WO2006016395A1

WO2006016395A1 - Udp-glucuronyl transferase and gene encoding the same

Info

Publication number: WO2006016395A1
Application number: PCT/JP2004/011437
Authority: WO
Inventors: Toru Nakayama; Shinya Sawada; Hirokazu Suzuki; Tokuzo Nishino
Original assignee: National University Corporation Tohoku University
Priority date: 2004-08-09
Filing date: 2004-08-09
Publication date: 2006-02-16
Also published as: JPWO2006016395A1

Abstract

UDP-glucuronyl transferase (BpUGT) derived from Bellis perennis; a gene coding for the same; and a transformant having the gene. Thus, glucuronylation of a flavonoid compound can be performed to thereby realize functional improvements for this compound, such as flavonoid compound stabilization, absorption increase, physiological activity control and active substance sustained-release. Further, through providing of the UDP-glucuronyl transferase (BpUGT) derived from Bellis perennis, gene coding for the same and transformant having the gene, there can be provided effective modification means applicable to metabolic engineering control, regulation of color or tone and stabilization of flower color development according to genetic engineering technique. In particular, there is provided an enzyme having the amino acid sequence of SEQ ID NO: 1 and an enzyme coded for by the gene specified in SEQ ID NO: 2.

Description

Specification

UDP-Dalcronyltransferase and its gene

Technical field

[0001] The present invention relates to a novel UDP-dalcronyltransferase and a gene thereof.

Background art

[0002] UDP-Daluronyltransferase (UGT) is responsible for sugar chain synthesis and detoxification of various compounds in the body of mammals, and its detoxification action has been extensively studied due to its pharmaceutical importance. I'm going. Dalcronyl transferase is an enzyme that plays a central role in glucuronidation and detoxification mechanisms, and by dalucuronylating various toxic compounds such as steroids, bilirubin and other hydrophobic endogenous substances, phenol and drugs. Increase the water solubility of these compounds and promote their excretion into urine and bile. These gnolechlorine buds are catalyzed by gnolecronyltransferase using UDP (uridine diphosphate) glucuronic acid as a sugar residue donor. There have been many findings on vertebrate-derived gnolecronyltransferase involved in such detoxification mechanisms, and this group of enzymes is a multimer, a membrane protein localized in the endoplasmic reticulum. It is known to require divalent metal ions (Mg ²⁺ , Mn ²⁺ ) for its catalytic activity, and to catalyze dalclonil, a variety of hydrophobic compounds with wide specificity for sugar residue receptors. (See, for example, Non-Patent Document 1-15).

[0003] In plants, such a mechanism of glycosylation exists and plays a role in solubilizing hydrophobic compounds. However, many of the secondary metabolite glycosides that have been found so far in plants are dalcosylated, dalcronyl compounds are rarely found, and molecules related to gnoleclonylation in plants. Little knowledge is available at the level.

Previous studies have shown that oleorelin-7-0-didalchronyl 4'-0-dalcronide derived from rye cotyledon, baicalein 7-0-dalcronide derived from the family Lamiaceae and gnolecronyl group transfer, which may be involved in the synthesis of soybean-derived saponin The enzyme was purified (see Non-Patent Document 6). In these studies, soybean-derived dalcuronyltransferase is It is thought to have properties similar to those of vertebrate dalcronyltransferase, such as requiring metal ions for the expression of activity in proteins. However, on the other hand, rye and Scarab dalcronyltransferases differ from known animal dalcronyltransferases in that they are water-soluble proteins and do not require metal cofactors for their expression of activity. The results are available (see Non-Patent Documents 7 and 8). From this, it is possible that these enzymes have different functions from the known mammalian gnolecronyltransferases. However, there have been no reports of successful gene cloning of gnorecronyltransferase involved in the synthesis of such secondary metabolites in plants, and the functions and evolution of these plant-derived gnorechronyltransferases in vivo. The above position remains unclear.

So far, cloning of dalcronyltransferase genes from plants has been attempted, and two genes presumed to be darcronyltransferases have been obtained. One is dalcronyltransferase (PsUGTl) derived from Pisum sativum (see Non-Patent Document 9), and the other is glucoronyltransferase (NpGUTl) derived from Nicotiana plumbaginifolia (NpGUTl) ( Non-Patent Document 10). However, with regard to the expression product of PsUGTl, UDP-glucuronic acid can be used as a substrate, and except for the fact that it is expressed in the mitotic phase of the root growth point, including the identity of its sugar residue receptor, NpGUTl is only speculated to be a sequence homology dalcronyltransferase, and no enzymatic knowledge of these expression products has been obtained.

[0004] On the other hand, anthocyanins are a type of flavonoid compound, and are a general term for anthocyanidins and compounds that are modified with sugars, methyl groups, acyl groups, sulfuric acid, gnoretathion, etc., such as red, blue, purple, and black purple. It is a pigment found in purple shiso, red cabbage and colored leaves.

[0005] The types of anthocyanins are extremely abundant, and hundreds of types of anthocyanins have been isolated from plants and their structures have been determined in previous studies. These anthocyanins exist in the living body in a glycosylated form. Most of them have been modified with glucose, and there are a few that have been modified with galactose-rhamnose. No one having a lucuronyl group has been found. However, cyanidin 3-0- (6 "-0-malonyl-2" -0-glucuronyl darcoside), isolated from the red daisy (Bellis perennis) petal in 1994, is the only anthocyanin and has a darcronyl group. It is an unusual dye in the structure (see Non-Patent Documents 11 and 12).

[0006] Plant flavonoid compounds such as anthocyanins have antioxidant, antibacterial, carcinogenic, arteriosclerotic, pile caries, halitosis, liver function, visual acuity, etc. In general, these actions are said to be expressed in the form of aglycones, but flavonoid aglycones are generally poorly water-soluble and in particular anthocyanin aglycones (antocyanidins). ) Is unstable in aqueous solution, etc. Intestinal absorption of these compounds is also carried out in the form of glycosides and their modified forms (eg, acylated forms) and has a half-life in vivo. Therefore, glycosides and their modified products have been shown to be longer. Therefore, glucosides of flavonoid compounds and their modifications are stabilized and absorbed by these compounds. These compounds can play an effective role in improving the functions of these compounds, such as improving their properties, controlling their physiological activities, and slowing the release of active substances.Gnoreclonylation is an effective flavonoid that can be used for such purposes. Although it is a decoration means, the enzyme catalyst and its gene that can achieve this have not been obtained yet.

[0007] In recent years, flower color modification techniques using genetic engineering techniques have become realistic. As described above, anthocyanins exist in modified forms in petal cells. This is because the modified form is more stable in physicochemical and biochemical ways, and can bring out subtle colors and tones (see Non-Patent Documents 11 and 12). By doing so, it can serve as a substrate for other modification reactions in the cell and conversely block it, and may play a role in metabolic control in vivo. It is an effective modification means that can be used for stabilization of flower color expression, color and tone control, and metabolic control by various genetic engineering techniques, but there are still no enzyme catalysts and their genes that can achieve this. Not obtained.

[0008] Non-Patent Document 1: C. D. King, G. R. Rios, M. D. Green, and T. R. Tephly.

UDP-Glucuronosyltransferases.Current Drug Metabolism, 1, 143-161 (2000) Non-Patent Document 2: Karl Walter Bock Vertebrate UDP-glucuronosyltransferases: functional and evolutionary aspects. Biochemical Pharmacology, 66, 691-696 (2003)

Non-Patent Document 3: Akira Iyanagi Biochemistry Vol. 75, No. 3, 234-241, (2003)

Non-Patent Document 4: Christopher D. King, Mitchell D. Green, Gladys R. Rios, Birgit L. CofFman, Ida S. Owens, Warren P. Bishop, and Thomas R. Tephly. The

glucuronidation of exogenous and inherent compounds by stably expressed rat and human udp— glucuronosyltransferase 1.1. Archives of Biochemistry and

Biophysics, 332, 92—100 (1996)

Non-Patent Document 5: Sahidan B. Senafi, Douglas J. Clarke, and Brian Burchell.

Investigation of the substrate specificity of a cloned expressed human bilirubin UDP-glucuronosyltransferase: UDP-suger specificity and involvement in steroid and xenobiotic glucuronidation. Biochem. J., 303, 233-240 (1994)

Non-Special Reference 6: Yasunori Kurosawa, Hidenari Takahara, and Masakazu Shiraiwa.UDP-glucuronicacid: soyasapogenol glucuronosyltransferase involved in saponin biosynthesis in germinating soybean seeds.Planta, 215, 620-629 (2002)

^^ Special Article 7: Margot Schulz and Gotttfried Weissenbock. Three specific

UDP-glucuronate: flavone-glcuronosyltransferases from primary leaves of Secale cereale.Phytochemistry, 27, 1261-1267 (1988)

Non-Special Terms 8: lgeyuki Nagashima, Masao Hiratani, and Takafumi Yoshikawa. Purification and characterization of UDP-glucuronate: baicalein

7-O-glucuronosyltransferase from Scutellaria baicalensis Georgi. Cell suspension cultures. Phytochemistry, 53, 533-538 (2000)

Non-Patent Document 9: Ho-Hyung Woo, Marc J. Orbach, Ann M. Hirsch, and Martha C. Hawes. Meristem— localized inducible expression of a UDP-glycosyltransferase gene is essential for growth and development in pea and alfalfa.Plant Cell , 11, 2303-2315 (1999)

Non-Patent Literature 0: Hiroaki Iwai, Nobutaka. Masaoka, Tadashi Ishii, and Shinobu Satoh. A pectin glucuronyltransferase gene is essentinal for intercellular attachment in the plantmeristem. PNAS, 99, 16319—16324 (2002)

Non-Patent Document 11: Kenjiro Toki, Norio Saito, and Toshio Honda.Three cyanidin 3-glucuronylglucosides from red flower of Bellis perennis.Phytochemistry, 30, 3769-3771 (1991)

Patent Document 12: Norio Saito, Kenjiro Toki, Toshio Honda, and Koshiro Kawase. And yanidin 3-malonylglucuronylglucoside in Bellis and Cyani in 3-malonylglucoside in

Dendranthema. Phytochemistry, 27, 2963-2996 (1988)

Disclosure of the invention

Problems to be solved by the invention

[0009] Therefore, in view of the conventional circumstances as described above, the present invention aims to obtain an enzyme capable of darcronylation of anthocyanin and its gene, and cyanidin which is a darcronylated anthocyanin accumulated in a red daisy petal. 3-0- (6 "_0-malonyl-2" -0-dalcronyl dalcoside), a gene that is expected to cause darcronylation of dalcronyl transferase (BpUGT) Cloning was also performed. Means for solving the problem

That is, the present invention has at least one amino acid sequence selected from SEQ ID NO: 1 (FIG. 1), SEQ ID NO: 2 (FIG. 2) and SEQ ID NO: 3 (FIG. 3) according to claim 1. Itfe child is.

According to the present invention, there is also provided a gene derived from red daisy, which is the base sequence represented by SEQ ID NO: 4 (FIG. 4) according to claim 2 and having the 1313th base sequence as an amino acid translation region. The

These genes are preferably genes encoding UDP-dalcronyltransferase.

Furthermore, according to the present invention, there is provided a transformant obtained by introducing the gene described above.

[0011] The present invention also includes at least one amino acid sequence selected from SEQ ID NO: 1 (FIG. 1), SEQ ID NO: 2 (FIG. 2) and SEQ ID NO: 3 (FIG. 3) according to claim 1. UDP—Gnoreclonyl transferase. Further, according to the present invention, the UD p -dalcronyl group transfer derived from red daisy comprising the base sequence represented by SEQ ID NO: 4 (FIG. 4) according to claim 2 and having the base sequence as an amino acid translation region An enzyme (BpUGT) is provided.

In the present invention, the reaction catalyzed by BpUGT is specifically represented by the following formula (1).

[0013] [Chemical 1]

The invention's effect

[0014] According to the present invention, there are provided a UDP-dalcronyltransferase (BpUGT) derived from red daisy and its gene. This enables the flavonoid compounds dalcroniol to improve the functions of these compounds, including stabilization of these compounds, improvement of absorbability, control of physiological activity, and sustained release of active substances. It provides effective modification means that can be used to stabilize flower color expression, adjust color and color tone, and control metabolic engineering using engineering techniques.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, specific embodiments of the present invention will be described.

II Gurukuro Maun 'Cochlea Measurement ¾

UDP-glucuronic acid, trifluoroacetic acid (TFA), and acetonitrile were purchased from Nacalai Testa. Potassium dihydrogen phosphate and dipotassium hydrogen phosphate were purchased from Daishin Chemical. The anthocyanin substrate used was the one isolated and purified at the Faculty of Horticulture, Minamikyushu University.

[0016] Reaction solution (0.5 mg / mL cyanidin 3-0-6 "-0-malonylglucoside) in an Epenpendorf tube I 0.01% trifluoroacetic acid 'methanol 5 / i L, 0.5 mg / mL UDP-glucuronic acid 3 μL of aqueous solution, 20 mM potassium phosphate buffer (pH 7.0, final volume of 100 μL) was prepared. The reaction solution was pre-incubated with a dry thermo unit DTU-2B (TAITEC) at 30 ° C for 5 min, and the reaction was started by adding the enzyme preparation. After incubating at the same temperature for 10 min, the reaction was stopped by adding 200 / i L of ice-cooled 0.5% trifluoroacetic acid aqueous solution. The produced reaction product was analyzed using reverse phase high performance liquid chromatography (HPLC). The amount of the enzyme preparation used for the enzyme reaction was adjusted so that the amount of the reaction product was 10% or less of the initial amount of the substrate.

[0017] The reaction product is reverse phase HPLC (DYNAMAX HPLC system; UV-VIS detector,

Quantification was performed using a SHIMADZU SPD-10A VP; column, flow rate 0.7 mL / min; detection wavelength, 520 nm. The separation conditions are as shown in Table 1. Solvents in 3G and 3G5G methods are A solution, 0.5% trifluoroacetic acid aqueous solution; B solution, 50% acetonitrile aqueous solution containing 0.5% trifluoroacetic acid, and Qu and IsoF method solvents are A solution, 0.1% trifluoroacetic acid aqueous solution; Liquid B, a 90% acetonitrile solution containing 0.1% trifluoroacetic acid.

[0018] [Table 1]

Time% B Time% B

[min] 3G 3G5G [min] Qu IsoF

0 35 30 0 18 15

3 35 30 3 18 15

17 55 55 18 100 100

18 100 100 19 100 100

23 100 100 20 18 15

24 35 30 25 18 15

30 35 30

Example 2 Complete Purification of Dizzy Petal-Derived UDP-Daluronyl Anthertransferase

Polybulupolypyrrolidone (PVPP), phenylmethylsulfonyl fluoride (PMSF), 2-mercaptoethanol (2-ME), ammonium sulfate, ethylenediamine tetraacetic acid (EDTA), ethylene glycol, glycerol, 2- [ (3-Cholamidopropyl) -dimethylammonio] -1-propanesulfonic acid (CHAPS) and sodium chloride sodium were purchased from Nacalai Tester, respectively. The distributor of the column for enzyme purification was described each time. The starting material, Digi Ichibana, was collected (stored immediately after flowering) and stored (-80 ° C) during the market period (late winter winter). The operation was performed at a total stroke of 4 ° C. BpUGT activity Evaluation was made based on dalcronyl group transfer activity using benzidine 3-0-6 "-0-malonyldarcoside and UDP-glucuronic acid as substrates.

[0020] 500 g of Daisy petals, 180 g of PVPP and 2,000 mL of buffer EX (100 mM potassium phosphate buffer, pH 7.5, 10% glycerol, 0.2% 2-ME, 5 mM EDTA, 0.5 mM PMSF) The petals were crushed and homogenized with a Warinda blender, and the fogenate crushed liquid was centrifuged (8000 X g, 20 minutes). The obtained supernatant was clarified by suction filtration (Whatman 114 filter paper) and recovered as a crude enzyme solution. The same operation was repeated for 2000 g of Daisy 1 petal to obtain 7000 mL of enzyme preparation.

[0021] 7000 mL of the crude enzyme solution was subjected to ammonium sulfate fractionation, and 30 70% fraction precipitates were collected. The resulting precipitate was dissolved in 2,500 mL of buffer EX. Polyethyleneimine was added to this solution to a final concentration of 0.3%, stirred for 10 minutes, and then centrifuged (8000 Xg, 20 minutes). The resulting supernatant was dialyzed against dialysis buffer (40 mM potassium phosphate buffer pH 7.5, 0.2% 2-ME).

[0022] Q Sepharose Fast Flow (300 mL, Amasham Amersham Bioscience, 5) equilibrated with buffer QS1.A (40 mM potassium phosphate buffer pH 7.5, 0.2% 2-ME) after dialysis The column was washed with buffer QS1.A under a load (5 mL / min). Subsequently, the protein bound to the column was eluted with buffer B (100 mM potassium phosphate buffer pH 7.5, 10% glycerol, 0.2% 2-ME) to obtain 1900 mL of enzyme preparation. The obtained sample was dialyzed against 20 mM potassium phosphate buffer pH 7.5, 0.2% 2-ME.

[0023] All dialyzed enzyme preparation was buffered with QS2.A (20 mM potassium phosphate buffer pH 7.5, 0.2%

Load (4 mL / min) onto a Q Sepharose Fast Flow packed column (100 mL, φ 2 cm x 20 cm) equilibrated with 2-ME, 10% glycerol, and wash the column with buffer QS2.A . Subsequently, the protein bound to the column was eluted with a 0 100% linear concentration gradient of buffer QS2.B (200 mM potassium phosphate buffer pH 7.5, 10% glyceronole, 0.2% 2-ME). The active fractions were collected to obtain 135 mL of enzyme preparation. This was concentrated to 25 mL by ultrafiltration.

[0024] A final concentration of 20% saturated ammonium sulfate was added to the total amount of the obtained enzyme preparation, and buffer PS.A (100 mM potassium phosphate buffer pH 7.5, 20% saturated ammonium sulfate, 0.2% 2-ME ) Equilibrated with Phenyl A Sepharose low sub packed column (15 mL Amersham Bioscience, HR16 / 10) was loaded (1 mL / min). An additional 100 mL was washed with buffer PS.A, and the active fraction passed through was collected.

[0025] Enzyme applied to column (AmashamHR10 / 10, Amersham Bioscience) packed with hydroxyapatite (BioRad CHT-typel) equilibrated with buffer CHT.A (5 mM potassium phosphate buffer pH 7.5, 0.2% 2-ME) The whole sample was loaded (1 mL / min), and the column was washed with 100 mL of buffer CHT.A. Elution is carried out by changing the mixing ratio of buffer CHT.A and buffer CHT.B (300 mM potassium phosphate buffer pH 7.5, 0.2% 2-ME), in steps of 10 mM from 10 mM force to 100 mM. Elution was carried out. At each stage, 50 mL of buffer was used. The active fraction eluted with 40 mM and 50 mM potassium phosphate buffer was collected and concentrated to 23 mL by ultrafiltration.

[0026] Mono Q HR 10/10 (equilibrated with buffer MQ.A (20 mM potassium phosphate buffer pH 7.5, 10% glycerol glycerol, 0.1% (w / v) CHAPS, 0.2% 2-ME) Amasham

The total amount of the enzyme preparation obtained in Amersham Bioscience) was loaded (1 mL / min), and the column was washed with buffer MQ.A (50 mL). Next, 0 to 100% linear concentration gradient of buffer MQ.B (300 mM potassium phosphate buffer H 7.5, 10% glycerol, 0.1% (w / v) CHAPS, 0.2% 2-ME) (for 350 min) The protein bound to the column was eluted.

[0027] Superdex 200 pg 26/60 (Amersham Bioscience) was added to buffer SPD (20 mM KPB pH 7.5,

After equilibration with 0.15 M NaCl, 0.2% 2-ME, 0.1% CHAPS), the entire amount of the enzyme preparation was loaded (0.9 mL / min), followed by separation with 500 mL of buffer SPD. The active fractions were collected and concentrated to 3 mL using Centricon (YM10).

[0028] RESOURCE PHE (Amasham Amersham) equilibrated with buffer RP.A (100 mM potassium phosphate buffer pH 7.5, 20% saturated ammonium sulfate, 0.2% 2-ME, 0.1% (w / v) CHAPS)

Bioscience, 6 mL) was loaded with the entire amount of enzyme preparation (1 mL / min), and the column was washed with buffer RP.A (6 mL). The active fractions passed through were collected and concentrated to 2 mL with a centricon.

[0029] SDS-PAGE was performed according to Laemmli's method, and a separation gel having an acrylamide concentration of 10% was used. Electrophoresis was performed at a constant current of 20 mA.

[0030] Protein was quantified using Protein Assay Kit (BioRad). Usi serum albumin (Sigma) was used as a standard protein. [0031] SDS-PAGE of the obtained purified enzyme preparation showed a uniform protein band corresponding to 54 kDa, and this protein band appeared and disappeared in SDS-PAGE in the fractions after each chromatography. Was consistent with that of BpUGT activity, so it was considered to be the main body of BpUGT activity responsible for the alklonylation of anthocyanins in red digi. In general, mammalian-derived dalcronyltransferase is a membrane protein localized in the endoplasmic reticulum, and it has been clarified in previous studies that plant-derived darcronyltransferase is also derived from Koganebana. Baicalein 7-0-Dalcronyltransferase and the like form dimers. However, this enzyme was considered to be a water-soluble protein because it did not require a protein solubilizing reagent such as a surfactant during extraction and could be easily extracted with an aqueous solvent. In addition, in gel filtration column chromatography of this protein, BpUGT activity was observed at a position corresponding to 49 kDa, suggesting that this enzyme is a monomeric protein with a molecular mass of 54 kDa.

Example 3 Gene Cloning of BOUGT

Poly (A) +-RNA was obtained from the daisy petals disrupted in liquid nitrogen using the Straight A's mRNA Isolation System (Novagen). The resulting poly (A) + RNA is in a bowl shape,

A double-stranded cDNA library was prepared using the ZAP-cDNA Synthesis Kit (STRATAGENE). The resulting 7-stranded cDNA was purified using SizeSep 400 Spin Columns (Amersham Pharmacia Biotech) and then cloned into the Uni-ZAP XR insertion vector according to the prescription of the ZAP-cDNA Synthesis Kit. This was packaged in phage using the Gigapack III Gold Cloning Kit (STRATAGENE) to construct a daisy petal cDNA fuzzy library.

[0033] Of the obtained purified enzyme preparation, 20 pmol of the enzyme was subjected to SDS-PAGE, and after staining with Coomassie Blue, the target band was cut out. This was treated with ricinole endopeptidase at 35 ° C overnight, and the resulting BpUGT digestion product was separated by reverse-phase HPLC for peptide mapping. Each peak was collected and subjected to amino acid sequence analysis using an amino acid sequencer (Procise 494 HT Protein Sequencing Systern). As a result, the three internal amino acid sequences shown in Table 2 below could be read. Based on # 1 and # 2 of these sequences, three types of degenerate primers (Fl, Rl, R2) as shown in Table 3 below were prepared. [0034] [Table 2]

BpUGT partial amino acid sequence read by Edman degradation method

# 1 Ser Gly Pro Asp Phe Glu Thr lie Leu lie Lys

# 2 Asn Met Glu Ala Glu Val Asp Gly lie Val lie Glu Asn Leu Val # 3 Arg Glu Glu lie Ala Ala Val Val Arg Lys

[0035] [Table 3] Degenerate primer designed from partial amino acid sequence primer F1 5 '-GGI CCI GAY TTY GAR ACI ATH TTR ATH AAR-3'

primer Rl 5 '-ARR TTY TCD ATI ACD ATI CCR TCI ACY TC-3,

primer R2 5 '-CCR TCI ACY TCI GCY TCC ATR TT-3,

I: Inosine, R: A or G, Y: C or T, H: A or C or T, D: A or G or T

[0036] RT-PCR using primers F1 and Rl was performed using poly (A) + RNA obtained from a daisy petal as a saddle type. In this experiment, the QIAGEN OneSt RT-PCR Kit was used, and the composition of the reaction solution was in accordance with the kit prescription. Reaction conditions are 50 ° C, 30 minutes; 94 ° C, 15 minutes; [94 ° C, 15 seconds; 50 ° C, 15 seconds; 72 ° C, 30 seconds] X 35 cycles, 72 ° C, 10 minutes went. About the obtained PCR product nested PCR (reaction mixture: 1 ^st PCR product, 100 ng; ExTaq buffer, 1 X; dNTPs, 200 mM; primer Fl, 1 μ 1; primer R2, 1 μM; ExTaq polymerase (TaKaRa ), 2.5 U; reaction conditions 94 ° C, 2 minutes; [94 ° C, 15 seconds; 55 ° C, 15 seconds; 72 ° C, 30 seconds] X 30 cycles, 72 ° C, 7 minutes) .

The amplified DNA fragment was TA cloned into the pCR2.1-TOPO vector (TOPO TA cloning Kit, Invitrogen), and the base sequence was analyzed by a DNA sequencer (BECKMAN COULTER CEQ 2000XL DNA Analysis System). As a result of homology search using the estimated amino acid sequence, the obtained 965 bp PCR product showed the highest homology with the known flavonoid glycosyltransferase gene. Based on this, it was judged that there is a high possibility that this partial cDNA fragment is a part of the BpUGT gene. Using this partial cDNA sequence as a cage, a DIG (digoxigenin) -labeled probe was prepared by PCR DIG Probe Synthesis Kit (Roche Biochemicals).

[0037] Plaque hybridization using the prepared DIG-labeled BpUGT-specific probe The cDNA library was screened by The daisy petal cDNA phage library was infected with E. coli XLl-Blue MRF ', and about 50,000 plaques were formed on each of the eight NZY agar media. The phage DNA of the obtained plaque was adsorbed on Hybond N (Amersham Pharmacia Biotech) and processed by the method recommended by the manufacturer. Hybridization buffer containing the above DIG-labeled probe (100 ng / mL) is used as a hybridization buffer (30% formamide, 5xSSC, 0.02% SDS, 2% blocking reagent (Roche Diagnostics), 0.1% N_ Lauroyl sarcosine) at 37 ° C for 16 hours. Wash the membrane with washing solution (0.1 X SSC, 0.1% SDS) (65.C, 15 minutes x 2), then use DIG Labeling and Detection KIT (Roche Diagnostics) to test positive plaques as recommended by the manufacturer. As a result, 16 positive clones were finally obtained. The obtained positive clone was subcloned into the pBluescript SK (-) phagemid vector by the method recommended by STRATAGENE (in vivo excision). As a result of base sequence analysis, BpUGT cDNA with 1317 bp ORF encoding all the partial amino acid sequences obtained from purified BpUGT was obtained (SEQ ID NO: 1: Figure 1). ).

[0038] The conserved sequence common to the glycosyltransferase superfamily (GT) was present in the deduced amino acid sequence of the BpUGT gene (SEQ ID NO: 2: Fig. 2). In Fig. 2, the black line is a conserved sequence of glycosyltransferase and is assumed to be a sugar nucleotide recognition site. This conserved sequence is thought to be the binding site for sugar nucleotides. Enzymes belonging to the GT family are broadly divided into GT-A and GT-B due to their structure. The biggest difference between GT-A and GT-B is the position of the conserved sequence that seems to be the sugar nucleotide binding site. It is known to exist on the N-terminal side in GT-A and on the C-terminal side in GT-B. ing. The conserved sequence of this enzyme is present on the C-terminal side, as is the case with the vertebrate-derived dalcuronyltransferase, and the predicted amino acid sequence of this enzyme also has the DXD motif on the N-terminal side that is characteristic of GT-A Because of the strength, I guess it belongs to GT-B.

[0039] So far, the glycosyltransferase superfamily has been classified into 73 families (James A. ampbell, Giaeon J. Davies, Vincent Bulone and Bernard Henrissat. A classification of nucleotide-diphospho-sugar glycosyltransferases based on amino acid Sequence similarities.See Biochem J. 326,929—942 (1997)).

(http: 〃 afinb.cnrs-mrs.fr/CAZY/index.html). These families are classified based on the deduced amino acid sequence of each enzyme gene, and enzymes belonging to the same family are presumed to retain similar three-dimensional structures. Using the BpUGT gene isolated in this study and the deduced amino acid sequences of the enzyme genes belonging to each family, a phylogenetic tree was created. As a result, this enzyme gene belongs to the same group as the family 1 enzyme. Therefore, we considered that BpUGT is classified into Family 1. Glycosyltransferases involved in the synthesis of secondary metabolites and vertebrate dalcronyltransferases involved in glucuronidation that have been found in plants so far belong to Family 1. However, there was no significant homology between this enzyme and animal-derived gnolecronyltransferase other than the sugar nucleotide binding site, which is low. In addition, the similarity to plant-derived anthocyanin glycosyltransferase is 15% to 40%, and the similarity is similar to the flavonoid 1,2 rhamnosyltransferase derived from pomelo (Fundan), which is the most similar to this enzyme. Was around 40%.

Next, as a result of creating an evolutionary phylogenetic tree based on the deduced amino acid sequences of enzyme genes belonging to Family 1, this enzyme is located away from mammalian-derived dalcuronyltransferases, and is a plant-derived GT population. Belonged (Figure 3). Based on this, it was considered that gnorechronyl transferase activity was obtained by evolution different from that of mammal-derived gnoleclonyl transferase known to have this enzyme power.

^ _Research (Tnomas Vogt and atnk Jones. Glycosyltransferases in plant natural product Synthesis: characterization of a supergene family. See Trends in Plant Science, 5, 380-386 (2000)). Clusters have been found and each has the following characteristics. Cluster I contains many enzymes that transfer the darcosyl group to the 3-position of flavonols or anthocyanins. Cluster II consists of an enzyme that transfers the dalcosyl group to the 5-position of anthocyanin and other enzymes with unknown functions. Although the commonality of cluster III has not been clarified, it is considered to be an enzyme having a broad substrate specificity and transferring a gnorecosinole group in a site-specific manner. Within each cluster, the homology is about 40-50%. The homology is about 20%. The BpUGT gene did not belong to any of these three clusters and was closely related to the flavonoid-rhamnosyltransferase. These transglycosylation reactions catalyzed by BpUGT and rhamnosyltransferase both have the property of adding a new sugar residue to the sugar residue already bound to the flavonoid aglycone. If more types of enzymes are discovered, there is a possibility of forming new clusters.

[0041] Example 4 Construction of ¾-species expression system in yeast of BOUGT

Agar, Galactose, Glucose, Polyethyleneimine (PEI), Glycerol, Adenine Sanoleate, Uracil, Lithium Acetate, EDTA, Polyethylene Glycol 3000, and various amino acids were purchased from Nakarai Tester. Yeast Nitrogen Base without amino acids, Bacto peptone, and yeast extract were purchased from DIFCO. Restriction enzymes were purchased from TaKaRa or New England Biolabs.

[0042] Primers Xhol-F and Kpnl-R, which introduce an Xhol site at the 5 'end and a Kpnl site at the 3' end of BpUGT cDNA, were synthesized. PBluescript-BpUGT having the ORF of BpUGT obtained in Example 3 was subjected to PCR (reaction solution composition: pBluescript-BpUGT, 400 ng; pfli buffer, 1 X; dNTPs, 200 mM; primer XhoI_F, 1 μΜ; primer

Kpnl-R, 1 μΜ; pfu Turbo polymerase (TOYOBO), 2.5 U; Reaction conditions: 96. C, 2 minutes, [96 ° C, 45 seconds; 55 ° C, 45 seconds; 72 ° C, 2 minutes] X 30 cycles, then 72 ° C 10 minutes) It refine | purified by the method of. The obtained DNA was subcloned into the CR4Blunt-TOPO vector using the Zero Blunt TOPO PCR Cloning Kit for Sequencing (Invitrogen) according to the method recommended by the manufacturer. As a result, to obtain a plasmid _(P CR4-BpUGTxk) for揷入full length BpUGT. Nucleotide sequence analysis confirmed that mutations occurred in the BpUGT gene.

Complete digestion of pCR4-BpUGTxk with Xhol and Kpnl, recover the generated approximately 1400 bp fragment, and use the resulting DNA fragment for Saccharomyces cerevisiae expression vector

Subcloned into the Xhol and Kpnl sites of pESC-TRP (STRATAGENE)

(pESC-TRP-BpUGT) ₀ pESC-TRP-BpUGT was used to transform E. coli XL1-Blue. [0043] Using 2 μg of pESC-TRP-BpUGT extracted from the cultured pESC_TRP-BpUGT XLl-Blue, the Saccharomyces cerevisiae YPH499 strain was transformed according to the method recommended by pESC Yeast Epitope Tagging Vectors (STRATAGENE).

[0044] The obtained transformant was cultured with shaking (30 ° C, 160 rpm) for 3 days in 50 mL of SD Trp dropout medium prepared by the method recommended by pESC Yeast Epitope Tagging Vectors (STRATAGENE). When cell turbidity (OD) reaches 0.5, centrifuge (6,000 X g, 15 minutes)

600

The cells were collected and washed twice with 0.1 mL of SG dropout medium prepared by the method recommended by the kit. The cells were resuspended in SG Trp dropout medium 50 mL, was inoculated suspension 4 mL to SG Trp dropout medium 4 L, the main culture was carried out (30 ° C, 160 rpm) 0 7 days in this condition After culturing for 10 days, when the OD force became S1.4-1.5, the cells were collected using centrifugation (6,000 X g, 15 minutes).

600

The cells were stored frozen at -30 ° C.

The following operations were performed at a total stroke of 4 ° C. 26 mL disruption buffer (100 mM potassium phosphate buffer pH 7.5, 10% glycerol, 0.5% 2-ME, 5 mM EDTA, 0.5 mM PMSF, 0.1% (w / v) CHAPS) per 12.6 g of cells And suspended well. A glass bead of φ 0.5 mm was added to the suspension until the beads were above the liquid level. This was set in a multi-bead shocker (Yasui Kikai), and the cells were crushed (crushing conditions: [2500 rpm, 1 minute; 0 rpm, 1 minute] X 5 times). The beads and the extract were separated by centrifugation (> 1000 X g, 1 minute), and the extract was centrifuged again (10,000 X g, 20 minutes). The supernatant was supplemented with PEI to a final concentration of 0.3%, stirred for about 10 minutes, and then centrifuged (10,000 × g, 20 minutes). The obtained supernatant was recovered as a crude enzyme solution. By the method already described, it was confirmed that BpUGT activity was present in the crude enzyme solution.

[0045] The total amount of the obtained crude enzyme solution was added to buffer QS3.A (20 mM potassium phosphate buffer pH 7.5, 0.2%

2-ME), load (1 mL / min) into a Q Sepharose Fast Flow packed column (30 mL, φ 1 cm x 30 cm) equilibrated with buffer QS3.A, and buffer QS3 The column was washed with A. Subsequently, the protein bound to the column was eluted with a 100% linear concentration gradient of buffer QS3.B (300 mM potassium phosphate buffer pH 7.5, 0.2% 2-ME). The active fractions were collected to obtain 140 mL of enzyme preparation. This is Amicon Ultra-15 10,000 MWCO

Concentrated to 12 mL using (MILLIPORE). [0046] Example 5: Identification of BOUGT recombinant reaction product

The enzyme reaction was carried out by the method described in Example 1, and the product was purified and recovered by HPLC. Then, acetonitrile was removed using an aspirator, loaded onto HPLC again and eluted with methanol. The obtained sample was concentrated to 1 mL with an evaporator, and a part of the obtained sample was subjected to HPLC analysis and FAB-MS analysis to identify BpUGT recombinant reaction products. In HPLC analysis, the retention time of the peak considered to be a reaction product was consistent with the main pigment in Daisy 1 petal. Based on this, it was presumed that this enzyme catalyzes the reaction of adding a gnorechloryl group to the 2 "position of cyanidin 3_0_6" _0-malonyl gnorecoside. As a result of fractionation of the peak considered to be a reaction product and structural analysis by FAB-MS spectrum, the molecular weight of cyanidin ₃ _ ₀ _ ( ₆ "-0-malonyl_2" _0_ glucuronyl glucoside) As a result, it was confirmed that the expression product of the gene isolated this time has BpUGT activity.

[0047] Example 6 BOUGT gene expression analysis

Daisy's tissues (petals, cylindrical flowers, gakuguki, leaves) were ground in liquid nitrogen, and total RNA was obtained using RNeasy Miniprep Kit (QIAGEN). Absorbance was measured to determine the concentration and purity of tota nore RNA.

[0048] Primers specific to the BpUGT cDNA sequence were designed so that the amplified fragment was 150 bp or less. This primer was designed in accordance with the conditions described in the QuantiTect SYBR Green RT-PCR Kit (QIAGEN) manual. For the RT-PCR reaction, QuantiTect SYBR Green RT-PCR Kit (QIAGEN) was used, and amplification of the PCR product was monitored by Light Cycler Quick System 330 (Roche). For the calibration curve, pET32a_BpUGT was used as a saddle type, and 0.2 μg of total RNA derived from each digitized tissue was used as a saddle type. PCR reaction conditions were (50 ° C, 20 min; 95 ° C, 15 min, then [94 ° C, 15 sec; 55 ° C, 20 sec; 72 ° C, 10 sec] X 35 cycles; then 95 ° C 0 seconds; 65 ° C, 15 seconds), calibration curves were prepared each time.

A calibration curve was prepared with the horizontal axis representing the number of cycles and the vertical axis representing fluorescence intensity, and the number of copies of the BpUGT gene transcript in each daisy tissue was determined using this standard curve.

[0049] Fig. 4 shows the transcription amount (A) and specific activity (B) of the BpUGT gene in each daisy tissue. As a result, the transcription distribution of this enzyme gene in each daisy tissue was in good agreement with the distribution of BpUGT activity. This strongly suggests that the expression product of the BpUGT gene obtained in this study is the main body of BpUGT activity in Red Digi. In addition, since this enzyme gene was specifically expressed in petals showing coloration, its physiological significance is thought to be specific to anthocyanin dalcronylation.

[0050] Example 7 Experimental material for BOUGT characterization

Acetic acid, glycine, hydrochloric acid (HC1), tris (hydroxymethyl) aminomethane (Tris), hydroxylated rhodium (KOH), N-ethylmaleimide (NEM), jetyl pyrocarbonate (DEPC), 30% Monia water and metal salts were purchased from Nacalai Testa. UDP-glucose, UDP-galactose, uridine, UMP, UDP, UTP, / 3-estradiol, 17-estradiol, 1_naphthol, 2-naphthol, 4-methylumbelliferone, p-nitrophenol, and human UDP-Dalcronyltransferase 1A1 (UGT1A1) was purchased from Sigma. Silica gel 60F (MERCK) was used for the TLC plate. UDP- [U_ ¹⁴ C] DARC

254

Ronic acid was purchased from Wako Pure Chemical Industries through the Japan Isotope Association. Cianidin

The purified enzyme preparation was characterized for dalcronyl group transfer activity using 3-0-6 "-0-malonyl darcoside and UDP-glucuronic acid as substrates.

[0051] Example 8 BOUGT reaction ϋΗ dependence and DH stability

The buffer solution in the reaction solution described above was changed, and the pH dependence of the BpUGT recombinant was evaluated. A 20 mM acetic acid-NaOH buffer solution was used at pH 4.0 ^ 6.0, and a 20 mM phosphoric acid-KOH buffer solution was used at H 6.0-7.5. For H 7.5-9.0, 20 mM Tris-HC1 buffer was used, and for pH 9.0-11.0, 20 mM glycine-KOH buffer was used.

The enzyme preparation was diluted (10 times) with the above-mentioned buffer solution at each pH and incubated at 20 ° C for 17 h. The residual activity of the enzyme preparation after the treatment was measured by the method described in Example 1, and the pH stability of the BpUGT recombinant was evaluated.

As shown in FIG. 5, the optimal reaction pH of this enzyme was 8.0 (FIG. 5A) and was stable at pH 7 (FIG. 5B).

[0052] Example 9 某晳 Characteristic Analysis (Part 1)

. The initial reaction rate of this enzyme was determined, and cyanidin was calculated from the reciprocal plot (1 / v vs. 1 / [S1]). 3-0-6 "-0-malonyl darcoside is calculated as K and V for UDP-glucuronic acid

m max

It was. In addition, instead of UDP-glucuronic acid, UDP-glucose and UDP-galactose were used for the enzymatic reaction. Also, instead of cyanidin 3-0-6 "-0_malonyl darcoside, pelral gonidine 3-〇-6, shi-malonyl darcoside, cyanidin 3-0-darcoside, delphidinidine 3-〇-darcoside, cyanidin 3-〇_3 ", 6" _0 -dimalonyl darcoside, pelargonidin 3,5-〇_diglucoside, pelargonidin 3-〇-6 "-〇_malonyl darcoside -5-0-darcoside, Cyanidine, genistein 7-0-Dalcoside, daidzein 7-0-Dalcoside, Geyustin 7-〇_6 "_0-malonyl darcoside, daidzein 7_0_6" _0-malonyl dalcoside, genistein, daidzein, quercetin 3-0 -darcoside , Quercetin 3-〇-6 ”-〇_malonyldarcoside was used for the enzymatic reaction.

[0053] (1) Specificity of sugar residue donor

Among the known dalcronyltransferases, there is also the ability to use sugars such as UDP-glucose as a sugar residue donor in addition to UDP-glucuronic acid. It is known to be a residue donor. The recognition of the sugar residue donor of this enzyme was very specific for UDP-gnorecronate, as well as many known gnorecronyltransferases.

[0054] (2) Sugar residue receptor specificity

K for Shianijin 3-0-6 "-0- malonyl Darco Sid is 32.4 ^s-1, Shianiji

cat

K for 3-0-6 "-0-malonyldarcoside is 19.4 μΜ, for UDP-glucuronic acid

m

The K was 455 μΜ. The V of this enzyme is 1,300 nmol / mg / s, which is already m max.

1,000 times greater than V of known vertebrate dalcronyltransferase

max

. This enzyme showed the highest activity against cyanidin 3-0-6 "-0_malonyl darcoside (relative activity 100%), and also showed an effective activity against the following anthocyanins: pelargonidin 3-〇 -6 "-0_malonyl darcoside (86% relative activity), cyanidin 3-0-darcoside (31%), delphidinidine ₃ _0 -darcoside (14%). However, the following flavonoid compounds accept dalcronyl groups Could not be a body (relative activity, less than 0.1%): cyanidin 3_0-3 ", 6" -〇 dimalonino legnorecoside, peranolegonidin 3, 5-〇_dignolecoside, peranolegonidin 3-〇-6,, -〇-malonyl darcoside-5-0 -darcoside, quercetin 3-0 -darcoside, quercetin 3-0-6, -O-malonyldarcoside, daidzin, genistin, daidzein 7-0-6, 0-malonyldarcoside, cyanidin, quercetin, daidzein, genistein. P-nitrophenyl J3 -D -Darcoside could not be a substrate.

[0055] Example 10 Analysis of cocoon characteristics (Part 2)

In order to compare the substrate specificity of BpUGT and known vertebrate-derived gnolecuronyltransferases, the presence or absence of glucoronyltransferase activity of BpUGT against compounds of the following formula, which is a good substrate for human liver-derived UGT1A1, was investigated. The detection of glucoronyltransferase activity using the described radioisotope is mainly performed by the method of Bansal et al. (Surendra K. Bansal and. Gessner. A unined method ror the assay of uridine.

diphosphoglucuronyltransferase activities toward various aglycondes using uridine diphospho [u-14c] glucuronic acid. Analytical Biochemistry, 109, 321-329 (1980)).

[0056] β-estradiol, 17a-estradiol, 1_naphthol as sugar residue receptors,

2-Naphthol, 4-methylumbelliferone, and p-nitrophenol were used. The definition of 1U is the amount that can catalyze 1 nmol of dalcronyl group transfer at 37 ° C for 1 minute.

UDP- [U- ¹⁴ C] glucuronic acid (0.02 μ Ci / μ L, 225 mCi / mmol) 5 μL and final concentration of 3 mM of each sugar residue receptor in 20 mM Tris-maleate buffer pH 7.4 Added (total volume 20 μL). UGT1AK 5 mg protein / mL) 10 μL, (0.034 U equivalent) was added to each tube to initiate the reaction. The reaction was stopped at 30 ° C for 30 minutes and 100% ethanol was added to stop the reaction.

Instead of UGT1A1 10 in a reaction mixture of the same composition, add 2 μΐ (0.093 U equivalent) of natural BpUGT (0.12 mg / mL), and add 20 mM Tris-maleic acid buffer pH 7.4 to make the total volume. Adjusted to 30 z L. This reaction solution was reacted at 30 ° C. for 30 minutes in the same manner as in (1), and the reaction was stopped by adding 100 μL of 100% ethanol.

[0057] 50 μL of each of the obtained reaction solutions was spotted on TLC. Separation was performed using η-butanol: acetone: acetic acid: 30% aqueous ammonia: water (70: 50: 18: 1.5: 60) as a developing solvent. The developed spot of the reaction product was observed by detecting ¹⁴ C radiation with a molecular weight detector (BioRad, GS525). [0058] As a result, for human UGT1A1, a force capable of confirming the dalcronyl group transfer activity for all these substrates was a force for which no activity was detected for BpUGT (Fig. 6). This force We considered that the physiological role of this enzyme is not related to detoxification like the known vertebrate-derived gnolecronyltransferase. Some vertebrate-derived dalcuronyltransferases exhibit dalcronyl group transfer activity against flavonoids such as anthocyanins, and UGT1A1 is also known to exhibit activity against flavonoid aglycones ( CD King, GR Rios, MD Green, and TR Tephly.

UDP-Glucuronosyltransferases. Current Drug Metabolism, 1, 143-161 (2000), and EJ Oliveira, DG Watson.In vitro glucuronidation or kaempferol and quercetin by human UGT-1A9 microsomes.FEBS Letters, 471, 1—6 ( 2000)). Therefore,

The activity of UGT1A1 against cyanidin 3-0-darcoside and cyanidin 3-〇-6 "-〇-malonylglucoside was not detected.

[0059] Example 11 阳.

The enzyme preparation was diluted (20 times) with a buffer containing final concentrations of 5 mM NEM and 1 mM DEPC and incubated at 20 ° C. for 20 minutes. As a control, the enzyme preparation was similarly treated with a buffer solution containing no NEM. By measuring the residual activity of each enzyme preparation after treatment and comparing it with the control, the effect of NEM, an alkyl group reagent specific for the SH group, was examined. In addition, the enzyme activity was measured with an Atsy system containing 0.1 mM of various divalent metal ions, and compared with the enzyme activity under the same conditions without the metal ion, each metal ion relative to the malonyl group transfer activity was measured. The effect was investigated. Furthermore, enzyme activity was also measured in the assembly containing 1 mM uridine, UMP, UDP, and UTP, and the product inhibition of BpUGT was examined by comparing it with the enzyme activity in the same assay that did not contain these. . In the Atsy system for evaluating the influence of these inhibitors, 20 mM potassium phosphate buffer pH 7.0 was used as the reaction buffer.

[0060] As a result, the degree of inhibition increased with the increase of the force S and phosphate group, which were not inhibited at all by uridine, and this enzyme activity was most strongly inhibited by UDP. This suggests that the phosphate group may be involved in the recognition of the sugar residue donor.

[0061] In addition, this enzyme contains malonic acid, which is a part of cyanidin 3_0-6 "-〇-malonyldarcoside, It was not disturbed by gnole course.

[0062] From the sequence comparison of enzymes belonging to Family 1, it has been found that histidine residues are important and highly conserved, and it is speculated that they play an important role in catalytic activity. In the enzyme, inhibition of catalytic activity was not observed by DEPC, which is a reagent for modifying histidine residues. The enzyme was completely inactivated by NEM, an alkylating agent specific for cysteine residues.

[0063] The effects of various metal ions on this enzyme were examined. Known vertebrate transferases require divalent metal ions (Mg ²⁺ , Mn ²⁺ ) for catalytic activity (Iyanagi Satoshi 75th No. 3, 234-241, 2003 , * 5 and Christopher D. King, Mitchell D. reen, Gladys R. Rios, Birgit L. CofFman, ida S. Owens, Warren P. Bishop, and Thomas R. Tephly.The glucuronidation of exogenous and endogenous compounds by stably expressed rat and human UDP-glucuronosyltransferase 1.1. Archives of Biochemistry and Biophysics, 332, 92-100 (1996)). However, this enzyme does not require these cofactors for its catalytic activity, and although inhibition of activity by metal ions was observed, activation was not observed. In addition, when the enzyme was treated with 5 mM EDTA for 20 minutes, the enzyme activity was not affected, suggesting that the enzyme does not require metal ions for its catalytic activity. . In addition, the activity was not detected in the presence of Hg ²⁺ and Cu ²⁺ , but in recent reports, anthocyanin force g ²⁺ or Cu ²⁺ forms a metal complex and precipitates, which makes it colorless. Therefore, these metal ions may not act on the enzyme but may act on the anthocyanin substrate.

Brief Description of Drawings

[0064] FIG. 1 shows the base sequence (SEQ ID NO: 1) of the enzyme gene according to the present invention. The first underline in the sequence indicates the translation start codon, and the last underline indicates the translation stop codon.

FIG. 2 is an amino acid sequence (SEQ ID NO: 2) of an enzyme according to the present invention. Underlined sequences represent the conserved sequences in glycosyltransferase series 1.

FIG. 3 is a phylogenetic tree of glycosyltransferase family 1. Figures I, II, and III show the cluster (s) of the plant glycosyltransferase family 1. In addition, the GeneBank accession number of each enzyme in FIG. 3 is as follows. Tobacco IslOa, U32643; Tobacco Is5a, U32644; tomato Twil, X85138; Dorotheanthus 5GT,

Y18871; Scutellaria 7GT, BAA83484; petunia RhamT, Z25802; Perilla 5GT1,

AB013596; Perilla 5GT2, AB013597; Verbena 5GT, BAA36423; Petunia 5GT, 027455; Barley 3GT, X15694; Maize 3GT, X13501; Grape 3GT, AF000371; Perilla 3GT, AB002818; Gentiana 3GT, D85186; Petunia 3GT, AB027454, Ipomea 3GT AF028237; citrus RhamT, AY048882.1; Eggplant 3GT, X77369; Gentiana 3, GT, BAC54092; Nierembergia RhamT, AB078511.1; UGT1A1, P22309; UGT1A3, P33503; UGT1A6, P19224; UGT2A1, CAB41974.1; U1662B UGT2B10, P36537; UGT2B15, A48633

Fig. 4] Comparison of BpUGT gene transcription and activity.

FIG. 5 is a graph showing the pH dependence and pH stability of BpUGT. Figure 5 (A) shows the pH dependence of BpUGT (30 ° C, 20 minutes), and Fig. 5 (B) shows the pH stability (20 ° C, 12 hours treatment). In addition, each plot shows age: 20 mM acetic acid-NaOH; ■: 20 mM phosphoric acid-KOH; A: 20 mM

Tris-HCl; X: 20 mM glycine-KOH.

FIG. 6 shows the specificity for the sugar residue donor.

Claims

The scope of the claims

[1] UDP-Daluronyl transferase having an action of catalyzing a reaction of acting on anthocyanin to produce the corresponding dalclonyl anthocyanin, (A) an amino acid sequence described in SEQ ID NO: 2 or ( B) It has a modified amino acid sequence obtained by inserting, deleting or substituting one or more amino acid residues within a range in which the catalytic activity can be maintained for the amino acid sequence. Item 3. The UDP-dalcronyl transferase according to Item 2.

[2] A nucleotide sequence encoding a UDP-durcronyltransferase having an action of catalyzing a reaction that acts on anthocyanin to produce a corresponding darcronyl anthocyanin, (a) described in SEQ ID NO: 1 in the sequence listing Base sequence or (b) Insertion, deletion or substitution of one or more bases within the range in which the action of the UDP-dalcronyltransferase encoded by the base sequence is maintained for the base sequence of SEQ ID NO: 1. A nucleotide sequence encoding a UDP-dalcronyltransferase, characterized in that it comprises a mutated nucleotide sequence obtained in the above manner.

[3] The base sequence according to claim 2, wherein the mutated base sequence hybridizes with the base sequence of SEQ ID NO: 1 under stringent conditions.

[4] The gene according to claim 2, wherein the gene is derived from red daisy (Bellis perennis).

[5] The UDP monodaluronyl transferase according to claim 1, which is derived from red daisy (Bellis perennis).

[6] A transformant, wherein the gene according to claim 2 or claim 3 or claim 4 is introduced.

[7] A process for producing dalclonylated anthocyanins using the UDP-gnolechronyltransferase according to claim 1 or 5

[8] A method for producing a gnoreclonylated anthocyanin, comprising using the transformant according to claim 6.