A flavone glycosyltransferase, its encoding gene and applications

Abstract

The present invention belongs to the technical fields of genetic engineering and enzyme engineering, and relates to a flavonoid glycosyltransferase derived from Stevia rebaudiana, its coding gene and applications. The flavonoid glycosyltransferase of the present invention is derived from Stevia rebaudiana, and its amino acid sequence is shown as SEQ ID NO.1; its nucleotide sequence is shown as SEQ ID NO.2. The flavonoid glycosyltransferase SrUGT72B1 of the present invention is a 3-, 5-, 7-O glycosyltransferase capable of catalyzing the glycosylation of flavonoid compounds, and has broad-spectrum catalytic activity, catalyzing the glycosylation of flavonols, flavones, dihydroflavones and dihydroxy chalcones. It has high catalytic efficiency for flavonols (quercetin, kaempferol, quercetin-3-O-glucoside), flavones (apigenin, luteolin), and can be used for the biosynthesis of 3-, 5-, 7-O glycosylated products of these compounds, and can glycosylate the 7-hydroxy group of quercetin-3-O-glucoside to obtain a disaccharide glycosylated product.

Description

Technical Field

[0001] The present invention relates to a flavonoid glycosyltransferase (UDP - glycosyltransferases, UGT) derived from Stevia rebaudiana, its encoding gene and application, belonging to the technical fields of genetic engineering and enzyme engineering. Background Art

[0002] Flavonoids are a class of secondary metabolites with rich contents and varieties in plants, and have various pharmacological activities such as anti - tumor, antioxidant, antibacterial, improving sugar and lipid metabolism, and protecting the cardiovascular and cerebrovascular systems. Glycosylation is one of the most common modification methods of flavonoids, which further improves the abundance of flavonoids and provides a huge material basis for drug screening. Glycosylation can also change the hydrophilicity and hydrophobicity, chemical stability, sub - cellular localization and other characteristics of flavonoid molecules, thereby regulating the transport and function of flavonoid molecules in cells and plants and participating in the stress response mechanism of plants. Over - expressing certain flavonoid glycosyltransferases in plants can eliminate the product inhibition in the flavonoid biosynthesis pathway, activate the flavonoid biosynthesis pathway, accumulate more secondary metabolites, and improve the stress response ability of plants.

[0003] Most flavonoid glycoside natural products are isolated from plants, but the large - scale preparation is limited by the complex plant extraction process and high separation and purification costs. Chemical synthesis of glycosides involves long - drawn - out group protection and de - protection steps to achieve regioselective and stereoselective glycosylation, resulting in low synthesis yields. The glycosylation of flavonoids in plants is catalyzed by glycosyltransferases (glycosyltransferase, GT, EC 2.4.x.y), which transfer glycosyl groups from activated glycosyl donors to glycosyl acceptors and form glycosidic bonds, generally with high regioselectivity. At present, there are few reports on the biosynthesis of flavonol 7 - O - glucoside, and the yields of flavone 7 - O - glucosides (apigenin - 7 - O - glucoside and luteolin - 7 - O - glucoside) are relatively low. The enzymes involved in flavonoid glycosylation in plants are glycosyltransferases of the GT1 family, which have been studied more in model organisms such as Arabidopsis thaliana and rice, but are relatively scarce in Stevia rebaudiana. Moreover, most known flavonoid glycosyltransferases can only glycosylate a single site of flavonoids, and some can glycosylate the 3 - or 5 - hydroxyl groups of flavonoids. At present, no glycosyltransferase that can simultaneously achieve glycosylation at the 3 - position, 5 - position, and 7 - position has been found.

[0004] Stevia rebaudiana contains various flavonoid glycosides, such as rutin, kaempferol glucoside, kaempferol rhamnoside, quercetin 3 / 7-O-glucoside, quercetin 3-O-arabinoside, etc. These flavonoid glycosides play important roles in the immune defense process of Stevia rebaudiana plants. Therefore, screening for UGTs with 3 / 5 / 7-O glycosylation functions in Stevia rebaudiana, elucidating their catalytic activities, and using them in the synthetic biology research of flavonoid glycosides or for the quality improvement of Stevia rebaudiana is of great significance.

[0005] The presence of various flavonoid glycosides in Stevia rebaudiana indicates that there are multiple glycosyltransferases involved in flavonoid glycosylation in Stevia rebaudiana. Liu Qiong et al. from Sichuan Agricultural University screened and studied the flavonoid glycosyltransferases in Stevia rebaudiana and found that SrUGT-741 has catalytic activity towards isoquercitrin, SrUGT-720 and SrUGT-752 have catalytic activity towards kaempferol, and SrUGT-634 has catalytic activity towards apigenin. Current research shows that plant glycosyltransferases involved have flexible substrate accommodation and can glycosylate multiple substrates, while the glycosyltransferases identified in the above research can only catalyze one substrate. From the liquid-phase results provided, the substrate peak is much higher than the product peak, and the enzyme catalytic activity is poor. Moreover, the glycosylation products of these enzymes are single, and the glycosylation sites are unknown. Therefore, flavonoid glycosyltransferases with high activity or multi-site glycosylation in Stevia rebaudiana still need to be further explored and functionally verified.

[0006] This patent provides a new flavonoid glycosyltransferase in Stevia rebaudiana. This glycosyltransferase has the characteristics of a broad substrate spectrum, high activity, and the ability to glycosylate the 3-position, 5-position, and 7-position hydroxyl groups of flavonoids, and can glycosylate flavonoids or flavonols such as 7-hydroxyflavone, apigenin, quercetin, kaempferol, and luteolin. Moreover, the glycosylation products are diverse, and it has potential application value for the in vitro preparation of glycosylation products at different positions of quercetin and kaempferol. Flavonoids are products of the phenylpropanoid metabolic pathway, and the phenylpropanoid metabolic pathway is related to plant disease resistance. The glycosylation of flavonoids promotes the positive progress of the phenylpropanoid pathway and is beneficial to improving plant disease resistance. Therefore, this enzyme also has potential application value in the quality improvement of Stevia rebaudiana. Summary of the Invention

[0007] The first object of the present invention is to provide a Stevia rebaudiana flavonoid glycosyltransferase gene and the protein encoded thereby. The second object of the present invention is to provide a preparation method for the above-mentioned flavonoid glycosyltransferase. The third object of the present invention is to provide the application of the Stevia rebaudiana flavonoid glycosyltransferase in the in vitro synthesis of flavonoid glycosides.

[0008] To achieve the above technical objectives, the technical solutions adopted by the present invention are as follows:

[0009] In the first aspect of the present invention, a flavonoid glycosyltransferase is provided, and its amino acid sequence has one or more of the following characteristics:

[0010] 1) having the amino acid sequence shown in SEQ ID NO.1;

[0011] MAEFAKRLVQHHHISATIIVPTTGAPPKTQISVLESLPENIHHLFLPPVSVDGLPEDTRPERIIT

[0012] FIMEASLSSLKDALSSLKSKTKLVALAFDMFGHVSMKVSQELNLLNFLFFPMSLMALTFTFI

[0013] LPKLDEETSGEYKDLSSPVKVPGSVSFNGPELMAPVQVRSDEVYKEYLLLSKKLFLLDGIM

[0014] VNSFKELEEESFRVLHDEIAGQTPIYPVGPLIRSDPNNGPDRHECLKWLDDQPSGSVLLVSFG

[0015] SAGTLSLEQVHELAHGLELSNHRFIWIVRSPNKGSNASFLTDYNEDDPSGFLPEGFLARTMN

[0016] RGFIVPLWGPQIKILSHESVGGFLTHCGWNSTLESVVHGVPMIAWPLYAEQQMNAKMMIEA

[0017] LDLALRLEIDEYGIYRKDEIKKVVKELMEGDEGKKIAKRLKELKLAAMKA

[0018] 2) having the amino acid residue sequence from the amino terminus at positions 1 - 424 of SEQ ID NO.1;

[0019] 3) an amino acid sequence having flavonoid glycosyltransferase activity formed by substitution and / or deletion and / or addition of one or more than two amino acids to the amino acid sequence shown in SEQ ID NO.1;

[0020] 4) an amino acid sequence having at least 80% identity with SEQ ID NO.1.

[0021] In the second aspect of the present invention, there is provided a flavonoid glycosyltransferase gene, the nucleotide sequence of which has one or more than two of the following characteristics:

[0022] 1) having the deoxyribonucleic acid sequence shown in SEQ ID NO.2;

[0023] ATGGCCGAGTTTGCTAAGCGACTCGTCCAACACCACCATATCTCCGCCACTATCATCGTT

[0024] CCTACCACCGGAGCTCCTCCCAAAACCCAAATATCCGTTCTTGAATCCTTGCCGGAAAA

[0025] CATCCATCACCTGTTTCTCCCTCCGGTTAGCGTTGACGGATTACCGGAGGATACTCGGCC

[0026] GGAGCGTATAATCACCTTCATCATGGAAGCTAGTCTTTCAAGTCTTAAAGATGCTTTAAG

[0027] TTCTTTGAAATCAAAAACCAAGCTTGTTGCATTAGCTTTTGATATGTTTGGACATGTTTCA

[0028] ATGAAAGTTTCACAAGAACTTAACCTCTTGAACTTCTTGTTCTTTCCAATGAGTTTAATG

[0029] GCTTTAACCTTCACTTTTATATTACCAAAGCTTGATGAAGAGACGTCCGGCGAGTATAAA

[0030] GACTTGTCGAGCCCGGTTAAAGTACCGGGCTCAGTTAGTTTTAACGGGCCAGAGCTGAT

[0031] GGCCCCAGTCCAGGTCCGGTCCGATGAAGTATACAAAGAGTATCTTTTACTTTCCAAGA

[0032] AATTGTTTTTGCTTGATGGTATCATGGTGAATAGCTTTAAGGAGTTGGAAGAAGAAAGCT

[0033] TTCGGGTTTTACATGATGAAATTGCGGGTCAAACACCAATTTACCCAGTCGGGCCACTCA

[0034] TTCGGTCTGATCCAAATAATGGGCCAGACCGACATGAGTGTTTGAAGTGGTTAGACGAT

[0035] CAACCTAGTGGATCGGTTTTGTTGGTGTCTTTTGGTAGTGCTGGGACCCTTTCTTTAGAA

[0036] CAAGTTCATGAGCTAGCCCATGGGTTAGAGCTGAGTAACCATAGGTTTATATGGATTGTT

[0037] AGAAGCCCTAATAAGGGTTCAAATGCTTCCTTTCTTACCGACTATAACGAAGATGATCCA

[0038] TCAGGGTTTTTACCCGAAGGGTTTCTAGCTAGGACGATGAATCGAGGCTTCATCGTGCCA

[0039] TTGTGGGGCCCGCAAATAAAGATACTGAGTCACGAGTCAGTCGGTGGGTTCTTGACTCA

[0040] TTGTGGATGGAACTCGACTCTAGAGAGTGTTGTTCATGGGGTGCCAATGATTGCTTGGCC

[0041] TCTATATGCAGAGCAACAAATGAATGCTAAAATGATGATTGAAGCACTTGATTTGGCATT

[0042] AAGACTTGAAATTGATGAATATGGAATATATCGAAAAGATGAGATCAAGAAGGTTGTGA

[0043] AGGAGTTAATGGAGGGAGATGAAGGGAAGAAGATAGCCAAAAGGTTGAAAGAATTGA

[0044] AGCTTGCTGCCATGAAAGCTTAA

[0045] 2) A deoxyribonucleic acid sequence encoding the amino acid sequence of SEQ ID NO.1;

[0046] 3) A nucleotide sequence obtained by substituting, deleting, and / or adding one or more nucleotides to the deoxyribonucleic acid sequence of SEQ ID NO.2 and encoding a flavone glycosyltransferase with activity;

[0047] 4) A deoxyribonucleic acid sequence having a homology of 80% or more with the deoxyribonucleic acid sequence defined by SEQ ID NO.2 and capable of encoding a flavone glycosyltransferase.

[0048] The third aspect of the present invention provides a recombinant expression plasmid containing the flavone glycosyltransferase gene.

[0049] The fourth aspect of the present invention provides a recombinant genetically engineered bacterium containing the flavone glycosyltransferase gene.

[0050] The fifth aspect of the present invention provides a method for preparing a flavone glycosyltransferase, which comprises cloning the aforementioned flavone glycosyltransferase gene into a recombinant expression vector, introducing it into a host cell, and obtaining a recombinantly expressed flavone glycosyltransferase.

[0051] In the above technical solutions, further, the recombinant expression vector includes one or more of an Escherichia coli expression vector, a yeast expression vector, a Bacillus subtilis expression vector, a Lactobacillus expression vector, a Streptomyces expression vector, a phage vector, a filamentous fungus expression vector, a plant expression vector, an insect expression vector, or a mammalian cell expression vector.

[0052] The host cell includes one of an Escherichia coli host cell, a yeast host cell, a Bacillus subtilis host cell, a Lactobacillus host cell, an actinomycete host cell, a filamentous fungus host cell, an insect cell, or a mammalian cell.

[0053] The sixth aspect of the present invention provides the application of the aforementioned flavone glycosyltransferase in catalyzing the glycosylation of flavonols, flavones, dihydroflavones, and dihydroxy chalcone compounds; the application in preparing flavonol glycosides, flavone glycosides, dihydroflavone glycosides, and dihydroxy chalcone glycoside compounds; and the application in preparing flavone diglycoside compounds.

[0054] In the above technical solutions, further, the flavonol glycoside compounds include quercetin-3-O-glucoside, quercetin-5-O-glucoside, quercetin-7-O-glucoside, quercetin-3,7-O-diglucoside, kaempferol-3-O-glucoside, kaempferol-5-O-glucoside, and kaempferol-7-O-glucoside; the flavone glycoside compounds include apigenin-5-O-glucoside, apigenin-7-O-glucoside, luteolin-5-O-glucoside, and luteolin-7-O-glucoside; and the flavone diglycoside compounds include quercetin-3,7-O-diglucoside.

[0055] In the above technical solution, further, the substrate glycosylation sites of the application include the 3-position and / or 5-position and / or 7-position hydroxyl groups of flavonoids.

[0056] The seventh aspect of the present invention provides the application of the aforementioned flavonoid glycosyltransferase gene in the genetic breeding of Stevia rebaudiana.

[0057] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0058] SrUGT72B1 provided by the present invention is a 3-, 5-, 7-O glycosyltransferase that is first discovered to be able to catalyze the glycosylation of flavonoid compounds in Stevia rebaudiana. The full-length sequence of the gene was obtained from cDNA by using PCR technology. After constructing the pET21a protein expression vector and transforming Escherichia coli BL21(DE3), the target protein was induced and purified. In vitro enzyme activity functional identification proved that SrUGT72B1 has broad-spectrum catalytic activity and can catalyze the glycosylation of flavonols, flavonoids, dihydroflavonoids, and dihydroxy chalcone compounds. It has relatively high catalytic efficiency for compounds such as flavonols (quercetin, kaempferol, quercetin-3-O-glucoside), flavonoids (apigenin, luteolin), etc., and the product types are rich. It can be used for the biosynthesis of 3-, 5-, 7-O glycosylation products of these compounds, and can glycosylate the 7-position hydroxyl group of quercetin-3-O-glucoside, the 3-position hydroxyl group of quercetin-5-O-glucoside, and the 3-position hydroxyl group of quercetin-7-O-glucoside to obtain disaccharide glycosylation products. Therefore, it has high economic value and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0060] Figure 1 : Classification of the glycosyltransferase family of the target gene SrUGT72B1.

[0061] Figure 2 : Electrophoresis diagram of the ORF amplification product of the target gene SrUGT72B1.

[0062] Figure 3 : SDS-PAGE electrophoresis diagram of SrUGT72B1 protein.

[0063] Wherein: M: Protein molecular weight standard; Lane 1: Purified SrUGT72B1 protein.

[0064] Figure 4: Ribbon diagrams of the three-dimensional structures of VvGT1 and SrUGT72B1 interacting with substrates. Kaempferol molecules and UDP are shown using stick models. Among them, Figure 4 A is VvGT1, Figure 4 B is SrUGT72B1.

[0065] Figure 5 : HPLC chromatogram and product MS spectrum of SrUGT72B1 catalyzing 2,4,5-trichlorophenol (TCP).

[0066] Figure 6 : HPLC chromatogram and product MS spectrum of SrUGT72B1 catalyzing 7-hydroxyflavone.

[0067] Figure 7 : HPLC chromatogram and product MS spectrum of SrUGT72B1 catalyzing quercetin.

[0068] Figure 8 : HPLC chromatogram and product MS spectrum of SrUGT72B1 catalyzing kaempferol.

[0069] Figure 9 : HPLC chromatogram and product MS spectrum of SrUGT72B1 catalyzing luteolin.

[0070] Figure 10 : HPLC chromatogram and product MS spectrum of SrUGT72B1 catalyzing quercetin-3-O-glucoside. Detailed implementation methods

[0071] The present invention will be described in detail below with reference to examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following implementation methods are all conventional methods, and the experimental reagents involved are all conventional biochemical reagents.

[0072] Example 1 Transcriptome sequencing of Stevia rebaudiana leaves of different varieties

[0073] Using Stevia rebaudiana varieties with high survival rate under drought treatment and Stevia rebaudiana varieties with low survival rate under drought treatment as experimental materials, collect the middle leaves of Stevia rebaudiana plants after 2 months of growth, and store them at -80 °C for later use. Extract RNA and perform detection, and use Illumina HiSeq TM 2000 sequencing platform for transcriptome library sequencing.

[0074] Example 2 Screening of UGTs genes based on Stevia rebaudiana transcriptome data

[0075] The sequencing results were aligned and annotated in the database to find the Unigene sequences annotated as UDP - glycosyltransferases and Flavonoid - glycosyltransferases for further analysis and screening. The FPKM values of the key genes in the flavonoid synthesis pathway and candidate glycosyltransferases of the two strains were obtained using transcriptome data, and the co - expression analysis of the candidate glycosyltransferase genes and the key genes in the flavonoid synthesis pathway was performed using the MeV4.9.0 biological software.

[0076] The glycosyltransferase genes co - expressed with the key genes in the flavonoid synthesis pathway were aligned with the known flavonoid glycosyltransferase genes in the NCBI database, and the sequence similarity with the flavonoid glycosyltransferase sequences from other known sources was less than 50%. The phylogenetic tree was constructed using MEGA 7.0 ( Figure 1 ), and the homology analysis of the candidate glycosyltransferase genes was carried out. The model of the candidate glycosyltransferase was constructed using SWISS - MODEL, and the substrate docking analysis with kaempferol and UDP - Glc was performed using the Auto Dock software.

[0077] Analysis of the phylogenetic tree ( Figure 1 ) found that the glycosyltransferases UGT88D7 and GmIFGT, which are evolutionarily close to SrUGT72B1, can glycosylate the 7 - hydroxy group of flavonoids and the 7 - hydroxy group of isoflavonoids respectively. The three - dimensional structure of SrUGT72B1 has a very high similarity with VvGT1, and the catalytic substrate of VvGT1 is kaempferol ( Figure 4 A). Docking analysis ( Figure 4 B) found that the distance between the hydroxy group of kaempferol in the substrate cavity and the anomeric carbon of UGP - Glc is relatively close, which is conducive to the SN2 substitution reaction to achieve the glycosylation of kaempferol. Therefore, it is inferred that SrUGT72B1 is a flavonoid glycosyltransferase in Stevia rebaudiana.

[0078] Example 3 Cloning and expression of the SrUGT72B1 - encoding gene

[0079] The software Bioxm 2.6 was used to find the ORF (Open Reading Frame) of SrUGT72B1. Full - length primers SrUGT72B1 - F / R were designed in the non - coding regions on both sides of the ORF to amplify the gene.

[0080] Full - length primers:

[0081] SrUGT72B1 - F: CGGCATATGATGGCCGAGTTTGCTAAGCG; (SEQ ID NO.3)

[0082] SrUGT72B1-R: ACGCTCGAGTTAAGCTTTCATGGCAGCAAGC; (SEQ ID NO.4)

[0083] Since NdeI and XhoI restriction sites were introduced into the designed upstream and downstream primers respectively, the PCR clean product and the expression vector pET21a were digested with NdeI and XhoI respectively. After the digestion products were cleaned and recovered, they were ligated with T4 DNA ligase (ligation system: (0.5 μL of 5 μL T4 DNA Ligase, 0.5 μL of 10 μL 10×T4 DNA Ligase Buffer, 2 μL of pET21a, 2 μL of PCR product), ligation condition: overnight ligation at room temperature). Take 5 μL of the ligation product to transform E. coli TOP10 competent cells, and spread them on solid Luria - Bertani medium containing 100 μg / mL ampicillin, and culture at 37 °C for 12 - 16 h. Pick monoclonal colonies, use the corresponding primers for colony PCR verification, inoculate the monoclonal colonies with correct amplification into liquid Luria - Bertani medium containing 100 μg / mL ampicillin for culture, and extract plasmids; use restriction enzymes NdeI and XhoI to perform double digestion on the extracted plasmids, and send the recombinant plasmids with correct results to BGI for sequencing. The sequencing results showed that the candidate gene shown in SEQ ID No.2 was inserted between the NdeI and XhoI restriction sites of pET21a, and the insertion direction was correct, proving that the recombinant plasmid was successfully constructed, and these recombinant plasmids were named pET21a - SrUGT72B1. The full - length nucleotide sequence of the flavonoid glycosyltransferase SrUGT72B1 gene is 1275 bp, and the nucleotide sequence is as shown in SEQ ID NO.2; it encodes 424 amino acids, and the amino acid sequence is as shown in SEQ ID NO.1, and the theoretical molecular weight of the protein is (47.40) kDa.

[0084] Transform pET21a - SrUGT72B1 into E. coli BL21(DE3), and perform induction expression and purification on it. Use polyacrylamide gel electrophoresis to detect the expression and purification of SrUGT72B1. The purified glycosyltransferase shows a single band on the electrophoresis gel, and its position coincides with the predicted molecular weight ( Figure 3 ).

[0085] Example 4 Detection of Flavonoid Substrates of SrUGT72B1

[0086] The purified flavonoid glycosyltransferase protein was functionally verified in vitro according to the following reaction system (200 μL):

[0087] 10 mM Tris (pH 7.5), 10 μg of purified protein, 1 mM UDP-Glucose, 1 mM of receptor substrate, and the receptor substrates include: 2,4,5-trichlorophenol (TCP), 7-hydroxyflavone, quercetin, kaempferol, luteolin, quercetin 3-O-glucoside. Add an equal volume of absolute ethanol to terminate the reaction, shake well, centrifuge at 12,000 rpm for 10 minutes, take the supernatant and filter it through a 0.22 μm organic filter membrane, and use liquid chromatography-mass spectrometry to detect the composition of the reaction products. Instrument model: Waters e2695. Injection volume: 10 μL, chromatographic column: Elite superil ODS2 (5 μm, 250×4.6 mm), column temperature: 40 °C. Chromatographic conditions: UV 256 nm, mobile phase: (A): water (containing 0.1% formic acid), (B): acetonitrile (1% formic acid), flow rate: 1 mL / min, elution program: 0 - 1.5 min, 20% B; 1.5 - 16 min, linearly increase to 50% B. Induce BL21(DE3) containing the empty plasmid pET21a, take the supernatant after cell disruption and react with different substrates under the above conditions, and use this reaction system as a control.

[0088] The results of liquid chromatography-mass spectrometry detection showed ( Figure 5 ), in the catalytic system of 2,4,5-trichlorophenol, there was an obvious product peak (a) at 9.4 min in the experimental group compared with the control group, and the substrate peak area decreased significantly, indicating that SrUGT72B1 can indeed glycosylate 2,4,5-trichlorophenol.

[0089] The results of liquid chromatography-mass spectrometry detection showed ( Figure 6 ), in the catalytic system of 7-hydroxyflavone, there was one more product peak (b) in the experimental group compared with the control group. The mass spectrometry detection results of the peak position b sample showed that its m / z was [M-H] - 399.0860, and the molecular weight of 7-hydroxyflavone was 238.24. The molecular weight of the new substance increased by 162 compared with 7-hydroxyflavone, indicating that SrUGT72B1 added a glucose molecule to the 7-hydroxy group of 7-hydroxyflavone.

[0090] The results of liquid chromatography-mass spectrometry detection showed ( Figure 7 ), in the catalytic system of quercetin, there were 3 more product peaks (c, d, e) in the experimental group compared with the control group. The mass spectrometry detection results of c, d, f showed that their m / z were [M-H] -463.0709, 463.0698, 463.0712, the molecular weights of c, d, and e are 162 higher than that of quercetin (MW: 302.23), and the relative abundance of the mass spectrometry is e > d > c, which is consistent with the size of the liquid-phase peak area, indicating that SrUGT72B1 can glycosylate the hydroxyl groups at three positions of quercetin, and the main product is quercetin-3-O-glucoside.

[0091] The liquid chromatography-mass spectrometry detection results show ( Figure 8 ), in the catalytic system of kaempferol, there are 3 more product peaks (f, g, h) in the experimental group than in the control group. The mass spectrometry detection results of f, g, and h show that their m / z are [M-H] - 447.0863, 447.0883, 447.1014, the molecular weights of f, g, and h are 162 higher than that of kaempferol (MW: 286.24), indicating that SrUGT72B1 can glycosylate the hydroxyl groups at three positions of quercetin to obtain kaempferol-3-O-glucoside, kaempferol-5-O-glucoside, and kaempferol-7-O-glucoside.

[0092] The liquid chromatography-mass spectrometry detection results show ( Figure 9 ), in the catalytic system of luteolin, there are 2 more product peaks (i, j) in the experimental group than in the control group. The mass spectrometry detection results of i and j show that their m / z are [M-H] - 447.0774 and 447.0783, the molecular weights of i and j are 162 higher than that of luteolin (MW: 286.24), indicating that SrUGT72B1 can glycosylate the hydroxyl groups at two positions of luteolin to obtain luteolin-3-O-glucoside and luteolin-7-O-glucoside.

[0093] The liquid chromatography-mass spectrometry detection results show ( Figure 10 ), in the catalytic system of quercetin 3-O-glucoside, there is 1 more product peak (k) in the experimental group than in the control group. The mass spectrometry detection result of k shows that its m / z is [M-H] - 625.1116, the molecular weight of k is 162 higher than that of quercetin 3-O-glucoside (MW: 464.4), indicating that SrUGT72B1 can glycosylate the hydroxyl group at one position of quercetin 3-O-glucoside to obtain quercetin 3,7-O-diglucoside.

[0094] In summary, SrUGT72B1 can glycosylate multiple hydroxyl groups of flavonoids such as 2,4,5-trichlorophenol, 7-hydroxyflavone, quercetin, kaempferol, luteolin, quercetin 3-O-glucoside, etc., and achieve the preparation of flavonoid diglycosides. 2,4,5-Trichlorophenol has one glycosylation product, and 7-hydroxyflavone has only one glycosylation product, which is flavone-7-O-glucoside; quercetin has three glycosylation products, and one main product, which is quercetin-3-O-glucoside; kaempferol has three main glycosylation products; luteolin has two glycosylation products, which are luteolin-3-O-glucoside and luteolin-5-O-glucoside; quercetin 3-O-glucoside has one product, which is quercetin-3,7-O-diglucoside.

[0095] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the technical principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A flavone glycosyltransferase, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

2. A flavonoid glycosyltransferase gene, characterized in that: Its nucleotide sequence encodes the flavone glycosyltransferase described in claim 1.

3. A recombinant expression plasmid containing the flavone glycosyltransferase gene described in claim 2.

4. A recombinant genetically engineered bacterium containing the flavone glycosyltransferase gene described in claim 2.

5. A method for preparing a flavone glycosyltransferase, characterized in that: Clone the flavone glycosyltransferase gene described in claim 2 into a recombinant expression vector, introduce it into a host cell, and obtain a recombinantly expressed flavone glycosyltransferase; The recombinant expression vector is one of an Escherichia coli expression vector, a yeast expression vector, a Bacillus subtilis expression vector, a lactic acid bacterium expression vector, a Streptomyces expression vector, and a phage vector; The host cell is one of an Escherichia coli host cell, a yeast host cell, a Bacillus subtilis host cell, a lactic acid bacterium host cell, and an actinomycete host cell.

6. Use of the flavone glycosyltransferase described in claim 1 in the preparation of flavonol glycoside compounds, flavone glycoside compounds, or flavone diglycoside compounds; The flavonol glycoside compounds are quercetin-3-O-glucoside, quercetin-5-O-glucoside, quercetin-7-O-glucoside, kaempferol-3-O-glucoside, kaempferol-5-O-glucoside, or kaempferol-7-O-glucoside; the flavone glycoside compounds are luteolin-5-O-glucoside and luteolin-7-O-glucoside; the flavone diglycoside compounds are quercetin-3,7-O-diglucoside.

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