TW201011106A - Glucuronyl transferase and polynucleotide encoding the same - Google Patents

Abstract

The present invention provides a novel glucuronyl transferase and a polynucleotide encoding the glucuronyl transferase (such as a polynucleotide containing a polynucleotide composed of the 1st to 1362th bases of a base sequence represented by SEQ ID NO: 7; or a polynucleotide containing a polynucleotide encoding a protein having an amino acid sequence represented by SEQ ID NO: 8) and the like. Thereby, a novel glucuronyl transferase with wide substrate specificity and the like is provided.

Description

201011106, VI. Description of the Invention: [Technical Field] The present invention relates to a glucuronyltransferase and a polynucleotide encoding the transferase, and a vector, a transformant and the like containing the above polynucleotide. [Prior Art] Flavonoid is a general term for secondary metabolites of plants of the phenyl propanoid system. One of them is anthocyanin which determines red, dials to blue-violet, etc. The main pigment of the suit. Gluco The glucoside of flavonoids or flavonols, which is also one of the flavonoids, although it is light yellow in color, is greatly affected by the formation of a complex with anthocyanins, so it is called It is a copigment. In general, the shift of the color of the flower caused by the secondary pigment to the blue wavelength is called copigmentation. In the petals of Antirrhinum majus, the genus of apigenin, one of the flavonoids, is known as glucuronide (also known as glucuronid glucoside, or grape). The uronic acid conjugate () is considered to act as a co-pigment (Ref. 1:

Asen, S. et al., Phytochemistry 11, 2739-2741, 1972). In addition, the pigment of the blue petal of the Centaurea cyanus forms a metal complex. The presence of the flavonoid 7-glucopic acid is also found in the complex (Reference 2: Shiono, M. Et al, Nature 436, 791-792, 2005). The root of Scute 11 aria baica lens is accumulated in the root of Scute 11 aria baica lens is 320590 201011106 flavonoid 7-glucuronide, which is called baicalin. It is used as a traditional Chinese medicine (Huangqi) with stomach function (Ref. 3: Gao, Z. et al., Biochemicaet)

Biophysica Acta 1472, 643-650, 1999). SbUBGAT (or Sb7GAT) which is used to transfer the enzyme of glucose aldehyde to the 7th place of baicalein from the root of Astragalus (Reference 4: Nagashima S. et al., Phytochemistry 53, 533-538, 2000) The gene corresponding to the Sb7GAT has been received in the gene pool (access; [〇n No. AB042277), however its function has not been confirmed. In addition, in the leaves of Perilla frutescens, which is eaten daily, it has recently been found that a variety of flavonoids are accumulated in the 7-glycoside, and it is expected to be in the health of human body (Ref. 5. Yamazaki, M. et al., Phytochemistry 62, 987-988, 2003). Although the above-mentioned flavonoid 7-glucuronide has become a secondary metabolite of plants which has attracted attention in the field of flower color and health food, there are many unknown parts of its biosynthetic enzymes (for example, glucuronyltransferase). • [Document] 1. Asen, S. et al. Phytochemistry 11, 2739-2741. 1972 2. Shiono, M. et al. Nature 436, 791-792. 2005 3. Gao, Z. et al., Biochemica et Biophysica Acta 1472, 643-650. 1999 4. Nagashima S. et al., Phytochemistry 53, 533-538, 2000 5. Yamazaki, M. et al. Phytochemistry 62, 987-998. 4 320590 201011106 '2003 Under the above circumstances, it is required to identify a matrix-specific and novel glucuronyltransferase and a gene encoding the transferase. The present invention has been made in view of the above circumstances, and provides a glucuronyltransferase, a polynucleotide encoding the transferase, a vector containing the polynucleotide, a transformant, and the like as described below. (1) A polynucleotide which is as described in any one of the following items (a) to (f): (a) a polynucleotide comprising a nucleotide sequence represented by SEQ ID NO: 7. a polynucleotide consisting of the nucleotide sequences of 1st to 1362; (b) a polynucleotide comprising a polynucleotide encoding a protein having the amino acid sequence of SEQ ID NO: 8; a polynucleotide comprising a polynucleotide encoding a protein, ^ which is deleted, substituted, inserted and/or added by from 1 to 15 amino acids in the amino acid sequence of SEQ ID NO: 8. The amino acid sequence is composed of, and the protein has UDP-glucuronyltransferase activity; (d) a polynucleotide comprising a polynucleotide encoding a protein having a sequence number of 8: An amino acid sequence having more than 80% homology of the amino acid sequence, and the protein has UDP-glucuronyltransferase activity; 320590 201011106. (e) a polynucleotide, the Contains: under severe conditions followed by serial number: 7 a polynucleotide in which the nucleotide sequences of the first to the first base sequences of the base sequence are complementary to each other, and a polynucleotide encoding a protein having UDP-glucuronyltransferase activity; or f) a polynucleotide comprising: a base sequence which is complementary to a base sequence encoding a polynucleotide consisting of a protein consisting of the amino acid sequence of SEQ ID NO: 8 under severe conditions构成 The constructed polynucleotide is hybridized and encodes a polynucleotide having a protein of UDP-glucuronyltransferase activity. (2) The polynucleotide according to the above (1), which is as described in any one of the following items (g) to (j): (g) a polynucleotide comprising a polynucleus encoding a protein Glycosyl acid, which is composed of an amino acid sequence in which at least 10 amino acids in the amino acid sequence of SEQ ID NO: 8 are deleted, substituted, inserted, and/or added, and the protein has UDP-glucuronyltransferase activity; (h) a polynucleotide comprising a polynucleotide encoding a protein having 90% or more relative to the amino acid sequence of SEQ ID NO: 8. a homogenous amino acid sequence, and the protein has UDP-glucuronyltransferase activity; (i) a polynucleotide comprising: a base sequence shown by SEQ ID NO: 7 under highly stringent conditions a polynucleotide having a base sequence of 1 to 1362 which is a complementary base sequence, a nucleic acid 6 320590 201011106 acid hybridized, and a polynucleotide encoding a protein having UDP-glucuronyltransferase activity; or (j a polynucleotide comprising a a hybridization of a polynucleotide consisting of a nucleotide sequence encoding a polynucleotide sequence of a protein consisting of the amino acid sequence of SEQ ID NO: 8 under a highly stringent condition, And a polynucleotide encoding a protein having UDP-glucuronyltransferase activity. (3) The polynucleic acid described in the above (1), which comprises a polynucleotide having a sequence of nucleotides 1 to 1362 of a nucleotide sequence represented by SEQ ID NO: 7. (4) The polynucleotide according to the above (1), which comprises a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 8. (5) The polynucleotide according to any one of the above items (1) to (3), which is a DNA 〇Λ (6) protein, which is any of the above items (1) to (5) Encoded by a polynucleotide encoded as described. (7) A vector comprising the polynucleotide according to any one of the items (1) to (5) above. (8) A transformant according to any one of the above (1) to (5). (9) A transforming body to which the carrier described in the above item (7) is introduced. (10) A method for producing a protein according to the above (6), which is characterized in that the transformant described in the above (8) or (9) is used to produce the 7 320590 201011106 protein of the above (6). (11) A method for producing a glucosaldehyde conjugate, which comprises the use of the protein described in the above (16) as a catalyst to form a glucuronic acid conjugate from a UDP-glucuronic acid and a sugar acceptor matrix. The polynucleotide of the present invention is useful for the manufacture of novel glucuronyltransferases, for example, by introduction into a transformant. The glucuronate transferase of the preferred embodiment of the present invention has a broad matrix specificity and has an activity of glucuronidating a plurality of sugar acceptor substrates.

[SEQ ID NO: Keywords] Serial number: 1 : Synthetic DNA 'Serial number: 2: Synthetic DNA Serial number: 3: Synthetic DNA Serial number: 4: Synthetic DNA Serial number: • 5: Synthetic DNA Serial number: 6 : Synthetic DNA Serial Number: 9: Synthetic DNA Serial Number: 10: Synthetic DM Sequence Number: 11: Synthetic DNA Serial Number: 12: Synthetic DNA Serial Number: 13: Synthetic DNA Serial Number: 14: Synthetic DM [Embodiment] The glucuronyltransferase of the invention, the polynucleotide encoding the enzyme, and the carrier, the transformant and the like containing the above polynucleotide are detailed. 8 320590 201011106 The blue flower is formed by the Persian genus Scrophularia Veronica persica's main anthocyanin bitter pigment is delphinidin 3-0 -(3-0-(6-0-coumarin)-glucosyl)_6-〇-fungyl-glycoside -5 -〇-glucoside (delphinidin 3-0-(3-0-(6-〇-coumaroy 1 )-glucos yl)-6-0-coumaroyl-gliicoside-5-0-glucoside)' and the main flavonoid is celery 7-0-(3_0_glucuronosylgluconate (apig enin 7-0-(3-0-glucuronosyl)-gl Ucuronide) (refer to the class of the family, Miho, Sakamoto Toyo University master's thesis, Heisei 15). The flavonoid 7-glucuronide system with the buds as the skeleton shows a significant auxiliary color effect on the main anthocyanins, so It is thought to be related to the color of Persian's mother-in-law. The inventors of the present invention isolated the flavonoid 7-glucuronyltransferase (Fiav〇n 〇id 7-) by PCR from the cDNA derived from the petals of Persian A polynucleotide of 0-glucuronosyltransferase, abbreviated as F7GAT), and a polynucleotide of the present invention (SEQ ID NO: 7) H 1. A polynucleotide of the present invention contains a base represented by the sequence number: 7.

The present invention first provides (a) - 320590 9 201011106 - code: 8 amino acid sequence in which the amino acid sequence of 1 to 15 amino acids is deleted, substituted, inserted and/or added, and A protein having UDP-glucuronyltransferase activity. The protein may be enumerated, for example, by the amino acid sequence of SEQ ID NO: 8, for example, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10 , 1 to 9, 1 to 8, 1 to 7, 1 to 6 (1 to several), 1 to 5, 1 to 4, 1 to 3, 1 to 2, 1 amine A protein consisting of an amino acid sequence in which a base acid residue is deleted, substituted, inserted, and/or added, and has a ❹ UDP-glucuronyltransferase activity protein. In general, the number of deletions, substitutions, insertions, and/or additions of the above amino acid residues is preferably as small as possible. Further, examples of the protein having the same function include (d) an amino acid sequence having a homogeneity of 80% or more with respect to the amino acid sequence of SEQ ID NO: 8, and having UDP-glucuronic acid transfer. An enzyme-active protein; examples of the protein include, for example, about 80% or more, 81% or more, 82% or more, 0 83% or more, and 84% with respect to the sequence number: 8 amino acid sequence. Above, 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% The above homologous amino acid sequence of 99.8% or more and 99.9% or more, and a protein having UDP-glucuronyltransferase activity. The above homogeneity values, in general, the higher the better. Here, "UDP-glucuronyltransferase activity" refers to the glucuronidation of the hydroxyl group of a sugar acceptor matrix such as ketone, stilbene, and lignan of chlorophyll 10 320590 201011106 (for example, flavonoids) The hydroxyl group at the 7 position is acidified by gluconic acid) to catalyze the catalytic activity of the reaction of the glucose disk and the acid conjugate. The UDP-glucose-transferase activity can be determined, for example, by reacting UDP-glucose and a sugar acceptor matrix (for example, flavonoids) in the presence of an enzyme to be evaluated, as determined by HPLC or the like. For details, please refer to the description of the following examples).

Further, the present invention also includes (e) a polynucleotide comprising: complementing the base sequence of the first to the 1362th base sequences of the nucleotide sequence shown by the sequence number of the sequence under severe conditions; The polynucleotide consisting of the sequence of the test sequence carries a heterogeneous parent and encodes a protein having UDP-glucuronyltransferase activity: a multinuclear "; and (1) a polynucleotide" which contains: in a harsh strip: Encoding an egg consisting of the amino acid sequence of the sequence number: 8: Si acid: the sequence of the test sequence is composed of a complementarity sequence, which is introduced into the parent, and the code has a glucuronide transferase property. A polynucleotide of a protein. In the second place, "multi-core * acid" means waking up or Xinjiang. Wherein, the base sequence is the first to the 1362th of the 7th non-base sequence, and the polynucleic acid, or the sequence, is complementary to the sequence of the purple complement of the sequence. ^The polynuclear Wei of the amino acid sequence is used as a probe, and the polynuclear (4) consisting of the sequence is all or part of the cloque 320590 11 201011106 hybridization or Southern hybridization. The polynucleotide obtained by the method of hybridization, for example, is available in the literature Sarabrook & Russel 1, Molecular Cloning · A Laboratory Manual Vol. 3, Cold Spring Harbor, Laboratory Press 2001, Ausubel, Current Protocols in Molecular

Methodology described in Biology' John Wiley & Sons 1987-1997", etc. "Severe conditions" in this specification refer to any of the conditions of low severity, moderate severity, and severe conditions. "Low severity conditions" are conditions such as 5xSSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, 32 °C. Further, "moderately harsh conditions" means conditions such as 5xSSC, 5x denhard solution, 0.5% SDS, 50% guanamine, and 42 °C. "Highly stringent conditions" means conditions such as 5xSSC, 5χDenhardt solution, 0.5% SDS, 50% formamide, 5〇°c. Among these conditions, the more the temperature is raised, the more efficiently the homogenous DNA is expected to be obtained. However, in terms of the factors affecting the harshness of hybridization, there are complex elements such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration, and = such factors can be appropriately selected as long as they are peers. To achieve the same strictness, 'When using a marketer's kit for hybridization, for example, A1 kphos Direct Labelling Reagents CAMERSHAM Pharmaci a company product can be used). At this time, refer to the agreement (pr〇t〇co 1) attached to the kit, and heat-culture with the labeled probe overnight, at 55 conditions, with 3 with 0.; U (W/ V) After washing the membrane with a washing buffer of SDS, 320590 12 201011106 was detected. Examples of the polynucleotide which can be hybridized in addition to the above, for example, when FASTA, BLAST, etc. are used to search for a homogenous soft body and calculated by a default parameter, the nucleotide sequence of the sequence number: 7 is The DNA of the sequence of the first to the first steps of 1362 to 1362, or the DNA of the amino acid sequence of the conjugated mother sequence number: 8 is 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% with ©, 83% or more, 84% or more, and 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 More than 8% or more than 99.9% of the homogenous DNA, and the homology of the amino acid sequence or the sequence of the test, can be performed using the algorithm of Carlin and the Aguirre (personal name) BLAST (refer to Proc. Natl. Acad.

Sci. USA 872264-2268, 1990; proc Natl Acad Sci USA 90: 5873, 1993). A program called BLASTN or BLASTX according to the BLAST algorithm has been developed (see Altschul SF, et al; J Mol Biol 215: 403, 1990). When using the BLASTN program to parse the base sequence, the parameters are, for example, score=100, wordlength=12. In addition, when the BLASTX program is used to resolve the amino acid sequence, the parameter system is used.

For example, score= 50, wordlength=3. When using the BLAST and Gapped BLAST programs, the default parameters for each program are used. 13 320590 201011106 The polynucleotide of the present invention described above can also be produced by well-known genetic engineering methods or well-known synthetic methods. 2. Protein of the present invention Another embodiment of the present invention also provides a protein encoded by the above-described polynucleotide of the present invention. The protein of the present invention is a protein consisting of the amino acid sequence of SEQ ID NO: 8. Another protein of the present invention has a protein f having the amino acid sequence of SEQ ID NO: 8. A further aspect of the present invention is the protein of the amino acid sequence having a deletion, substitution, insertion and/or addition of 1 to 15 amino acids in the amino acid sequence of SEQ ID NO: 8. A protein having UDp_glucuronyltransferase activity. Examples of the protein include a protein having an amino acid sequence having the homogeneity as described above and having the amino acid sequence shown in the sequence No. 8. and having UDP-glucuronyltransferase activity. The protein can be obtained from the literature "3 coffee 131' 〇〇 1 ^ & 1 ^ ^ 8 & 11, 1 | 1 〇 16 〇 11 & 1 · - Cloning : A Laboratory Manual Vol. 3, Cold Spring ❹ Harbor, Laboratory Press 2001^ > ^Ausubel, Current Protocols in Molecular Biology, John Wiley & Sons 1987-1997", "Nuc. Acids Res·, 10, 6487 (1982)", "Proc· Natl. Acad. Sci. USA, 79, 6409 (1982)", Gene, 34, 315 (1985)", "Nuc. Acids. Res., 13, 4431 (1985)", "proc. Natl. Acad. Sci. USA, 82, 488 (1985 In the amino acid sequence of the protein of the present invention, "one or more (for example, 1 to 15 and preferably 1 or less) amines are produced. Deletion of a base acid residue 14 320590 201011106 Loss, substitution, insertion and/or addition" means that one or a plurality of amine groups are present at any position in the same sequence and in one or more amino acid sequences. Deletion, substitution, insertion, and/or addition of acid residues, and two or more cases may occur simultaneously in deletion, substitution, insertion, and addition. The amino acid residues which are mutually replaceable are as follows. The amino acid residues contained in the same group may be substituted with each other. Group A: leucine, isoleucine, norleucine, lysine, ortho-amine, alanine, 2-aminobutyric acid, guanidine thioglycol, o-mercaptosine Acid, tert-butylglycine, triterpenic acid, cyclohexylalanine; group B: aspartic acid, glutamic acid, isoaspartic acid, iso Glutamic acid, 2-aminoadipate, 2-aminooctanedioic acid; Group C: Asparagine, glutamine; Group D: lysine, arginine, bird Amino acid, 2,4-diaminobutyric acid, 2,3-diaminopropionic acid; Group E: proline, 3-hydroxyproline, 4-hydroxyproline; Group F: serine , threonine, homosine; group G: phenylalanine, Λ vatar. Further, the protein of the present invention can also be produced by a chemical synthesis method such as Fmoc method (mercaptomethoxycarbonyl method) or tBoc method (third-butoxycarbonyl method). In addition, chemical synthesis can also be carried out using a peptide synthesizer of companies such as Advanced Chemtec, Parkin-Elmer, Pharmacia, Protein Technology Instrument, Synthservegar, Parseptive, and Shimadzu Corporation. Here, the protein of the present invention is a glucuronic acid transferase. The "glucuronic acid transferase" catalyzes the reaction of a glucuronic acid conjugate by transferring a glucuronic acid residue from a sugar donor 320590 15 201011106 to a sugar acceptor matrix. The 'sugar acceptor matrix' in the present invention is, for example, a yellow-yellow I, a diphenylethyl, a coumarin, and a lignan. Further, the sugar supply system is, for example, UDp_g uronic acid. The protein of one aspect of the invention catalyzes the reaction of transferring a glucuronic acid residue from UDP-glucuronic acid to a sugar acceptor matrix to produce a glucuronic acid complex and UDP. The flavonoids of the sugar acceptor matrix include flavonoids, flavonols, flavanones, isoflavones, flavonoid c-glucosides, aurone and catechins. Among them, examples of yellow _ can be exemplified by yellow sputum, scutellarein, apigenin, iute 〇lin, tricotin, diosmetin, and chrysoeriol. )Wait. Examples of the flavonols include quercetin, myricetin, and kaempfer〇1. Examples of the yellow burned steel include naringenin. Examples of the isoflavones include genistein, daidzein, and fordonone. Examples of the flavonoid C glycoside include, for example, vitexin, isovitexin, and orientin. Examples of the sputum may be, for example, aureusidin. Examples of the catechins include catechins and epigallocatechin gallate. The distyryl group includes resveratrol and its glycoside piceid. Muscarin includes (+)-rosinol ((+)-pinoresinol), (+)-spinal menthol ((+)~Piperitol), (+)-sesenophenol 16 320590 201011106 ((+)- Sesaminol), ( + )-linolenic lignan (( + )-secoisolariciresinol), (+)-sesame catechin 1 (SC1) ((+)-sesamin catechol 1), (+) sesame Consider 2 (SC2), (+)-episin catechol 2 (EC2) and borax sin (matairesinol). In one aspect of the invention, the sugar acceptor matrix is yellowish. In another aspect of the invention, the sugar acceptor matrix is a xanthone having a hydroxyl group at the 4' position of the B ring. In still another aspect of the present invention, the ruthenium receptor substrate is at least one selected from the group consisting of scutellarin, sproutin, luteolin, geranin, sirolimus, kaempferol, and naringenin. Sugar acceptor matrix. For example, the gluconate transferase (VpF7GAT) consisting of the amino acid sequence of the sequence number: 8 is when the sugar acceptor matrix is wild avidin, quercetin, apigenin, luteolin, geranin and Flavonoids such as ginsengin, flavonols such as quercetin and kaempferol, and flavanones such as naringenin show activity; especially when the sugar acceptor matrix is wild baicalein, apigenin, luteolin, and fragrant When lignin, stilbene, kaempferol and naringenin show more potent activity than other sugar receptor substrates. 3. Carrier and a transformant obtained by introducing the vector In another embodiment, the present invention provides a performance vector comprising 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 the above items (a) to (?)). Preferably, the present invention is preferably a A. Glycosyl acid containing any one of the above (g) to (].) towels. The expression carrier of the present invention preferably comprises a polynuclear glycoside as described below. , which comprises a polynucleotide consisting of the nucleotide sequences 1 to 1362 of the sequence of the sequence: sequence 320590 17 201011106; or a polynucleotide comprising a polynucleotide encoding a protein, The protein system consists of the amino acid sequence shown in SEQ ID NO: 8. The vector of the present invention generally comprises: (1) a promoter which is transcribed in a host cell; (ii) a polynucleotide of the present invention which binds to the promoter (for example, the above (a) a polynucleotide of any one of (j)); and (iii) a transcriptional termination of RNA molecules and polyadenylation, and comprising a beta signal capable of functioning in a host cell as a constituent element Expression cassette. The vector of the above-described table is introduced into the host cell. The preparation method of the expression carrier can be carried out by a method using a plasmid, a bacteriophage or a cosmid, and is not limited to the above method. The specific kind of the vector is not particularly limited, and a vector which can be expressed in the host cell can be appropriately selected. That is, in order to accurately display the polynucleotide of the present invention, a suitable promoter sequence is selected, and the polynucleotide of the present invention is combined with the polynucleotide of the present invention in various plastids to prepare a vector. The vector can be used as a performance vector. The expression vector of the present invention depends on the type of host to be introduced, and includes a field of expression regulation (e.g., a promoter, a terminator, and/or a replication origin). For the promoter of the expression vector for bacteria, a commonly used promoter (for example, a trc promoter, a tac promoter, a lac promoter, etc.) can be used; a yeast promoter can be used, for example, a glyceraldehyde-3-phosphate dehydrogenase promoter. The PH05 promoter and the like; examples of the promoter for the filamentous fungus include, for example, amylase, trpC and the like. Further, examples of the promoter for animal cell host include a viral promoter (Example 18 320590 201011106 such as SV40 initial promoter, SV40 late promoter, etc.). The expression vector is preferably one containing at least one selectable marker. The marker can utilize auxotrophic markers (ura5, niaD), drug resistance markers (hygromyCine, Zeocin), Geneticin tolerance gene (G418r), copper tolerance gene (CUP1) (Marin et al., Proc. Natl. Acad. Sci. USA, 81,337, 1984), cerulenin tolerance gene (fas2m, PDR4) (refer to pig lumbosacral feeding, et al., Biochemistry, 64, 660, 1992; Hussain et al. People, gene, ® 101, 149, 1991), etc. Further, the present invention provides a transformant into which a polynucleotide of the present invention (for example, a polynucleotide having any one of the above items (a) to (j) is introduced) is provided. The method for producing the transforming body (production method) is not particularly limited, and examples thereof include a method of transforming the recombinant vector into a host. The host cell used in the '- is not particularly limited, and various cells which have been known in the past can be suitably used. Specifically, for example, bacterium such as Escherichia coli, yeast (saccharomyces cerevisiae, schizosaccharomyces pombe), nematodes (Caenorhabditis elegans), oocytes of xen〇pUS iaevis, and the like can be used. Suitable media and culture conditions for the above host cells are well known in the relevant art and need not be described. Further, the species to be transformed is not particularly limited, and for example, various microorganisms, plants, animals, and the like exemplified in the above host cells can be used. The method of transformation of the host cell can utilize a generally known method. For example, the electroporation method can be used (匕16(:1:1'〇卩〇&1:丨011) (refer to "3〇1^1^^6〇.八等320590 19 201011106人, Appl. Environ. Microbiol, 66, 4655-4661, 2000), particle delivery (refer to the method described in JP-A-2005-287403 "Method for Breeding Lipid-producing Bacteria"), Protoplast Spheroplast (Spheroplast) Refer to proc. Natl. Acad. Sci. USA, 75 pl929 (1978)), acetic acid clock method (see j. Bacteriology, 153, pl63 (1983)) ^ Proc. Natl. Acad. Sci. USA, 75 pl929 (1978) , Methods in yeast genetics, 2000 edition: A Cold

The method described in the Spring Harbor Laboratory Course Manual, etc., is not limited to the above method. In another aspect of the invention, the transformant can be a plant transform. The plant transformation system of this embodiment is produced by introducing a recombinant vector containing the polynucleotide of the present invention into a plant in such a manner that a polypeptide encoded by the polynucleotide is expressed. When a recombinant expression vector is used, the recombinant expression vector used for the transformation of the plant body is not particularly limited as long as it is a carrier which can express the polynucleotide of the present invention in the plant. Examples of the substrate include, for example, a vector having a promoter capable of displaying a polynucleic acid in a plant cell (for example, a 35S promoter of cauliflower mosaic I), or an activation-inducing activation by external stimulation. Carrier of the child. In the present invention, a plant to be transformed into a whole refers to a whole plant body, a plant organ (for example, a leaf, a petal, a stem, a root, a seed, etc.), a plant tissue (such as a skin phlet (ph 1 〇em), a thin body). Parenchyma, xylem, '..A beam-grating, spongy tissue, etc., or plant culture cells, or plant cells of various forms (eg, suspension culture cells), protoplasts 320590 20 201011106 (cal lus Any of the above may be any of a monocot (protoplast), a slice of a leaf, or a callus. The plant used for the transformation is not particularly limited, and the plant belonging to the class or the dicotyledon. The method of introducing a gene into a plant can make a method of transformation (such as Agrobacterium method, gene grabbing method, PEG method, electroporation method, etc.) known to the same person in the field of (4). For example, a method by the method of soil (4) and a method of directly introducing into a plant cell is known. When the soil rod method is used, the constructed plant is guided to a suitable soil rod g (for example, Agrobacterium tumefaciens), and the strain is subjected to a leaf-disk method. (Refer to Nei Gong Bowen, Plant-based Handbook (10) 0), pp. 27-31, Kodansha Science and Technology, Tokyo, Japan Basic ^ Infected with sterile cultured leaves, and transformed plants. In addition, it can also be used

The method of Nagel et al. (cf. Micribiol. Lett., 67, 325 (1990)). The method is first, for example, introducing a expression vector into Agrobacterium, followed by 'transformation according to the method described in the literature Plant Molecular Bi〇i〇gy Manuai (s. B. Gelvin et al., Academic Press Publishers). A method of introducing Agrobacterium into a plant cell or plant tissue. Here, the "plant tissue" includes a healing tissue obtained by cultivating plant cells. When transforming using the Agrobacterium tumefaciens method, a binary vector (pBI121 or pPZP202, etc.) can be used. Further, a method of directly introducing a gene into a plant cell or a plant tissue is known as an electroporation method or a gene gun method. When using the gene grab, the plant body, plant organ, and plant tissue itself can be directly used, and can also be used after slicing and can be used as a protoplast. The sample 320590 21 201011106 can be processed using a gene introduction device (for example, PDS_1000 (product of BIO_RAD)). The treatment conditions vary depending on the plant or the sample, and are generally carried out at a pressure of about 45 to 2000 psi and a distance of about 4 to 12 cm. The cells or plant tissues into which the gene has been introduced are first selected by a drug resistance agent such as hygromycin resistance, and then regenerated into a plant according to a predetermined method. The method of regenerating from a transformed cell into a plant can be carried out in accordance with a method known in the art depending on the type of plant cell. When a plant culture cell is used as a host, the recombinant vector is introduced into a cultured cell by a gene gun or an electroporation method to perform transformation. As a result of the transformation, callus or sputum germination (sh〇〇t), hairy roots and the like can be directly used for cell culture, tissue culture or organ culture. Further, an appropriate concentration of phytohormone (auxin, cytokinin, gibberellin, abscisi-c acid, or the like) can be administered using a conventional plant tissue culture method. Ethylene, brassinolide, etc., etc. are regenerated into plant bodies. Whether the 10 gene is actually introduced into plants can be confirmed by PCR, Southern hybridization, Northern hybridization, and the like. For example, modulating DM from a transforming plant and designing a DM-specific primer for PCR can be carried out under the same conditions as those used in the preparation of the above plastid. Then, 'proliferation products are subjected to agarose electrophoresis, polyacrylamide gel electrophoresis or capillary electrophoresis, etc., and stained with ethidium bromide (Eidium bi*〇mide), SYBR green solution, etc., by detecting the proliferation by a zone. The product was confirmed to be transformed. Further, PCR can be carried out by using a fluorescent dye or the like as a primer to detect a proliferating product. It is also possible to combine the growth products of 22 320590 201011106 with a solid phase such as a microplate, and to recognize the proliferation product by fluorescence or enzyme reaction. - If you get the system? A nuclear thief combines a transformed plant body into a genome to obtain the offspring of the plant using sexual reproduction or asexual reproduction. Further, for example, seeds, fruits, earings, tubers, roots, plants, callus, protoplasts, etc. may be obtained from the plant or its progeny, or the clonal (done), and such The plant body is produced on a large scale. Accordingly, the present invention also encompasses a plant body into which a polynucleic acid of the present invention is introduced, or a progeny of the plant having the same trait as the plant, or a tissue derived therefrom. Also, there have been reports on the transformation methods of various plants. Examples of the transforming plant of the present invention include sesame, rice, grass, barley, wheat, rapeseed potato, poplar, camphor, eucalyptus tree (卽(五)乂 buckle), potato: soybean, t , iupine, jade, broccoli, rose, chrysanthemum, carnation, snapdragon, cyclamen, orchid, platy platycodon, freesia, gerbera, gladiolus , gypsophila, kaianch〇e, lily, pelargonium, geranium, Arabidopsis thaliana, and L〇tus corniculatusvar Japonicus), etc., is not limited to the above range. According to the form of the present invention, the plant body of the functional plant material of the transformed plant system is used. According to still another aspect of the present invention, the transformed plant system flower 320590 23 201011106 =:::1 color changed plant Zhao, flower one 4 · Benfa @之蛋自胄的财财法 The present invention is Another method for the preparation of the protein f of the present invention is to carry out the method of manufacturing a ❹ or = cell _ body or culture. ^The separation of eggs (four), fine 养 养 养 养 ' 当 当 当 当 当 当 当 当 当 当 当 当 当 当 当 当 当 当 当 当 当 当 当 当 当 当 当 当 当 当 当 当 当 当 当 当 当 当 当 当 当 当 当 当 当After the cells or cells are disrupted, the crude extract of the protein of the present invention is obtained according to a general method (J, u, filtration, etc.). When the protein of the present month is accumulated in the culture solution, after the completion of the culture, the cells or cells are cultured in accordance with a general method (for example, centrifugation, filtration, etc.) to obtain a protein containing the present invention. Cultivate the supernatant. The purification of the protein of the present invention contained in the extract obtained by the above method or the culture supernatant can be carried out according to a general separation and purification method. The separation and purification method may be, for example, ammonium sulfate precipitation method, gel filtration chromatography, ion exchange chromatography, affinity chromatography, reverse phase high-speed liquid chromatography, dialysis, ultrafiltration, etc., alone or in moderation. Use in combination. 5. Method for Producing Gluconic Acid Conjugates Further, the present invention provides a method for producing a glucosulphate complex of Portuguese 320590 24 201011106 using the protein of the present invention. Since the protein of the present invention catalyzes the reaction of transferring glucuronic acid from a sugar donor (eg, UDP-glucuronic acid) to a sugar acceptor matrix (eg, flavonoids, di-vinyl or lignan), The protein of the invention can produce a glucuronic acid conjugate by using a sugar acceptor matrix and a sugar donor as raw materials. The sugar acceptor matrix is preferably a flavonoid. For example, the reaction can be carried out at 3 ° C by modulating a solution containing a lol-containing sugar acceptor matrix, a 2 mM sugar donor, a 50 mM calcium phosphate buffer (pH 7 5), and 2 〇/zM of the protein of the present invention. 30 counterclockwise, and the production of glucuronate tandem. From this solution, a glucuronic acid conjugate can be isolated and purified by a known method. Specifically, for example, ammonium sulfate precipitation method, gel filtration chromatography, ion parent chromatography, affinity chromatography, reverse phase high speed liquid chromatography, dialysis, ultrafiltration, etc., may be combined individually or in moderation. And use. The glucose sulphate conjugate thus obtained can be used as a raw material for functional foods, a reagent for investigating its function in the living body, or an antioxidant (refer to Gao, Z., Huang, K., Yang, X). ., and Xu, H. (1999) φ Biochimica et Biophysica Acta 1472, 643-650.). [Examples] The present invention is illustrated in more detail by the following examples, which are not limited thereto. [Example 1] Gene cloning The molecular biology method used in this example, unless otherwise detailed, is described in the literature Molecular Cloning (Sambrook et al., Cold Spring Harbour Laboratory Press, 2001). The method of loading. 25 320590 201011106

For the glucuronyltransferase SbUBGAT of the Labiatae (Nagashima S. et al., Phytochemistry 53, 533-538, 2000), based on the homology search by Blast analysis, it was found that at the amino acid sequence level, 55% of the sequence was found. The homogeneity of the ginsengaceae ginseng glucoside enzyme gene (ie, AmUGTcglO, registration number AB362988) (see Ono, E. et al., Proc. Natl. Acad. Sci. USA 103, 11075-11080, 2006) 〇 for separation The gene encoding the flavonoid 7-glucuronyltransferase VpF7GAT, which belongs to the genus Scrophularia, is based on the sequence of the Snapdragon, which is also belonging to the genus Scrophulariaceae. The two primers shown below are designed. Sequence numbers 1 and 2). Serial number]

AmF7GAT-Fl : 5,-GTG ATA GAT TTC TTT TGC AAT-3, - Serial Number 2

AmF7GAT-R3 : 5'-ACC CTA TTC ATC CTC TGC TCC-3' ❿ Use the RNeasy plant mini kit (QIAGEN) to extract total RNA from the petals of Persian, and use it according to the conditions recommended by the manufacturer. RT-PCR cDNA was synthesized from total RNA of l#g using Super Script First-Strand Synthesis System (product of Invitrogen). Using this cDNA as a template' attempts to isolate the gene encoding vpF7GAT by PCR using the primers with sequence numbers 1 and 2 above. Specifically, the PCR reaction solution (50//1) is composed of Persian Possina cDNA1 #1, 1 xExTaq buffer (product of TaKaRa Bio), 0.2 mM dNTPs, and primers (SEQ ID NO: 1 and 2). 4 pmol / μ 1, ExTaq poly 26 320590 201011106 synthase 2. 5U composition. The PCR reaction was carried out at 94 ° C for 3 minutes, and then subjected to amplification for 35 cycles of 1 minute at 94 ° C for 1 minute, 50 ° C for 1 minute, and 72 ° C for 2 minutes. The PCR reaction solution was separated by a 0.8% agarose electrophoresis, and the amplified fragment was obtained at a size of about 1.0 kb. This amplified fragment was inserted into a multi-cloning site of a p CR-T0P0II vector (product of Invitrogen), and was synthesized using a DNA sequence analyzer 31 (Applied Biosystems). The primer sequence of the nuclear acid primer is used to determine the sequence of the insert. The obtained sequence of the test sequence was analyzed by the CLUSTAL-W program (MACVECT0R 7.2 software, product of Accerly) to show the sequence homology of the Snapdragon AmUGTcg 1 〇 and the Yellow S SbtiBGAT. As a candidate gene of VpF7GAT. However, as a result of the alignment with AmlIGTcglO, the resulting amplified cDNA fragment was confirmed to be an incomplete open reading frame (also known as 0RF) in the 5' and 3' fields. Therefore, using the gene racer kit (11^1:1'€^11 company product) and according to the manufacturer's recommended method for rapid amplification of cDNA end (hereinafter referred to as RACE), The 5' and 3' domains of the cDNA fragment were amplified. RACE uses the primer set specific for the vPF7GAT gene shown in the following sequence numbers 3 to 6.

GR-VpF7GAT-RV : -TTC CAG GAG GGT TTC GAA CGG ACC 27 320590 201011106 ΑΤΑ-3, SEQ ID NO: 4 GR-VpF7GAT-nest-RV: 5, -CTA GAG GTG CAA CGA ΑΤΑ AAA CTT-3, Serial Number 5 GR-VpF7GAT-Fw : 5? -TAT GGT CCG TTC GAA ACC CTC CTG GAA-3' Serial number 6

® GR-VpF7GAT-nest-Fw : 5' -AGG ATC CTG ACC TGG AAA CA-3, for the amplified fragment obtained by RACE, the base sequence is determined according to the primer step method using the synthetic oligonucleotide primer. The VpF7GAT candidate gene containing the full length of ORF and its amino acid sequence (SEQ ID NO: 7 (cDNA sequence of VpF7GAT), SEQ ID NO: 8 (amino acid sequence of VpF7GAT)) was obtained. ❸ The VpF7GAT candidate gene showed sequence homogeneity of 61% and 51% with the A. sinensis AmUGTcgl0 and Yellow A SbUBGAT at the amino acid sequence level, respectively. [Example 2] Construction of the cut-off body In order to understand the biochemical function of the VpF7GAT candidate protein (hereinafter simply referred to as the present enzyme) obtained in Example 1, a coliform expression vector which expresses the enzyme cDM was constructed. The cDNA containing the entire length of the sputum was amplified by PCR using the VpF7GAT candidate gene-specific primer set shown in SEQ ID NO: 9 and 10 and 320590 28 201011106. The template was a cDNA synthesized using the total RNA extracted from the petals of Persian. Shore number 9 CACC-NdeI-VpF7GAT-Fw : 5* -CAG CCA TAT GGA AGA CAC AAT CAT CCT-3’ Sequence number 10

XhoI-VpF7GAT-Rv: 5, -CTC GAG TTT TTA CCC AAT AAC CAA CTT GAT-3, O PCR reaction (using KOD Plus polymerase, product of Τ0Υ0Β0) After heat denaturation at 94 ° C for 2 minutes, The cycle was carried out for 35 cycles at 94 ° C for 15 seconds, at 50 ° C for 30 seconds, and at 68 ° C for 1.5 minutes. The amplified DNA fragment was subcloned to the pCR-Blunt ΙΙ-Τ0Ρ0 vector (using the Zero Blunt Τ0Ρ0 PCR selection kit, manufactured by Invitrogen) and was prepared by the ABI3100Avant Genetic Analyzer (Applied Biosystems) ) Carry out the marrying of the base sequence. The resulting system is completely digested with a restriction enzyme of Ndel and Xhol, and the resulting DNA fragment of about 1.5 kb containing the full length of ORF is ligated to the Ndel of the coliform expression vector pET-15b (product of Novagen).

Xhol site, and the coliform expression vector. [Example 3] The performance of the wood-intestinal heavy-box protein was as follows. Using the above-obtained plastids, the Escherichia coli BL2KDE3)® strain was transformed according to a predetermined method. The obtained transformant is contained in an ampicillin containing 5_/ιη1 29 320590 201011106 • (6) called LB medium (containing 1 §/1 membrane protein Yuedong, 5g/i enzyme extract, lg/1 gas In the ω, in the immersion vibration 4 culture - night. The culture medium that reaches the stationary phase - inoculated in the same composition of the medium, incubated at 37t, when the concentration of bacteria _00) Upon arrival at 〇7, IPTG' with a final concentration of 〇.μ was added and shaken at 22 °C for 2 hrs. All of the following operations are at 4. (: proceed under '. The cultured transformant is collected using a centrifuge (7, 〇 _, u minutes), and a buffer s [containing 20 mM sodium phosphate buffer (pH 7.4), 20 gaze, 0.5M sodium chloride, i-dish stone-mercaptoethanol] 2ml/g cells, suspended, followed by ultrasonication (15 seconds X8 times), centrifugation (15, (10) 〇 xg , 1 minute). Add the final concentration of 聚. 12% (w/v) of the acetamiprid in the supernatant and suspend it for 30 minutes. Centrifugal treatment (丨5) , 〇〇〇xg, 10 is the clock), and the supernatant liquid is recovered as a crude enzyme solution. The crude enzyme solution is treated with a His SpinTrap (GE Healthcare) product which is equilibrated with a buffer S, and then centrifuged. (70xg, 30 seconds) treatment. After washing with 6 〇〇β 1 buffer S, use buffers S containing 1 〇〇, 2 〇〇, 5 mM imidazole each 600 / 1 according to the stage The protein bound to the column was eluted by using a Microcon YM-30 (manufactured by Amicon). Each eluted fraction was treated with 20 遽 potassium phosphate buffer (|) 117.5), 141^/5-mercaptoethanol. Buffer substitution. As a result of SDS-PAGE analysis, it was confirmed that there was a purified expression protein in the vicinity of about 50 kDa estimated from the amino acid sequence of VpF7GAT in the 200Mm taste eluting fraction, so this fraction was used for enzyme analysis (see 320590 30 201011106 As indicated by the arrow in Figure 1: the target protein). [Example 4] The reaction conditions and the standard reaction conditions were as follows. Prepare a reaction solution of 5〇# 1 (containing 2 mM UDP-glucuronic acid, 1 〇〇βΜ sugar acceptor matrix, 5 mM potassium phosphate buffer (pH 7.5), enzyme solution) to add enzyme The reaction was started, and the reaction was carried out for 3 minutes at 3 (TC). The reaction was terminated by adding cyanide decane 5〇仏1 containing 5%.5% TFA, and the reverse phase HpLC (LC-2〇1〇 system, Shimadzu Corporation product) was used. Analytical. The HPLC conditions are as follows. The column is used in the column oven 4〇〇c

Develosil C30-UG-5 (4·6mmxl50mni, manufactured by Nomura Chemical Co., Ltd.), mobile phase A is 0.1% TFA/H2O, mobile phase β is 〇. TFA/9〇% cyanide methane. The elution conditions were followed by a linear concentration gradient of 15 minutes (B2〇%_B7〇%) and then held at B70% for 1 minute. Then, return to B2〇% and equilibrate for 20 minutes. The flow rate was carried out at 1 ml/min. The detection system was carried out using a spD-Ml〇A type Photodiode kray detector (product of Shimadzu Corporation) at a wavelength of 280 nm and 360 nm. Under these conditions, the standard apigenin (provided by the company), the buds 7-glucuronide and the apigenin 7-glycoside (a product of the company) are replenished by the petals of the snapdragon. 11.75 minutes, 8.36 minutes, and 8.22 minutes of washing (refer to Figure 2 (A): Sproutin, Figure 2 (C): 7-glycolic acid in the order]. As a result of HPLC analysis of the enzyme reaction solution using phytosin as a sugar acceptor matrix and UDP-glucuronic acid as a sugar donor, it was confirmed that the retention time of the 7-glycoside acid of the standard product was the same. The new product is stored 320590 31 201011106 ' (see Figure 2 (B)). The conditions of LC-MS were based on Develosil C30-UG-3 column (manufactured by Nomura Chemical Co., Ltd., 3. 0mmxl50mm), and mobile phase A liquid using water containing citric acid 'B liquid system containing yttrium. 1% formic acid 1% acetonitrile. The linear concentration gradient of 2 〇 minutes (20% - 70% of solution B) was eluted, and then an isocratic elution was performed for 5 minutes with 7 〇% b. (The flow rate was 0·2 ml/min' The column oven temperature was 4 (TC). The detection was carried out using a Photodiode Array detector (SPD-Ml0A type, manufactured by Shimadzu Corporation) to collect data of a wavelength of 230 to 500 nm, and a chromatogram of A337 nm was measured. Further, after the pda detector, a TOF-MS detector (Q-TOF Premier, Micromass, UK) was connected, and the molecular weight of the product was measured according to the following conditions. The measurement conditions of the MS were carried out in a negative mode (ion source was ESI, Lock). Spray reference: leucine enkephalin 以此 is this - Enkephalin) (m/z 554. 2615 [Μ-ΗΓ), capillary: 2. 7kV, Cone: 30V, MS/MS collision energy · 2〇eV) . Under these conditions, the substrate of the substrate was washed at a holding time of 17.51 minutes to impart m/z 269. 0441 [Μ-ΗΓ molecular ion. On the other hand, the product produced by washing at a holding time of 11.72 minutes was given m/z 445. 0759 [Μ-ΗΓ molecular ion, which was confirmed to be added to a glucose by acid to apigenin. (Refer to Figure 2 (D)). Further, according to the MS/MS analysis, a fragment ion of m/z 269. 0450 [M-H] which is consistent with apigenin can be detected from the molecular ion of the product. From the above results, it was revealed that this enzyme is a protein having the activity of Persian. 32 320590 201011106 [Example 5] μι^ατ冬蹲jfc function is also measured according to the literature (MogU (:hi, A. et al., plant % 145_427, 2008/) g-method' determination of this enzyme for Liaotang The selectivity of the body (the sugar acceptor matrix is the sprouting illusion, the result is the relative activity of 1%% of the _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ High specificity for UDp-glucuronic acid. Further, the substrate specificity (Km) of the enzyme for spingin and UDP-glucose acid was 10.7±1.7βΜ and 36.6±8.7/zM, respectively, for apigenin. The catalytic activity (kcat) is 8.64 S-1. As a result of the selectivity of the sugar acceptor matrix of the enzyme (sugar donor is UDp glucuronic acid), the apigenin which is an intrinsic matrix shows the most Still active (refer to Figure 3). The relative activity of the activity of apigenin is 1%, and belongs to the flavonol which belongs to the flavonoids of wild scutellaria, scutellaria, luteolin, sylvestin and sirolimus. The bark _ and the mountain age; ❺ and the naringenin belonging to the flavanone are 88.3%, respectively. 1%, 14 9%, 12.9%, 34. 〇%, 1. 〇%, 13.3% and 18.0%. Especially for the flavonoid B ring 4, the hydroxyl group is methylated The geranin, or the B ring has two or more hydroxyl groups such as yellow _, and the activity of the enzyme is lower than that of the single base at 4, and further, because there is no The activity of the hydroxyl group baicalein is low, so it is known that the hydroxyl group of the B ring of the jaundice is extremely important in identifying the sugar acceptor matrix of the enzyme. In addition, under the conditions of the enzyme reaction, it cannot be confirmed. For resveratrol belonging to diphenyl 320590 33 201011106 ethylene, esculetin belonging to coumarin, sesaminol belonging to lignan, daidzein belonging to isoflavone, genistein and belonging The glucuronide transfer activity of isoflavin and valerin of flavonoid C glycosides. Therefore, the enzyme exhibits potent activity against flavonoids, particularly flavonoids having a hydroxyl group at the 4' position of the B ring. Flavonoids: apigenin, Luteolin, geranium, ruthenium, avocin, and flavonoids Iso Vitexin, Cursive hormone and ❹, hereinafter Glc represents glucose.

❿ 芽 木 木 木 木 香 金 金 金 金 金 金 金 金 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( 〇CH〇(R3'=OCH3,R4'=0H) (R6=0H) (R4, =0H, R6=0H) (R4'=0H, R6=C-Glc) (R3'=0H, R4'= 0H,R8=C-Glc) 34 320590 201011106 'Flavone alcohol: quercetin and kaempferol R3,

Shannai ('R4' = 0H) 槲 quercetin (R3' = 0H, R4' = 0H) xanthone: naringenin

OH 〇 柚 naringenin [Example 6]

The expression of the VpF7GAT gene was analyzed according to the same method as described in the literature (Noguchi, Α. et al., Plant J. 54, 415-427, 2008), based on the quantitative RT- using 7500 Real Time PCR Systein (Applied Biosystems). The PCR method was used to analyze the expression patterns of different organs of the VpF7GAT gene. From the organs of Persian mother-in-law (ie, leaves, flowers, fruits, stems, roots), extract the total amount according to the method of 35 320590 201011106 in Example 1, and take (8) the reverse transcription reaction (10) to obtain the cDNA of each organ. Use this as a template for PCR.疋1 PCR-specific gene-specific primers use pHmer

The Express 3.0 program (product of Applied Bi〇systems) is designed in the following four categories. The VpF7GAT specific primer uses the sequence number 丨丨 and 12. The internal standard gene system was amplified using Persian Borneo ribosomal RNA (AF5〇9785) and using the gene-specific primers of SEQ ID NOs: 13 and 14 below.序列 SEQ ID NO: 11 qVpF7GAT-Fw : 5'-GCG GTT TCG GCC TCT GT-3, SEQ ID NO: qVpF7GAT-Rv : 5' -TCC GAT ATC TTC AGG GAT GAT TTC-3, SEQ ID NO: qVprRNA-Fw : 5 '-GCG GAA GGA TCA TTG TCG AT-3' SEQ ID NO: 14 φ qVprRNA-Rv : 5'-CTA GCG GGC GGA GCT TAT TA-3' The amount of VpF7GAT is normalized by the amount of internal standard gene expression. Applied Biosystems obtained relative performance. As a result, it was revealed that the VpF7GAT gene was highly expressed in the petals (see Fig. 4). Because the accumulation of the flavonoid 7-glucuronic acid imparted by this enzyme is consistent with the field of expression of the enzyme gene, it is strongly demonstrated that this enzyme participates in the performance of Persian Posner by the formation of a secondary pigment. Further, although the expression of VpF7GAT was also confirmed in the blade, however, it was not considered to be a significant coloration, and this phenomenon may be caused by a significant amount of the main pigment of the flower 36 320590 201011106. Glucose = to be able to be isolated from Persian Possina to use the enzyme to breed out %_) ° So 'the functional body can change the plant body (such as blue flowers) and research [in the industry] Utilization] Lufan grape acid transfer enzyme, whose matrix specificity is ❹: various salicylic acid conjugates of the invention. Therefore, if an acid transfer enzyme is used, it is possible to develop a plant body (e.g., blue flower) or a functional food material such as a flower color change. [Simple description of the diagram] Figure 1 shows that the coliform protein has not been confirmed. The Μ indicates the size mark (sizem == 2 (pellet) part, the C line indicates the lack of this ▲ H does not sink the φ point indicates 5_W elution two points q = indicates the non-adsorption part elution part, 5〇〇 indicates _ 'The target protein f for the expression of Escherichia coli (Vpp7 (^k^, arrow indicates that Figure 2 (A) shows the eight diagrams of apigenin (8) showing the enzyme reaction solution (Graph of the order of the fruit. 2nd HPLC analysis) Fig. 2 is a diagram showing the results of HPLC analysis of the guanylate of the glucosinolate, which shows the 7th position of celery (grassin and UDP-glucose age, Fig. 2 (Fig. 2) D) shows the enzyme reaction diagram.) Figure 3 shows the results of the VpF7GAT ^ 'Matrix-specific matrix-specific solution 320590 37 201011106 ' by the results of the recapture. Figure 4 shows A graph showing the results of quantitative RT-PCR analysis of the different organs of the VpF7GAT gene. [Key element symbol description] None 0 ❹ 〇38 320590

Claims (1)

201011106 VII. Patent Application Range: 1. A polynucleotide, as described in any one of the following items (a) to (f): (a) a polynucleotide comprising the sequence number: 7 a polynucleotide consisting of the nucleotide sequences of the first to the first steps in the base sequence; (b) a polynucleotide comprising a polynucleotide encoding a protein having the amino acid of the sequence number: 8. O(c)-polynucleotide comprising a polynucleotide encoding a protein which is deleted, substituted by 1 to 15 amino acids in the amino acid sequence of SEQ ID NO: 8. An amino acid sequence inserted and/or added, and the protein has UDP-glucuronyltransferase activity; (d) a polynucleotide comprising a polynucleotide encoding a protein, the protein having SEQ ID NO: 8 shows an amino acid sequence of homogeneity of 80% or more with respect to the amine-based acid sequence, and the protein has UDP-glucuronyltransferase activity; (e) a polynucleotide, Contains: under harsh conditions Column number: The nucleotide sequence of the base sequence of 1 to 1362 of the nucleotide sequence shown in FIG. 7 is a polynucleotide composed of a complementary base sequence, and a multinuclear encoding a protein having UDP-glucuronyltransferase activity (f) a polynucleotide comprising: a base sequence of a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 8 under severe conditions and encoded by 39 320590 201011106 ' A polynucleotide consisting of a complementary base sequence is hybridized, and a polynucleotide having a protein having UDP-glucuronyltransferase activity is encoded. 2. The polynucleotide of claim 1, wherein the polynucleotide is one of the following (g) to (j): (g) a polynucleotide comprising a polynucleotide acid encoding a protein The protein consists of an amino acid sequence in which at least 10 amino acids in the amino acid sequence of SEQ ID NO: 8 are deleted, substituted, inserted and/or added, and the protein has UDP-glucose. (1) A polynucleotide comprising a polynucleotide encoding a protein having an amine having a homogeneity of 90% or more for the amino acid sequence of SEQ ID NO: 8. a guanidine acid sequence having UDP-glucuronyltransferase activity; (i) a polynucleic acid comprising: under the most severe conditions, the first nucleotide sequence of sequence number: 7 a polynucleotide in which a base sequence of 1362 is a complementary base sequence hybridizes and encodes a protein having a UDP-glucuronyltransferase activity; or (j) a polynucleic acid; Its system contains: Hybridization under the severe conditions with a polynucleotide consisting of a base sequence encoding a polynucleotide sequence of a polynucleotide of 40 320590 201011106 ^ which is represented by the amino acid sequence of SEQ ID NO: 8. And a multinuclear ophthalmic acid encoding a protein having UDP-glucuronyltransferase activity. 3. The polynucleotide of claim 1, which comprises the polynucleotide consisting of the nucleotide sequences of the first to the first 1362 of the nucleotide sequence of SEQ ID NO: 7. 4. A polynucleotide according to claim 1, which comprises a polynucleotide encoding a protein ® consisting of the amino acid sequence of SEQ ID NO: 8. 5. The polynucleotide of any one of claims 1 to 3, which is deoxyribonucleic acid (DM). 6. A protein characterized by a multi-nucleotide acid as claimed in any one of claims 1 to 5. A carrier comprising a polynucleotide according to any one of claims 1 to 5. A morphosome of the invention, which is characterized in that the polynucleotide of any one of claims 1 to 5 is introduced. A transforming body characterized by introducing a carrier of claim 7 of the patent application. A method for producing a protein, which is characterized in that a protein as claimed in claim 6 is produced using a transformant as disclosed in claim 8 or 9. A method for producing a glucuronic acid conjugate, characterized in that glucuronic acid is formed from a UDP-glucuronic acid and a sugar acceptor matrix by using a protein of the sixth item of the patent scope of claim 41 320590 201011106 as a catalyst. Joint body. ❿ 42 320590 201011106 Sequence Listing &lt;110> Suntory Co., Ltd. &lt;120&gt; Glucuronyltransferase and polynucleotide encoding the transferase &lt;130&gt; G08-0075TW &lt;160&gt; 14 &lt;170&gt; Patent In Version 3.4 &lt;210&gt; 1 &lt;211&gt; 21 <212> DNA &lt;213> Artificial sequence &lt;220> &lt;223> Synthetic DNA &lt;400&gt; 1 21 gtgatagatt tcttttgcaa t &lt;210> 2 &lt;211&gt; 21 &lt;212> DNA &lt;213>artificial sequence &lt;220〉 &lt;223>Synthetic DNA <400> 2 21 accctattca tcctctgctc c &lt;210> 3 &lt;211&gt; 27 &lt;212> DNA <213> artificial sequence &lt;220> &lt;223>synthetic DNA 1 320590 201011106 ' &lt; 400> 3 ttccaggagg gtttcgaacg gaccata 27 &lt;210&gt; 4 &lt;211&gt; 24 &lt;212>DNA .&lt;213>artificial sequence&lt;220&gt;&lt;223&gt;&gt;223>synthetic DNA &lt;400> 4 ctagaggtgc aacgaataaa actt 24 〇&lt ; 210> 5 &lt; 211 &gt; 27 &lt; 212 &gt; DNA &lt; 213 &gt; artificial sequence &lt; 220 &lt; 223 &gt; 223 &gt; synthetic DMA &lt; 400 &gt; 4 &gt; 27 27 tatggtccgt tcgaaaccct cctggaa &lt;210> 6 &lt;211&gt; 20 ❹ &lt;212&gt; DNA <213> artificial sequence &lt;220> s &lt;223>synthetic DNA &lt;400> 6 aggatcctga cctggaaaca 20 &lt;210> 7 &lt;211&gt; 1365 &lt;212> DNA &lt;213> Veronica persica 2 320590 48 201011106 &lt;220&gt;&lt;221&gt; CDS &lt;222>(1)..(1365) &lt;400&gt; 7 atg gaa gac aca ate ate etc tat get tea tee gtg cac ctg aac tet Met Glu Asp Thr Lie lie Leu Tyr Ala Ser Ser Val His Leu Asn Ser 1 5 10 15 96 Gtg eta gta ata gee aag ttc ata aac aaa cat cat cct tet ate tee Val Leu Val lie Ala Lys Phe lie Asn Lys His His Pro Ser lie Ser 20 25 30 144 ata ate ata etc age aat get cct gat tea gee gca tet tee Att acc lie lie lie Leu Ser Asn Ala Pro Asp Ser Ala Ala Ser Ser Ile Thr Tet gaa gee tea tea ate act tac cat ega etc cct act ccc gac att Ser Glu Ala Ser Ser lie Thr Tyr His Arg Leu Pro Thr Pro Asp lie 50 55 60 cct ccc aac ate ate act aat cca gtc gaa ett ett ttc gag gtt Cca Pro Pro Asn lie Me Thr Asn Pro Val Glu Leu Leu Phe Glu Val Pro 65 70 75 80 192 240 ego etc aac aat ccc aat gtc aaa caa tac ett gaa caa ate tee caa Arg Leu Asn Asn Pro Asn Val Lys Gin Tyr Leu Glu Gin lie Ser Gin 85 90 95 288 Aaa act aat gtc aaa gca ttc ate att gat ttc ttt tgc aac tea get Lys Thr Asn Val Lys Ala Phe lie lie Asp Phe Phe Cys Asn Ser Ala 100 105 110 336 ttt gaa gtt tet aeg agt ttg aac att cca acc tac ttc tac Gtc age Phe Glu Val Ser Thr Ser Leu Asn Me Pro Thr Tyr Phe Tyr Val Ser 115 120 125 agt ggc ggt ttc ggc etc tgt get ttc etc cac ttc cca acc aeg gac Ser Gly Gly Phe Gly Leu Cys Ala Phe Leu His Phe Pro Thr Thr Asp 130 135 140 384 432 gaa ate ate cct caa gat ate gga gac ttg aac gat tat ctg gaa ate 480 3 320590 528201011106 Glu lie lie Pro Gin Asp lie Gly Asp Leu Asn Asp Tyr Leu Giu lie 145 150 155 160 cca ggc Tgc cca ccc gtt cac tct tta gat ttc cct aaa gga atg ttt Pro Gly Cys Pro Pro Val His Ser Leu Asp Phe Pro Lys Gly Met Rie 165 170 175 ttc agg cac act aat acc cac aat cat ttc ctt gac act gcc aga aac Phe Arg His Thr Asn Thr His Asn His Phe Leu Asp Thr Ala Arg Asn 180 185 190 atg agg aaa gcc aat ggg att ctg gtg aac teg ttc gat get ctt gag Met Arg Lys Ala Asn Gly Ile Leu Val Asn Ser Ph e Asp Ala Leu Glu 195 200 205 576 624 Tat aga tct aaa gca get tta ttg aac gga att tgc gtt ccg aac ggt Tyr Arg Ser Lys Ala Ala Leu Leu Asn Gly lie Cys Val Pro Asn Gly 210 215 220 672 cca aca ccc caa gtt tta ttc gtt gca cct eta gtt act gga Atg aac Pro Thr Pro 6ln Val Leu Phe Val Ala Pro Leu Val Thr Gly Met Asn 225 230 235 240 720 agt aga aaa ggc gac teg gag cat gaa tgt tta age tgg ctt gac tea Ser Arg Lys Gly Asp Ser Glu His Glu Cys Leu Ser Trp Leu Asp Ser 245 250 255 caa cca agt aag agt gta att ttc eta tgt ttt ggc aga aag ggt ttt Gin Pro Ser Lys Ser Val lie Phe Leu Cys Phe Gly Arg Lys Gly Phe 260 265 270 ttc tcc aaa caa cag ttg caa Gaa ata gca act ggc ttg gaa aac agt Phe Ser Lys Gin Gin Leu Gin Glu lie Ala Thr Gly Leu Glu Asn Ser 275 280 * 285 ggc cat agg ttt eta tgg tcc gtt ega aac cct cct gga att aat aat Gly His Arg Phe Leu Trp Ser Val Arg Asn Pro Pro Gly lie Asn Asn 290 295 300 gag gat cct gac ctg gaa aca ctt ctt cca gag ggt ttt ctg gaa agg Glu Asp Pro Asp Leu Glu Thr Leu Leu Pro Glu Gly Phe Leu Qlu Arg 305 310 315 32 0 768 816 864 912 960 act aaa gaa ega gga ttc gtg ata aag tea tgg geg cct cag aaa gaa 1008 4 320590 1056201011106 Thr Lys 6lu Arg Gly Phe Val He Lys Ser Trp Ala Pro Gin Lys Glu 325 330 335 gta eta age cat gag Tee gtt gga ggg ttc gtg aca cat tgt ggt agg Val Leu Ser His Glu Ser Val Gly Gly Phe Val Thr His Cys Gly Arg 340 345 350 agt teg ata tta gaa gca gtg tea ttc ggt gtg cct atg att ggt ttt Ser Ser lie Leu Glu Ala Val Ser Phe Gly Val Pro Met lie Giy Phe 355 360 365 cca ata tac geg gag caa agg atg aat egg gta ttc atg gtt gag gaa Pro lie Tyr Ala Glu Gin Arg Met Asn Arg Val Phe Met Val Glu Glu 370 375 380 1104 1152 Atg aaa gtg tea ttg ccg tta gat gag get ggt gat gga ett gtt aeg Met Lys Val Ser Leu Pro Leu Asp Glu Ala Gly Asp Gly Leu Val Thr 385 390 395 400 1200 tcc ggt gag etc gaa aag ega gtg aag gaa ttg atg ggt Teg gtt agt Ser Gly Glu Leu Glu Lys Arg Val Lys Glu Leu Met Gly Ser Val Ser 405 410 415 1248 ggg aaa geg att ega caa ega gtt aat gag ttg aaa gtt teg ggc gag Gly Lys Ala lie Arg Gin Arg Val Asn Glu Leu Lys Val Ser Gly 6lu 420 425 430 gca geg gtg aag gaa ggt ggt tet tea gtg gtt gat ctg gac aag ttc Ala Ala Val Lys Glu Gly Gly Ser Ser Val Val Asp Leu Asp Lys Phe 435 440 445 ate aag ttg gtt att ggg taa Ile Lys Leu Val Ile Gly 450 1296 1344 1365 &lt;210&gt; 8 &lt;211&gt; 454 &lt;212&gt; PRT &lt;213&gt; Veronica persica &lt;400&gt; 8 Met Glu Asp Thr lie Me Leu Tyr Ala Ser Ser Val His Leu Asn Ser 5 320590 201011106 1 ίο 15 Val Leu Val lie Ala Lys Phe lie Asn Lys His His Pro Ser lie Ser 20 25 30 lie lie lie Leu Ser Asn Ala Pro Asp Ser Ala Ala Ser Ser lie Thr 35 40 45 Ser Glu Ala Ser Ser lie Thr Tyr His Arg Leu Pro Thr Pro Asp lie 50 55 60 〇Pro Pro Asn lie lie Thr Asn Pro Val Glu Leu Leu Phe Glu Val Pro 65 70 75 80 Arg Leu Asn Asn Pro Asn Val Lys Gin Tyr Leu Glu Gin Ile Ser Gin 85 90 95 Lys Thr Asn Val Lys Ala Phe lie lie Asp Phe Phe Cys Asn Ser Ala 100 105 110 Phe Glu Val Ser Thr Ser Leu Asn lie Pro Thr Tyr Phe Tyr Val Ser 115 120 125 Ser Gly 6ly Phe Gly Leu Cys Ala Phe Leu His Phe Pro Thr Thr Asp 130 135 140 Glu lie lie Pro 61n Asp lie Gly Asp Leu Asn Asp Tyr Leu Glu lie 145 150 155 160 Pro Gly Cys Pro Pro Val His Ser Leu Asp Phe Pro Lys Gly Met Phe 165 170, 175 Phe Arg His Thr Asn Thr His Asn His Phe Leu Asp Thr Ala Arg Asn 6 320590 201011106 180 185 190 Met Arg Lys Ala Asn Gly lie Leu Val Asn Ser Phe Asp Ala Leu 6lu 195 200 205 Tyr Arg Ser Lys Ala Ala Leu Leu Asn Gly He Cys Val Pro Asn Gly 210 215 220 Pro Thr Pro Gin Val Leu Phe Val Ala Pro Leu Val Thr Gly Met Asn 225 230 235 240 Q Ser Arg Lys Gly Asp Ser Glu His Glu Cys Leu Ser Trp Le u Asp Ser 245 250 255 Gin Pro Ser Lys Ser Val lie Phe Leu Cys Phe Gly Arg Lys Gly Fhe 260 265 270 Phe Ser Lys Gin Gin Leu Gin Glu lie Ala Thr Gly Leu Glu Asn Ser 275 280 285 Gly His Arg Phe Leu Trp Ser Val Arg Asn Pro Pro Gly Ile Asn Asn 290 295 300 Glu Asp Pro Asp Leu Glu Thr Leu Leu Pro Glu Gly Phe Leu Glu Arg 305 310 315 320 Thr Lys Glu Arg Gly Phe Val lie Lys Ser Trp Ala Pro Gin Lys Glu 325 330 335 Val Leu Ser His Glu Ser Val Gly Gly Phe Val Thr His Cys Gly Arg 340 345 350 Ser Ser lie Leu Glu Ala Val Ser Phe Gly Val Pro Met lie Gly Phe 7 320590 201011106 355 360 365 Pro lie Tyr Ala Glu Gin Arg Met Asn Arg Val Phe Met Val Glu Glu 370 375 380 Met Lys Val Ser Leu Pro Leu Asp Glu Ala Gly Asp Gly Leu Val Thr 385 390 395 400 Ser Gly Glu Leu Glu Lys Arg Val Lys Glu Leu Met Gly Ser Val Ser 405 410 415 Gly Lys Ala lie Arg Gin Arg Val Asn Glu Leu Lys Val Ser Gly Glu 420 425 430 Ala Ala Val Lys Glu Gly Gly Ser Ser Val Val Asp Leu Asp Lys Phe 435 440 445 Ile Lys Leu Val Ile Gly 450 &lt;210> 9 &lt;211&gt; 27 &lt;212&gt; DNA &lt; 213 &gt; artificial sequence &lt;220 &lt; 223 &gt; 223 &gt; synthetic DNA &lt;400&gt; 9 cacccatatg gaagacacaa tcatcct &lt;210&gt; 10 &lt;211&gt; 30 &lt;212> DNA &lt;213&gt; artificial sequence 201011106 &lt;220&gt;&lt;223>synthetic DNA &lt;400> 10 ctcgagtttt tacccaataa ccaacttgat &lt;210> 11 &lt;211&gt; 17 &lt;212&gt; DNA &lt;213&gt; artificial sequence &lt;220&gt;&lt;223> synthetic DNA &lt;400&gt; 11 gcggtttcgg cctctgt &lt;210> 12 &lt;211&gt; 24 <212> DNA &lt;213> artificial sequence &lt;220> &lt; 223> Synthetic DNA &lt;400〉 12 tccgatatct tgagggatga tttc &lt;210> 13 &lt;211&gt; 20 &lt;212&gt; DNA <213> artificial sequence &lt;220> &lt;223>synthetic DNA &lt;400&gt; 13 gcggaaggat cattgtcgat &lt;210> 14 201011106 ' &lt;211> 20 &lt ;212&gt; DNA &lt;213>Artificial Sequence&lt;220&gt;&lt;223>Synthetic DNA &lt;400> 14 ctagcgggcg gagcttatta 20 ❹ 10 320590