Application of citrus flavonoid glycosyl transferase CitUGT73A78 in biosynthesis of flavonoid glycoside

Abstract

The invention provides an application of citrus flavonoid glycosyl transferase CitUGT73A78 in biosynthesis of flavonoid glycoside, and belongs to the technical field of gene engineering and enzyme engineering. According to the present invention, the function of the CitUGT73A78 gene is cloned and verified, and the in vitro enzyme activity test proves that the CitUGT73A78 recombinant protein has broad-spectrum catalytic activity, and can efficiently catalyze the glucosylation of a variety of flavonoids so as to generate the corresponding flavonoids 7-O-glucoside and / or flavonoids 4 '-O-glucoside. An in-vivo transient overexpression test also proves that the CitUGT73A78 gene has a promoting effect on the accumulation of the content of the citrus flavonoid 7-O-glucoside. The invention not only provides a preparation method for the production of flavonoid 4 '-O-glucoside, has the advantages of simplicity, convenience, high catalytic efficiency, low cost, greenness and safety, but also can be applied to nutritional quality improvement and germplasm innovation of citrus fruits.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and enzyme engineering, and in particular relates to application of citrus flavonoid glycosyltransferase CitUGT73A78 in biosynthesis of flavonoid glycosides. Background Art

[0002] Citrus is one of the world's most common fruits, and it is also an important plant with both medicinal and edible properties, with extremely high economic and medicinal value. For example, dried tangerine peel and tangerine peel made from dried citrus peel can be used as raw materials for Chinese medicinal materials. They have multiple functions such as anti-oxidation, anti-inflammatory, lowering blood lipids, and relieving cough and phlegm, and are widely used in food, health products, and medicine. These health effects are mainly due to the rich active ingredients in citrus, such as flavonoids, vitamin C, and carotenoids. At present, more than 80 flavonoids have been isolated and identified in citrus, mainly in the form of glycosides, and the most abundant content is flavanone-O-glycosides, flavonoid-O-glycosides, and flavonoid-C-glycosides. In the metabolic synthesis pathway of flavonoids, naringenin, as the central precursor, is modified by hydroxylases or oxymethoxytransferases to form flavonoid aglycones such as hesperidin, eriodictyol and isosakurantei, which are then converted into corresponding flavonoid 7-O-glucosides by 7-O-glucoside transferase (7GT), and then converted into bitter neohesperidin (such as naringin) and non-bitter rutin (such as hesperidin) by 1,2-rhamnosyltransferase (1,2-RhaT) or 1,6-rhamnosyltransferase (1,6-RhaT).

[0003] Flavonoid 7-O-glucoside is a compound formed by the hydroxyl group at the 7th position of the A ring of the flavonoid molecule and the sugar molecule connected by a glycosidic bond. Flavonoid 4'-O-glucoside is a compound formed by the hydroxyl group at the 4' position of the B ring of the flavonoid molecule and the sugar molecule connected by a glycosidic bond. Flavonoid 7-O-glucoside or flavonoid 4'-O-glucoside compounds not only play an important role in plant growth and development and stress response, but also have multiple pharmacological activities such as antioxidant, anti-inflammatory, hypolipidemic and anti-cancer. For example, apigenin 7-O-glucoside has antioxidant and anti-inflammatory effects, and luteolin 7-O-glucoside has hypoglycemic, hypolipidemic and cardiovascular protection effects. At present, there are three main ways to obtain this type of compound: although the extraction and separation method can be directly obtained from plants, the content of flavonoid 7-O-glucoside or flavonoid 4'-O-glucoside in most plants is very low and the composition is complex, which makes separation difficult and the yield is low, making it difficult to meet market demand; the chemical synthesis method requires strict reaction conditions, involves cumbersome hydroxyl protection and deprotection processes, and requires the use of specific reagents and expensive catalysts, which is easy to cause environmental pollution; in contrast, the in vitro enzymatic method has the advantages of simple operation, mild conditions, green environmental protection, good stereoselectivity and regioselectivity. Therefore, the discovery and development of efficient flavonoid glycosyltransferases is of great significance for the industrial production of flavonoid 7-O-glucoside or flavonoid 4'-O-glucoside.

[0004] The glycosylation modification of flavonoids in plants is mainly catalyzed by UDP-glycosyltransferase (UGT). This enzyme can transfer the sugar group from the UDP-sugar donor molecule to the flavonoid receptor molecule. Its typical structural feature is that the C-terminus contains a conserved motif (PSPGbox) composed of 44 amino acid residues, which is responsible for binding to the UDP-sugar donor, while the N-terminal sequence is highly variable and specifically recognizes and binds to specific substrate molecules. With the rapid development of genomics and bioinformatics, the study of the plant UGT family has made important progress: 107 UGT family members were found in Arabidopsis and divided into 14 phylogenetic groups (AN), and then four new phylogenetic groups O, P, Q and R were found in peach, grape, corn and tea. The citrus UGT members reported so far are mainly distributed in groups A, C, E, F, H and R. Group D contains a large number of citrus UGT family members, and it is still unclear whether the UGT members of this group are involved in the biosynthesis of flavonoid glycosides. CsUGT76F1 isolated from sweet orange is distributed in the H group, and it prefers to catalyze the 3-OH and 7-OH positions of flavonols, while CgUGT90A31 found in grapefruit is distributed in the C group, and it prefers to catalyze the 7-OH, 4'-OH and 3'-OH positions of flavanones. The catalytic efficiency and regioselectivity of both are poor. There are few reports on key UGT members in citrus that efficiently catalyze the glycosylation modification of flavonoids at the 7-OH and 4'-OH positions. Summary of the invention

[0005] In view of this, the object of the present invention is to provide an application of citrus flavonoid glycosyltransferase CitUGT73A78 in the biosynthesis of flavonoid glycosides.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides an application of a citrus flavonoid glycosyltransferase CitUGT73A78 in the biosynthesis of flavonoid glycosides. The amino acid sequence of the CitUGT73A78 is shown in SEQ ID No.2.

[0008] Preferably, the flavonoid glycoside comprises flavonoid 7-O-glucoside and / or flavonoid 4'-O-glucoside.

[0009] The present invention provides a use of a citrus flavonoid glycosyltransferase CitUGT73A78 or a biomaterial containing CitUGT73A78 in at least one of the following a to e:

[0010] a. catalyzing the glycosidation of flavonoids containing 7-OH and / or flavonoid compounds containing 4'-OH;

[0011] b. biosynthesis of flavonoid 7-O-glucoside and / or flavonoid 4'-O-glucoside compounds;

[0012] c. Cultivating transgenic plants rich in flavonoid 7-O-glucoside and / or flavonoid 4'-O-glucoside compounds;

[0013] d. Preparation of medicines, foods, cosmetics or health products containing flavonoid 7-O-glucoside and / or flavonoid 4'-O-glucoside compounds;

[0014] e. Preparation of enzyme catalysts;

[0015] The amino acid sequence of CitUGT73A78 is shown in SEQ ID No.2.

[0016] Preferably, the flavonoid compounds containing 7-OH include at least one of flavonols, flavones and flavanones;

[0017] The flavonoid 7-O-glucoside compound includes at least one of flavonol 7-O-glycosides, flavonoid 7-O-glycosides and flavanone 7-O-glycosides.

[0018] Preferably, the flavonoid compound containing 4'-OH includes one or two compounds of flavonols and flavones;

[0019] The flavonoid 4'-O-glucoside compound includes one or both of flavonol 4'-O-glucoside and flavonoid 4'-O-glucoside.

[0020] Preferably, the flavonoids include one or more of apigenin, luteolin, diosgenin, acacetin and euphorbia cerifera;

[0021] The flavonols include one or more of quercetin, kaempferol and isorhamnetin;

[0022] The flavanones include one or more of naringenin, hesperetin, eriodictyol and isosakurantei.

[0023] Preferably, the flavonoids include one or both of apigenin and luteolin;

[0024] The flavonols include one or both of quercetin and kaempferol.

[0025] Preferably, the biological material comprises at least one of the following: a recombinant expression vector, an expression cassette, a recombinant cell and a transformant.

[0026] Preferably, the recombinant expression vector is selected from one or both of a prokaryotic expression vector and a eukaryotic expression vector;

[0027] The host cell of the recombinant cell is selected from at least one of plant cells, bacteria and fungi.

[0028] Preferably, the biological material comprises the coding gene of CitUGT73A78;

[0029] The coding gene of CitUGT73A78 is shown in the following S1 and / or S2:

[0030] S1, a gene with a nucleotide sequence as shown in SEQ ID No. 1;

[0031] S2. A gene that has been codon-optimized based on the gene described in S1 and whose amino acid sequence is not changed.

[0032] The present invention provides a method for preparing flavonoid 7-O-glucoside and / or flavonoid 4'-O-glucoside, comprising the following steps:

[0033] The flavonoid containing 7-OH and / or the flavonoid compound containing 4'-OH and the UDP sugar donor are used as substrates to carry out a catalytic reaction in at least one of the following enzyme systems B1 to B3:

[0034] B1, CitUGT73A78 mentioned above;

[0035] B2, a recombinant cell expressing the above-mentioned CitUGT73A78;

[0036] B3, recombinant protein isolated and extracted from recombinant cells through recombinant expression;

[0037] The recombinant cell described in item B3 contains the above-mentioned CitUGT73A78 encoding gene or the expression vector of the above-mentioned CitUGT73A78 encoding gene.

[0038] Preferably, the temperature of the catalytic reaction is 40 to 65°C; the time of the catalytic reaction is 30 minutes to 2 hours; the pH value of the catalytic reaction system is 6.0 to 7.5;

[0039] The catalytic reaction system is 4-6 μL of 0.3-1.6 mM UDP sugar donor, 3-5 μL of 360-440 μM flavonoid compound containing 7-OH and / or 4'-OH, 18-22 μL of 0.4-0.6 μg / μL CitUGT73A78, and 70-72 μL of 90-110 mM Tris-HCl buffer at pH 7.5 containing 2.0 mM dithiothreitol;

[0040] The UDP glycosyl donor includes UDP-glucose;

[0041] The flavonoids containing 7-OH include at least one type of compound selected from the group consisting of flavonols, flavones and flavanones;

[0042] The flavonoid compounds containing 4'-OH include one or both of flavonols and flavones.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] The present invention provides an application of citrus flavonoid glycosyltransferase CitUGT73A78 in the biosynthesis of flavonoid glycosides. The present invention verifies the function of citrus CitUGT73A78 for the first time. The CitUGT73A78 recombinant protein obtained by recombinant expression exhibits broad-spectrum catalytic activity in vitro, and can efficiently catalyze the glycosylation of various flavonoid compounds to generate corresponding flavonoid 7-O-glucoside and / or flavonoid 4'-O-glucoside. In vivo transient overexpression and gene silencing experiments also confirmed that CitUGT73A78 has a significant promoting effect on the accumulation of flavonoid 7-O-glucoside content in citrus. The present invention not only provides a new preparation method for the production of flavonoid 7-O-glucoside and / or flavonoid 4'-O-glucoside, which has the advantages of simplicity, high catalytic efficiency, low cost and green safety, but also can be applied to the improvement of citrus fruit nutritional quality and germplasm innovation. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is the identification and phylogenetic analysis of the citrus UGT gene family members;

[0046] Figure 2 This is an analysis diagram of the expression pattern of CitUGT73A78 gene in different tissues of Ponkan;

[0047] Figure 3 The SDS-PAGE electrophoresis diagram of the recombinant protein of CitUGT73A78, M is the protein Maker; C is the crude protein after induction without purification; P is the purified CitUGT73A78 protein;

[0048] Figure 4 The schematic diagram (A), HPLC spectrum (B), standard reference substance (C) and high-resolution mass spectrum of the product (D) of the in vitro catalytic naringenin by the recombinant protein CitUGT73A78 are shown;

[0049] Figure 5 The diagram (A), HPLC spectrum (B), standard reference substance (C) and high-resolution mass spectrum of the product (D) catalyzed by the recombinant protein CitUGT73A78 are shown;

[0050] Figure 6The diagram (A), HPLC spectrum (B), standard reference substance (C) and high-resolution mass spectrum of the product (D) catalyzed by CitUGT73A78 recombinant protein are shown;

[0051] Figure 7 The schematic diagram (A), HPLC spectrum (B), standard reference substance (C) and high-resolution mass spectrum of the product (D) catalyzed by the recombinant protein CitUGT73A78 are shown;

[0052] Figure 8 The diagram (A), HPLC spectrum (B), standard reference substance (C) and high-resolution mass spectrum of the product (D) catalyzed by the recombinant protein CitUGT73A78 are shown;

[0053] Fig. 9 The schematic diagram (A), HPLC spectrum (B), standard reference substance (C) and high-resolution mass spectrum of the product (D) of luteolin catalyzed by the recombinant protein CitUGT73A78 are shown;

[0054] Fig.10 The schematic diagram (A), HPLC spectrum (B), standard reference substance (C) and high-resolution mass spectrum of the product (D) catalyzed by CitUGT73A78 recombinant protein.

[0055] Fig.11 The schematic diagram (A), HPLC spectrum (B), standard reference substance (C) and high-resolution mass spectrum of the product (D) catalyzed by CitUGT73A78 recombinant protein for acacetin;

[0056] Fig.12 The schematic diagram (A), HPLC spectrum (B), standard reference substance (C) and high-resolution mass spectrum of the product (D) catalyzed by CitUGT73A78 recombinant protein are shown;

[0057] Fig.13 The schematic diagram (A), HPLC spectrum (B), standard reference substance (C) and high-resolution mass spectrum of the product (D) catalyzed by CitUGT73A78 recombinant protein for quercetin;

[0058] Fig.14 The schematic diagram (A), HPLC spectrum (B), standard reference substance (C) and high-resolution mass spectrum of the product (D) catalyzed by CitUGT73A78 recombinant protein for kaempferol;

[0059] Fig.15 The schematic diagram (A), HPLC spectrum (B), standard reference substance (C) and high-resolution mass spectrum of the product (D) catalyzed by the CitUGT73A78 recombinant protein are shown;

[0060] Fig.16The effects of different temperatures (A) and pH (B) on the catalytic activity of CitUGT73A78 recombinant protease;

[0061] Fig.17 The results of enzyme reaction kinetic analysis of the recombinant protein CitUGT73A78;

[0062] Fig.18 Figure A shows the detection result of the relative expression level of the CitUGT73A78 gene in the transiently overexpressed sugar orange peel; Figure B shows that transient overexpression of the CitUGT73A78 gene increases the content of flavonoid glycosides in the sugar orange peel. DETAILED DESCRIPTION

[0063] The present invention provides an application of a citrus flavonoid glycosyltransferase CitUGT73A78 in the biosynthesis of flavonoid glycosides. The amino acid sequence of the CitUGT73A78 is shown in SEQ ID No.2.

[0064] In the present invention, the flavonoid glycoside includes flavonoid 7-O-glucoside and / or flavonoid 4'-O-glucoside.

[0065] The present invention provides an application of a citrus flavonoid glycosyltransferase CitUGT73A78 or a biomaterial containing CitUGT73A78 in at least one of the following a to e:

[0066] a. catalyzing the glycosidation of flavonoids containing 7-OH and / or flavonoid compounds containing 4'-OH;

[0067] b. biosynthesis of flavonoid 7-O-glucoside and / or flavonoid 4'-O-glucoside compounds;

[0068] c. Cultivating transgenic plants rich in flavonoid 7-O-glucoside and / or flavonoid 4'-O-glucoside compounds;

[0069] d. Preparation of medicines, foods, cosmetics or health products containing flavonoid 7-O-glucoside and / or flavonoid 4'-O-glucoside compounds;

[0070] e. Preparation of enzyme catalysts;

[0071] The amino acid sequence of CitUGT73A78 is shown in SEQ ID No.2.

[0072] In the present invention, the amino acid sequence of CitUGT73A78 is as follows:

[0073] MGSKIPQLHVFFFPFMAHGHMIPIVDMAKLFATRGVKASVITTPANAPYVSKSVERAN

[0074] ELGIELDVKTIKFPSVEAGLPDGCENLDAITNEVNKELIVKFLGATTKLQEPLEQLLRDHKPD

[0075] CLVADIFFPWATDAAAKFGIPRLVFHGTSFFSLCASNCLRLYEPHKKVSSDSEPFVMPHFPGEI

[0076] KLTRNQLPDFVKQDMGDNDLSRLLKATNESESRSYGVAVNSFYELEPAYADHYRKALGRRA

[0077] WHIGPVSLCNRNFEDKALRGKQASIDELECLKWLNSKQPNSVVYICFGSLANFTSAQLMEI

[0078] ATGLEASGRNFIWVVRKNKNDGGEGGKEDWLPEGFEKRMEGKGLIIRGWAPQVLILDHEA

[0079] VGGFVTHCGWNSTIEAVAAGVPLVTWPVSAEQFYNEKMVNEVLKIGVGVGIQKWCRIVGD

[0080] FVKREKIEKAVNEIMVGDRAEEMRSRAKALGKMAKRAVENGGSSYSDLSALIEELRLSRHQ

[0081] SS(SEQ ID No.2)。

[0082] In the present invention, the CitUGT73A78 has a broad spectrum of catalytic activity in the biosynthesis of flavonol 7-O-glucosides, flavonoid 7-O-glucosides, flavanone 7-O-glucosides, flavonol 4'-O-glucosides, and flavonoid 4'-O-glucosides. The biosynthesis is catalyzed by using CitUGT73A78 as an enzyme catalyst, UDP glucose as a sugar donor, and flavonoid compounds containing 7-OH and / or 4'-OH as substrates to obtain flavonoid glycoside compounds. The flavonoid compounds containing 7-OH include at least one type of compounds among flavonols, flavones, and flavanones; the flavonoid compounds containing 4'-OH include one or two compounds among flavonols and flavones. Among the flavonoid compounds containing 7-OH, the flavonoids include one or more of apigenin, luteolin, diosmetin, acacetin and scutellarin; the flavonols include one or more of quercetin, kaempferol and isorhamnetin; the flavanones include one or more of naringenin, hesperidin, eriodictyol and isosakurantei. Among the flavonoid compounds containing 4'-OH, the flavonoids include one or both of apigenin and luteolin; the flavonols include one or both of quercetin and kaempferol.

[0083] In the present invention, the flavonoid glycoside compound includes a flavonoid 7-O-glucoside compound or a flavonoid 4'-O-glucoside compound.

[0084] In the present invention, the flavonoid 7-O-glucoside compound includes at least one of flavonol 7-O-glucosides, flavonoid 7-O-glucosides and flavanone 7-O-glucosides. When the flavonoid substrate is one or more of apigenin, luteolin, diosmetin, acacia or acacia, and UDP-glucose is the sugar donor, the flavonoid 7-O-glycoside preferably includes one or more of apigenin 7-O-glucoside, luteolin 7-O-glucoside, diosmetin 7-O-glucoside, acacia 7-O-glucoside or acacia 7-O-glucoside. When the flavonol substrate is one or more of quercetin, kaempferol or isorhamnetin, and UDP-glucose is the sugar donor, the flavonol 7-O-glycosides preferably include one or more of quercetin 7-O-glucoside, kaempferol 7-O-glucoside or isorhamnetin 7-O-glucoside. When the flavanone substrate is one or more of naringenin, hesperetin, eriodictyol and isosakuretene, and UDP-glucose is the sugar donor, the flavanone 7-O-glycosides preferably include one or more of naringenin 7-O-glucoside, hesperetin 7-O-glucoside, eriodictyol 7-O-glucoside or isosakuretene 7-O-glucoside.

[0085] In the present invention, the flavonoid 4'-O-glucoside compound includes one or two compounds of flavonol 4'-O-glucoside and flavonoid 4'-O-glucoside. When the flavonoid substrate is one or two of apigenin and luteolin, and UDP-glucose is the sugar donor, the flavonoid 4'-O-glucoside is one or two of apigenin 4'-O-glucoside and luteolin 4'-O-glucoside. When the flavonol substrate is one or two of quercetin and kaempferol, and UDP-glucose is the sugar donor, the flavonol 4'-O-glucoside is one or two of quercetin 4'-O-glucoside and kaempferol 4'-O-glucoside.

[0086] In the present invention, the biological material includes at least one of the following: a recombinant expression vector, an expression cassette, a recombinant cell and a transformant. The recombinant expression vector is selected from one or both of a prokaryotic expression vector and a eukaryotic expression vector, and the prokaryotic expression vector preferably includes pET-32a(+). The present invention does not impose any special restrictions on the construction method of the recombinant expression vector, and a construction method well known in the art can be used, such as artificial synthesis or cloning by homologous recombination. The host cell of the recombinant cell is selected from at least one of a plant cell, a bacterium and a fungus. The plant cell preferably includes a citrus cell. The bacteria preferably include Escherichia coli, Agrobacterium tumefaciens, etc. The fungus preferably includes yeast. The present invention does not impose any special restrictions on the construction method of the recombinant cell, and a construction method well known in the art can be used, such as electroporation transformation or Agrobacterium-mediated transformation.

[0087] In the present invention, the biological material comprises the coding gene of CitUGT73A78; the coding gene of CitUGT73A78 is as shown in the following S1 and / or S2: S1, a gene with a nucleotide sequence as shown in SEQ ID No.1; S2, a gene with a codon optimized sequence and without changing the amino acid sequence based on the gene in S1. The nucleotide sequence of the coding gene of CitUGT73A78 is as follows:

[0088] atgggcagtaaaatccctcagcttcacgtttttttctttccattcatggctcacggccacatgattccaattgtggacatggcgaagttgtttgc

[0089] aactcgtggggtgaaagcaagtgtcattacaactccagccaatgcgccttacgtctccaaatcagtagaaagagccaatgaattgggcatcgaat

[0090] tggatgtgaagaccatcaagttcccttctgtggaggccggtttgccagacggatgtgaaaatcttgacgcaatcacaaatgaagtgaacaaggaa

[0091] ttgattgtcaagtttttgggggccacaacgaagctccaagaacctcttgagcagctgttgcgagatcataaaccagattgtttggtagccgacatat

[0092] tcttcccttgggccactgacgctgctgccaagtttggtattcccaggctggtgtttcacggcactagtttcttttctttgtgtgcatcaaattgcctgag

[0093] gctatatgagccacacaagaaggtttcatcggattcagagccatttgtgatgcctcattttccgggtgagatcaagctgacgagaaaccaattgcc

[0094] agattttgtaaaacaggatatgggagataatgacttgagcaggttgttgaaagcaactaacgagtcagagtcgaggagctatggtgttgccgtga

[0095] acagcttttatgagctggagcctgcttatgctgaccattacaggaaggctttgggaaggagggcatggcatattggccctgtttccttatgcaatag

[0096] gaattttgaagataaagctttgaggggaaagcaagcctctattgatgaactggagtgcttgaagtggttgaattcaaagcaacccaattctgttgttt

[0097] acatttgctttggaagcctggccaactttacttctgctcagctaatggagattgcaacgggcctggaagcttcagggagaaatttcatttgggttgtg

[0098] aggaaaaacaagaatgatggcggtgaaggaggaaaggaagattggttgcctgaaggatttgagaagagaatggagggaaaagggctaatca

[0099] ttagaggatgggcgccacaggttttgatacttgatcacgaagctgttggaggatttgtgacccattgcggatggaattcaactattgaagcggtgg

[0100] ctgctggggtacctctggtgacatggcccgtctctgcggagcaattttacaatgaaaagatggtgaatgaagttctgaagattggcgttggtgttg

[0101] gaatccagaaatggtgtagaattgtgggggattttgtgaagagagagaaaatagaaaaggcagtgaatgagataatggtgggtgatagggcag

[0102] aagaaatgaggagcagagccaaggctttggggaagatggcaaaaagggctgttgaaaatggaggttcatcgtactctgatttgagtgctcttatt

[0103] gaggagttgcgtttaagtcgccatcaaagcagctaa (SEQ ID No. 1). The coding gene of CitUGT73A78 contains 1473 nucleotides, encodes a protein of 490 amino acids, and belongs to the UGT73 family. The present invention has no particular limitation on the method of codon optimization, and the corresponding codon optimization method can be selected according to the type of host cell.

[0104] In the present invention, the method of cultivating transgenic plant lines rich in flavonoid 7-O-glucoside compounds and / or flavonoid 4'-O-glucoside compounds preferably uses a genetic engineering method to overexpress CitUGT73A78 in plants to obtain transgenic plants. For example, CitUGT73A78 is connected to a pGreenII002962_SK vector to obtain a Ci tUGT73A78-SK recombinant plasmid, which is then transfected with Agrobacterium, and the CitUGT73A78-SK Agrobacterium suspension is injected into rock sugar orange. Furthermore, the CitUGT73A78 target gene was constructed into the pGreenII002962_SK vector to obtain the CitUGT73A78-SK recombinant plasmid, which was transferred into the Agrobacterium EHA105 strain by chemical transformation. The positive single clones were selected and cultured in LB liquid medium containing 50 μg / mL Kan and 25 μg / mL Rif at 28°C for 1 to 2 days. The cells were collected by centrifugation at 5000 rpm for 10 min at room temperature and resuspended in infiltration buffer (10 mM MgCl2, 10 mM MES, 200 mM acetosyringone, pH 5.6) to an OD of 0. 600 The CitUGT73A78-SK Agrobacterium suspension was injected into the sugar orange peel. The overexpression of CitUGT73A78 can significantly increase the content of flavonoid 7-O-glucoside and / or flavonoid 4'-O-glucoside products.

[0105] In the present invention, the enzyme catalyst is a flavonoid glycosyltransferase catalyst, such as a flavonoid 7-O-glycosyltransferase catalyst.

[0106] The present invention provides a method for preparing flavonoid 7-O-glucoside and / or flavonoid 4'-O-glucoside, comprising the following steps:

[0107] The flavonoid containing 7-OH and / or the flavonoid compound containing 4'-OH and the UDP sugar donor are used as substrates to carry out a catalytic reaction in at least one of the following enzyme systems B1 to B3:

[0108] B1, CitUGT73A78 mentioned above;

[0109] B2, a recombinant cell expressing the above-mentioned CitUGT73A78;

[0110] B3, recombinant protein isolated and extracted from recombinant cells through recombinant expression;

[0111] The recombinant cell described in item B3 contains the above-mentioned CitUGT73A78 or the expression vector of the above-mentioned CitUGT73A78.

[0112] In the present invention, the temperature of the catalytic reaction is preferably 40 to 65°C, more preferably 45 to 55°C, more preferably 47, 48, 49, 50, 51, 52, 53 or 54°C; the time of the catalytic reaction is preferably 30min to 2h, more preferably 55 to 65min, more preferably 1h; the pH value of the catalytic reaction system is 5.5 to 7.5, more preferably 7.0 to 7.3, more preferably 7.0, 7.1, 7.2 or 7.3. The present invention adopts the above-mentioned catalytic reaction temperature, time and pH, and the enzyme activity of CitUGT73A78 as an enzyme catalyst is high.

[0113] In the present invention, the catalytic reaction system is 4 to 6 μL of 0.3 to 1.6 mM UDP sugar donor, 3 to 5 μL of 360 to 440 μM flavonoid compound containing 7-OH and / or 4'-OH, 18 to 22 μL of 0.4 to 0.6 μg / μL CiTUGT73A78, and 70 to 72 μL of 90 to 110 mM pH 7.5 Tris-HCl buffer containing 2.0 mM dithiothreitol;

[0114] The UDP glycosyl donor includes UDP-glucose;

[0115] The flavonoids containing 7-OH include at least one type of compound selected from the group consisting of flavonols, flavones and flavanones;

[0116] The flavonoid compounds containing 4'-OH include one or both of flavonols and flavones.

[0117] In the present invention, the flavonoids in the flavonoid compounds containing 7-OH include one or more of apigenin, luteolin, dioscorea linalool, acacetin and aurea linalool; the flavonols in the flavonoid compounds containing 7-OH include one or more of quercetin, kaempferol and isorhamnetin; the flavanones in the flavonoid compounds containing 7-OH include one or more of naringenin, hesperidin, aurea linalool and isosakurantei; the flavonoids in the flavonoid compounds containing 4'-OH include one or both of apigenin and luteolin; the flavonols in the flavonoid compounds containing 4'-OH include one or both of quercetin and kaempferol.

[0118] In the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0119] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0120] Example 1

[0121] Cloning, expression vector construction and phylogenetic analysis of citrus flavonoid glycosyltransferase CitUGT73A78 gene

[0122] 1. Experimental methods

[0123] The reference sequence of CitUGT73A78 gene (ID: Ciclev10028305m) was obtained by analyzing the citrus pan-genome database (http: / / www.citrusgenomedb.org / ), and a specific primer pair containing the recombinant expression vector pET-32a (+) was designed. The upstream primer: gtatcggatccgaattcatgggcagtaaaatccctcagc (SEQ ID No. 3) and the downstream primer: tggtggtgctcgaggctgctttgatggcgacttaaac (SEQ ID No. 4) were used as templates for PCR amplification. The PCR reaction system (total volume 30 μL) was 2×PhantaMaxMasterMix (DyePlus) 15 μL, 10 μM upstream and downstream primers 1.2 μL each, cDNA template 1 μL, DEPC 11.6 μL. The PCR reaction program was 95°C pre-denaturation for 3 min; 95°C for 15 s, 58°C for 15 s, 72°C for 1 min, 35 cycles; 72°C for 5 min, and stored at 4°C. After agarose gel electrophoresis analysis and PCR product recovery and purification, it was ligated to the target vector pET-32a(+) digested with restriction endonucleases EcoRI and XhoI, and transformed into Escherichia coli competent DH5α by heat shock method. Positive colonies were selected and sequenced to obtain CitUGT73A78-pET-32a(+) recombinant plasmid.

[0124] (II) Experimental results

[0125] 1. After sequencing verification, the CitUGT73A78 gene sequence SEQ ID No. 1 that matches the citrus genome database was obtained, which contains 1473 nucleotides and encodes a protein of 490 amino acids, as shown in SEQ ID No. 2.

[0126] 2. If Figure 1 As shown in the figure, based on the Citrus clementine genome (v1.0), a total of 136 citrus UGT family members were identified and divided into 18 phylogenetic groups and 25 families. It is worth mentioning that CitUGT73A78 is distributed in the UGT73 family in group D, and is clustered in the same branch with flavonoid 7-O-glycosyltransferases identified in plants such as Arabidopsis (AtUGT73C6), rainbow chrysanthemum (Db7GlcT), strawberry (FaGT7) and tobacco (NTGlcT2).

[0127] Example 2

[0128] Analysis of the expression pattern of CitUGT73A78 gene in different tissues of Ponkan

[0129] 1. Experimental methods

[0130] 1. Materials: The 'Ponkan' plant was used as the material and divided into 9 tissue parts, including roots, stems, leaves, flowers, oil cell layer, white cortex, cyst, juice cell and seeds. After cutting into small pieces, the pieces were quickly frozen with liquid nitrogen and stored in a -80℃ refrigerator.

[0131] 2. RNA extraction and cDNA synthesis: RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit was used to extract total RNA from 9 different tissues. The PrimeScriptTM RTreagent Kit with gDNAEraser (Takara, Japan) was used to remove genomic DNA and then reverse transcribed into cDNA. The cDNA was diluted 10 times for subsequent gene expression analysis.

[0132] 3. Gene expression analysis: Primers were designed using NCBI / Primer-BLAST online software (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / index.cgi?LINK_LOC=BlastHome), and primer specificity was verified by melting curve analysis, agarose gel electrophoresis analysis and qPCR product resequencing. Real-time quantitative PCR technology was used, with citrus β-actin as the internal reference gene, the upstream primer was catccctcagcaccttcc (SEQ ID No.5) and the downstream primer was ccaaccttagcacttctcc (SEQ ID No.6), and the upstream primer of CitUGT73A78 qPCR was gctgctgccaagtttggtatt (SEQ ID No.7) and the downstream primer was ctcacccggaaaatgaggca (SEQ IDNo.8). The total volume of the qPCR reaction system was 20 μL, and the qPCR reaction system was 10 μL 2×ChemQ UniversalSYBR Master Mix (Weizan, China), 0.2 μL of 10 μM upstream and downstream primers, 2 μL of diluted cDNA, and 7.6 μL of DEPC water; the reaction program was 95°C for 3 min; 95°C for 10 s, 60°C for 30 s, and 45 cycles; 95°C for 10 s, and the melting curve was from 65°C to 95°C, increasing by 0.5°C every 5 s. The qPCR instrument was a CFX96 real-time fluorescence quantitative PCR instrument (Bio-Rad, USA), and a negative control without cDNA was included in each test. 2-ΔΔCt Methods The relative expression levels of the CitUGT73A78 gene in Example 1 in different tissues of Ponkan were calculated.

[0133] (II) Experimental results

[0134] Figure 2 The results showed that the expression level of CitUGT73A78 gene was higher in the oil cell layer of the fruit, while the expression level in the other nine tissues including roots, stems, leaves, flowers, white cortex, orange peel, pericarp, juice cells and seeds was lower. It was speculated that the CitUGT73A78 gene may be involved in the biosynthesis of flavonoid glycosides in the oil cell layer of the fruit.

[0135] Example 3

[0136] Expression and purification of CitUGT73A78 recombinant protein

[0137] 1. Experimental methods

[0138] 1. The CitUGT73A78-pET-32a(+) recombinant plasmid of Example 1 was transformed into the Escherichia coli BL21(DE3) pLysS expression host bacteria. After colony PCR verification, positive colonies were picked and inoculated into 500 mL LB(Amp+) liquid culture medium for overnight culture (37°C, 200 rpm) until OD 600 is 0.6-0.8, and recombinant bacteria are obtained.

[0139] 2. Add IPTG at a final concentration of 1mM to the above recombinant bacteria, induce recombinant protein expression at 16℃, 150rpm overnight, transfer the bacterial solution to a 500mL centrifuge tube. Centrifuge at 4℃, 4000g for 15min, discard the supernatant, and collect the bacteria. Add 25mL 1×PBS buffer, fully resuspend the bacteria, and place at -80℃ for more than 24h. Transfer the cold stored bacteria to a 30℃ water bath to thaw, and use an ultrasonic disruptor for 5min. Centrifuge at 4℃, 10000rpm for 30min, and collect the supernatant. The supernatant was passed through a Meillx-HV sterilization membrane (0.45 μm, diameter 33 mm, Millipore USA) to remove bacteria and impurities, and the protein was purified using a HisTALONTM (Takara) gravity column. 1 μL of the purified recombinant protein was subjected to SDS-PAGE electrophoresis detection, desalted using a PD-10 (GE Healthcare UK) desalting column, and stored in Tris-HCl buffer (100 mM Tris, 2 mM DTT, pH 7.5) containing 10% glycerol and stored at -80°C for use.

[0140] (II) Experimental results

[0141] like Figure 3As shown, SDS-PAGE electrophoresis analysis showed that the CitUGT73A78 recombinant protein containing a 6× histidine tag had an obvious protein band at about 70 kDa, which was consistent with the predicted protein molecular weight (73.54 kDa), and the purified band was single. Therefore, it can be used for subsequent enzyme activity analysis.

[0142] Example 4

[0143] In vitro enzymatic activity and functional identification of CitUGT73A78 recombinant protein

[0144] 1. Experimental methods

[0145] 1. In vitro enzyme activity reaction: 12 flavonoid substrates including naringenin, hesperidin, eriodictyol, isosakurantei, apigenin, luteolin, diosmetin, acacetin, euphorbia quinata, quercetin, kaempferol and isorhamnetin were selected to verify the glycosylation function of the CitUGT73A78 recombinant protein of Example 3; the reaction system was 71 μL Tris-HCl buffer (100 mM, pH 7.5, 2.0 mM DTT), 5 μL UDP-glucose (1.5 mM), 4 μL substrate (400 μM), 20 μL CitUGT73A78 recombinant protein (0.5 μg / μL); the enzyme reaction was incubated at 30°C for 16 h and an equal volume of pre-cooled methanol was added to terminate the reaction. The reaction was all carried out with empty protein as a control to obtain the enzyme reaction product. After centrifugation at room temperature for 30 min at 13000 rpm, the supernatant was aspirated for HPLC and mass spectrometry analysis.

[0146] 2. HPLC enzyme activity detection and product identification: The activity of the enzyme reaction product was detected by HPLC using an Agilent 1290 Infinity HPLC liquid chromatograph (Agilent Technologies, USA) and an Inertsil C18 ODS (4.6×250 mm, 5 μm) column. Chromatographic conditions: 0.1% phosphoric acid water (solution A) and chromatographic acetonitrile (solution B) were used as mobile phases, the flow rate was 1 mL / min, and the gradient elution conditions were: 0-5 min, 20% B; 5-10 min, 20%-27% B; 10-15 min, 27% B; 15-25 min, 27%-40% B; 25-35 min, 40%-60% B; 35-40 min, 60%-80% B; 40-42 min, 80%-100% B; 42-45 min, 100% B; 45-49 min, 80%-20% B; 49-55 min, 20% B. The detection wavelength was 280 nm, the column temperature was 25° C., and the injection volume was 10 μL. The enzyme reaction products were analyzed by mass spectrometry using an AB TripleTOF 5600 time-of-flight liquid-mass spectrometer (AB SCIEX, USA). The negative ion scanning mode was as follows: ion source temperature (TEM) 550°C, voltage (IS) -4500V; scanning range (m / z) 100-1500Da; nebulizer gas (GS1), nebulizer gas (GSI2), and curtain gas (CUR) pressures were 55psi, 55psi, and 35psi, respectively. The products were identified based on the retention time and ion fragmentation information of the enzyme activity products and the corresponding standards.

[0147] (II) Experimental results

[0148] The results of in vitro enzyme activity experiments showed that, with UDP-glucose as the glycosyl donor, the recombinant protein CitUGT73A78 could specifically catalyze the glycosylation modification of 7-OH position of flavanones such as naringenin, hesperetin, eriodictyol, and isosakurantein to produce naringenin 7-O-glucoside, hesperetin 7-O-glucoside, eriodictyol 7-O-glucoside, and isosakurantein 7-O-glucoside that were consistent with the standard reference substances (see Figures 4 to 7 ); when flavonoids (apigenin, luteolin, dioscorea, acacetin and aureus) and flavonols (quercetin, kaempferol and isorhamnetin) were used as substrates, the CitUGT73A78 recombinant protein could not only catalyze the glycosylation of flavonoids (apigenin, luteolin, dioscorea, acacetin and aureus) and flavonols (quercetin, kaempferol and isorhamnetin) compounds at the 7-OH position, but also catalyze the glycosylation of flavonoids such as apigenin and luteolin and flavonols such as quercetin and kaempferol at the 4'-OH position (see Figures 8 to 15), generating the corresponding flavonoid 7-O-glucoside and flavonoid 4'-O-glucoside.

[0149] Example 5

[0150] Determination of kinetic parameters of CitUGT73A78 enzyme reaction

[0151] 1. Experimental methods

[0152] 1. Optimization of enzyme reaction conditions: Naringenin was selected as the substrate, and the effects of different temperatures and pH values ​​on the reaction efficiency of the CitUGT73A78 recombinant protein of Example 3 were determined.

[0153] Determination of the optimal temperature of enzymatic reaction: set 13 temperature gradients at 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃ and 80℃ respectively, and terminate the reaction after 1h in 100mM pH 7.5 Tris-HCl buffer.

[0154] Determination of the optimal pH for enzymatic reaction: prepare 100 mM Tris-HCl buffer with different pH values, and set the pH gradient to 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 and 10.0. The reaction was terminated after incubation at 30°C for 1 h. The sample was centrifuged at 13000 rpm for 30 min.

[0155] For specific in vitro enzyme activity reaction experimental methods, see Example 4.

[0156] The supernatant was aspirated for HPLC analysis, and the analysis conditions were consistent with the HPLC enzyme activity detection and product identification in Example 4.

[0157] 2. Determination of enzyme reaction kinetic parameters: using UDP-Glo TMGlycosyltransferase Assay kit was used to determine the affinity and catalytic efficiency of CitUGT72AZ4 recombinant protein for different substrates; the reaction system was 50 μL, including 100 mM pH 7.0 Tris-HCl, 650 μM UDP-glucose, 2 μg recombinant protein, 0-500 μM flavonoid substrate (0, 50, 100, 150 μM naringenin; 0, 100, 200, 300 μM hesperetin; 0, 100, 200, 300 μM eriodictyol; 0, 100, 200, 300 μM isosakurantei; 0, 50, 100, 150 μM apigenin; 0, 20, 40, 60, 80 μM luteolin; 0, 100, 200, 300 μM auretin; 0, 100, 200, 300 μM quercetin or 0, 100, 200, 300 μM kaempferol), incubate at 37 °C for 30 min, transfer 25 μL of the reaction solution to a white flat-bottom 96-well plate, and add an equal volume of UDPDetection Reagent detection reagent, gently shake the 96-well plate to mix, and incubate at room temperature for 60 minutes. The luminescence value was measured using an enzyme reader, and the graph was plotted using GraphPad Prism v8.0 software. The Michaelis-Menten constant (K) was calculated by nonlinear curve fitting using the Michalis-Menten equation. m ) and catalytic constant (k cat )value.

[0158] (II) Experimental results

[0159] Fig.16 The optimal reaction conditions experiment showed that with naringenin as substrate, the CitUGT73A78 recombinant protein exhibited the highest catalytic activity at a reaction temperature of 50°C and pH 7.0 (100 mM Tris-HCl buffer).

[0160] Table 1 Enzyme kinetic parameters of different substrates

[0161]

[0162] Fig.17 The enzyme reaction kinetic results shown in Table 1 showed that the CitUGT73A78 recombinant protein exhibited high catalytic efficiency for 9 flavonoid substrates, among which the catalytic efficiency for luteolin was the highest, followed by apigenin and quercetin, and the catalytic efficiency for eriodictyol was the lowest.

[0163] Example 6

[0164] Transient overexpression of CitUGT73A78 in the peel of rock sugar orange

[0165] 1. Experimental methods

[0166] EcoRⅠ and XhoI were selected as restriction enzyme sites, and specific primer pairs were designed using the Takara online website (https: / / www.takarabio.com / learning-centers / cloning / primer-design-and-other-tools), including the upstream primer gagctgcaggaattcatgggcagtaaaatccctcag (SEQ ID No.9) and the downstream primer tatagccccccctcgagttagctgctttgatggcgac (SEQ ID No.10). The amplified CDS sequence shown in SEQID No.1 was then ligated to the pGreenII002962_SK vector (SK vector) to obtain the CitUGT73A78-SK recombinant plasmid. The CitUGT73A78-SK recombinant plasmid and SK empty vector were transformed into Agrobacterium EHA105 (pSoup) competent cells by chemical transformation, and the bacterial solution was evenly spread on LB solid medium containing 50μg / mL kanamycin (Kan) and 25μg / mL rifampicin (Rif). The positive single clone was selected and placed in LB liquid medium (containing 50μg / mL Kan and 25μg / mL Rif) for 1-2 days at 28℃, centrifuged at 5000rpm for 10min at room temperature to collect the bacteria, and resuspended the bacteria in infiltration buffer (10mMMgCl2, 10mM MES, 200mM acetosyringone, pH 5.6) to OD 600 The ratio of β-glucose to β-glucose was 0.6-0.8. The sugar orange fruits with uniform size, relatively uniform maturity and no pests and diseases were selected, and CitUGT73A78-SK and empty Agrobacterium suspension were injected on both sides of the equator of the same fruit, first cultured in the dark at 25℃ for 1 day, and then transferred to normal light conditions (photoperiod: 16h light / 8h dark). Five days after injection, the peel of the injected area was taken for flavonoid glycoside content detection and gene expression analysis.

[0167] (II) Experimental results

[0168] like Fig.18 As shown in A, the relative expression level of CitUGT73A78 gene was significantly upregulated in the transiently overexpressed sugar orange peel (P<0.05).

[0169] In view of the instability of flavonoid 7-O-glucoside in citrus, these glycosides are easily converted into more stable disaccharide structures such as neohesperidin or rutin by 1,2-rhamnosyltransferase and 1,6-rhamnosyltransferase. Therefore, the final products of flavonoid 7-O-glucoside (such as naringenin 7-O-glucoside, hesperidin 7-O-glucoside and isosakurantei 7-O-glucoside) in the peel of rock sugar orange were analyzed and determined, such as naringenin rutin (Nar7Rut), hesperidin (Hes7Rut) and melissa glycoside (Did7Rut) content.

[0170] Fig.18 The results in Figure B showed that the total content of rutinosides in the peel of the rock sugar orange transiently overexpressing CitUGT73A78 was significantly increased (P<0.05), which was 1.29 times higher than that of the empty control group (EV). The above results indicate that CitUGT73A78 plays an important role in promoting the biosynthesis of flavonoid glycosides in citrus peel.

[0171] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An application of citrus flavonoid glycosyltransferase CitUGT73A78 in the biosynthesis of flavonoid glycosides, characterized in that: The amino acid sequence of CitUGT73A78 is shown in SEQ ID No.

2.

2. The use according to claim 1, characterized in that: The flavonoid glycosides include flavonoid 7-O-glucoside and / or flavonoid 4'-O-glucoside.

3. Use of a citrus flavonoid glycosyltransferase CitUGT73A78 or a biomaterial containing CitUGT73A78 in at least one of the following a to e: a. catalyzing the glycosidation of flavonoids containing 7-OH and / or flavonoid compounds containing 4'-OH; b. biosynthesis of flavonoid 7-O-glucoside and / or flavonoid 4'-O-glucoside compounds; c. Cultivating transgenic plants rich in flavonoid 7-O-glucoside and / or flavonoid 4'-O-glucoside compounds; d. Preparation of medicines, foods, cosmetics or health products containing flavonoid 7-O-glucoside and / or flavonoid 4'-O-glucoside compounds; e. Preparation of enzyme catalysts; The amino acid sequence of CitUGT73A78 is shown in SEQ ID No.

2.

4. The use according to claim 3, characterized in that: The flavonoid compounds containing 7-OH include at least one of flavonols, flavones and flavanones; The flavonoid 7-O-glucoside compound includes at least one of flavonol 7-O-glycosides, flavonoid 7-O-glycosides and flavanone 7-O-glycosides.

5. The use according to claim 3, characterized in that: The flavonoid compounds containing 4'-OH include one or two compounds of flavonols and flavones; The flavonoid 4'-O-glucoside compound includes one or both of flavonol 4'-O-glucoside and flavonoid 4'-O-glucoside.

6. The use according to claim 4, characterized in that: The flavonoids include one or more of apigenin, luteolin, diosgenin, acacetin and euphorbia cerifera; The flavonols include one or more of quercetin, kaempferol and isorhamnetin; The flavanones include one or more of naringenin, hesperetin, eriodictyol and isosakurantei.

7. The use according to claim 5, characterized in that: The flavonoids include one or both of apigenin and luteolin; The flavonols include one or both of quercetin and kaempferol.

8. The use according to claim 3, characterized in that: The biological material comprises at least one of the following: a recombinant expression vector, an expression cassette, a recombinant cell and a transformant; The biological material comprises the coding gene of CitUGT73A78; The coding gene of CitUGT73A78 is shown in the following S1 and / or S2: S1, a gene with a nucleotide sequence as shown in SEQ ID No. 1; S2. A gene that has been codon-optimized based on the gene described in S1 and whose amino acid sequence is not changed.

9. A method for preparing flavonoid 7-O-glucoside and / or flavonoid 4'-O-glucoside, characterized in that: The following steps are involved: The flavonoid containing 7-OH and / or the flavonoid compound containing 4'-OH and the UDP sugar donor are used as substrates to carry out a catalytic reaction in at least one of the following enzyme systems B1 to B3: B1, CitUGT73A78 according to claim 1; B2, a recombinant cell expressing CitUGT73A78 according to claim 1; B3, recombinant protein isolated and extracted from recombinant cells through recombinant expression; The recombinant cell described in item B3 contains the coding gene of CitUGT73A78 described in claim 8 or an expression vector containing the coding gene of CitUGT73A78 described in claim 8.

10. The preparation method according to claim 9, characterized in that: The temperature of the catalytic reaction is 40 to 65°C; the time of the catalytic reaction is 30 minutes to 2 hours; the pH value of the catalytic reaction system is 5.5 to 7.5; The catalytic reaction system is 4-6 μL of 0.3-1.6 mM UDP sugar donor, 3-5 μL of 360-440 μM flavonoid compound containing 7-OH and / or 4'-OH, 18-22 μL of 0.4-0.6 μg / μL CitUGT73A78, and 70-72 μL of 90-110 mM Tris-HCl buffer at pH 7.5 containing 2.0 mM dithiothreitol; The UDP glycosyl donor includes UDP-glucose; The flavonoid compounds containing 7-OH include at least one of flavonols, flavones and flavanones; The flavonoid compounds containing 4'-OH include one or both of flavonols and flavones.

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