99.9% or more of the amino acid sequence and UDP-galactose Examples include proteins having transferase activity and UDP-glucose transferase activity. In general, the larger the homology value, the better. In the present invention, the functionally equivalent protein of (d) above has (i) an amino acid residue corresponding to the 19th amino acid residue (proline) in the amino acid sequence represented by SEQ ID NO: 4. Similarly, an amino acid sequence consisting of an amino acid sequence that is proline, and (ii) an amino acid sequence in which the amino acid residue corresponding to the 373rd amino acid residue (glutamine) in the amino acid sequence represented by SEQ ID NO: 4 is also glutamine It is preferably composed of an amino acid sequence satisfying both the characteristics (i) and (ii). This preferred embodiment is the same for the polynucleotide (h).

Here, “UDP-galactose transferase activity” as used in the present invention means that galactose is transferred to the hydroxyl group at the 3-position of a flavonoid serving as a sugar acceptor substrate, depending on the UDP-galactose of the sugar donor, It means an enzyme having an activity of catalyzing a reaction that produces a body, ie, flavonoid 3-O-galactosyltransferase (F3GalT) activity.
For UDP-galactose transferase activity, for example, UDP-galactose and a sugar receptor substrate flavonoid (eg, flavonol) are reacted in the presence of the enzyme to be evaluated, and the resulting reaction product is analyzed by HPLC, etc. (Specifically, refer to the description of Examples described later).

In addition, “UDP-glucose transferase activity” as used in the present invention means that glucose is transferred to the hydroxyl group at the 3-position of a flavonoid serving as a sugar acceptor substrate in a UDP-glucose-dependent manner as a sugar donor. It means an enzyme having an activity of catalyzing a reaction that produces flavonoid, ie, flavonoid 3-O-glucosyltransferase (F3GlcT) activity.
For UDP-glucose transferase activity, for example, UDP-glucose and a flavonoid that is a sugar receptor substrate (for example, flavonol) are reacted in the presence of the enzyme to be evaluated, and the resulting reaction product is analyzed by HPLC or the like. (Specifically, refer to the description of Examples described later).
The above-mentioned UDP-galactose transferase activity and UDP-glucose transferase activity can also be measured simultaneously using two types of sugar donors (UDP-galactose and UDP-glucose).

The present invention also relates to (e) a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 3 under stringent conditions, and UDP-galactose transferase activity and UDP-glucose transferase activity. Also included are polynucleotides containing polynucleotides that encode proteins having: In the present invention, the polynucleotide (e) above is (i) “CCC” having the same base corresponding to the 55th to 57th bases (CCC) in the base sequence represented by SEQ ID NO: 3. It contains a polynucleotide that is either “CCU (CCT)”, “CCA” or “CCG” even if it is not the same base, and (ii) the base represented by SEQ ID NO: 3 The base corresponding to the 1117th to 1119th bases (CAA) in the sequence is preferably the same “CAA” or a polynucleotide that is not the same base but is “CAG”, Particularly preferably, the polynucleotide contains a polynucleotide that satisfies both the characteristics (i) and (ii). Here, the 55th to 57th bases (CCC) are bases (codons) encoding the 19th amino acid residue (proline) in the amino acid sequence represented by SEQ ID NO: 4. “CCU (CCT)”, “CCA” and “CCG” are also bases (codons) encoding proline. The 1117th to 1119th base (CAA) is a base (codon) encoding the 373rd amino acid residue (glutamine) in the amino acid sequence represented by SEQ ID NO: 4. "Is also a base (codon) encoding glutamine. This preferred embodiment is the same for the polynucleotide (i).
The present invention further includes (f) hybridization under stringent conditions with a polynucleotide comprising a base sequence complementary to a base sequence of a polynucleotide encoding a protein comprising the amino acid sequence represented by SEQ ID NO: 4, and Also included are polynucleotides that contain a polynucleotide that encodes a protein having UDP-galactosyltransferase activity and UDP-glucose transferase activity. In the present invention, the polynucleotide of (f) above is (i) “CCC” in which the bases corresponding to the 55th to 57th bases (CCC) in the base sequence represented by SEQ ID NO: 3 are the same. It contains a polynucleotide that is either “CCU (CCT)”, “CCA” or “CCG” even if it is not the same base, and (ii) the base represented by SEQ ID NO: 3 The base corresponding to the 1117th to 1119th bases (CAA) in the sequence is preferably the same “CAA” or a polynucleotide that is not the same base but is “CAG”, Particularly preferably, the polynucleotide contains a polynucleotide that satisfies both the characteristics (i) and (ii). Here, the 55th to 57th bases (CCC) are bases (codons) encoding the 19th amino acid residue (proline) in the amino acid sequence represented by SEQ ID NO: 4. “CCU (CCT)”, “CCA” and “CCG” are also bases (codons) encoding proline. The 1117th to 1119th base (CAA) is a base (codon) encoding the 373rd amino acid residue (glutamine) in the amino acid sequence represented by SEQ ID NO: 4. "Is also a base (codon) encoding glutamine. This preferred embodiment is the same for the polynucleotide (j).
In the present specification, “polynucleotide” means DNA or RNA. Preferably, it is DNA.

In the present specification, the “polynucleotide hybridizing under stringent conditions” means, for example, a polynucleotide comprising a base sequence complementary to the base sequence of SEQ ID NO: 3 or the amino acid sequence represented by SEQ ID NO: 4. A polynucleotide obtained by using a colony hybridization method, a plaque hybridization method, a Southern hybridization method, or the like using as a probe all or part of a polynucleotide consisting of a base sequence complementary to the base sequence of the encoding polynucleotide Say. Examples of hybridization methods include "Sambrook & Russell, Molecular Cloning: A Laboratory Manual Vol. 3, Cold Spring Harbor, Laboratory Press 2001", "Ausubel, Current Protocols in Molecular Biology, John Wiley & Sons 1987-1997". Can be used.
In the present specification, “stringent conditions” may be any of low stringent conditions, moderately stringent conditions, and highly stringent conditions. “Low stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, 32 ° C. The “medium stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, and 42 ° C. “High stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, 50 ° C. Under these conditions, it can be expected that DNA having higher homology can be efficiently obtained as the temperature is increased. However, multiple factors such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration can be considered as factors that affect hybridization stringency. Those skilled in the art will select these factors as appropriate. It is possible to achieve similar stringency.

In addition, when using a commercially available kit for hybridization, Alkphos Direct Labeling Reagents (made by Amersham Pharmacia) can be used, for example. In this case, follow the protocol attached to the kit, incubate with the labeled probe overnight, and then wash the membrane with a primary wash buffer containing 0.1% (w / v) SDS at 55 ° C. , Hybridized DNA can be detected.
In addition to the above, the hybridizable polynucleotide includes DNA of the nucleotide sequence of SEQ ID NO: 3 or the amino acid represented by SEQ ID NO: 4, when calculated using homology search software such as FASTA and BLAST using default parameters. About 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79 % Or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5 % Or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more of DNA having homology.

The homology between the amino acid sequence and the base sequence is determined by the algorithm BLAST (Proc. Natl. Acad. Sci. USA, vol. 87, p. 2264-2268, 1990; Proc. Natl. Acad. Sci. USA, vol. 90, p. 5873, 1993). Programs called BLASTN and BLASTX based on the BLAST algorithm have been developed (Altschul SF, et al., J. Mol. Biol., Vol. 215, p. 403, 1990). When analyzing a base sequence using BLASTN, parameters are set to, for example, score = 100 and word length = 12. When analyzing an amino acid sequence using BLASTX, the parameters are set to, for example, score = 50 and word length = 3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used.
The polynucleotide of the present invention described above can be obtained by a known genetic engineering technique or a known synthesis technique.

2. In another embodiment, the present invention also provides a protein encoded by the above-described polynucleotide of the present invention. A protein of an embodiment of the present invention is a protein consisting of the amino acid sequence represented by SEQ ID NO: 4. Another embodiment of the present invention is a protein having the amino acid sequence represented by SEQ ID NO: 4.
The protein of still another embodiment of the present invention comprises an amino acid sequence in which 1 to 15 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 4, and UDP-galactose transferase It is a protein having activity and UDP-glucose transferase activity. Examples of such a protein include a protein having the amino acid sequence having the homology as described above with the amino acid sequence represented by SEQ ID NO: 4 and having UDP-galactose transferase activity and UDP-glucose transferase activity. Such proteins are described in "Sambrook & Russell, Molecular Cloning: A Laboratory Manual Vol. 3, Cold Spring Harbor, Laboratory Press 2001", "Ausubel, Current Protocols in Molecular Biology, John Wiley & Sons 1987-1997", "Nuc Acids. Res., Vol. 10, p. 6487, 1982 "," Proc. Natl. Acad. Sci. USA, vol. 79, p. 6409, 1982 "," Gene, vol. 34, p. 315, 1985 "," Nuc. Acids. Res., Vol. 13, p. 4431, 1985 "," Proc. Natl. Acad. Sci. USA, vol. 82, p. 488, 1985 "etc. It can be obtained using a mutagenesis method. In addition, the above-described protein of still another embodiment includes (i) an amino acid in which the amino acid residue corresponding to the 19th amino acid residue (proline) in the amino acid sequence represented by SEQ ID NO: 4 is also proline And (ii) those consisting of an amino acid sequence in which the amino acid residue corresponding to the 373rd amino acid residue (glutamine) in the amino acid sequence represented by SEQ ID NO: 4 is also glutamine are preferred, Particularly preferably, it consists of an amino acid sequence satisfying both of the characteristics (i) and (ii). That is, the deletion, substitution, insertion and / or addition of the amino acid residue is performed for the amino acid other than the 19th amino acid and / or the 373rd amino acid in the amino acid sequence represented by SEQ ID NO: 4. Preferably there is.

In the amino acid sequence of the protein of the present invention, 1 or more (for example, 1 to 15, preferably 10 or less) amino acid residues are deleted, substituted, inserted and / or added. Means that there is a deletion, substitution, insertion and / or addition of one or more amino acid residues at a position in a plurality of amino acid sequences, and two or more of deletion, substitution, insertion and addition occur simultaneously. May be.
Examples of amino acid residues that can be substituted with each other are shown below. Amino acid residues contained in the same group can be substituted for each other. Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, o-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine; Group B: aspartic acid, glutamic acid, isoaspartic acid , Isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid; group C: asparagine, glutamine; group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid; group E : Proline, 3-hydroxyproline, 4-hydroxyproline; Group F: serine, threonine, homoserine; Group G: phenylalanine, tyrosine.

The protein of the present invention can also be produced by chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method). It can also be chemically synthesized using peptide synthesizers such as Advanced Chemtech, Perkin Elmer, Pharmacia, Protein Technology Instrument, Syntheselve Vega, Perceptive, Shimadzu, etc. .
Here, the protein of the present invention is a flavonoid 3-position galactose-glucosyltransferase. “Galactose-glucosyltransferase” refers to a reaction in which a galactose residue is transferred from a sugar donor to a sugar acceptor substrate to produce a galactose glycoside, and a glucose residue is transferred from the sugar donor to a sugar acceptor substrate. Both reactions that produce glycosides are catalyzed. In the present invention, the sugar acceptor substrate is a flavonoid, and the sugar donors are UDP-galactose and UDP-glucose. A protein according to an embodiment of the present invention is a reaction in which a galactose residue is transferred from UDP-galactose to a hydroxyl group at the 3-position of a flavonoid (eg, flavonol) as a sugar acceptor substrate to generate a galactose glycoside and UDP. To catalyze. In another embodiment of the present invention, the protein transfers a glucose residue from UDP-glucose to a hydroxyl group at the 3-position of a flavonoid (eg, flavonol) that is a sugar receptor substrate. Catalyze the reaction that yields Furthermore, the protein according to another aspect of the present invention includes a galactose residue and a glucose residue from a combined UDP-galactose and UDP-glucose to a hydroxyl group at the 3-position of a flavonoid (eg, flavonol) as a sugar receptor substrate. To catalyze the reaction that produces galactose glycosides, glucose glycosides and UDP.

  Preferred flavonoids for the sugar receptor substrate include flavonols, flavanones, isoflavones, anthocyanidins, flavone C glycosides, aurones and catechins. Among these, examples of the flavonol include quercetin, myricetin, laricitrine, isorhamnetin, syringethin, kaempferol and the like. Examples of flavanones include naringenin. Examples of isoflavones include genistein, daidzein, and formononetin. Examples of anthocyanidins include cyanidin, delphinidin and pelargonidin. Examples of flavone C glycosides include vitexin, isovitexin and orientin. Examples of auron include aureusidin and the like. Examples of catechins include catechin and epigallocatechin gallate. In the present invention, among the flavonoids of the sugar receptor substrate, flavonol is more preferable among the above, and quercetin, kaempferol and isorhamnetin are particularly preferable.

3. Vector and Transformant Introducing the Vector In another embodiment, the present invention also provides an expression vector containing the polynucleotide of the present invention. The expression vector of the present invention contains the polynucleotide of the present invention (for example, the polynucleotide of any one of (a) to (j) above). Preferably, the expression vector of the present invention contains any one of the polynucleotides (g) to (j). More preferably, the expression vector of the present invention contains a polynucleotide comprising a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 3 or a polynucleotide encoding a protein comprising the amino acid sequence represented by SEQ ID NO: 4.
The vector of the present invention usually contains (i) a promoter capable of being transcribed in a host cell; (ii) a polynucleotide of the present invention bound to the promoter (for example, any of the polynucleotides (a) to (j) above). Nucleotides); and (iii) with respect to transcription termination and polyadenylation of RNA molecules, configured to include an expression cassette containing as a component a signal that functions in the host cell. The vector thus constructed is introduced into a host cell. As a method for producing an expression vector, a method using a plasmid, phage, cosmid or the like can be mentioned, but it is not particularly limited.

The specific type of vector is not particularly limited, and a vector that can be expressed in a host cell can be appropriately selected. That is, according to the type of the host cell, a promoter sequence is appropriately selected in order to reliably express the polynucleotide of the present invention, and a vector in which this and the polynucleotide of the present invention are incorporated into various plasmids or the like is used as an expression vector. Good.
The expression vector of the present invention contains an expression control region (for example, a promoter, a terminator and / or an origin of replication) depending on the type of host to be introduced. Conventional promoters (eg, trc promoter, tac promoter, lac promoter, etc.) are used as promoters for bacterial expression vectors. Examples of yeast promoters include glyceraldehyde 3-phosphate dehydrogenase promoter, PH05 promoter, etc. Examples of the promoter for filamentous fungi include amylase and trpC. Examples of promoters for animal cell hosts include viral promoters (eg, SV40 early promoter, SV40 late promoter, etc.).
The expression vector preferably contains at least one selectable marker. Such markers include auxotrophic markers (ura5, niaD), hygromycin resistance gene, zeocin resistance gene, geneticin resistance gene (G418r), copper resistance gene (CUP1) (Marin et al., Proc. Natl. Acad Sci. USA, vol. 81, p. 337, 1984), cerulenin resistance gene (fas m, PDR4) (respectively, Ogura et al., Biochemistry, vol. 64, p. 660, 1992; Hussain et al. Gene, vol. 101, p. 149, 1991) can be used.

The present invention also provides a transformant into which the polynucleotide of the present invention (for example, any one of the polynucleotides (a) to (j) above) is introduced.
A method for producing a transformant (production method) is not particularly limited, and examples thereof include a method for transformation by introducing the above-described recombinant vector into a host. The host cell used here is not particularly limited, and various conventionally known cells can be suitably used. Specifically, for example, bacteria such as Escherichia coli, yeasts (budding yeast Saccharomyces cerevisiae, fission yeast Schizosaccharomyces pombe), nematodes (Caenorhabditis elegans), Xenopus laevis oocytes, etc. Can do. Appropriate culture media and conditions for the above-described host cells are well known in the art. In addition, the organism to be transformed is not particularly limited, and examples thereof include various microorganisms, plants and animals exemplified in the host cell.
As a method for transforming a host cell, a publicly known method can be used. For example, an electroporation method (Mackenxie DA et al., Appl. Environ. Microbiol., Vol. 66, p. 4655-4661, 2000), a particle delivery method (Japanese Patent Application Laid-Open No. 2005-287403, “Method of Breeding Lipid-Producing Bacteria”) Method), spheroplast method (Proc. Natl. Acad. Sci. USA, vol. 75, p. 1929, 1978), lithium acetate method (J. Bacteriology, vol. 153, p. 163, 1983) , Methods in yeast genetics, 2000 Edition: A Cold Spring Harbor Laboratory Course Manual, etc.), but is not limited thereto.

In another embodiment of the present invention, the transformant can be a plant transformant. The plant transformant according to this embodiment is obtained by introducing a recombinant vector containing the polynucleotide according to the present invention into a plant so that the polypeptide encoded by the polynucleotide can be expressed.
When a recombinant expression vector is used, the recombinant expression vector used for transformation of the plant body is not particularly limited as long as it is a vector capable of expressing the polynucleotide according to the present invention in the plant. Examples of such a vector include a vector having a promoter that constitutively expresses a polynucleotide in plant cells (for example, the cauliflower mosaic virus 35S promoter) or a promoter that is inducibly activated by an external stimulus. The vector which has is mentioned.
Plants to be transformed in the present invention include whole plants, plant organs (eg leaves, petals, stems, roots, seeds, etc.), plant tissues (eg epidermis, phloem, soft tissue, xylem, vascular bundle, It means any of a palisade tissue, a spongy tissue, etc.) or a plant culture cell, or various forms of plant cells (eg, suspension culture cells), protoplasts, leaf sections, callus, and the like. The plant used for transformation is not particularly limited, and may be any plant belonging to the monocotyledonous plant class or the dicotyledonous plant class.

  For the introduction of genes into plants, transformation methods known to those skilled in the art (for example, Agrobacterium method, gene gun, PEG method, electroporation method, etc.) are used. For example, a method using Agrobacterium and a method for directly introducing it into plant cells are well known. When the Agrobacterium method is used, the constructed plant expression vector is introduced into an appropriate Agrobacterium (for example, Agrobacterium tumefaciens), and this strain is introduced into the leaf disk method (Hirofumi Uchimiya). Author, Plant Gene Manipulation Manual (1990), pp. 27-31, Kodansha Scientific, Tokyo) and the like can be used to infect sterile cultured leaf pieces to obtain transformed plants. The method of Nagel et al. (Micribiol. Lett., 67, 325 (1990)) can also be used. In this method, for example, an expression vector is first introduced into Agrobacterium, and then transformed Agrobacterium is introduced into plant cells or plant tissues by the method described in the Plant Molecular Biology Manual (SBGelvin et al., Academic Press Publishers). It is a method to introduce. Here, “plant tissue” includes callus obtained by culturing plant cells. In the case of performing transformation using the Agrobacterium method, a binary vector (such as pBI121 or pPZP202) can be used.

As a method for directly introducing a gene into a plant cell or plant tissue, an electroporation method and a gene gun method are known. When using a gene gun, a plant body, a plant organ, and a plant tissue itself may be used as they are, or may be used after preparing a section, or a protoplast may be prepared and used. The sample thus prepared can be processed using a gene transfer apparatus (for example, PDS-1000 (BIO-RAD)). Treatment conditions vary depending on the plant or sample, but are usually performed at a pressure of about 450 to 2000 psi and a distance of about 4 to 12 cm.
A cell or plant tissue into which a gene has been introduced is first selected for drug resistance such as hygromycin resistance, and then regenerated into a plant by a conventional method. Regeneration of a plant body from a transformed cell can be performed by a method known to those skilled in the art depending on the type of plant cell.
When plant cultured cells are used as a host, transformation is carried out by introducing a recombinant vector into the cultured cells using a gene gun, electroporation method or the like. Callus, shoots, hairy roots, etc. obtained as a result of transformation can be used as they are for cell culture, tissue culture or organ culture, and can be used at a suitable concentration using conventionally known plant tissue culture methods. It can be regenerated into plants by administration of plant hormones (auxin, cytokinin, gibberellin, abscisic acid, ethylene, brassinolide, etc.).

Whether or not a gene has been introduced into a plant can be confirmed by PCR, Southern hybridization, Northern hybridization, or the like. For example, DNA is prepared from a transformed plant, PCR is performed by designing DNA-specific primers. PCR can be performed under the same conditions as those used for preparing the plasmid. After that, the amplified product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis or capillary electrophoresis, stained with ethidium bromide, SYBR Green solution, etc., and the amplified product is detected as a single band, It can be confirmed that it has been transformed. Moreover, PCR can be performed using a primer previously labeled with a fluorescent dye or the like to detect an amplification product. Furthermore, it is possible to employ a method in which the amplification product is bound to a solid phase such as a microplate and the amplification product is confirmed by fluorescence or enzyme reaction.
Once a transformed plant in which the polynucleotide of the present invention is integrated into the genome is obtained, offspring can be obtained by sexual or asexual reproduction of the plant. Further, for example, seeds, fruits, cuttings, tubers, tuberous roots, strains, callus, protoplasts, etc. can be obtained from the plant or its progeny, or clones thereof, and the plant can be mass-produced based on them. it can. Therefore, the present invention also includes a plant body into which the polynucleotide according to the present invention is introduced so that it can be expressed, or a progeny of the plant body having the same properties as the plant body, or a tissue derived therefrom.

In addition, transformation methods for various plants have already been reported. Examples of the transformant plant according to the present invention include grape, sesame, rice, tobacco, barley, wheat, rapeseed, potato, tomato, poplar, banana, eucalyptus, sweet potato, tides, alfalfa, lupine, corn, cauliflower, rose , Chrysanthemum, carnation, snapdragon, cyclamen, orchid, eustoma, freesia, gerbera, gladiolus, gypsophila, kalanchoe, lily, pelargonium, geranium, petunia, torenia, tulip, forsythia, Arabidopsis thaliana and Miyakogusa.
According to an aspect of the present invention, the transformed plant body is a functional food material plant body.

4). In another embodiment, the present invention also provides a method for producing the protein of the present invention using the transformant described above.
Specifically, the protein of the present invention can be obtained by separating and purifying the protein of the present invention from the culture of the transformant. Here, the culture means any of a culture solution, cultured cells or cultured cells, or crushed cells of cultured cells or cultured cells. Separation and purification of the protein of the present invention can be carried out according to a usual method.
Specifically, when the protein of the present invention accumulates in cultured cells or cells, the cells or cells are disrupted after culturing by an ordinary method (for example, ultrasound, lysozyme, freeze-thawing, etc.). After that, a crude extract of the protein of the present invention can be obtained by a usual method (for example, centrifugation, filtration, etc.). When the protein of the present invention is accumulated in the culture solution, the cells or cells and the culture supernatant are separated from each other by the usual method (for example, centrifugation, filtration, etc.) after completion of the culture. A culture supernatant containing the protein can be obtained.
Purification of the protein of the present invention contained in the extract or culture supernatant thus obtained can be performed according to a usual separation / purification method. Separation / purification methods include, for example, ammonium sulfate precipitation, gel filtration chromatography, ion exchange chromatography, affinity chromatography, reverse phase high performance liquid chromatography, dialysis method, ultrafiltration method, etc., alone or in appropriate combination. be able to.

5). Method for Producing Galactose Glycoside and Glucose Glycoside Further, the present invention provides a method for producing a galactose glycoside and / or a glucose glycoside using the protein of the present invention.
The protein of the present invention catalyzes a reaction for transferring galactose from UDP-galactose as a sugar donor to a flavonoid as a sugar acceptor substrate, and a reaction for transferring glucose from UDP-glucose as a sugar donor (whichever This reaction is also a reaction that transfers to the hydroxyl group at the 3-position of the flavonoid). Therefore, by using the protein of the present invention, a galactose glycoside and / or a glucose glycoside can be produced using a sugar acceptor substrate and a sugar donor as raw materials. When only UDP-galactose is used as a sugar donor, a galactose glycoside can be produced. When only UDP-glucose is used, a glucose glycoside can be produced. When UDP-galactose and UDP-glucose are used at the same time. Can simultaneously produce a galactose glycoside and a glucose glycoside. Among the flavonoids, flavonol is preferable as the sugar receptor substrate, and quercetin, kaempferol and myricetin are particularly preferable.

For example, prepare a solution containing 1 mM sugar acceptor substrate, 2 mM sugar donor, 50 mM calcium phosphate buffer (pH 7.5) and 20 μM of the protein of the present invention, and react at 30 ° C. for 30 minutes. Thus, a galactose glycoside and / or a glucose glycoside can be produced. From this solution, the galactose glycoside can be separated and purified by a known method. Specifically, for example, ammonium sulfate precipitation, gel filtration chromatography, ion exchange chromatography, affinity chromatography, reverse phase high performance liquid chromatography, dialysis method and ultrafiltration method may be used alone or in appropriate combination. it can.
The galactose glycoside thus obtained is useful as a functional food material, a reagent for examining its function in vivo, or an antioxidant.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

Gene Cloning Molecular biological techniques used in this example were according to the method described in Molecular Cloning (Sambrookra, Cold Spring Harbor Laboratory Press, 2001) unless otherwise specified.
Based on the full-length sequence of VvGT1 (Offen, W. et al., EMBO J., vol. 25, p. 1396-1405, 2006), a grape-derived flavonoid 3-position glucose transferase gene, Istituto Agrario San Michele all Blast homology search was performed against grape genome database (http://genomics.research.iasma.it/iasma/) provided by 'Adige (IASMA). As a result, seven candidate UGT genes with high homology were found (FIG. 1). These are Cluster I, which is a cluster of flavonoid 3-position UGT by NJ phylogenetic tree analysis using MEGA4 program (Tamura, K. et al., Mol. Biol. Evol., Vol. 24, p. 1596-1599, 2007). (Fig. 1). Furthermore, the synteny on the chromosome was clarified by comparing the candidate gene sequence with the chromosome physical map (FIG. 2). VvGT3, VvGT5 and VvGT6 are located in the same transcriptional orientation on chromosome 11, and a flavonol sulfotransferase-like gene was found in common between them (Varin, L. et al., Proc Natl. Acad. Sci. USA, vol. 89, p. 1286-1290, 1992). VvGT5 and VvGT6 were particularly highly homologous and were considered to be paralogs generated by gene duplication. In addition, VvGT2 and VvGT4 that were located in the same transcription direction were found on chromosome 6. A copy of the set of VvGT2 and VvGT4 was found downstream in the same transcription direction, and named VvGT2-like and VvGT4-like, respectively. On the other hand, the known VvGT1 was located on chromosome 16.

Next, in order to clone these VvGT genes, grape leaf-derived cDNA was prepared by the following method, and amplification by PCR was performed using it as a template. Total RNA was extracted from 0.1 g of grape leaves (Vitis vinifera, cultivar: Cabernet Sauvignon) using FruitMate for RNA purification and Fast Pure RNA kit (TaKaRa Bio) according to the manufacturer's recommended method. CDNA was synthesized using the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen) according to the conditions recommended by the manufacturer.
The procedure for cDNA cloning and expression vector construction is shown below using the VvGT6 gene as an example. An attempt was made to isolate VvGT6 by PCR using the cDNA obtained by reverse transcription as a template and the following VvGT6 gene-specific primers (SEQ ID NOs: 1 and 2) designed based on genomic sequence information.
Specifically, PCR reaction solution (50 μl) was obtained from grape leaf-derived cDNA 1 μl, 1 × ExTaq buffer (TaKaRaBio), 0.2 mM dNTPs, primers (SEQ ID NOs: 1 and 2) each 0.4 pmol / μl, ExTaq polymerase 2.5 unit. Become. The PCR reaction was carried out at 94 ° C. for 3 minutes, and then a total of 35 cycles of amplification reaction was performed with 94 ° C. for 1 minute, 50 ° C. for 1 minute, and 72 ° C. for 2 minutes as one cycle.

CACC-NdeI-VvGT6-Fw:
5'-CAC CCA TAT GAC TGC CAC CGC GAG CTC CAT G-3 '(SEQ ID NO: 1)

BglII-VvGT6-RV:
5'- AGA TCT CTA CTT ATT GGT ATC CAA ATG TAA CT -3 '(SEQ ID NO: 2)

When the PCR reaction solution was separated by 0.8% agarose gel electrophoresis, an amplified fragment having a size of about 1.4 kb was obtained. This amplified fragment is subcloned into the pENTR-Directional-TOPO vector (Invitrogen), and the base sequence of the inserted fragment is determined by the primer walking method using synthetic oligonucleotide primers using DNA Sequencer model 3100 (Applied Biosystems). The sequence was determined, and this sequence and the sequence derived from the Pinot Noir varieties registered in the database (IASMA accession number: VV78X045394.7) Compared with 4), it was confirmed that the cDNA inserted into the vector was VvGT6 (SEQ ID NOs: 3 and 4).

VvGT6 cDNA sequence:
(SEQ ID NO: 3)

Amino acid sequence of VvGT6:
MTATASSMDRHVAVLGFPPHAATLLKLLRRLASAAPTTIFSFFNTAKANNSIFSPQSPHGLHNLRVYDVADGVPEGHVLSANPLERIDLFFKATPGNFYDAIQVAEAEIGRKISCLVSDAFLWFTADMAEEMRVPWLAIWTSALCSLSVHIYTDAIREAVKVVGRVQDQTLDFIPGFSAIKVEDLPEGIVFGDIESPFACMLHKMGLTLPRATAVATNSFEELEPIVTNDPKSKLQKVLAVGPFDLSSPPQLILDASGCLPWLDNKKEASVAYVSFGSIATPPPNEIVALAEALEATGIPFLWSLREHAMDNLPKGFLERTTAHGKVVSWAPQPQILAHASVGVFITHSGWNSVIESIVGGVPMICRPFFGDQCIDKRMVEDVWGIGVGVEGGVLTKSGVMSALGLILSHEGNKMREKIRVLKELARRAVEPNGSSTQNLSNLLEVITTSKLHLDTNK (SEQ ID NO: 4)

  An approximately 1.4 kb VvGT6 fragment was excised from the pENT-Directional-TOPO vector using the NdeI and BglII restriction enzyme sites added within the primer sequence, and the NdeI and BamHI sites of pCold I (TaKaRa Bio), an E. coli expression vector. To obtain an E. coli expression vector of VvGT6. When constructing the E. coli vector, the His tag upstream of the NdeI site of this vector was matched with the open reading frame of VvGT6, and the chimeric VvGT6 protein fused with the His tag was designed to be expressed.

Analysis of enzyme function In order to clarify the biochemical function of this enzyme, this enzyme was expressed in E. coli.
Using the plasmid obtained above, E. coli BL21 (DE3) strain was transformed according to a conventional method. The obtained transformant was shaken at 37 ° C. overnight in 4 ml of LB medium (10 g / l typtone pepton, 5 g / l yeast extract, 1 g / l NaCl) containing 50 μg / ml ampicillin. Cultured. 4 ml of the culture solution that reached the stationary phase was inoculated into 80 ml of the medium having the same composition and cultured with shaking at 37 ° C. When the bacterial turbidity (OD600) reached approximately 0.5, a cold shock was performed at 15 ° C. for 30 minutes, and then 0.5 mM IPTG was added, followed by shaking culture at 18 ° C. for 20 hours.
All the following operations were performed at 4 ° C. The cultured transformants are collected by centrifugation (5,000 × g, 10 minutes), and Buffer S [20 mM HEPES buffer (pH 7.5), 20 mM imidazole, 14 mM β-mercaptoethanol] 1 ml / g cell Was added and suspended. Subsequently, ultrasonic crushing (15 seconds × 8 times) was performed, followed by centrifugation (15,000 × g, 15 minutes). The obtained supernatant was recovered as a crude enzyme solution. In order to purify a VvGT6-expressing protein having a His tag, a crude enzyme solution was loaded on His SpinTrap (GE Healthcare) equilibrated with Buffer S and centrifuged (70 × g, 30 seconds). After washing with Buffer, the protein bound to the column was eluted stepwise with 5 ml each of Buffer S containing 100 mM and 500 mM imidazole. Each elution fraction was substituted with 20 mM HEPES buffer (pH 7.5) and 14 mM β-mercaptoethanol using Microcon YM-30 (Amicon) (dialysis magnification 1000 times).

As a result of SDS-PAGE analysis, in the fraction eluted with 500 mM imidazole, protein was confirmed in the vicinity of an estimated molecular weight of VvGT6 of 49.68 kDa, and this fraction was used for enzyme analysis (FIG. 3).
Standard enzyme reaction conditions are as follows. Prepare 50 μl of the reaction solution (1 mM sugar donor, 200 μM sugar acceptor substrate, 20 mM HEPES buffer (pH 7.5), purified enzyme about 2 μg), start the reaction by adding the enzyme solution, Reacted for 1 minute. The reaction was stopped by adding equal amounts of 0.1% TFA and 40% CH ₃ CN, and the supernatant obtained by centrifugation at 15000 rpm for 2 minutes at 4 ° C. was analyzed by reverse phase HPLC.
The reverse phase HPLC system consists of the following instruments. MODEL305 (Gilson) as Pump A, MODEL302 (Gilson) as Pump B, MODEL231 (Gilson) as Sample Injector, 401 (Gilson) as Dilutor, 811B (Gilson) as DYNAMIC MIXER, Pressure Module (RAININ), DGU-12A (Shimadzu Corporation) was used as DEGASSER, and SPD-M20A DIODE ARRAY DETECTOR (Shimadzu Corporation) was used as the detector.

The HPLC conditions for flavonols are as follows. The column used was J'sphere ODS-M80 (4.6 x 150 mm, YMC) at room temperature, and mobile phase A (0.2% formic acid / 10% CH ₃ CN) and mobile phase B (0.2% formic acid / 90% CH ₃ CN) Was used. Elution conditions were equilibrated with B10% for 3 minutes, followed by a 10-minute linear concentration gradient (B10% → B40%), followed by a further 10-minute linear concentration gradient (B40% → B90%), followed by B90% for 1 minute. Retained. Thereafter, it was returned to B10% again and equilibrated for 10 minutes. The flow rate was 0.7 ml / min. Detection was performed at 360 nm using SPD-M20A DIODE ARRAY DETECTOR. Under these conditions, the standard products (standard products) quercetin, quercetin 3-galactoside and quercetin 3-glucoside (all from SIGMA) are eluted in order of retention times of about 17.8 minutes, 11.3 minutes and 11.5 minutes, respectively.
As a result of HPLC analysis of an enzyme reaction solution using quercetin as a sugar acceptor and UDP-galactose as a sugar donor, a new product A was confirmed at a retention time of about 11.3 minutes, which was the same as that of quercetin 3-galactoside ( FIG. 4). Furthermore, as a result of HPLC analysis of an enzyme reaction solution using quercetin as a sugar acceptor and UDP-glucose as a sugar donor, a new product B was confirmed at a retention time of about 11.5 minutes, which was the same as that of quercetin 3-glucoside. (FIG. 4). This indicated that the VvGT6 gene encodes F3GalT and F3GlcT (ie, F3Gal / GlcT) having the activity of transferring galactose and glucose to the 3rd position of the flavonoid.

Next, the optimum pH of the enzyme was determined. As shown in FIG. 5, various buffers from pH 3 to pH 10.5 were prepared, and 100 μM quercetin as a sugar acceptor, 1 mM UDP-glucose as a sugar donor, 50 mM buffer, 1 mM DTT, and 100 ng of the purified VvGT6 enzyme, 30 The reaction was carried out at 0 ° C. for 10 minutes. The reaction was started by adding quercetin and stopped by adding a 40% acetonitrile solution containing an equal amount of 0.1% TFA. 100 μl of the supernatant obtained by centrifuging the reaction stop solution (15000 rpm, 2 minutes, 4 ° C.) was subjected to HPLC analysis, and the amount of product under each buffer condition was compared. As a result, strong enzyme activity was observed in the neutral to basic region, and the optimum pH was 8.0 (FIG. 6).
Next, the optimum temperature of the enzyme was determined. 100 μM quercetin as a sugar acceptor, 1 mM UDP-glucose, 50 mM Tricine-NaOH (pH 8.0) buffer, 1 mM DTT and 100 ng of the purified VvGT6 enzyme as a sugar donor, 10, 20, 30, 35, 40, 45, 50 And at 60 ° C. for 15 minutes each. The reaction was started by adding quercetin and stopped by adding a 40% acetonitrile solution containing an equal amount of 0.1% TFA. 100 μl of the supernatant obtained by centrifuging the reaction stop solution (15000 rpm, 2 minutes, 4 ° C.) was subjected to HPLC analysis, and the amount of product under each temperature condition was compared. As a result, the optimum reaction temperature was 35 ° C. (FIG. 7).

Next, the sugar donor specificity of this enzyme was examined. 100 μM quercetin as each sugar acceptor, 1 mM each sugar donor (UDP-glucuronic acid, UDP-glucose and UDP-galactose (all from SIGMA)), 50 mM Tricine-NaOH (pH 8.0) buffer, 1 mM DTT and the above 100 ng of purified VvGT6 enzyme was reacted at 30 ° C. for 15 minutes. The reaction was started by adding a sugar receptor and stopped by adding a 40% acetonitrile solution containing an equal amount of 0.1% TFA. 100 μl of the supernatant obtained by centrifuging the reaction stop solution (15000 rpm, 2 minutes, 4 ° C.) was subjected to HPLC analysis, and the amount of product in each sugar donor was compared (FIG. 8). As a result, a product was obtained only when UDP-glucose and UDP-galactose were used as sugar donors. This indicates that VvGT6 does not use at least UDP-glucuronic acid as a sugar donor.
Next, the specificity of the enzyme for the sugar receptor was examined. Genistein and daidzein were purchased from Fujicco, Naringenin was purchased from Nacalai Tesque, Capsaicin was purchased from SIGMA, and the others were purchased from Funakoshi. Each sugar acceptor 100 μM shown in FIG. 9, 1 mM UDP-glucose, 50 mM Tricine-NaOH (pH 8.0) buffer, 1 mM DTT and 100 ng of the purified VvGT6 enzyme were reacted at 30 ° C. for 15 minutes. The reaction was started by adding a sugar receptor, and stopped by adding a 40% acetonitrile solution containing an equal amount of 0.1% TFA. 100 μl of the supernatant obtained by centrifuging the reaction stop solution (15000 rpm, 2 minutes, 4 ° C.) was subjected to HPLC analysis, and the amount of product in each sugar receptor was compared. The specificity for UDP-galactose was evaluated for the sugar receptor whose activity was detected. As a result, it was shown that VvGT6 is specific to flavonol (FIG. 9). The kinetic parameters for various flavonols are shown in FIG.

In order to confirm the bifunctional sugar donor selectivity of VvGT6, the reaction was performed using UDP-galactose and UDP-glucose simultaneously. 100 μM quercetin as each sugar acceptor, 1 mM each sugar donor (UDP-glucose and UDP-galactose), 50 mM Tricine-NaOH (pH 8.0) buffer, 1 mM DTT and 1000 ng of the purified VvGT6 enzyme reacted at 30 ° C. for 10 minutes. It was. The reaction was started by adding a sugar receptor and stopped by adding a 40% acetonitrile solution containing an equal amount of 0.1% TFA. 100 μl of the supernatant obtained by centrifuging the reaction stop solution (15000 rpm, 2 minutes, 4 ° C.) was subjected to HPLC analysis, and the amount of product in each sugar donor was compared (FIG. 10). The HPLC analysis conditions are as follows.
Column J'sphere ODS-M80 (4.6 × 150 mm, Ltd. YMC) using at room temperature, mobile phase A (0.2% formic acid / 10% CH ₃ CN) and a mobile phase B (0.2% formic acid / 90% CH ₃ CN) was used. Elution conditions were equilibrated with B10% for 3 minutes, held for 12 minutes with a linear concentration gradient (B10% → B20%), and further for 8 minutes with a linear concentration gradient (B20% → B90%), followed by 1 minute with B90%. . Then, it returned to B10% again and equilibrated for 10 minutes. The flow rate was 0.7 ml / min. Detection was performed at 360 nm using SPD-M20A DIODE ARRAY DETECTOR (manufactured by Shimadzu Corporation). Under these conditions, standard quercetin, quercetin 3-glucoside and quercetin 3-galactoside are eluted in this order at retention times of about 23.3 minutes, 12.5 minutes and 12.1 minutes, respectively. As a result of the analysis, the same degree of quercetin 3-galactoside and quercetin 3-glucoside were detected in the simultaneous reaction solution (FIG. 10).

  As described above, VvGT6 was shown to be a bifunctional novel flavonoid 3-position glycosyltransferase (F3Gal / GlcT) showing specificity for two types of UDP sugar donors, UDP-glucose and UDP-galactose.

Gene expression analysis
In order to identify the functional region of VvGT6, a grape organ of the VvGT6 gene was obtained in the same manner as described in “Noguchi, A. et al., Plant J., vol. 54, p. 415-427, 2008”. Another gene expression pattern was analyzed by SYBR-Green quantitative RT-PCR using 7500 Real Time PCR System (Applied Biosystems).
For grapes, total RNA was extracted from the organs (leaves, petiole, seeds, fruits, and peels) of the Pinot Noir and Cabernet Sauvignon varieties in the same manner as in Example 1, and 0.5 μg of this was reverse transcribed with Random Hexamer primer. (RT) By reaction, cDNA of each organ was obtained and used as a PCR template.
The following 8 types of gene-specific primers used for quantitative PCR were designed using Primer Express 3.0 program (Applied Biosystems). As VvGT6 gene-specific primers, those shown in SEQ ID NOs: 5 and 6 were used. As comparative controls, an anthocyanin 3-glucose transferase VvGT1 and a VvGT6 homologue VvGT5 were also designed and analyzed, respectively (VvGT1 gene-specific primers: SEQ ID NOs: 7 and 8, VvGT5). Gene-specific primer: SEQ ID NO: 9 and 10). Grape ubiquitin elongase (GenBank accession number: CAO48597) was adopted as an internal standard gene and amplified using the following gene-specific primers (SEQ ID NOs: 11 and 12). The expression level of each VvGT gene was normalized with the expression level of the internal standard gene, and the relative expression level was obtained by the ΔΔCt method (Applied Biosystems).

VvGT6-Fw:
5'-GGT TCC CTG GTT GGC AAT TT-3 '(SEQ ID NO: 5)

VvGT6-Rv:
5'- GCA CCC GCC CCA CAA CCT T-3 '(SEQ ID NO: 6)

VvGT1-Fw:
5'- CCC ACC GCC GGT TAT ACC -3 '(SEQ ID NO: 7)

VvGT1-Rv:
5'- CGA CCG AGG TGG GTT TTC T -3 '(SEQ ID NO: 8)

qVvGT5-Fw1:
5'- GCT CCA TCT CCT CTG CTC AAA -3 '(SEQ ID NO: 9)

qVvGT5-Rv2:
5′-GAA AGC ACA AGG TCC TCT-3 ′ (SEQ ID NO: 10)

VvUBQ2-Fw:
5'-TCC AGG ACA AGG AAG GGA TTC-3 '(SEQ ID NO: 11)

VvUBQ2-Rv:
5'- GCC ATC CTC AAG CTG CTT TC -3 '(SEQ ID NO: 12)

  As a result, it was confirmed that the VvGT6 gene was remarkably expressed in the leaves where the product accumulated (FIG. 11). On the other hand, the VvGT1 gene is specifically expressed in the pericarp and is described in known literature (Ford, CM, et al., J. Biol. Chem., Vol. 273, p. 9224-9233, 1998). Consistent with the results. In addition, since the VvGT6 gene was also strongly expressed in the peel, it was strongly shown that the flavonol 3-position galactose glycoside and glucose glycoside present in the peel and wine were produced by VvGT6. The VvGT5 and VvGT6 genes show similar gene expression patterns, and it is speculated that the locus of these genes in close proximity on the genome affects the coordinated gene expression pattern of both genes ( Figure 2).

Identification of amino acid residues involved in sugar donor selectivity
VvGT1, a grape-derived flavonoid 3-position glucose transferase, is used to identify the amino acid residues that determine the distinctive sugar donor selectivity of VvGT6 (the grape-derived flavonoid 3-position galactose-glucosyltransferase). (See “Offen, W. et al. (2006) EMBO J. 25, 1396-1405.”) Grape-derived flavonoid 3-glucuronyltransferase VvGT5, Udo-derived flavonoid galactose transferase ACGalT (“Kubo , A. et al. (2004) ABB 429, 198-203. ”) And PhF3GalT, a petunia-derived flavonoid 3-position galactose transferase (“ Miller, KD et al. (1999) J. Biol. Chem. 274 , 34011-34019. ”), Multiple alaminate was performed by Clustal-W. The results are shown in FIG.
As shown in FIG. 12, VvGT6 had a unique proline residue (P19) immediately before the 20th histidine residue (H20) of the active catalytic residue VvGT1 (“Offen, W. et al (2006) EMBO J. 25, 1396-1405. ”). Since this position (P19) is next to the active catalyst residue, it was considered to be located near the active pocket into which the substrate enters. In addition, VvGT6 does not have the characteristic histidine residue found in flavonoid galactosyltransferase in the PSPG box that is highly conserved in plant UDP sugar-dependent glycosylase (UGT). The glutamine residue (Q373) found in the UGT enzyme that translocates glucose and glucuronic acid was conserved.
In order to elucidate the role of these two amino acid residues (P19 and Q373) in the sugar donor selectivity of VvGT6, VvGT6-P19T mutant and VvGT6-Q373H mutant were prepared, and the sugar donor selectivity It was investigated. The VvGT6-P19T mutant is a mutant protein in which the 19th proline residue (P) in the amino acid sequence of wild-type VvGT6 is replaced with a threonine residue (T), and the VvGT6-Q373H mutant is a wild-type mutant. This is a mutant protein in which the 373rd glutamine residue (Q) of VvGT6 in the amino acid sequence of VvGT6 is substituted with a histidine residue (H).
Each amino acid substitution mutation was introduced by PCR according to the method described in “Noguchi, A. et al. (2009) Plant Cell. 21, 1556-1572” using the following primers (SEQ ID NOs: 13 to 18). PCR conditions were as follows: wild-type VvGT6 gene plasmid as a template, KOD plus DNA polymerase (TOYOBO) with heat denaturation at 96 ° C for 30 seconds, 95 ° C, 30 seconds → 58 ° C, 1 minute → 68 ° C, 3 A total of 25 reactions were performed with one minute as the cycle.
Specifically, for the VvGT6-P19T mutation, first, using a VvGT6 gene plasmid as a template, PCR using the primers of SEQ ID NOs: 13 and 16 and PCR using the primers of SEQ ID NOs: 14 and 15 were performed. The two PCR fragments obtained were mixed. Then, the mutation (VvGT6-P19T mutation) was introduced by PCR using the diluted mixture as a template and the primers of SEQ ID NOs: 13 and 14.
Similarly, the VvGT6-Q373H mutation was obtained by first performing PCR using the primers of SEQ ID NOs: 13 and 18 and PCR using the primers of SEQ ID NOs: 14 and 17 using the VvGT6 gene plasmid as a template. Two kinds of PCR fragments were mixed. Next, the mutation (VvGT6-Q373H mutation) was introduced by performing PCR using the diluted mixture as a template and using the primers of SEQ ID NOs: 13 and 14.
The PCR products (2 types) obtained by mutagenesis in this way must be subcloned into the pENTR-Directional-TOPO vector (Invitrogen) using the method recommended by the manufacturer, and the desired mutation has been introduced. Was confirmed by sequencing.

CACC-NdeI-VvGT6-Fw:
5'- CACC CATATG ACTGCCACCGCGAGCTC -3 '(SEQ ID NO: 13)

BglII-VvGT6-Rv:
5′- AGATCT CTACTTATTGGTATCCAA-3 ′ (SEQ ID NO: 14)

VvGT6-P19T-Fw:
5'-TTCCCCACCCATGCAGCTA-3 '(SEQ ID NO: 15)

VvGT6-P19T-Rv:
5'-TAGCTGCATGGGTGGGGAA-3 '(SEQ ID NO: 16)

VvGT6-Q373H-Fw:
5′-TTCTTTGGAGATCATTGTATCGACA-3 ′ (SEQ ID NO: 17)

VvGT6-Q373H-Rv:
5'-TGTCGATACAATGATCTCCAAAGAA-3 '(SEQ ID NO: 18)

Using the restriction enzyme sites of NdeI and BglII added in the primer sequence (sequences with the underlined portion in the base sequences shown in SEQ ID NOs: 13 and 14 above), an approximately 1.4 kb VvGT6 fragment was pENT-Directional- Cut out from the TOPO vector and ligated between the NdeI and BamHI sites of pET15b (Novagen), an E. coli expression vector, to obtain an E. coli expression vector for each VvGT6. In the construction of the E. coli vector, the His tag upstream of the NdeI site of the vector was matched with the open reading frame of VvGT6, and the chimeric VvGT6 protein fused with the His tag was designed to be expressed.
The prepared VvGT6-P19T and VvGT6-Q373H mutants were transformed into Escherichia coli BL21 by the same method as in Example 2, and after addition of 1 mM IPTG, cultured at 18 ° C. for 20 hours to prepare as His-tagged proteins. did. In the same manner as in Example 2, the sugar donor selectivity of the protein was examined.
As a result, the VvGT6-P19T mutant had a slightly shifted sugar donor selectivity from UDP-galactose to UDP-glucose, indicating that the P19 residue is a residue involved in UDP-galactose recognition. (Table 1 below). In addition, since the VvGT6-Q373H mutant hardly uses UDP-glucose as a sugar donor and UDP-galactose as the main sugar donor, the Q373 residue is an amino acid residue essential for the recognition of UDP-glucose in VvGT6. (Table 1 below).
From the above results, amino acid residues (P19 and Q373) involved in the selectivity for two characteristic sugar donors of VvGT6 were clarified.

As described above, a UGT enzyme (F3Gal / GlcT) having a novel activity of transferring glucose and galactose from grape to the 3rd position of flavonoid could be isolated. By utilizing the present invention, it is possible to artificially glycosidate flavonoid 3-position with glucose and galactose, contributing to the development of new functional food materials and plants capable of producing useful compounds. it can.
Therefore, the present invention is extremely useful in that it can be widely used in agriculture, food industry, pharmaceutical industry and related industries.

SEQ ID NO: 1 synthetic DNA
Sequence number 2: Synthetic DNA
Sequence number 5: Synthetic DNA
Sequence number 6: Synthetic DNA
Sequence number 7: Synthetic DNA
Sequence number 8: Synthetic DNA
Sequence number 9: Synthetic DNA
SEQ ID NO: 10: synthetic DNA
SEQ ID NO: 11: synthetic DNA
SEQ ID NO: 12: synthetic DNA
SEQ ID NO: 13: synthetic DNA
SEQ ID NO: 14: synthetic DNA
SEQ ID NO: 15: synthetic DNA
SEQ ID NO: 16: synthetic DNA
Sequence number 17: Synthetic DNA
Sequence number 18: Synthetic DNA

Claims (15)

The polynucleotide according to any one of the following (a) to (f):
(A) a polynucleotide comprising a polynucleotide comprising the base sequence represented by SEQ ID NO: 3;
(B) a polynucleotide containing a polynucleotide encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 4;
(C) consisting of an amino acid sequence in which 1 to 15 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 4, and UDP-galactose transferase activity and UDP-glucose transferase activity A polynucleotide comprising a polynucleotide encoding a protein having
(D) a polyprotein encoding a protein having an amino acid sequence having 80% or more homology to the amino acid sequence represented by SEQ ID NO: 4 and having UDP-galactose transferase activity and UDP-glucose transferase activity A polynucleotide containing nucleotides;
(E) a protein that hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 3 and has UDP-galactosyltransferase activity and UDP-glucose transferase activity Or (f) a polynucleotide comprising a base sequence complementary to the base sequence of a polynucleotide encoding a protein comprising the amino acid sequence represented by SEQ ID NO: 4 and stringent conditions A polynucleotide comprising a polynucleotide that hybridizes with and encodes a protein having UDP-galactosyltransferase activity and UDP-glucose transferase activity. The polynucleotide according to claim 1, which is any of the following (g) to (j):
(G) consisting of an amino acid sequence in which no more than 10 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 4, and exhibit UDP-galactose transferase activity and UDP-glucose transferase activity A polynucleotide containing a polynucleotide encoding the protein having;
(H) a polyprotein encoding a protein having an amino acid sequence having 90% or more homology with the amino acid sequence represented by SEQ ID NO: 4 and having UDP-galactose transferase activity and UDP-glucose transferase activity A polynucleotide containing nucleotides;
(I) hybridizes with a polynucleotide comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 3 under highly stringent conditions and has UDP-galactose transferase activity and UDP-glucose transferase activity A polynucleotide containing a polynucleotide encoding a protein; or (j) a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 4 and a highly stringent A polynucleotide comprising a polynucleotide that hybridizes under conditions and encodes a protein having UDP-galactose transferase activity and UDP-glucose transferase activity. The polynucleotide according to claim 1 or 2, wherein the polynucleotides described in (c) to (j) are the polynucleotides described in (c ') to (j') below, respectively:
(C ′) consisting of an amino acid sequence in which 1 to 15 amino acids excluding the 19th and / or 373rd amino acid in the amino acid sequence represented by SEQ ID NO: 4 have been deleted, substituted, inserted and / or added, And a polynucleotide comprising a polynucleotide encoding a protein having UDP-galactose transferase activity and UDP-glucose transferase activity;
(D ′) an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 4 and corresponding to the 19th amino acid in the amino acid sequence represented by SEQ ID NO: 4 Contains a polynucleotide encoding a protein that is proline and / or has an amino acid sequence in which the amino acid corresponding to the 373rd amino acid is glutamine and has UDP-galactosyltransferase activity and UDP-glucose transferase activity A polynucleotide to:
(E ′) a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 3 in the base sequence represented by SEQ ID NO: 3; The polynucleotide corresponding to the 57th base is a polynucleotide encoding proline and / or the base corresponding to the 1117th to 1119th bases is a base encoding glutamine, and UDP-galactosyltransferase A polynucleotide comprising a polynucleotide encoding a protein having activity and UDP-glucose transferase activity;
(F ′) a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to the base sequence of a polynucleotide encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 4; The base corresponding to the 55th to 57th bases in the base sequence of the polynucleotide encoding the protein consisting of the amino acid sequence represented by 4 is a base encoding proline and / or the 1117th to 1119th bases A polynucleotide comprising a polynucleotide wherein the corresponding base is a glutamine-encoding base and the protein encodes a protein having UDP-galactose transferase activity and UDP-glucose transferase activity;
(G ′) consisting of an amino acid sequence in which no more than 10 amino acids excluding the 19th and / or 373rd amino acid are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 4; A polynucleotide comprising a polynucleotide encoding a protein having UDP-galactosyltransferase activity and UDP-glucose transferase activity;
(H ′) an amino acid sequence having 90% or more homology with the amino acid sequence represented by SEQ ID NO: 4 and corresponding to the 19th amino acid in the amino acid sequence represented by SEQ ID NO: 4 Contains a polynucleotide encoding a protein that is proline and / or has an amino acid sequence in which the amino acid corresponding to the 373rd amino acid is glutamine and has UDP-galactosyltransferase activity and UDP-glucose transferase activity A polynucleotide to:
(I ′) a polynucleotide that hybridizes with a polynucleotide comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 3 under highly stringent conditions, the 55 th of the base sequence represented by SEQ ID NO: 3 The base corresponding to the 57th base is a polynucleotide encoding proline and / or the base corresponding to the 1117th to 1119th base is a base encoding glutamine, and UDP-galactose transfer A polynucleotide comprising a polynucleotide encoding a protein having enzymatic activity and UDP-glucosyltransferase activity; or (j ′) complementary to the nucleotide sequence of a polynucleotide encoding a protein comprising the amino acid sequence represented by SEQ ID NO: 4 Under a highly stringent condition with a polynucleotide consisting of a simple base sequence Whether the base corresponding to the 55th to the 57th base in the base sequence of the polynucleotide encoding the protein comprising the amino acid sequence represented by SEQ ID NO: 4 is a base encoding proline, / Or a polynucleotide in which the base corresponding to the 1117th to 1119th bases is a base encoding glutamine, and a polynucleotide encoding a protein having UDP-galactosyltransferase activity and UDP-glucosyltransferase activity Polynucleotide.   The polynucleotide according to any one of claims 1 to 3, wherein the UDP-galactosyltransferase activity is a flavonoid 3-position galactosyltransferase activity.   The polynucleotide according to any one of claims 1 to 4, wherein the UDP-glucose transferase activity is a flavonoid 3-position glucose transferase activity.   The polynucleotide according to claim 1, comprising a polynucleotide comprising the base sequence represented by SEQ ID NO: 3.   The polynucleotide according to claim 1, comprising a polynucleotide encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 4.   The polynucleotide according to any one of claims 1 to 7, which is DNA.   A protein encoded by the polynucleotide according to any one of claims 1 to 8.   The vector containing the polynucleotide of any one of Claims 1-8.   A transformant into which the polynucleotide according to any one of claims 1 to 8 has been introduced.   A transformant into which the vector according to claim 10 has been introduced.   The method for producing a protein according to claim 9, wherein the transformant according to claim 11 or 12 is used.   A galactose glycoside and / or glucose which produces galactose glycoside and / or glucose glycoside from UDP-galactose and / or UDP-glucose and a sugar acceptor substrate using the protein according to claim 9 as a catalyst. A method for producing glycosides.   15. A method according to claim 14, wherein the sugar receptor substrate is a flavonoid.