Screening identification and application of key enzyme participating in triterpenoid saponin glycosylation modification

Abstract

The invention provides screening identification and application of a key enzyme participating in triterpenoid saponin glycosylation modification. Belongs to the technical field of gene engineering. According to the invention, a glycosyl transferase gene is screened based on the multi-omics research of a traditional Chinese medicine clinopodium polycephalum-based original plant-calamint, a pET28a-MBP-CcGT-g51295 prokaryotic expression vector is constructed, the glycosyl transferase is successfully obtained, and the research finds that the glycosyl transferase disclosed by the invention can be used for catalyzing Saikogenin A and Saikogenin F by utilizing a glycosyl donor UDP-fucose to respectively generate Prosakogenin A and Prosakogenin F; the method comprises the following steps: catalyzing Saikogenin A by using UDP-glucose so as to generate clinoposide I and clinoposide X, and further catalyzing Saikogenin A by using UDP-glucose so as to generate the clinoposide I and the clinoposide X, the preparation method comprises the following steps: catalyzing Prosakogenin A by using UDP-glucose so as to generate Saikosaponin B1, Saikosaponin c1 and Saikosaponin X, and synthesizing the Saikosaponin B1, the Saikosaponin c1 and the Saikosaponin X by using UDP-glucose; uDP-glucose is utilized to catalyze Prosakigenin F to generate Sasakiponin A and Buddlejasponin IV, the catalytic function of glycosyl transferase is disclosed, a gene component is provided for biological research on synthesis of triterpenoid saponin components, and the problem of biological resources of rare saponin is solved.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and more specifically, to the screening, identification and application of a key enzyme involved in the glycosylation modification of triterpenoid saponins. Background Art

[0002] Triterpenoid saponins are widely present in medicinal plants and are the main active ingredients of many precious traditional Chinese medicines. The content of triterpenoid saponins in natural medicinal materials is low, and they face problems such as high chemical extraction costs and serious pollution in the purification process, which to a certain extent limit their new drug research and development and clinical applications. Therefore, the biosynthesis of triterpenoid saponins has become a research hotspot. Dysosma versipellis is one of the "Top Ten Anhui Medicines", has significant pharmacological activities and has been developed into drugs, and triterpenoid saponins (such as bupleurum saponins and buddlejasaponin) are its important active ingredients.

[0003] Triterpenoid saponins are a class of natural compounds, and their basic structure includes: a triterpene skeleton and a sugar chain. Among them, the sugar chain part is connected to the triterpene skeleton through a glycosidic bond, and the type, number and connection position of the sugar chain determine the diversity and function of triterpenoid saponins. Glycosyltransferase is an enzyme that catalyzes glycosylation reactions and is responsible for transferring activated sugar donors, such as UDP-fucose, UDP-glucose, UDP-rhamnose, etc., to the triterpene skeleton to form glycosidic bonds. Different glycosyltransferases can recognize different sugar donors and receptors, thereby producing triterpenoid saponins with diverse structures. Glycosylation not only increases the structural complexity of triterpenoid saponins but also significantly affects their physicochemical properties and biological activities. Therefore, glycosyltransferase is crucial for the synthesis of triterpenoid saponins.

[0004] However, the current research on glycosyltransferases that catalyze the fucosylation and glucosylation modifications at the C-3 position of triterpenoid compounds has not yet made a breakthrough, and systematic research is urgently needed.

[0005] Therefore, how to provide a glycosyltransferase that can catalyze the fucosylation and glucosylation modifications at the C-3 position of triterpenoid compounds is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides the screening, identification and application of a key enzyme involved in the glycosylation modification of triterpenoid saponins, successfully discovers a glycosyltransferase that can catalyze the fucosylation and glucosylation modifications at the C-3 position, provides gene components for the synthetic biology research of triterpenoid saponin components, and solves the biological resource problem of rare saponins.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A glycosyltransferase involved in the glycosylation modification of triterpenoid saponins, which is composed of the amino acid sequence shown in SEQ ID NO.6 or an amino acid sequence with the same enzymatic catalytic performance after substitution, deletion and / or addition of one or more amino acids to the amino acid sequence shown in SEQ ID NO.6.

[0009] Another object of the present invention is to provide: A gene encoding the above glycosyltransferase, which is composed of the nucleotide sequence shown in SEQID NO.3 or a nucleotide sequence with the same enzymatic catalytic performance after substitution, deletion and / or addition of one or more bases to the nucleotide sequence shown in SEQ ID No.3.

[0010] Another object of the present invention is to provide: A primer set for amplifying the above glycosyltransferase gene, which includes CcGT-g51295-F and CcGT-g51295-R, and the specific sequences are as follows:

[0011] CcGT-g51295-F: 5’-ATGGCGATCAATGGAAAACCGCCTC-3’, SEQ IDNo.1;

[0012] CcGT-g51295-R: 5’-TCACTGGACTAGGATTTGAGAAGAAATGTC-3’, SEQ ID No.2.

[0013] Another object of the present invention is to provide: A biological material containing the above glycosyltransferase gene, and the biological material is a recombinant vector, expression cassette, transgenic cell line or recombinant bacterium.

[0014] Another object of the present invention is to provide the application of the above glycosyltransferase or the above gene encoding the glycosyltransferase or the above biological material, and the application is any one of the following directions:

[0015] (1) Application in the preparation of glycosyltransferase and / or products containing glycosyltransferase-related;

[0016] (2) Application in the preparation of glycosyltransferase mutants and / or products containing glycosyltransferase mutant-related;

[0017] (3) Application in the preparation of recombinant glycosyltransferase and / or products containing recombinant glycosyltransferase-related;

[0018] (4) Application in the fucosylation and glucosylation modification of the C-3 position of triterpenoid saponins;

[0019] (5) Application in the glycosylation modification of triterpenoid saponins;

[0020] (6) Application in the biosynthesis of triterpenoid saponins.

[0021] Preferably, in the applications of steps (4)-(6), glycosyltransferases use the glycosyl donor UDP-fucose to catalyze the formation of Prosaikogenin A and Prosaikogenin F from Saikogenin A and Saikogenin F, respectively;

[0022] Use UDP-glucose to catalyze the formation of clinopodiside I and clinopodiside X from Saikogenin A;

[0023] Use UDP-glucose to catalyze the formation of Saikosaponin B1, Saikosaponin c1 and Saikosaponin X from Prosaikogenin A;

[0024] Use UDP-glucose to catalyze the formation of Saikosaponin A and Buddlejasaponin IV from Prosaikogenin F.

[0025] From the above technical solutions, it can be seen that compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention uses liquid chromatography-mass spectrometry technology to detect the content differences of 8 target components in the roots, stems and leaves of Clinopodium chinense. Then, based on the Clinopodium chinense genome, members of the glycosyltransferase GT gene family of Clinopodium chinense are identified, and an evolutionary tree is constructed to analyze the evolutionary relationship between Clinopodium chinense and GT of other species, and the key enzymes involved in the glycosylation modification of triterpenoid saponins are preliminarily screened. Finally, based on the transcriptome data of different tissue parts of Clinopodium chinense, the differential gene expression is analyzed, and the key enzyme genes involved in the glycosylation modification of triterpenoid saponins are further screened. The glycosyltransferase gene was successfully screened by the above method.

[0027] Using the above-mentioned gene, the prokaryotic expression vector pET28a-MBP-CcGT-g51295 was constructed, and the glycosyltransferase was successfully obtained. Using Saikogenin A, Saikogenin F, Prosaikogenin A, and Prosaikogenin F as substrates, the catalytic function of the enzyme in vitro was verified. The study found that the glycosyltransferase of the present invention can use the glycosyl donor UDP-fucose to catalyze the formation of Prosaikogenin A and Prosaikogenin F from Saikogenin A and Saikogenin F, respectively; use UDP-glucose to catalyze the formation of clinopodiside I and clinopodiside X from Saikogenin A; use UDP-glucose to catalyze the formation of Saikosaponin B1, Saikosaponin c1, and Saikosaponin X from Prosaikogenin A; use UDP-glucose to catalyze the formation of Saikosaponin A and Buddlejasaponin IV from Prosaikogenin F. In summary, the glycosyltransferase of the present invention catalyzes the fucosylation and glucosylation modifications at the C-3 position of triterpenoid compounds, provides gene components for the synthetic biology research of triterpenoid saponin components, and solves the problem of biological resources of rare saponins. Description of the Drawings

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0029] Figure 1 It is: metabolomics study of triterpenoid saponin components in different tissue parts of Clinopodium chinense (Benth.) O. Ktze.; 1A-1F are the contents of triterpenoid saponin components in different tissue parts of Clinopodium chinense (Benth.) O. Ktze., respectively.

[0030] Figure 2 It is: screening and phylogenetic analysis of key glycosyltransferases for the biosynthesis of triterpenoid saponins in Clinopodium chinense (Benth.) O. Ktze.; 2A shows 11 candidate genes screened in Example 2; 2B is the ML phylogenetic tree.

[0031] Figure 3It is: in vitro functional verification of CcGT-g51295 using UDP-fucose to catalyze the formation of Prosaikogenin A and Prosaikogenin F from Saikogenin A and Saikogenin F respectively; Figures 3A and 3B are the reaction route diagrams of CcGT-g51295 catalyzing the formation of Prosaikogenin A and Prosaikogenin F from Saikogenin A and Saikogenin F respectively; Figures 3C and 3D are the mass spectrometry detection results of CcGT-g51295 catalyzing the formation of Prosaikogenin A and Prosaikogenin F from Saikogenin A and Saikogenin F respectively; Figures 3E and 3F are the mass spectrometry fragmentation pattern diagrams of the catalytic products Prosaikogenin A and Prosaikogenin F respectively.

[0032] Figure 4 It is: in vitro functional verification of CcGT-g51295 catalyzing the formation of clinopodiside I and clinopodiside X from Saikogenin A; Figure 4A is the reaction route diagram; Figure 4B is the liquid phase detection result diagram.

[0033] Figure 5 It is: of clinopodiside I 1 H NMR and 13 C NMR mass spectrometry detection results; Figure 5A is the 1 H NMR mass spectrometry detection result of clinopodiside I (600 MHz, measured in pyridine-d 5 ); Figure 5B is the 13 C NMR mass spectrometry detection result of clinopodiside I (150 MHz, measured in pyridine-d 5 ).

[0034] Figure 6 It is: HSQC and HMBC mass spectrometry detection results of clinopodiside I; Figure 6A is the HSQC mass spectrometry detection result of clinopodiside I measured in pyridine-d 5 ); Figure 6B is the HMBC mass spectrometry detection result of clinopodiside I measured in pyridine-d 5 ).

[0035] Figure 7 It is: NOESY of clinopodiside I and 1H- 1 H COSY mass spectrometry results; 7A, NOESY mass spectrometry results of clinopodiside I (measured in pyridine-d 5 ); 7B, 1 H- 1 H COSY mass spectrometry results (measured in pyridine-d 5 ).

[0036] Figure 8 are: 1 H NMR and 13 C NMR mass spectrometry results; 8A, 1 H NMR mass spectrometry results (600 MHz, measured in pyridine-d 5 ); 8B, 13 C NMR mass spectrometry results (150 MHz, measured in pyridine-d 5 ).

[0037] Figure 9 are: 5 HSQC and HMBC mass spectrometry results of clinopodiside X; 9A, HSQC mass spectrometry results of clinopodiside I (measured in pyridine-d 5 ); 9B, HMBC mass spectrometry results of clinopodiside I (measured in pyridine-d

[0038] Figure 10 are: 1 H- 1 H COSY mass spectrometry results; 10A, NOESY mass spectrometry results of clinopodiside I (measured in pyridine-d 5 ); 10B, 1 H- 1 H COSY mass spectrometry results (measured in pyridine-d 5 ).

[0039] Figure 11It is: in vitro functional verification of CcGT-g51295 catalyzing Prosaikogenin A to generate Saikosaponin B1, Saikosaponinc1 and SaikosaponinX; 11A, catalytic route map; 11B, liquid phase detection result map; C, catalytic product mass spectrometry fragmentation pattern map.

[0040] Figure 12 It is: in vitro functional verification of CcGT-g51295 catalyzing Prosaikogenin F to generate Saikosaponin A and Buddlejasaponin IV; 12A and 12B are respectively the route maps of CcGT-g51295 catalyzing ProsaikogeninF to generate Saikosaponin A and Buddlejasaponin IV; 12C and 12D are respectively the mass spectrometry detection results of CcGT-g51295 catalyzing Prosaikogenin F to generate Saikosaponin A and Buddlejasaponin IV; 12E and 12F are respectively the mass spectrometry fragmentation pattern maps of the catalytic products Saikosaponin A and Buddlejasaponin IV. Specific implementation mode

[0041] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative work shall fall within the protection scope of the present invention.

[0042] Example 1

[0043] Metabolomics study on triterpenoid saponin components in different tissue parts of Clinopodium chinense (Benth.) O. Kuntze

[0044] Take out the Clinopodium chinense (Benth.) O. Kuntze root, stem and leaf samples stored at -80°C and place them in liquid nitrogen, grind them into fine powder, quickly weigh 1.0 g and put it into a centrifuge tube, add 10 mL of methanol for ultrasonic extraction; after the extract is concentrated, add 1.0 mL of methanol for reconstitution, and use liquid chromatography-mass spectrometry technology to determine and analyze the relative contents of 8 compounds (Saikogenin A, Saikogenin F, ProsaikogeninA, ProsaikogeninF, Saikosaponin B1, Saikosaponin A, Buddlejasaponin IV, Buddlejasaponin IVb) in different tissue parts of Clinopodium chinense (Benth.) O. Kuntze.

[0045] The measurement conditions are as follows: The instrument models are Agilent Technologies 1290 Infinity Ⅱ and 6545 Q-TOF liquid chromatography-mass spectrometry; Waters Acquity BEH C18 column (1.7 μm, 100×2.1 mm) was used, and the column temperature was 40 °C;

[0046] Chromatographic conditions: Mobile phase: A - water (containing 0.1% formic acid), B - acetonitrile (containing 0.1% formic acid), flow rate: 0.2 mL / min, elution program: 0 - 5 min (25% B to 40% B), 5 - 13 min (40% B to 42.5% B), 13 - 14 min (42.5% B to 100% B), 14 - 20 min (100% B), 20 - 21 min (100% B to 25% B), 21 - 26 min (25% B);

[0047] Mass spectrometry conditions: The drying gas temperature was 350 °C (flow rate 8.0 L / min), the sheath gas was 250 °C (flow rate 11.0 L / min), the nebulizer was 45 psig, and the VCap was 4000 V;

[0048] Agilent MassHunter Qualitative Analysis 10.0 was used to compare and analyze the relative contents of 8 compounds in different tissue parts.

[0049] Result analysis: The research results showed that: The content of Buddlejasaponin IVb was relatively high in the leaves of Clinopodium chinense (Benth.), and its biosynthetic precursor compounds Prosaikogenin A and Saikosaponin B1 were present in the roots of Clinopodium chinense (Benth.)( Figure 1 A - 1C); The content of Buddlejasaponin IV was relatively high in the leaves of Clinopodium chinense (Benth.), its direct biosynthetic precursor Saikosaponin A mainly accumulated in the leaves, and the indirect biosynthetic precursor Prosaikogenin F had a relatively high content in the roots of Clinopodium chinense (Benth.)( Figure 1 D - 1F); Saikogenin A and Saikogenin F were not detected.

[0050] Example 2

[0051] Screening of the key CcGT enzyme - encoding gene for triterpenoid saponin biosynthesis based on the multi - omics research of Clinopodium chinense (Benth.)

[0052] Based on the Clinopodium chinense genome data, the UGT protein sequences of Arabidopsis thaliana were extracted from the TAIR database and used for BLASTP against Clinopodium chinense to search for homologous genes. The protein sequences were aligned using the Mega software, and an ML phylogenetic tree was constructed with 1000 bootstrap replicates. The RNA-Seq transcriptome data of Clinopodium chinense roots, stems, and leaves were aligned to the Clinopodium chinense genome using HiSAT2, and the gene expression FPKM values were calculated using Cufflinks to further screen for UGT genes that were specifically highly expressed in Clinopodium chinense.

[0053] Result analysis: A total of 214 GT-encoding genes were annotated in the genome using bioinformatics techniques, and 11 candidate genes ( Figure 2 A) were screened out by combining gene differential expression analysis, among which g51295 was significantly highly expressed in plant leaves; phylogenetic analysis showed that g51295 belongs to the UGT73 subfamily, and it is speculated that it has the activity of glycosylating the C-3 position of the triterpenoid saponin in Clinopodium polycephalum Figure 2 B).

[0054] Example 3

[0055] Synthesis of the glycosyltransferase CcGT-g51295 involved in the glycosylation modification of triterpenoid saponins

[0056] (1) Cloning of the target gene

[0057] The RNA of Clinopodium chinense leaves and roots was extracted using the Tiangen Polysaccharide Polyphenol Plant Total RNA Extraction Kit, and the cDNA template was synthesized using Takara's PrimeScriptTM II 1st Strand cDNA Synthesis Kit for the cloning of the CcGT target gene.

[0058] According to the results of bioinformatics analysis and combined with the sequence characteristics of CcGT-g51295, primers for cloning CcGT-g51295 were designed, and the target gene was cloned using Novoprotein 2×Phanta Flash MasterMix.

[0059] The sequences of the amplification primers are as follows:

[0060] CcGT-g51295-F: 5’-ATGGCGATCAATGGAAAACCGCCTC-3’, SEQ ID No.1;

[0061] CcGT-g51295-R: 5’-TCACTGGACTAGGATTTGAGAAGAAATGTC-3’, SEQ ID No.2.

[0062] The cloning system is as follows: Primer: 2×Phanta Flash MasterMix: 25 μL; CcGT-g51295-F (10 μM): 2 μL; Primer CcGT-g51295-R (10 μM): 2 μL; cDNA: 2 μL; ddH 2 O: 19 μL.

[0063] PCR reaction parameters: Denaturation at 98 °C for 30 s; 98 °C for 10 s; Annealing at 54 °C for 10 s; Extension at 72 °C for 20 s; 40 cycles; Finally, extension at 72 °C for 1 min.

[0064] Detect using DNA electrophoresis to obtain the target gene band. Use the Tiangen ordinary agarose gel DNA recovery kit to recover the target gene band. Use Takara's DNA A-Tailing Kit to add an A tail to the target gene fragment; Use Takara's pMD TM 18-T Vector Cloning Kit to ligate the target gene to the T vector; Verify the sequence accuracy of the amplified gene by first-generation sequencing; For the pMD18-T-CcGT-g51295 with correct sequencing, extract the plasmid using the Tiangen plasmid miniprep kit.

[0065] The nucleotide sequence of the target gene CcGT-g51295 is as follows:

[0066]

[0067] (2) Construction of expression vector

[0068] The pET28a (Cat. No. 69864-3) expression vector was purchased from Novagen; the MBP·Tag was synthesized into the NcoI and NheI restriction enzyme cleavage sites of the pET28a vector by gene chemical synthesis method to form the pET28a-MBP vector. The pET28a-MBP vector was digested with EcoR I and Xho I restriction enzymes of Takara to obtain a linearized vector fragment. The pET28a-MBP-CcGT-g51295 prokaryotic expression vector was constructed based on the homologous recombination strategy using the Novoprotein ClonExpress II One Step Cloning Kit.

[0069] The accuracy of the constructed expression vector was verified by first-generation sequencing technology, and the plasmid with correct sequencing of pET28a-MBP-CcGT-g51295 was extracted using the Tiangen plasmid miniprep kit.

[0070] The primer sequences for amplifying the expression vector are as follows:

[0071] pET28a-MBP-CcGT-g51295-F: 5’-atgggtcgcggatccgaattcATGGCGATCAATGGAAAACCG-3’, SEQ ID No. 4;

[0072] pET28a-MBP-CcGT-g51295-R: 5’-gtggtggtggtggtgctcgagTCACTGGACTAGGATTTGAGAAGAAA-3’, SEQ ID No. 5.

[0073] (3) Synthesis of glycosyltransferase CcGT-g51295

[0074] The recombinant expression vector pET28a-MBP-CcGT-g51295 was transformed into the Escherichia coli expression strain BL21(DE3). Take 2 mL of the overnight-expanded culture broth and add it to 40 mL of LB liquid culture (containing 50 μg / mL kanamycin resistance), activate at 37 °C and 200 rpm for 3 h, add IPTG with a final concentration of 0.3 mM, and induce at 16 °C and 160 rpm for 24 h. Centrifuge to collect the bacteria and obtain the crude enzyme by ultrasonic cell disruption. After measurement, the amino acid sequence of the obtained glycosyltransferase CcGT-g51295 is as follows:

[0075] MAINGKPPHFALLPFLAQGHLIPMVDIAKLLAKRGAAVSFLVTPQNFNRVKKVLDRAVDSGLSIRVCLLTLPGAEAGLPEDCENFDMLPSMNYVLNFFKATAMMGGQVEDLLVELNPTCLIADMCFPWATDVAMKLRIPRLVFHGTSCFSLVCMNVLKNSKLLEGVNGDLEYFVVPDLPDRIEITKAQLRGSAEDISPEWMEIRQQIFSSEEDAAGTVANTFHELEPEYVRAYIKLRNKNLWCIGPVSLCNVDDSDKAERGNTAAIDGHDCLEWLDSHAPESVVYACLGSISRVVTTQLKEMGLGLEASDRPFIWVIREAPDEFNAWLTSEKFEERVKGKGLLIRGWAPQVLILSHPSVGGFVTHCGWNSTLEGVSAGLPMITWPVFAEQFCNEKFIVNVIKTGVRVGVEVPVLLGMDEGAQVQVKSDDVKMAIDRLMEGGEEGEERRERARKLGEAAKRAVEEGGSSHLNMGQLIQDMVALKAKYGEKSVDGAKLDDISSQILVQ, SEQ ID NO.6.

[0076] Example 4

[0077] Functional verification of the glycosyltransferase CcGT - g51295 involved in triterpenoid saponin glycosylation modification

[0078] Using the crude enzyme of the protein obtained in Example 3, an in vitro test tube reaction was carried out (200 μL reaction system: 100 mM Tris - HCl at pH 8.5, 100 μM substrates (Saikogenin A, Saikogenin F, Prosaikogenin A, Prosaikogenin F), 5 mM glycosyl donor (UDP - fucose or UDP - glucose), 50 μL crude protein; reaction at 30 °C for 3 h) to verify its catalytic activity. For substrates and products with standards, liquid chromatography - mass spectrometry technology was used for qualitative analysis (such as the above - mentioned metabolomics detection method). For unknown products, product preparation was carried out, and structural characterization was combined with nuclear magnetic resonance spectroscopy technology.

[0079] Result analysis: CcGT - g51295 has the following functions: using the glycosyl donor UDP - fucose to catalyze Saikogenin A and Saikogenin F to generate Prosaikogenin A and Prosaikogenin F respectively (Figure 3 )

[0080] Using UDP-glucose to catalyze the formation of clinopodiside I and clinopodiside X from Saikogenin A Figures 4 - 10 )

[0081] Using UDP-glucose to catalyze the formation of Saikosaponin B1, Saikosaponin c1 and Saikosaponin X from Prosaikogenin A Figure 11 )

[0082] Using UDP-glucose to catalyze the formation of Saikosaponin A and Buddlejasaponin IV from Prosaikogenin F Figure 12 )

[0083] The various embodiments in the specification are described in a progressive manner. Each embodiment focuses on the differences from other embodiments. For the same or similar parts among the embodiments, reference can be made to each other.

[0084] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A glycosyltransferase involved in the glycosylation modification of triterpenoid saponins, characterized in that: The glycosyltransferase is composed of the amino acid sequence shown in SEQ ID NO.6 or an amino acid sequence having the same enzymatic catalytic performance after substitution, deletion and / or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO.

6.

2. A gene encoding the glycosyltransferase according to claim 1, characterized in that: The gene is composed of the nucleotide sequence shown in SEQ ID NO.3 or a nucleotide sequence having the same enzyme catalytic performance after the nucleotide sequence shown in SEQ ID No.3 is replaced, deleted and / or added with one or more bases.

3. A primer set for amplifying the glycosyltransferase gene according to claim 2, characterized in that: The primer set includes CcGT-g51295-F and CcGT-g51295-R, and the specific sequences are as follows: CcGT-g51295-F: 5'-ATGGCGATCAATGGAAAACCGCCTC-3', SEQ IDNo.1; CcGT-g51295-R: 5'-TCACTGGACTAGGATTTGAGAAGAAATGTC-3', SEQ ID No.

2.

4. A biological material containing the glycosyltransferase gene according to claim 2, characterized in that: The biological material is a recombinant vector, an expression box, a transgenic cell line or a recombinant bacterium.

5. Use of the glycosyltransferase according to claim 1 or the gene encoding the glycosyltransferase according to claim 2 or the biomaterial according to claim 4, characterized in that: The application is any of the following: (1) Application in the preparation of glycosyltransferases and / or glycosyltransferase-related products; (2) Application in the preparation of glycosyltransferase mutants and / or products containing glycosyltransferase mutants; (3) Application in the preparation of recombinant glycosyltransferases and / or products containing recombinant glycosyltransferases; (4) Application in the modification of C-3 fucosylation and glucosylation of triterpenoid saponins; (5) Application in glycosylation modification of triterpenoid saponins; (6) Application in the biosynthesis of triterpenoid saponins.

6. The use according to claim 5, characterized in that: In the application of steps (4)-(6), the glycosyltransferase utilizes the glycosyl donor UDP-fucose to catalyze Saikogenin A and Saikogenin F to generate Prosaikogenin A and Prosaikogenin F, respectively; Saikogenin A is catalyzed by UDP-glucose to produce clinopodiside I and clinopodiside X; Prosaikogenin A is catalyzed by UDP-glucose to produce Saikosaponin B1, Saikosaponin c1 and Saikosaponin X; Prosaikogenin F is catalyzed by UDP-glucose to produce Saikosaponin A and Buddlejasaponin IV.

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