Use of a citrus flavonoid glycosyltransferase CitUGT73A78 in biosynthesis of flavonoid glycosides

Abstract

The application provides application of citrus flavonoid glycosyltransferase CitUGT73A78 in biosynthesis of flavonoid glycosides, and belongs to the technical field of genetic engineering and enzyme engineering.The application clones and verifies the function of the CitUGT73A78 gene, and in-vitro enzyme activity tests prove that the recombinant protein of CitUGT73A78 has broad-spectrum catalytic activity, can efficiently catalyze glucosylation of various flavonoids, and generate corresponding flavonoid 7-O-glucoside and / or flavonoid 4'-O-glucoside.In-vivo transient overexpression tests also prove that the CitUGT73A78 gene has a promoting effect on accumulation of the content of citrus flavonoid 7-O-glucoside.The application not only provides a preparation method for production of flavonoid 4'-O-glucoside, has the advantages of simplicity, high catalytic efficiency, low cost, and green safety, but also can be applied to improvement of nutritional quality of citrus fruits and germplasm innovation.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzyme engineering technology, and in particular relates to the application of a citrus flavonoid glycosyltransferase CitUGT73A78 in the biosynthesis of flavonoid glycosides. Background Technology

[0002] Citrus fruits are among the world's most abundant fruits and also important medicinal and edible plants, possessing extremely high economic and medicinal value. For example, dried citrus peel, processed into tangerine peel (Chenpi) and tangerine peel (Huajuhong), can be used as raw materials for traditional Chinese medicine, possessing various effects such as anti-oxidation, anti-inflammation, lowering blood lipids, and relieving cough and phlegm, and are widely used in food, health products, and pharmaceuticals. These health effects are mainly attributed to the rich content of flavonoids, vitamin C, carotenoids, and other active ingredients in citrus fruits. Currently, more than 80 kinds of flavonoids have been isolated and identified in citrus fruits, mainly existing in the form of glycosides, with flavanone-O-glycosides, flavonoid-O-glycosides, and flavonoid-C-glycosides being the most abundant. In the flavonoid metabolic synthesis pathway, naringenin serves as a central precursor. After modification by hydroxylases or oxymethoxytransferases, it forms flavonoid aglycones such as hesperidin, sennaol, and isosinol. These aglycones are then converted into corresponding flavonoid 7-O-glucosides by 7-O-glucoside transferase (7GT). Under the catalysis of 1,2-rhamnosyltransferase (1,2-RhaT) or 1,6-rhamnosyltransferase (1,6-RhaT), they respectively form bitter-tasting new hesperidin glycosides (such as naringin) and non-bitter rutin glycosides (such as hesperidin).

[0003] Flavonoid 7-O-glucosides are compounds formed by the hydroxyl group at the 7-position of the A ring of a flavonoid molecule linked to a sugar molecule via a glycosidic bond. Flavonoid 4'-O-glucosides are compounds formed by the hydroxyl group at the 4' position of the B ring of a flavonoid molecule linked to a sugar molecule via a glycosidic bond. Flavonoid 7-O-glucosides and flavonoid 4'-O-glucoside compounds not only play important roles in plant growth, development, and stress responses, but also possess various pharmacological activities such as antioxidant, anti-inflammatory, lipid-lowering, and anticancer effects. For example, apigenin 7-O-glucoside has antioxidant and anti-inflammatory effects, while luteolin 7-O-glucoside has hypoglycemic, lipid-lowering, and cardiovascular protective effects. Currently, there are three main approaches to obtaining these compounds: extraction and separation methods, while allowing direct extraction from plants, often result in low concentrations and complex compositions of flavonoid 7-O-glucoside or flavonoid 4'-O-glucoside in most plants, making separation difficult and yielding low quantities that cannot meet market demand; chemical synthesis requires strict reaction conditions, involves cumbersome hydroxyl protection and deprotection processes, and necessitates the use of specific reagents and expensive catalysts, easily causing environmental pollution; in contrast, in vitro enzymatic methods offer advantages such as simple operation, mild conditions, environmental friendliness, and good stereoselectivity and regioselectivity. Therefore, the discovery and development of efficient flavonoid glycosyltransferases are 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 uridine diphosphate glycosyltransferase (UGT). This enzyme transfers glycosyl groups from UDP-glycodon donor molecules to flavonoid acceptor molecules. Its typical structural feature is a conserved motif (PSPGbox) of 44 amino acid residues at the C-terminus, responsible for binding to the UDP-glycodon, while the N-terminal sequence exhibits high variability, specifically recognizing and binding to specific substrate molecules. With the rapid development of genomics and bioinformatics, significant progress has been made in the study of the plant UGT family: 107 UGT family members were discovered in Arabidopsis thaliana and classified into 14 phylogenetic groups (ANs). Subsequently, four new phylogenetic groups (O, P, Q, and R) were discovered in peach, grape, maize, and tea. Currently reported citrus UGT members are mainly distributed in groups A, C, E, F, H, and R. Group D contains a relatively large number of citrus UGT family members; whether these UGT members participate in the biosynthesis of flavonoid glycosides remains unclear. Currently, CsUGT76F1 isolated from sweet orange is distributed in group H, and it preferentially catalyzes the 3-OH and 7-OH positions of flavonols, while CgUGT90A31 found in grapefruit is distributed in group C, and it preferentially catalyzes the 7-OH, 4'-OH, and 3'-OH positions of flavanones. Both exhibit poor catalytic efficiency and regioselectivity. Key UGT members that efficiently catalyze glycosylation modification of flavonoids at the 7-OH and 4'-OH positions in citrus fruits have been rarely reported. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide an application of the citrus flavonoid glycosyltransferase CitUGT73A78 in the biosynthesis of flavonoid glycosides.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides the application of citrus flavonoid glycosyltransferase CitUGT73A78 in the biosynthesis of flavonoid glycosides, wherein the amino acid sequence of CitUGT73A78 is shown in SEQ ID No. 2.

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

[0009] This invention provides the use of citrus flavonoid glycosyltransferase CitUGT73A78 or biological material containing CitUGT73A78 in at least one of the following a to e:

[0010] a. Catalyzing the glycosylation 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. Cultivate transgenic plants rich in flavonoid 7'-O-glucoside and / or flavonoid 4'-O-glucoside compounds;

[0013] d. Preparation of pharmaceuticals, food, 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 compound containing 7-OH includes at least one class of compounds selected from flavonols, flavones, and flavanones;

[0017] The flavonoid 7-O-glucoside compounds include at least one class of compounds selected from 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 selected from flavonols and flavones;

[0019] The flavonoid 4'-O-glucoside compounds include one or two compounds of flavonol 4'-O-glucosides and flavonoid 4'-O-glucosides.

[0020] Preferably, the flavonoids include one or more of apigenin, luteolin, geraniol, acaciain, and sennain.

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

[0022] The flavanones include one or more of naringenin, hesperidin, sennaol, and isochoride.

[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 biomaterial includes 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 two of prokaryotic expression vectors and eukaryotic expression vectors;

[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 contains the encoding gene of CitUGT73A78;

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

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

[0031] S2. Genes that have undergone codon optimization based on the genes described in S1 without altering the amino acid sequence.

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

[0033] Catalytic reactions are carried out in at least one enzyme system from B1 to B3 using flavonoids containing 7-OH and / or flavonoid compounds containing 4'-OH and UDP glycosyl donors as substrates:

[0034] B1. The aforementioned CitUGT73A78;

[0035] B2. Recombinant cells expressing the aforementioned CitUGT73A78;

[0036] B3. Recombinant proteins isolated and extracted from recombinant cells through recombinant expression;

[0037] The recombinant cells described in item B3 contain the encoding gene of CitUGT73A78 or the expression vector of the encoding gene of CitUGT73A78.

[0038] Preferably, the temperature of the catalytic reaction is 40–65°C; the time of the catalytic reaction is 30 min–2 h; and the pH value of the system of the catalytic reaction is 6.0–7.5.

[0039] The catalytic reaction system consists of 4–6 μL of 0.3–1.6 mM UDP glycosyl 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 class of compounds among 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] This invention provides the application of the citrus flavonoid glycosyltransferase CitUGT73A78 in the biosynthesis of flavonoid glycosides. This invention is the first to verify the function of citrus CitUGT73A78. The recombinant CitUGT73A78 protein obtained through recombinant expression exhibits broad-spectrum catalytic activity in vitro, efficiently catalyzing the glycosylation of various flavonoid compounds to generate corresponding flavonoid 7-O-glucosides and / or flavonoid 4'-O-glucosides. In vivo transient overexpression and gene silencing experiments also confirmed that CitUGT73A78 significantly promotes the accumulation of flavonoid 7-O-glucosides in citrus. This invention not only provides a new preparation method for the production of flavonoid 7-O-glucosides and / or flavonoid 4'-O-glucosides, with advantages of simplicity, high catalytic efficiency, low cost, and environmental safety, but can also be applied to the improvement of citrus fruit nutritional quality and germplasm innovation. Attached Figure Description

[0045] Figure 1 A diagram illustrating the identification and phylogenetic analysis of members of the citrus UGT gene family;

[0046] Figure 2 A diagram showing the expression pattern of the CitUGT73A78 gene in different tissues of Ponkan orange.

[0047] Figure 3 This is an SDS-PAGE electrophoresis image of the recombinant CitUGT73A78 protein. M represents the protein marker; C represents the crude protein after induction without purification; and P represents the purified CitUGT73A78 protein.

[0048] Figure 4 The diagram (A), HPLC chromatogram (B), standard reference (C), and product (D) of the in vitro catalysis of naringenin by recombinant CitUGT73A78 protein are shown.

[0049] Figure 5 The diagram (A), HPLC chromatogram (B), standard reference (C), and product (D) of the CitUGT73A78 recombinant protein catalyzing hesperidin are shown.

[0050] Figure 6The diagram (A), HPLC chromatogram (B), standard reference (C), and product (D) of the CitUGT73A78 recombinant protein catalyzing sennaol are shown.

[0051] Figure 7 The diagram (A), HPLC chromatogram (B), standard reference (C), and product (D) of the CitUGT73A78 recombinant protein catalyzing isopyrrolizine are shown.

[0052] Figure 8 The diagram (A), HPLC chromatogram (B), standard reference (C), and product (D) of the CitUGT73A78 recombinant protein catalyzing apigenin are shown.

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

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

[0055] Figure 11 The diagram (A), HPLC chromatogram (B), standard reference (C), and high-resolution mass spectrum (D) of the product catalyzed by the CitUGT73A78 recombinant protein are shown.

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

[0057] Figure 13 The diagram (A), HPLC chromatogram (B), standard reference (C), and high-resolution mass spectrum (D) of the product catalyzing quercetin by the CitUGT73A78 recombinant protein are shown.

[0058] Figure 14 The diagram (A), HPLC chromatogram (B), standard reference (C), and high-resolution mass spectrum (D) of the product catalyzed by the CitUGT73A78 recombinant protein are shown.

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

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

[0061] Figure 17 The results are from the enzyme reaction kinetics analysis of the recombinant protein CitUGT73A78.

[0062] Figure 18 In Figure A, the relative expression level of the CitUGT73A78 gene was detected in the peel of the sugar orange after transient overexpression; in Figure B, the content of flavonoid glycosides in the peel of the sugar orange was increased by transient overexpression of the CitUGT73A78 gene. Detailed Implementation

[0063] This invention provides the application of citrus flavonoid glycosyltransferase CitUGT73A78 in the biosynthesis of flavonoid glycosides, wherein the amino acid sequence of CitUGT73A78 is shown in SEQ ID No. 2.

[0064] In this invention, the flavonoid glycosides include flavonoid 7'-O-glucoside and / or flavonoid 4'-O-glucoside.

[0065] This invention provides the use of citrus flavonoid glycosyltransferase CitUGT73A78 or biological material containing CitUGT73A78 in at least one of the following a to e:

[0066] a. Catalyzing the glycosylation 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. Cultivate transgenic plants rich in flavonoid 7'-O-glucoside and / or flavonoid 4'-O-glucoside compounds;

[0069] d. Preparation of pharmaceuticals, food, 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 this 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 this invention, the CitUGT73A78 exhibits broad-spectrum 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 utilizes CitUGT73A78 as an enzyme catalyst, UDP glucose as a sugar donor, and flavonoids containing 7-OH and / or 4'-OH as substrates to catalyze the synthesis of flavonoid glycosides. The 7-OH-containing flavonoids include at least one class of compounds selected from flavonols, flavones, and flavanones; the 4'-OH-containing flavonoids include one or both of flavonols and flavones. Among flavonoids containing 7-OH, the flavonoids include one or more of apigenin, luteolin, geraniol, farnesin, and sennatin; the flavonols include one or more of quercetin, kaempferol, and isorhamnetin; and the flavanones include one or more of naringenin, hesperidin, sennatin, and isorhamnetin. Among flavonoids containing 4'-OH, the flavonoids include one or both of apigenin and luteolin; and the flavonols include one or both of quercetin and kaempferol.

[0083] In this invention, the flavonoid glycosides include flavonoid 7-O-glucoside compounds or flavonoid 4'-O-glucoside compounds.

[0084] In this invention, the flavonoid 7-O-glucoside compounds include at least one class of compounds selected from flavonol 7-O-glucosides, flavonoid 7-O-glucosides, and flavanone 7-O-glucosides. When the flavonoid substrate is one or more of apigenin, luteolin, geraniol, farnesin, or sennain, and UDP-glucose is the sugar donor, the flavonoid 7-O-glucosides preferably include one or more of apigenin 7-O-glucoside, luteolin 7-O-glucoside, geraniol 7-O-glucoside, farnesin 7-O-glucoside, or sennain 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-glucose, kaempferol 7-O-glucose, or isorhamnetin 7-O-glucose. When the flavanone substrate is one or more of naringenin, hesperidin, sennarol, or isorhamnetin, and UDP-glucose is the sugar donor, the flavanone 7-O-glycosides preferably include one or more of naringenin 7-O-glucose, hesperidin 7-O-glucose, sennarol 7-O-glucose, or isorhamnetin 7-O-glucose.

[0085] In this invention, the flavonoid 4'-O-glucoside compound includes one or two compounds selected from flavonol 4'-O-glucosides and flavonoid 4'-O-glucosides. 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 this invention, the biological material comprises at least one of the following: a recombinant expression vector, an expression cassette, recombinant cells, and a transformant. The recombinant expression vector is selected from one or both of prokaryotic and eukaryotic expression vectors, with the prokaryotic expression vector preferably including pET-32a(+). This invention does not impose any special limitations on the construction method of the recombinant expression vector; any construction method well-known in the art can be used, such as artificial synthesis or cloning via homologous recombination. The host cell of the recombinant cell is selected from at least one of plant cells, bacteria, and fungi. The plant cells preferably include citrus cells. The bacteria preferably include *Escherichia coli*, *Agrobacterium tumefaciens*, etc. The fungi preferably include yeast. This invention does not impose any special limitations on the construction method of the recombinant cell; any construction method well-known in the art can be used, such as electroporation transformation or *Agrobacterium-mediated transformation.

[0087] In this invention, the biological material comprises the coding gene of CitUGT73A78; the coding gene of CitUGT73A78 is as shown in S1 and / or S2 below: S1, a gene with a nucleotide sequence as shown in SEQ ID No. 1; S2, a gene based on the gene described in S1, but with codon optimization and without altering the amino acid sequence. 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, encoding a protein of 490 amino acids, belonging to the UGT73 family. This invention does not specifically limit the method of codon optimization; the appropriate codon optimization method can be selected according to the type of host cell.

[0104] In this invention, the method for cultivating transgenic plant lines rich in flavonoid 7'-O-glucoside and / or flavonoid 4'-O-glucoside compounds using CitUGT73A78 preferably employs genetic engineering methods to overexpress CitUGT73A78 in plants to obtain transgenic plants. For example, CitUGT73A78 is ligated into the pGreenII002962_SK vector to obtain the CitUGT73A78-SK recombinant plasmid, which is then transfected into Agrobacterium and injected into the resulting CitUGT73A78-SK Agrobacterium suspension into sugar oranges. Furthermore, the target gene CitUGT73A78 was constructed into the pGreenII002962_SK vector, and the resulting CitUGT73A78-SK recombinant plasmid was transformed into Agrobacterium EHA105 strain via chemical transformation. Positive single clones were selected and cultured in liquid medium containing 50 μg / mL Kan and 25 μg / mL Rif LB at 28°C for 1–2 days. The bacterial cells were collected by centrifugation at 5000 rpm for 10 min at room temperature and resuspended in osmosis buffer (10 mM MgCl2, 10 mM MES, 200 mM acetylsylcholine, pH 5.6) to OD. 600 The concentration of CitUGT73A78-SK Agrobacterium suspension was 0.6–0.8% when injected into the peel of rock sugar oranges. Overexpression of CitUGT73A78 significantly increased the content of flavonoid 7'-O-glucoside and / or flavonoid 4'-O-glucoside products.

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

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

[0107] Catalytic reactions are carried out in at least one enzyme system from B1 to B3 using flavonoids containing 7-OH and / or flavonoid compounds containing 4'-OH and UDP glycosyl donors as substrates:

[0108] B1. The aforementioned CitUGT73A78;

[0109] B2. Recombinant cells expressing the aforementioned CitUGT73A78;

[0110] B3. Recombinant proteins isolated and extracted from recombinant cells through recombinant expression;

[0111] The recombinant cells described in item B3 contain the above-mentioned CitUGT73A78 or the above-mentioned CitUGT73A78 expression vector.

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

[0113] In this invention, the catalytic reaction system consists of 4–6 μL of 0.3–1.6 mM UDP glycosyl 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.

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

[0115] The flavonoids containing 7-OH include at least one class of compounds among flavonols, flavones, and flavanones;

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

[0117] In this invention, the flavonoids in the 7-OH flavonoid compounds include one or more of apigenin, luteolin, geraniol, farnesin, and sennatin; the flavonols in the 7-OH flavonoid compounds include one or more of quercetin, kaempferol, and isorhamnetin; the flavanones in the 7-OH flavonoid compounds include one or more of naringenin, hesperidin, sennatin, and isorhamnetin; the flavonoids in the 4'-OH flavonoid compounds include one or both of apigenin and luteolin; and the flavonols in the 4'-OH flavonoid compounds include one or both of quercetin and kaempferol.

[0118] In this 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 will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0120] Example 1

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

[0122] (I) Experimental Methods

[0123] The reference sequence of the CitUGT73A78 gene (ID: Ciclev10028305m) was obtained from the Citrus pangenome database (http: / / www.citrusgenomedb.org / ). Specific primer pairs containing the recombinant expression vector pET-32a(+) were designed: upstream primer: gtatcggatccgaattcatgggcagtaaaatccctcagc (SEQ ID No. 3) and downstream primer: tggtggtgctcgaggctgctttgatggcgacttaaac (SEQ ID No. 4). PCR amplification was performed using cDNA from the oil cell layer of Ponkan orange fruit as a template. The PCR reaction system (total volume 30 μL) consisted of 15 μL of 2×PhantaMaxMasterMix (DyePlus), 1.2 μL each of 10 μM upstream and downstream primers, 1 μL of cDNA template, and 11.6 μL of DEPC. The PCR reaction program was as follows: 95℃ pre-denaturation for 3 min; 95℃ for 15 s, 58℃ for 15 s, 72℃ for 1 min, 35 cycles; 72℃ for 5 min, and storage at 4℃. After agarose gel electrophoresis analysis and purification of the PCR product, it was ligated into the target vector pET-32a(+) digested with restriction endonucleases EcoRI and XhoI. The vector was then transformed into competent E. coli DH5α cells using the heat shock method. Positive colonies were picked and sequenced for verification, thus obtaining the CitUGT73A78-pET-32a(+) recombinant plasmid.

[0124] (II) Experimental Results

[0125] 1. Sequencing verification yielded the CitUGT73A78 gene sequence SEQ ID No.1, which matches the citrus genome database. It contains 1473 nucleotides and encodes a protein with 490 amino acids, as shown in SEQ ID No.2.

[0126] 2. For example Figure 1 As shown, based on the citrus Clementine genome (v1.0), a total of 136 members of the citrus UGT family were identified and divided into 18 phylogenetic groups and 25 families. Notably, CitUGT73A78, distributed in group D of the UGT73 family, clusters in the same branch as flavonoid 7-O-glycosyltransferases identified in plants such as Arabidopsis thaliana (AtUGT73C6), chrysanthemum (Db7GlcT), strawberry (FaGT7), and tobacco (NTGlcT2).

[0127] Example 2

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

[0129] (I) Experimental Methods

[0130] 1. Materials: Using 'Ponkan' plants as materials, divide them into 9 tissue parts, including roots, stems, leaves, flowers, oil cells, white peel, sacs, juice sacs and seeds. After cutting them into small pieces, freeze them thoroughly with liquid nitrogen and store them in a -80℃ freezer.

[0131] 2. RNA extraction and cDNA synthesis: Total RNA was extracted from nine different tissues using the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit. Genomic DNA was removed using the PrimeScript™ RTreagent Kit with gDNAEraser (Takara, Japan), and the RNA was reverse transcribed into cDNA. The cDNA was then diluted 10-fold for subsequent gene expression analysis.

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

[0133] (II) Experimental Results

[0134] Figure 2 The results showed that the CitUGT73A78 gene was highly expressed in the oil cell layer of the fruit, while its expression level was low in nine other tissues, including roots, stems, leaves, flowers, white peel, pith, sac, juice sacs, and seeds. It is 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] CitUGT73A78 recombinant protein expression and purification

[0137] (I) Experimental Methods

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

[0139] 2. Add IPTG to the recombinant bacteria to a final concentration of 1 mM and incubate overnight at 16°C and 150 rpm to induce recombinant protein expression. Transfer the bacterial culture to a 500 mL centrifuge tube. Centrifuge at 4°C and 4000 g for 15 min, discard the supernatant, and collect the bacterial cells. Add 25 mL of 1×PBS buffer to fully resuspend the bacterial cells and incubate at -80°C for at least 24 h. After thawing the refrigerated bacterial cells in a 30°C water bath, sonicate them for 5 min. Centrifuge at 4°C and 10000 rpm for 30 min, and collect the supernatant. The supernatant was passed through a Meillx-HV sterilization membrane (0.45μm, diameter 33mm, Millipore USA) to remove bacteria and impurities. The protein was purified using a HisTALON™ (Takara) gravity column. 1 μL of the purified recombinant protein was subjected to SDS-PAGE electrophoresis and then desalted using a PD-10 (GE Healthcare UK) desalting column. The protein was then stored in Tris-HCl buffer (100mM Tris, 2mM DTT, pH 7.5) containing 10% glycerol and kept at -80°C for later use.

[0140] (II) Experimental Results

[0141] like Figure 3As shown, SDS-PAGE electrophoresis analysis revealed a distinct protein band at approximately 70 kDa for the recombinant CitUGT73A78 protein containing a 6×histidine tag, 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 enzyme activity and functional identification of CitUGT73A78 recombinant protein

[0144] (I) Experimental Methods

[0145] 1. In vitro enzyme activity reaction: Twelve flavonoid substrates, including naringenin, hesperidin, sennaol, isochoridin, apigenin, luteolin, geraniol, acaciain, sennaol, quercetin, kaempferol, and isorhamnetin, were selected to verify the glycosylation function of the CitUGT73A78 recombinant protein in Example 3. The reaction system consisted of 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), and 20 μL CitUGT73A78 recombinant protein (0.5 μg / μL). The enzyme reaction was incubated at 30°C for 16 h, and the reaction was terminated by adding an equal volume of pre-cooled methanol. Empty protein was used as a control in all reactions to obtain the enzyme reaction products. After centrifugation at 13000 rpm for 30 min at room temperature, the supernatant was collected for HPLC and mass spectrometry analysis.

[0146] 2. HPLC Enzyme Activity Detection and Product Identification: The enzyme reaction products were subjected to HPLC activity detection using an Agilent 1290 Infinity HPLC system (Agilent Technologies, USA) with an Inertsil C18 ODS (4.6×250mm, 5μm) column. The chromatographic conditions were as follows: 0.1% phosphoric acid solution (solution A) and chromatographic acetonitrile (solution B) were used as the mobile phase, with a flow rate of 1 mL / min. 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. Enzyme reaction products were analyzed by mass spectrometry using an AB TripleTOF 5600 time-of-flight LC-MS / MS system (AB SCIEX, USA), in negative ion scanning mode: ion source temperature (TEM) 550℃, voltage (IS) -4500V; scan range (m / z) 100–1500 Da; nebulizer gas (GS1), nebulizer gas (GSI2), and curtain gas (CUR) pressures were 55 psi, 55 psi, and 35 psi, respectively. Product identification was performed based on the retention time and ion fragmentation information of the enzyme active products and corresponding standards.

[0147] (II) Experimental Results

[0148] In vitro enzyme activity assays showed that, using UDP-glucose as a glycosyl donor, the CitUGT73A78 recombinant protein specifically catalyzed glycosylation modification at the 7-OH position of flavanones such as naringenin, hesperidin, sennarol, and isospermum erythritol, generating naringenin 7-O-glucoside, hesperidin 7-O-glucoside, sennarol 7-O-glucoside, and isospermum erythritol 7-O-glucoside, consistent with the standard reference (see [link to standard reference]). Figures 4-7 When flavonoids (apigenin, luteolin, geraniol, farnesin, and senna) and flavonols (quercetin, kaempferol, and isorhamnetin) are used as substrates, the CitUGT73A78 recombinant protein can catalyze the glycosylation of flavonoids (apigenin, luteolin, geraniol, farnesin, and senna) and flavonols (quercetin, kaempferol, and isorhamnetin) at the 7-OH position, and 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 8-15This generates the corresponding flavonoid 7'-O-glucoside and flavonoid 4'-O-glucoside.

[0149] Example 5

[0150] Determination of CitUGT73A78 enzyme reaction kinetic parameters

[0151] (I) Experimental Methods

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

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

[0154] Determination of the optimal pH for the enzymatic reaction: Prepare 100mM Tris-HCl buffer solutions with different pH values, with pH gradients set at 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. Terminate the reaction after incubation at 30℃ for 1 h; centrifuge the samples at 13000 rpm for 30 min.

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

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

[0157] 2. Determination of enzyme reaction kinetic parameters: using UDP-Glo TMThe Glycosyltransferase Assay kit was used to determine the affinity and catalytic efficiency of the CitUGT72AZ4 recombinant protein for different substrates; the reaction system consisted of 50 μL of 100 mM pH 7.0 Tris-HCl and 650 μM... UDP-glucose, 2 μg recombinant protein, 0–500 μM flavonoid substrates (0, 50, 100, 150 μM naringenin; 0, 100, 200, 300 μM hesperidin; 0, 100, 200, 300 μM sennaol; 0, 100, 200, 300 μM isospermum; 0, 50, 100, 150 μM apigenin; 0, 20, 40, 60, 80 μM luteolin; 0, 100, 200, 300 μM sennaol; 0, 100, 200, 300 μM quercetin or 0, 100, 200, 300 μM kaempferol), incubated at 37°C for 30 min. Then, 25 μL of the reaction mixture was transferred to a white flat-bottomed 96-well plate, and an equal volume of UDPDetection was added. Reagent assay reagent, gently shake the 96-well plate to mix, and incubate at room temperature for 60 min. Spectrophotometer readings were used to determine the luminescence value, and graphs were plotted using GraphPadPrism v8.0 software. The Michaelis constant (K) was calculated by fitting a nonlinear curve using the Michaelis-Menten equation. m ) and catalytic constant (k cat )value.

[0158] (II) Experimental Results

[0159] Figure 16 Optimal reaction conditions experiments showed that, using naringenin as a substrate, the recombinant CitUGT73A78 protein exhibited the highest catalytic activity at a reaction temperature of 50℃ and pH 7.0 (100mM Tris-HCl buffer).

[0160] Table 1 Enzyme kinetic parameters for different substrates

[0161]

[0162] Figure 17 The enzyme reaction kinetics results in Table 1 show that the CitUGT73A78 recombinant protein exhibits high catalytic efficiency for nine flavonoid substrates, with the highest efficiency for luteolin, followed by apigenin and quercetin, and the lowest efficiency for sennaol.

[0163] Example 6

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

[0165] (I) 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). The upstream primer was gagctgcaggaattcatgggcagtaaaatccctcag (SEQ ID No. 9), and the downstream primer was tatagccccccctcgagttagctgctttgatggcgac (SEQ ID No. 10). The CDS sequence amplified as shown in SEQ ID No. 1 was then ligated into the pGreenII002962_SK vector (SK vector) to obtain the CitUGT73A78-SK recombinant plasmid. The CitUGT73A78-SK recombinant plasmid and the SK empty vector were transformed into Agrobacterium EHA105 (pSoup) competent cells using a chemical transformation method. The bacterial culture was then evenly spread on LB solid medium containing 50 μg / mL kanamycin (Kan) and 25 μg / mL rifampin (Rif). 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 1–2 days. The cells were collected by centrifugation at 5000 rpm for 10 min at room temperature and resuspended to OD500 with osmosis buffer (10 mM MgCl2, 10 mM MES, 200 mM acetylsyleugenol, pH 5.6). 600 The concentration range was 0.6–0.8. Uniformly sized, relatively uniformly ripe, and disease-free *CitUGT73A78-SK* and *Agrobacterium* suspension were selected. CitUGT73A78-SK and an empty vector of *Agrobacterium* suspension were injected into both sides of the equator of the same fruit, respectively. The fruit was first incubated in the dark at 25°C for 1 day, then transferred to normal light conditions (photocycle: 16h light / 8h dark). Five days after injection, the peel from the injected area was collected for flavonoid glycoside content detection and gene expression analysis.

[0167] (II) Experimental Results

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

[0169] Given the instability of flavonoid 7-O-glucosides in citrus fruits, these glycosides are readily converted into more stable disaccharide structures, such as neohesperidin or rutin, by catalysis of 1,2-rhamnosperidin and 1,6-rhamnosperidin enzymes. Therefore, this study validated the results by analyzing the content of the final products of flavonoid 7-O-glucosides (such as naringenin 7-O-glucoside, hesperidin 7-O-glucoside, and isosarcinin 7-O-glucoside) in the peel of rock sugar oranges, including naringenin (Nar7Rut), hesperidin (Hes7Rut), and lemon balm glycoside (Did7Rut).

[0170] Figure 18 The results in section B showed that the total content of rutin in the peel of the sugar orange was significantly increased (P<0.05) by transient overexpression of CitUGT73A78, increasing by 1.29 times compared with the empty control group (EV). These results indicate that CitUGT73A78 plays an important promoting role in the biosynthesis of flavonoid glycosides in citrus peel.

[0171] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a 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; The flavonoid glycoside is a flavonoid 7-O-glucoside; The flavonoid 7-O-glucoside is at least one of the following compounds: flavonol 7-O-glucoside, flavonoid 7-O-glucoside, and flavanone 7-O-glucoside. Specifically, the flavonoid 7-O-glucoside is one or more of apigenin 7-O-glucoside, luteolin 7-O-glucoside, geraniol 7-O-glucoside, farnesin 7-O-glucoside, or sennatin 7-O-glucoside; the flavonol 7-O-glucoside is one or more of quercetin 7-O-glucoside, kaempferol 7-O-glucoside, or isorhamnetin 7-O-glucoside; and the flavanone 7-O-glucoside is one or more of naringenin 7-O-glucoside, hesperidin 7-O-glucoside, sennatin 7-O-glucoside, or isorhamnetin 7-O-glucoside.

2. The application of a 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; The flavonoid glycoside is a flavonoid 4'-O-glucoside; The flavonoid 4'-O-glucoside compound is one or two of flavonol 4'-O-glucoside and flavonoid 4'-O-glucoside; the flavonoid 4'-O-glucoside is one or two of apigenin 4'-O-glucoside and luteolin 4'-O-glucoside; the flavonol 4'-O-glucoside is one or two of quercetin 4'-O-glucoside and kaempferol 4'-O-glucoside.

3. The application of a 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; The flavonoid glycosides are flavonoid 7'-O-glucoside and flavonoid 4'-O-glucoside; The flavonoid 7-O-glucoside compound is one or more of apigenin 7-O-glucoside, luteolin 7-O-glucoside, quercetin 7-O-glucoside, or kaempferol 7-O-glucoside; the flavonoid 4'-O-glucoside compound is one or more of apigenin 4'-O-glucoside, luteolin 4'-O-glucoside, quercetin 4'-O-glucoside, and kaempferol 4'-O-glucoside.

4. The application of a citrus flavonoid glycosyltransferase CitUGT73A78 in catalyzing the glycosylation of flavonoid compounds containing 7-OH, characterized in that, The amino acid sequence of CitUGT73A78 is shown in SEQ ID No. 2; The flavonoid compound containing 7-OH is at least one of the following classes: flavonols, flavones, and flavanones. The flavonoids are one or more of apigenin, luteolin, geraniol, acaciain, and sennain; the flavonols are one or more of quercetin, kaempferol, and isorhamnetin; and the flavanones are one or more of naringenin, hesperidin, sennain, and isorhamnetin.

5. The application of a citrus flavonoid glycosyltransferase CitUGT73A78 in catalyzing the glycosylation of flavonoid compounds containing 4'-OH, characterized in that, The amino acid sequence of CitUGT73A78 is shown in SEQ ID No. 2; The flavonoid compound containing 4'-OH is one or two compounds selected from flavonols and flavones; the flavones are one or two selected from apigenin and luteolin; the flavonols are one or two selected from quercetin and kaempferol.

6. The application of a citrus flavonoid glycosyltransferase CitUGT73A78 in catalyzing the glycosylation of flavonoids containing 7'-OH and flavonoid compounds containing 4'-OH, characterized in that, The amino acid sequence of CitUGT73A78 is shown in SEQ ID No. 2; The flavonoids containing 7-OH and the flavonoid compounds containing 4'-OH are one or two compounds selected from flavonols and flavonoids; the flavonoids are one or two selected from apigenin and luteolin; the flavonols are one or two selected from quercetin and kaempferol.

7. The application of a biomaterial containing the encoding gene CitUGT73A78 in the biosynthesis of the flavonoid 7-O-glucoside, characterized in that, The amino acid sequence of CitUGT73A78 is shown in SEQ ID No. 2; The flavonoid 7-O-glucoside is at least one of the following compounds: flavonol 7-O-glucoside, flavonoid 7-O-glucoside, and flavanone 7-O-glucoside. Specifically, the flavonoid 7-O-glucoside is one or more of apigenin 7-O-glucoside, luteolin 7-O-glucoside, geraniol 7-O-glucoside, farnesin 7-O-glucoside, or sennatin 7-O-glucoside; the flavonol 7-O-glucoside is one or more of quercetin 7-O-glucoside, kaempferol 7-O-glucoside, or isorhamnetin 7-O-glucoside; and the flavanone 7-O-glucoside is one or more of naringenin 7-O-glucoside, hesperidin 7-O-glucoside, sennatin 7-O-glucoside, or isorhamnetin 7-O-glucoside.

8. The application of a biomaterial containing the encoding gene CitUGT73A78 in the biosynthesis of flavonoid 4'-O-glucoside, characterized in that, The amino acid sequence of CitUGT73A78 is shown in SEQ ID No. 2; The flavonoid 4'-O-glucoside compound is one or two of flavonol 4'-O-glucoside and flavonoid 4'-O-glucoside; the flavonoid 4'-O-glucoside is one or two of apigenin 4'-O-glucoside and luteolin 4'-O-glucoside; the flavonol 4'-O-glucoside is one or two of quercetin 4'-O-glucoside and kaempferol 4'-O-glucoside.

9. The application of a biomaterial containing the encoding gene CitUGT73A78 in the biosynthesis of flavonoid 7'-O-glucoside and flavonoid 4'-O-glucoside, characterized in that, The amino acid sequence of CitUGT73A78 is shown in SEQ ID No. 2; The flavonoid 7-O-glucoside compound is one or more of apigenin 7-O-glucoside, luteolin 7-O-glucoside, quercetin 7-O-glucoside, or kaempferol 7-O-glucoside; the flavonoid 4'-O-glucoside compound is one or more of apigenin 4'-O-glucoside, luteolin 4'-O-glucoside, quercetin 4'-O-glucoside, and kaempferol 4'-O-glucoside.

10. The application according to any one of claims 7 to 9, characterized in that, The biomaterials include at least one of the following: recombinant expression vector, expression cassette, and recombinant cells; The coding gene for CitUGT73A78 is shown in either S1 or S2 below: S1, a gene with a nucleotide sequence as shown in SEQ ID No. 1; S2. Genes that have undergone codon optimization based on the genes described in S1 without altering the amino acid sequence.

11. A method for preparing flavonoid 7'-O-glucoside and / or flavonoid 4'-O-glucoside, characterized in that, Includes the following steps: Catalytic reactions were carried out in the following B1 or B2 enzyme systems using flavonoids containing 7-OH and / or flavonoid compounds containing 4'-OH and UDP glycosyl donors as substrates: B1. The CitUGT73A78 as described in claim 1; B2. Recombinant cells expressing the CitUGT73A78 of claim 1; When preparing flavonoid 7-O-glucoside, the flavonoid 7-O-glucoside is at least one of the following compounds: flavonol 7-O-glucoside, flavonoid 7-O-glucoside, and flavanone 7-O-glucoside. Specifically, the flavonoid 7-O-glucoside is one or more of apigenin 7-O-glucoside, luteolin 7-O-glucoside, geraniol 7-O-glucoside, farnesin 7-O-glucoside, or sennatin 7-O-glucoside; the flavonol 7-O-glucoside is one or more of quercetin 7-O-glucoside, kaempferol 7-O-glucoside, or isorhamnetin 7-O-glucoside; and the flavanone 7-O-glucoside is one or more of naringenin 7-O-glucoside, hesperidin 7-O-glucoside, sennatin 7-O-glucoside, or isorhamnetin 7-O-glucoside. When preparing flavonoid 4'-O-glucoside, the flavonoid 4'-O-glucoside compound is one or two compounds selected from flavonol 4'-O-glucoside and flavonoid 4'-O-glucoside; the flavonoid 4'-O-glucoside is one or two compounds selected from apigenin 4'-O-glucoside and luteolin 4'-O-glucoside; the flavonol 4'-O-glucoside is one or two compounds selected from quercetin 4'-O-glucoside and kaempferol 4'-O-glucoside. When preparing flavonoid 7-O-glucoside and flavonoid 4'-O-glucoside, the flavonoid 7-O-glucoside compound is one or more of apigenin 7-O-glucoside, luteolin 7-O-glucoside, quercetin 7-O-glucoside, or kaempferol 7-O-glucoside; the flavonoid 4'-O-glucoside compound is one or more of apigenin 4'-O-glucoside, luteolin 4'-O-glucoside, quercetin 4'-O-glucoside, and kaempferol 4'-O-glucoside.

12. The preparation method according to claim 11, characterized in that, The temperature of the catalytic reaction is 40~65℃; the time of the catalytic reaction is 30min~2h; the pH value of the system of the catalytic reaction is 5.5~7.5; The catalytic reaction system consists of 4-6 μL of 0.3-1.6 mM UDP glycosyl 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 is UDP-glucose; The flavonoid compound containing 7-OH is at least one of flavonols, flavones, and flavanones; the flavonoid is one or more of apigenin, luteolin, geraniol, farnesin, and sennatin; the flavonol is one or more of quercetin, kaempferol, and isorhamnetin; and the flavanone is one or more of naringenin, hesperidin, sennatin, and isorhamnetin. The flavonoid compound containing 4'-OH is one or two compounds selected from flavonols and flavones; the flavones are one or two selected from apigenin and luteolin; the flavonols are one or two selected from quercetin and kaempferol.

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