Glycosyl transferase UGT73DY2 and coding gene and application thereof

Abstract

The invention belongs to the technical field of natural product biosynthesis, and particularly relates to glycosyl transferase UGT73DY2 as well as a coding gene and application thereof. Diosgenin-3-O-alpha-L-rhamnopyranosyl-(1-> 4)-[alpha-L-rhamnopyranosyl]-(1-> 2)-beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl-(1-> 4)-[alpha-L-rhamnopyranosyl]-(1-> 2) synthesized under catalysis of glycosyl transferase UGT73DY2 The-beta-D-glucoside is a key intermediate compound for biosynthesis of the polyphyllin II or the polyphyllin VII, the compounds are catalytically synthesized in vitro under the action of glycosyl transferase UGT73DY2, and a foundation is laid for industrial production of the polyphyllin II and the polyphyllin VII. Meanwhile, a new ophiopogonin compound can be synthesized under the action of the glycosyl transferase UGT73DY2, and a new direction is provided for further application research of the glycosyl transferase UGT73DY2.

Description

Technical Field

[0001] The invention belongs to the technical field of natural product biosynthesis, and specifically relates to a glycosyltransferase UGT73DY2 and a coding gene and application thereof. Background Art

[0002] Paris polyphylla, also known as Paris polyphylla, is a plant of the Veratrum family (Melanthiaceae) of the genus Paris. my country is one of the distribution centers of this genus, with about 17 species and 10 varieties, distributed in most provinces and regions, among which the diversity center is located in the Yunnan-Guizhou Plateau and Qionglai Mountain area of ​​China. Paris polyphylla Smith var. Yunnanensis and Paris polyphylla Smith var. Chinansis (French.) Hara are included in the "Chinese Pharmacopoeia". Their rhizomes are mainly used for medicinal purposes and are included in the "Pharmacopoeia of the People's Republic of China". The main effects are clearing away heat and detoxification, reducing swelling and relieving pain, cooling the liver and calming the nerves. They are used for furuncles, carbuncles, snake and insect bites, falls and injuries, and convulsions.

[0003] Paris polyphylla saponins are the main active ingredients of Paris polyphylla, which have a wide range of pharmacological activities such as anti-tumor, anti-inflammatory, analgesic, antibacterial, hemostatic, antioxidant and immunomodulatory. Paris polyphylla saponins have a complex structure and are difficult to chemically synthesize, and the internal formation mechanism of the plant has not yet been fully clarified. With the expansion of market demand, wild Paris polyphylla has been severely excavated and is in an extremely endangered state, making it increasingly difficult to obtain its active ingredients. Heterologous biosynthesis is one of the most reliable methods to provide a stable source of active substances.

[0004] Glycosyltransferases are involved in the synthesis, modification, and transport of plant secondary metabolites. The glycosylation function changes the water solubility, stability, and transport characteristics of compounds in plants in cells and plants. Secondly, glycosyltransferases can regulate hormone homeostasis in plants and affect plant growth and development. Glycosylation modification is a key step in the biosynthesis of Paris saponins, which requires the catalysis of glycosyltransferases. However, the biosynthetic pathways of Paris saponins II and Paris saponins VII, the main active ingredients of Paris, remain to be resolved. Once the biosynthetic pathways of Paris saponins II and Paris saponins VII are clarified, it is expected to achieve the goal of heterologous biosynthesis and solve the problem of increasingly difficult acquisition of Paris saponins. Summary of the invention

[0005] The purpose of the present invention is to provide a key glycosyltransferase in the biosynthesis pathway of paridis saponin II and paridis saponin VII, and utilize the catalytic reaction to synthesize the key intermediate compounds in the biosynthesis process of paridis saponin II or VII, and can also catalyze the synthesis of other steroidal saponin compounds such as ophiopogon saponin compounds, so as to realize the heterologous synthesis of steroidal saponin compounds such as paridis saponin compounds and ophiopogon saponin compounds.

[0006] The present invention provides a glycosyltransferase UGT73DY2, whose amino acid sequence is shown in SEQ ID NO.2.

[0007] The present invention also provides a gene encoding the aforementioned glycosyltransferase UGT73DY2, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] The present invention also provides a recombinant vector, an expression cassette or a recombinant bacterium comprising the aforementioned gene.

[0009] Furthermore, the recombinant vector is a plasmid connected to a gene with a nucleotide sequence as shown in SEQ ID NO.1; the plasmid includes pCold-TF, pESC-URA or pEAQ-HT.

[0010] Furthermore, the recombinant bacteria include recombinant Escherichia coli, recombinant Saccharomyces cerevisiae or recombinant Agrobacterium;

[0011] The recombinant Saccharomyces cerevisiae includes Saccharomyces cerevisiae into which the aforementioned genes, as well as DHCR7 gene, DHCR24 gene, RHM gene, CYP90B27 gene, CYP90G4 gene, CYP94D109 gene, UGT80A41 gene, UGT73CE1 gene, and cas9 gene are integrated; the expression of HMGR gene and ERG9 gene of the Saccharomyces cerevisiae is enhanced; the Saccharomyces cerevisiae includes Saccharomyces cerevisiae BY4742;

[0012] The recombinant Agrobacterium includes Agrobacterium integrated with the aforementioned genes, as well as CAS gene, HMGR gene, CYP94D109 gene, C5-SD2 gene, CPI-5 gene, CYP51 gene, SMO1-3 gene, SMO2-2 gene, SSR1-3 gene, CYP90B71 gene, CYP90G6 gene, CYP94N8 gene, CYP72A616 gene, CYP90G4 gene, CYP90B27 gene, UGT80A41 gene and UGT73CE1 gene; the Agrobacterium includes Agrobacterium LBA4404;

[0013] The accession number of the HMGR gene in NCBI is OP243233.1;

[0014] The accession number of the ERG9 gene in NCBI is WNM97123.1;

[0015] The accession number of the DHCR7 gene in NCBI is NP_958487.2;

[0016] The accession number of the DHCR24 gene in NCBI is NP_001008645.1;

[0017] The accession number of the RHM gene in NCBI is CAD92667.1;

[0018] The accession number of the CYP90B27 gene in NCBI is KX904822.1;

[0019] The accession number of CYP90G4 in NCBI is QDS03628.1;

[0020] The accession number of CYP94D109 in NCBI is QDS03630.1;

[0021] The accession number of UGT80A41 in NCBI is WIL59759.1;

[0022] The accession number of UGT73CE1 in NCBI is WIL59760.1;

[0023] The accession number of the CAS gene in NCBI is MN368727.1;

[0024] The accession number of the C5-SD2 gene in NCBI is OP243261.1;

[0025] The accession number of the CPI-5 gene in NCBI is OP243252.1;

[0026] The accession number of the CYP51 gene in NCBI is OP243253.1;

[0027] The accession number of the SMO1-3 gene in NCBI is OP243242.1

[0028] The accession number of the SMO2-2 gene in NCBI is OP243245.1;

[0029] The accession number of the SSR1-3 gene in NCBI is OP243236.1;

[0030] The accession number of the CYP90B71 gene in NCBI is QPZ88854.1;

[0031] The accession number of the CYP90G6 gene in NCBI is QPZ88855.1;

[0032] The accession number of the CYP94N8 gene in NCBI is QPZ88856.1;

[0033] The accession number of the CYP72A616 gene in NCBI is MK636705.1;

[0034] The nucleotide sequence of the cas9 gene is shown in SEQ ID NO.11.

[0035] The present invention also provides the use of the aforementioned glycosyltransferase UGT73DY2 or the aforementioned gene or the aforementioned recombinant vector, expression cassette or recombinant bacteria in the synthesis of steroidal saponin compounds, wherein the steroidal saponin compounds include Paris polyphylla saponin compounds and Ophiopogon japonicus saponin compounds.

[0036] Furthermore, the Paris polyphylla saponin compounds include diosgenin-3-O-α-L-rhamnose-(1→4)-[α-L-rhamnose pyranosyl]-(1→2)-β-D-glucoside and diosgenin-3-O-α-L-rhamnose-(1→4)-[α-L-rhamnose pyranosyl]-(1→2)-β-D-glucoside;

[0037] The ophiopogon japonicus saponin compound includes ophiopogon japonicus saponin-3-O-α-L-rhamnopyranosyl-(1→4)-[α-L-rhamnopyranosyl]-(1→2)-β-D-glucoside;

[0038] The chemical structural formula of the diosgenin-3-O-α-L-rhamnose-(1→4)-[α-L-rhamnose pyranosyl]-(1→2)-β-D-glucoside is shown in Formula I, the chemical structural formula of the diosgenin-3-O-α-L-rhamnose-(1→4)-[α-L-rhamnose pyranosyl]-(1→2)-β-D-glucoside is shown in Formula II, and the chemical structural formula of the ophiopogon japonicus saponin compound including ophiopogon japonicus saponin-3-O-α-L-rhamnose-(1→4)-[α-L-rhamnose pyranosyl]-(1→2)-β-D-glucoside is shown in Formula III;

[0039]

[0040] The present invention also provides a method for synthesizing steroidal saponin compounds, comprising the following steps:

[0041] Using Paris polyphylla saponin or Ophiopogon japonicus saponin-3-O-α-L-pyranorhamnose-(1→2)-β-D-glucoside as substrate and UDP-rhamnose as sugar donor, the reaction is catalyzed by the aforementioned glycosyltransferase UGT73DY2.

[0042] Or: ferment the aforementioned recombinant Saccharomyces cerevisiae in YPDA medium.

[0043] Furthermore, when the substrate is Paris saponin V, the catalytic reaction produces diosgenin-3-O-α-L-rhamnose-(1→4)-[α-L-rhamnose pyranosyl]-(1→2)-β-D-glucoside; when the substrate is Paris saponin VI, the catalytic reaction produces diosgenin-3-O-α-L-rhamnose-(1→4)-[α-L-rhamnose pyranosyl]-(1→2)-β-D-glucoside; when the substrate is Ophiopogonin-3-O-α-L-rhamnose-(1→2)-β-D-glucoside, the catalytic reaction produces Ophiopogonin-3-O-α-L-rhamnose-(1→4)-[α-L-rhamnose pyranosyl]-(1→2)-β-D-glucoside.

[0044] The present invention also provides a use of the aforementioned glycosyltransferase UGT73DY2 or the aforementioned gene or the aforementioned recombinant vector, expression cassette or recombinant bacteria in the preparation of a transgenic organism having the function of biosynthesizing Paris polyphylla saponin compounds.

[0045] Further, the transgenic organism includes transgenic tobacco;

[0046] The transgenic tobacco is Nicotiana benthamiana, in which the aforementioned genes, as well as CAS gene, HMGR gene, CYP94D109 gene, C5-SD2 gene, CPI-5 gene, CYP51 gene, SMO1-3 gene, SMO2-2 gene, SSR1-3 gene, CYP90B71 gene, CYP90G6 gene, CYP94N8 gene, CYP72A616 gene, CYP90G4 gene, CYP90B27 gene, UGT80A41 gene and UGT73CE1 gene are integrated into the genome;

[0047] The Paris polyphylla saponin compound includes diosgenin-3-O-α-L-rhamnopyranose-(1→4)-[α-L-rhamnopyranose]-(1→2)-β-D-glucoside.

[0048] The invention uses Paris polyphylla as research material and discovers a glycosyltransferase gene named UGT73DY2. It was verified that the UGT73DY2 protein expressed by this gene can catalyze the glycosylation of Paris saponin V, Paris saponin VI and Ophiopogon saponin-3-O-α-L-rhamnose-(1→2)-β-D-glucoside to produce diosgenin-3-O-α-L-rhamnose-(1→4)-[α-L-rhamnose]-(1→2)-β-D-glucoside, diosgenin-3-O-α-L-rhamnose-(1→4)-[α-L-rhamnose]-(1→2)-β-D-glucoside and Ophiopogon saponin-3-O-α-L-rhamnose-(1→4)-[α-L-rhamnose]-(1→2)-β-D-glucoside.

[0049] Diosgenin-3-O-α-L-rhamnose-(1→4)-[α-L-rhamnose]-(1→2)-β-D-glucoside and diosgenin-3-O-α-L-rhamnose-(1→4)-[α-L-rhamnose]-(1→2)-β-D-glucoside, which are synthesized by glycosyltransferase UGT73DY2, are key intermediate compounds in the biosynthesis of Parisopsis saponin II or VII. These compounds are synthesized in vitro by glycosyltransferase UGT73DY2, laying a foundation for the industrial production of Parisopsis saponin II and Parisopsis saponin VII. At the same time, a new ophiopogon saponin compound can be synthesized by glycosyltransferase UGT73DY2, which provides a direction for further application research of glycosyltransferase UGT73DY2.

[0050] The glycosyltransferase UGT73DY2 gene of the present invention is introduced into saccharomyces cerevisiae or Agrobacterium, and on the basis of the introduction of corresponding genes, such as DHCR7 gene, DHCR24 gene, RHM gene, CYP90B27 gene, CYP90G4 gene, CYP94D109 gene, UGT80A41 gene, UGT73CE1 gene, cas9 gene, etc., the recombinant saccharomyces cerevisiae constructed can produce paridis saponin compounds after fermentation and culture, and the transgenic tobacco obtained by transfecting the constructed recombinant Agrobacterium with the Agrobacterium-mediated method can also synthesize paridis saponin compounds by itself, and finally the heterologous biosynthesis of paridis saponin compounds is realized.

[0051] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0052] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 It is the PCR agarose gel electrophoresis diagram of the amplified product of the gene encoding glycosyltransferase UGT73DY2, where lane M is DL2000 DNA Marker and lane 1 is the target band (1485 bp);

[0054] Figure 2 Schematic diagram of the E. coli expression plasmid structure for the expression of glycosyltransferase UGT73DY2 for the synthesis of diosgenin-3-O-α-L-rhamnose-(1→4)-[α-L-rhamnose]-(1→2)-β-D-glucoside, pyranosapogenin-3-O-α-L-rhamnose-(1→4)[α-L-rhamnose](1→2)-β-D-glucoside and ophiopogonin-3-O-α-L-rhamnose-(1→4)-[α-L-rhamnose]-(1→2)-β-D-glucoside;

[0055] Figure 3 Schematic diagram of the recombinant yeast expression plasmid structure for expressing the glycosyltransferase UGT73DY2 for synthesizing diosgenin-3-O-α-L-rhamnopyranosyl-(1→4)-[α-L-rhamnopyranosyl]-(1→2)-β-D-glucoside;

[0056] Figure 4 Schematic diagram of the tobacco heterologous synthesis expression plasmid structure for expressing the glycosyltransferase UGT73DY2 for the synthesis of diosgenin-3-O-α-L-rhamnopyranosyl-(1→4)-[α-L-rhamnopyranosyl]-(1→2)-β-D-glucoside;

[0057] Figure 5 UPLC analysis chart of the enzymatic reaction product; wherein A is the catalytic reaction of glycosyltransferase UGT73DY2; B is the liquid chromatogram and mass spectrometry analysis chart of the enzymatic activity product of UGT73DY2 with Paris polyphylla saponin V (1) as substrate and UDP-rhamnose as sugar donor; C is the liquid chromatogram and mass spectrometry analysis chart of the enzymatic activity product of UGT73DY2 with Paris polyphylla saponin VI (2) as substrate and UDP-rhamnose as sugar donor; D is the liquid chromatogram and mass spectrometry analysis chart of the enzymatic activity product of UGT73DY2 with Ophiopogon japonicus saponin-3-O-α-L-pyranosyl-(1→2)-β-D-glucoside (3) as substrate and UDP-rhamnose as sugar donor;

[0058] Figure 6 The Michaelis-Menten regression curve of initial rate and sugar receptor concentration was fitted using origin, and the Michaelis constant (Km) was calculated; A and B are the nonlinear Michaelis-Menten regression curves of UGT73DY2 using Paris polyphylla saponin V and Paris polyphylla saponin VI as substrates, respectively.

[0059] Figure 7 Schematic diagram of the production pathway of diosgenin-3-O-α-L-rhamnopyranosyl-(1→4)-[α-L-rhamnopyranosyl]-(1→2)-β-D-glucoside in Saccharomyces cerevisiae.

[0060] Figure 8 The production of diosgenin-3-O-α-L-rhamnopyranosyl-(1→4)-[α-L-rhamnopyranosyl]-(1→2)-β-D-glucoside after transient expression of different co-expressed gene combinations in Nicotiana benthamiana, where the gray boxes indicate the absence of biosynthetic genes and the green boxes indicate the inclusion of biosynthetic genes in the co-expression experiment. DETAILED DESCRIPTION

[0061] The materials, reagents, etc. used in the specific embodiments of the present invention, unless otherwise specified, are all obtained from commercial sources or conventional preparation methods in the field. The structural formula of the steroidal saponin involved is as follows:

[0062]

[0063] Ophiopogon japonicus saponin-3-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucoside

[0064]

[0065] Diosgenin-3-O-α-L-rhamnopyranosyl-(1-4)-[α-L-rhamnopyranosyl]-(1-2)-β-D-glucoside

[0066]

[0067] Pianosaponin-3-O-α-L-rhamnopyranosyl-(1→4)[α-L-rhamnopyranosyl](1→2)-β-D-glucoside

[0068]

[0069] Ophiopogon japonicus saponin-3-O-α-L-rhamnopyranosyl-(1→4)-[α-L-rhamnopyranosyl]-(1→2)-β-D-glucoside

[0070]

[0071] Example 1 Discovery, cloning and expression of the gene encoding glycosyltransferase UGT73DY2

[0072] In the early stage of the study on Paris polyphylla, a glycosyltransferase gene was found in the transcriptome of Paris polyphylla (Pairs polyphylla Smith var.yunnanensis (Franch.) Hand.-Mazz.) of the family Pairs polyphylla Smith var.yunnanensis (Franch.) Hand.-Mazz. by Blast homology comparison. The open reading frame of the gene contains 1485 bases and is named UGT73DY2. The nucleotide sequence is shown in SEQ ID NO.1:

[0073]

[0074]

[0075] TRIzol Plus RNA Purification Kit was used to extract RNA from different tissues of Paris polyphylla (including rhizomes, fibrous roots, leaves and stems). III 1st Strand cDNA Synthesis Kit Instructions Reverse transcription of Paris polyphylla total RNA was performed to synthesize cDNA. The synthesized cDNA was used as a template, and PCR amplification was performed using the designed glycosyltransferase UGT73DY2 specific primers UGT73DY2-F: 5'-ATGGAGTCCCAACCAGAG-3' (SEQ ID NO.3) and UGT73DY2-R: 5'-TCAATTGCAACCAGAACAATCATAA3' (SEQ ID NO.4), and 2×Phanta MaxMaster Mix high-fidelity enzyme (vazyme, catalog number: 515-02). The amplified UGT73DY2 gene fragment ( Figure 1 ) was connected to pClone007 Blunt Simple Vector (Beijing Qingke Biotechnology, Catalog No.: TSV-007BS), the ligation product was transformed into Escherichia coli DH5a, and the pClone007-UGT73DY2 plasmid was extracted using the Oriscience Plasmid Mini Extraction Kit (Oriscience, Catalog No.: NB101-200T).

[0076] Using pClone007-UGT73DY2 plasmid as template, PCR amplification was performed using the designed primer pairs: pCold-UGT73DY2-F (SEQID NO.5): 5'-ATGGAGCTCGGTACCCTCGAGA TGGAGTCCCAACCAGAG-3'; pCold-UGT73DY2-R (SEQID NO.6): 5'-CTA TCTAGACTGCAGGTCGACTCAATTGCAACCAGAACAATCATAA-3', and 2×PhantaMax Master Mix high-fidelity enzyme (vazyme, catalog number: 515-02). The amplified UGT73DY2 gene fragment was connected to the pCold-TF expression vector containing a His tag at the N-terminus (the vector was double-digested with restriction endonucleases XhoI (TaKaRa, Catalog No.: 1094A) and SalI (TaKaRa, Catalog No.: 1080A)) through 2×Clonexpress Mix (vaeyme, Catalog No.: C115-02), and then transformed into Escherichia coli DH5a, and single clones were picked. After identification by colony PCR, the positive single clones were sequenced, and the correctly sequenced pCold-UGT73DY2 was extracted from the plasmid.

[0077] pCold-UGT73DY2 plasmid ( Figure 2) was transformed into the E. coli expression strain BL21 (DE3), and a single clone was selected for PCR verification. The correct single clone colony was inoculated into 6 mL of LB liquid medium containing Amp resistance and placed on a shaker at 37°C, 200 rpm overnight. The activated bacteria were inoculated into 100 mL of LB liquid medium containing Amp resistance, and cultured on a shaker at 37°C, 200 rpm to OD600 = 0.6, IPTG was added to a final concentration of 0.3 mM, and then protein expression was induced at 16°C, 200 rpm for 12-16 hours. The cells were collected and reselected with 8 mL of buffer 1 (0.5 M NaCl, 10 mM imidazole, 5 mM DTT, 20 mM Tris-HCl, pH 8.0), and then ultrasonically disrupted. 100 μL was taken as a control, and the remaining samples were centrifuged at 4°C and 12,000 rpm for 10 min. The supernatant was taken for protein purification. The crude enzyme after disruption was adsorbed with Ni-NTA agarose (Qiagen), and the protein was sequentially eluted with buffer 2 (0.5 M NaCl, 10 mM imidazole, 5 mM DTT, 20 mM Tris-HCl, 20 mM imidazole, pH 8.0) and buffer 3 (0.5 M NaCl, 10 mM imidazole, 5 mM DTT, 20 mM Tris-HCl, 250 mM imidazole, pH 8.0), and the buffer was collected at the same time. Buffer 3 was the purified protein. The protein eluted by buffer 3 was concentrated by 30kDa ultrafiltration tube, and the protein concentration was determined by BCA protein concentration determination kit. The eluted protein was added with an equal volume of glycerol and stored at -80°C. The protein solubility and size were analyzed by 10% SDS-PAGE analysis. The results showed that the UGT73DY2 protein had good solubility and the protein size was about 101kDa. The UGT73DY2 protein was sequenced, and its amino acid sequence is shown in SEQ ID NO.2:

[0078] MESQPELLHLVFFPFLARSHMIPMLETARLAVERGVKTTLVTTPANAHLIRPALHRTNSSLLPSHPPMQLQLIPFPSAEFGIPEGCENLTSLPLPLAAAFFNAIFALRAPLGALLRELSAHAL VADALFPWATGLAAEMGIPRLIFQVTGLFPLCAAHDLDAHRPHEAVGGDDEEFTIPGFPDPVTLTRGQIPEVFRHNFMLALLRDAELTSYGVIVNSFYALEPSYAEHYYKVAPRKVFLLGPVAL AGSNPSPPPLESGDPCITWLDSKPDDSVLYLSFGTLCRFSNEQLVELAEGLASSGHNFVWVVVARPESSGGPGEEWLPEGYERNVAGRGLLVSGWAPQTAILNHRAVGGFVIHCGWNSVMEAVAA EVPMATWPLHSEQFVIEKLLCDVLHVAVPMWEGWKSIWDDQKEVVRAGTVAASVKRLMGGGDEVEAMRRRVRELGELGRAAVAESGSSHSDMGRLIDVLTEERSKANEMIDKKGVSYDCSGCN*

[0079] In the experiment, pClone007-UGT73DY2 plasmid was used as a template, and the designed primer pairs were used: pESC-URA-UGT73DY2-F' (SEQ ID NO.7): 5'-GGAGAAAAAACCCCGGATCCAATGGAGTCCCAACCAGAG-3, pESC-URA-UGT73DY2-R (SEQ ID NO.8): 5'-CAACTTCTGTTCCATGTCGACTCAATTGCAACCAGAACAATCATAA-3', and 2×Phanta Max Master Mix high-fidelity enzyme (vazyme, catalog number: 515-02) for PCR amplification. The amplified UGT73DY2 gene fragment was connected with the Saccharomyces cerevisiae expression vector pESC-URA double-digested with restriction endonucleases BamHI (New England Biolabs, catalog number: R3136V) and SalI (TaKaRa, catalog number: 1080A) to obtain a recombinant vector ( Figure 3 ), transformed into Saccharomyces cerevisiae, and can also express the UGT73DY2 protein with a synthetic amino acid sequence as shown in SEQ ID NO.2.

[0080] Using pClone007-UGT73DY2 plasmid as template, PCR amplification was performed using the designed primer pair: pEAQ-HT-UGT73DY2-F (SEQ ID NO.9): 5'-CTGCCCAAATTCGCGACCGGTATGGAGTCCCAACCAGAG-3' and pEAQ-HT-UGT73DY2-R (SEQ ID NO.10): 5'-ACCAGAGTTAAAGGCCTCGAGTCAATTGCAACCAGAACAATCATAA-3', and 2×Phanta Max Master Mix high-fidelity enzyme (vazyme, catalog number: 515-02). The amplified UGT73DY2 gene fragment was connected to the tobacco expression vector pEAQ-HT double-digested with restriction endonucleases XhoI (TaKaRa, catalog number: 1094A) and AgeI (NewEngland Biolabs, catalog number: R3136V) to obtain a recombinant vector ( Figure 4 ), transformed into tobacco, can also express the synthetic amino acid sequence UGT73DY2 protein shown in SEQ ID NO.2.

[0081] Example 2 In vitro enzyme activity test and product analysis of glycosyltransferase UGT73DY2

[0082] The UGT73DY2 protein obtained in Example 1 and the cleavage product of the Pcold-TF empty vector were tested for in vitro enzyme activity. Specifically, Paris saponin V, Paris saponin VI and Ophiopogon saponin-3-O-α-L-pyranorhamnose-(1→2)-β-D-glucoside were used as substrates, and UDP-rhamnose was used as a sugar donor. The reaction was carried out in a reaction system containing UGT73DY2 protein or Pcold-TF empty vector, and the enzyme product after the reaction was tested and analyzed by liquid chromatography-mass spectrometry:

[0083] Reactions were performed in a final volume of 200 μL with a formulation of 50 mM Tris-HCl, 50 μM substrate, 100 μM UDP-rhamnose, and 185 μL of UGT73DY2 protein or empty vector. The reaction mixture was incubated at 37°C for 6 hours. After concentration using a vacuum freeze dryer, the sample was dissolved in 100 μL of methanol. The sample was centrifuged at 12,000 rpm for 25 minutes, and 25 μL was injected into a UPLC (ultra-performance liquid chromatography) using an Agilent 1260 system, using a ZORBAX SB-C18 analytical column, with a flow rate of 1 mL / min and a column temperature of 35°C. The product of Paris saponin V was separated from the enzyme product by water / acetonitrile (0-22 min linear gradient from 10% to 100% acetonitrile), washed with 100% acetonitrile for 5 min, and detected at a wavelength of 195 nm; the product of Paris saponin VI was separated from the enzyme product by water / acetonitrile (0-22 min linear gradient from 30% to 70% acetonitrile, 22-23 min from 70% to 100% acetonitrile), washed with 100% acetonitrile for 5 min, and detected at a wavelength of 195 nm; the product of Ophiopogon japonicus saponin-3-O-α-L-pyranosyl-rhamnosyl-(1→2)-β-D-glucoside was separated from the enzyme product by water / acetonitrile (0-22 min linear gradient from 30% to 70% acetonitrile, 22-23 min from 70% to 100% acetonitrile), washed with 100% acetonitrile for 5 min, and detected at a wavelength of 195 nm.

[0084] The reaction solution was detected by HPCL. Figure 5 Waters SynaptTM XS High Definition Mass Spectrometry equipped with an ESI source was used for mass spectrometry analysis. The mass spectrometry conditions were as follows: data acquisition in positive ion mode, capillary voltage 2.8 kV, sampling cone voltage 35.0 V, extraction cone voltage 3.0 V, mass spectrometry source temperature 110 °C, desolvation temperature 350 °C, desolvation gas flow rate 650 L / h, collision energy set to low energy scan 10-30 eV, high energy scan 20-45 eV, and scan mass range set to m / z 50 to 1200.

[0085] from Figure 5As shown in B to D, compared with the control, when glycosyltransferase UGT73DY2 used Parisopsis saponin V, Parisopsis saponin VI and Ophiopogon saponin-3-O-α-L-rhamnose-(1→2)-β-D-glucoside as substrates, specific product peaks were produced respectively. The comparison of liquid phase retention time and mass spectrometry molecular weight with those of the standard product confirmed that Parisopsis saponin V was synthesized into diosgenin-3-O-α-L-rhamnose-(1-4)-[α-L-rhamnose-pyranosyl]-(1-2)-β-D-glucoside under the catalysis of UGT73DY2 protein. -glucoside; Paris polyphylla saponin VI is synthesized into parenosapogenin-3-O-α-L-rhamnose-(1-4)-[α-L-rhamnose]-(1-2)-β-D-glucoside under the catalysis of UGT73DY2 protein, and Ophiopogon japonicus saponin-3-O-α-L-rhamnose-(1→2)-β-D-glucoside is synthesized into Ophiopogon japonicus saponin-3-O-α-L-rhamnose-(1→4)-[α-L-rhamnose]-(1→2)-β-D-glucoside under the catalysis of UGT73DY2 protein.

[0086] Example 3 Analysis of enzyme kinetic parameters of glycosyltransferase UGT73DY2

[0087] Enzyme kinetic parameters are important tools for studying the rate of enzyme-catalyzed reactions. The Michaelis constant (Km) is a measure of the affinity between the enzyme and the substrate, which can be used to evaluate the activity of the enzyme and determine the specificity of the enzyme.

[0088] In order to determine the enzyme kinetic parameters of UGT73DY2 protein, a final volume of 100 μL was prepared containing 50 mM Tris-HCl (pH 8.0), 100 μg of UGT73DY2 protein obtained in Example 1, 2 mM UDP-rhamnose and substrates at different concentrations, with Paris polyphylla saponin V as the substrate concentration gradient of 2.5, 5, 10, 20, 40, 60, 80, 100, 200 and 400 μM, and Paris polyphylla saponin VI as the substrate concentration gradient of 2.5, 5, 10, 20, 40, 60, 80, 100, 200, 400, 600, 800, 1200 μM. The mixture was incubated at 37°C for 30 minutes. The reaction was stopped with 100 μL of methanol and concentrated. The product was dissolved in 100 μL of methanol, centrifuged at 12,000 rpm for 25 min, and 25 μL of the supernatant was taken for UPLC analysis.

[0089] UPLC (ultra-performance liquid chromatography) using an Agilent 1260 system, with a ZORBAX SB-C18 analytical column, a flow rate of 1 mL / min, a column temperature of 35°C, and a mobile phase of water (A) and acetonitrile (B): For the product of UGT73DY2, a linear gradient of B from 10% to 100% (0 to 22 minutes) was used, followed by an isocratic gradient of 100% B (22.01 to 30 minutes). All experiments were performed in triplicate. Using the same method, calibration curves of Parisopsin V and Parisopsin VI were generated by UPLC in seven gradients of standard samples (2.5, 5, 10, 20, 40, 80, 100 μM). The equations and correlation coefficients obtained from the linear studies are as follows: Parisopsin V is y=8.4808x+6.1172(R 2 =0.99), Paris polyphylla saponin VI is y=8.3667x+14.28401(R 2 =0.99). The content of the reaction product was calculated according to the equation, and the nonlinear Michaelis-Menten regression curve of the initial rate and sugar receptor concentration was fitted using origin ( Figure 6 ), calculate the Michaelis constant (Km).

[0090] The above linear equation was used to calculate that the Km value of glycosyltransferase UGT73DY2 was 45.27791±5.4 when using Paris polyphylla saponin V as substrate; the Km value of glycosyltransferase UGT73DY2 was 80.04102±11.8 when using Paris polyphylla saponin VI as substrate.

[0091] The smaller the Km value, the greater the affinity between the enzyme and the substrate, and the higher the enzyme activity. From the above results, it can be seen that the glycosyltransferase UGT73DY2 has a strong affinity for both Parisopsin V and Parisopsin VI, among which the affinity for Parisopsin V is the strongest, and it has a high enzyme activity in the process of catalyzing Parisopsin V to synthesize diosgenin-3-O-α-L-pyranorhamnosyl-(1-4)-[α-L-pyranorhamnosyl]-(1-2)-β-D-glucoside.

[0092] Example 4 Recombinant Saccharomyces cerevisiae for Synthesis of Diosgenin-3-O-α-L-rhamnose-(1→4)-[α-L-rhamnose]-(1→2)-β-D-glucoside

[0093] In preliminary experiments, the UGT73DY2 gene was integrated into Saccharomyces cerevisiae. The recombinant Saccharomyces cerevisiae obtained was able to express the glycosyltransferase UGT73DY2. On this basis, the Saccharomyces cerevisiae was further genetically modified in order to obtain a grain wine yeast that can synthesize Paris polyphylla saponin compounds by itself.

[0094] The experiment used CRISPR gene editing technology to perform gene recombination on Saccharomyces cerevisiae BY4742, so that the expression of HMGR gene and ERG9 gene of Saccharomyces cerevisiae was enhanced, and the cas9 gene was integrated. The genetically recombined Saccharomyces cerevisiae was used as the initial Saccharomyces cerevisiae, and CRISPR gene editing technology was continued to be used to integrate DHCR7, DHCR24, RHM, CYP90B27, CYP90G4, CYP94D109, UGT80A41, UGT73CE1 and UGT73DY2 into its genome to obtain recombinant Saccharomyces cerevisiae.

[0095] The specific steps for obtaining recombinant Saccharomyces cerevisiae are as follows: 25 mL of initial Saccharomyces cerevisiae solution (OD 600 =0.8-1.0) centrifugation (2500-3000rpm, 4°C, 3 minutes) resuspend the brewer's yeast with the same volume of sterile water, wash the cells and centrifuge, resuspend the cells with an appropriate volume of 1.1×LiAc to make yeast competent, add 500ng sgRNA, 1μg donorDNA, and then add 500μL PEG / LiAc premix (8mL 50% PEG3350, 1mL 10×TE buffer, 1mL10×LiAc), place the above system in a 30°C water bath for 30 minutes, heat shock at 42°C for 15 minutes, centrifuge at 5000rpm for 2 minutes after the water bath, discard the supernatant, and resuspend in 100μL sterile ddH2O, spread the resuspended yeast on SD-URA plates and culture for 3 days, pick single clones for bacterial P verification, and then expand the yeast after successful sequencing in YPDA medium, and extract the fermentation broth by ultrasonic extraction. The extract was analyzed by UPLC-MS / MS, wherein the chromatographic conditions were the same as in Example 3, and the mass spectrometry conditions were as follows: positive ion mode, capillary voltage of 2.8 kV, sampling cone voltage of 35.0 V, and extraction cone voltage of 3.0 V. The UPLC-MS / MS source temperature was set to 110°C, the desolvation gas temperature was 350°C, and the desolvation gas flow rate was 650 L / h. The scanning mass range was set to m / z50 to 1200. The collision energy was adjusted to 10-30 eV during low energy scanning and to 20-45 eV during high energy scanning. Leucine-enkephalin was used to calibrate the data quality during the analysis. All data were collected in centroid mode and analyzed using Masslynx TM (V4.1) software for control.

[0096] Genes involved in CRISPR gene editing technology

[0097]

[0098]

[0099] Note: Nucleotide sequence of the cas9 gene (SEQ ID NO.11):

[0100] atggactataaggaccacgacggagactacaaggatcatgatattgattacaaagacgatgacgataagatggcccc

[0101] aaagaagaagcggaaggtcggtatccacggagtcccagcagccgacaagaagtacagcatcggcctggacatcg

[0102] gcaccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattcaaggtgctgggca

[0103] acaccgaccggcacagcatcaagaagaacctgatcggagccctgctgttcgacagcggcgaaacagccgaggcc

[0104] acccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatctgcaagagatcttc

[0105] agcaacgagatggccaaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataaga

[0106] agcacgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtaccccaccatctacc

[0107] acctgagaaagaaactggtggacagcaccgacaaggccgacctgcggctgatctatctggccctggcccacatgat

[0108] caagttccggggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggacaagctgttcatccag

[0109] ctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaaggccatcctg

[0110] tctgccagactgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgagaagaagaatggcctg

[0111] ttcggcaacctgattgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaa

[0112] actgcagctgagcaaggacacctacgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccg

[0113] acctgtttctggccgccaagaacctgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcacc

[0114] aaggcccccctgagcgcctctatgatcaagagatacgacgagcaccaccaggacctgaccctgctgaaagctctcg

[0115] tgcggcagcagctgcctgagaagtacaaagagattttcttcgaccagagcaagaacggctacgccggctacattga

[0116] cggcggagccagccaggaagagttctacaagttcatcaagcccatcctggaaaagatggacggcaccgaggaact

[0117] gctcgtgaagctgaacagagaggacctgctgcggaagcagcggaccttcgacaacggcagcatcccccaccaga

[0118] tccacctgggagagctgcacgccattctgcggcggcaggaagatttttacccattcctgaaggacaaccgggaaaa

[0119] gatcgagaagatcctgaccttccgcatcccctactacgtgggccctctggccaggggaaacagcagattcgcctgg

[0120] atgaccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtggacaagggcgcttccgccca

[0121] gagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcccaagcacagcctgctg

[0122] tacgagtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcct

[0123] gagcggcgagcagaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctga

[0124] aagaggactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcctcc

[0125] ctgggcacataccacgatctgctgaaaattatcaaggacaaggacttcctggacaatgaggaaaacgaggacattct

[0126] ggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatcgaggaacggctgaaaacctatgcccac

[0127] ctgttcga

[0128] Analysis of the components in the fermentation broth revealed that the recombinant Saccharomyces cerevisiae was able to synthesize a polysaccharide compound, diosgenin-3-O-α-L-rhamnose-(1→4)-[α-L-rhamnose-pyranosyl]-(1→2)-β-D-glucoside, during the fermentation process. The specific synthesis pathway is shown in Figure 7 .

[0129] Example 7 Synthesis of diosgenin-3-O-α-L-rhamnose-(1→4)-[α-L-rhamnose]-(1→2)-β-D-glucoside from tobacco

[0130] In the early stage of the experiment, the UGT73DY2 gene was integrated into tobacco to obtain tobacco that can express the glycosyltransferase UGT73DY2. On this basis, the tobacco was further transfected using the Agrobacterium-mediated method and more genetic modifications were performed on the tobacco in order to obtain tobacco that can synthesize Paris polyphylla saponin compounds on its own.

[0131] The experiment involved the expression of genes CAS (MN368727.1), HMGR (OP243233.1), CYP94D109 (QDS03630.1), C5-SD2 (OP243261.1), CPI-5 (OP243252.1), CYP51 (OP243253.1), SMO1-3 (OP243242.1), SMO2-2 (OP243245.1), SSR1-3 (OP243236.1), CYP90B71 (QPZ88854 .1), CYP90G6 (QPZ88855.1), CYP94N8 (QPZ88856.1), CYP72A616 (MK636705.1), CYP90G4 (QDS03628.1), CYP90B27 (KX904822.1), UGT80A41 (WIL59759.1), UGT73CE1 (WIL59760.1) and UGT73DY2 plasmids were transformed into Agrobacterium LBA4404 competent cells by liquid nitrogen rapid freezing. Transformants were screened on LB agar plates containing 50 μg / mL kanamycin, 50 μg / mL gentamicin and 50 μg / mL rifampicin at 28°C. The inoculated Agrobacterium strain was cultured in liquid LB medium containing 50 μg / mL kanamycin, 50 μg / mL gentamicin and 50 μg / mL rifampicin at 28°C and 200 rpm for 24-36 hours. The culture solution was then diluted at a ratio of 1:10 and cultured for another 24 hours. The cells were collected by centrifugation at 5000 rpm for 5 minutes and washed once with 20 mL MMA buffer (10 mM MES (pH 5.6, adjusted with KOH), 10 mM MgCl2, 100 μM acetosyringone). Afterwards, the cells were resuspended in MMA buffer and the OD was adjusted. 600to 0.2. When infiltrating with Agrobacterium, the suspensions of each strain were mixed in equal volumes. Before infiltration, 6-week-old Nicotiana benthamiana plants were watered and placed in the shade for about 2 hours. After infiltration, the plants were kept in the shade and transferred to normal growth conditions the next day and continued to grow in a greenhouse. After about 6 days, the infiltrated leaves were collected and freeze-dried using a vacuum freeze dryer. The freeze-dried samples were analyzed by UPLC-MS / MS, wherein the chromatographic conditions were the same as in Example 3 and the mass spectrometry conditions were the same as in Example 4.

[0132] Results Figure 8 ,from Figure 8 It can be seen that in the positive ion mode, the main ion fragment m / z869.48 produced by the cleavage of the standard can be found in the transgenic tobacco sample, which corresponds to the fragment ion peak of diosgenin-3-O-α-L-pyranorhamnosyl-(1→4)-[α-L-pyranorhamnosyl]-(1→2)-β-D-glucoside standard, indicating that diosgenin-3-O-α-L-pyranorhamnosyl-(1→4)-[α-L-pyranorhamnosyl]-(1→2)-β-D-glucoside exists in the sample, including CAS, HMGR, C5-SD2, CPI-5, CYP51, SMO1-3, SMO The transient expression effect of the combination of 2-2, SSR1-3, CYP94N8, CYP90G6, CYP90B27, UGT80A41, UGT73CE1 and UGT73DY2 was the best. According to the peak area of ​​the diosgenin-3-O-α-L-rhamnose-(1→4)-[α-L-rhamnose]-(1→2)-β-D-glucoside standard, the content of diosgenin-3-O-α-L-rhamnose-(1→4)-[α-L-rhamnose]-(1→2)-β-D-glucoside in the infected tobacco leaves was finally calculated to be 2.63 μg / g (dry weight).

[0133] In summary, the UGT73DY2 protein expressed by the glycosyltransferase gene UGT73DY2 of the present invention can catalyze the glycosylation of Paris saponin V, Paris saponin VI and Ophiopogon saponin-3-O-α-L-rhamnose-(1→2)-β-D-glucoside to generate diosgenin-3-O-α-L-rhamnose-(1→4)-[α-L-rhamnose pyranosyl]-(1→2)-β-D-glucoside, diosgenin-3-O-α-L-rhamnose-(1→4)-[α-L-rhamnose pyranosyl]-(1→2)-β-D-glucoside and Ophiopogon saponin-3-O-α-L-rhamnose-(1→4)-[α-L-rhamnose pyranosyl]-(1→2)-β-D-glucoside.

[0134] The glycosyltransferase UGT73DY2 gene of the present invention is introduced into Saccharomyces cerevisiae or Agrobacterium, and the corresponding gene is further introduced thereon. The recombinant Saccharomyces cerevisiae constructed can produce Paris saponin compounds after fermentation and culture. The transgenic tobacco obtained by transfecting the recombinant Agrobacterium constructed by Agrobacterium-mediated method can also synthesize Paris saponin compounds by itself, and finally the heterologous biosynthesis of Paris saponin compounds is realized.

Claims

1. A glycosyltransferase UGT73DY2, characterized in that: The amino acid sequence is shown in SEQ ID NO.

2.

2. The gene encoding the glycosyltransferase UGT73DY2 according to claim 1, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

1.

3. A recombinant vector, expression cassette or recombinant bacterium comprising the gene according to claim 2.

4. The recombinant bacterium according to claim 3, characterized in that: It includes recombinant Escherichia coli, recombinant Saccharomyces cerevisiae or recombinant Agrobacterium; The recombinant Saccharomyces cerevisiae includes a Saccharomyces cerevisiae into which the gene of claim 2, as well as the DHCR7 gene, the DHCR24 gene, the RHM gene, the CYP90B27 gene, the CYP90G4 gene, the CYP94D109 gene, the UGT80A41 gene, the UGT73CE1 gene, and the cas9 gene are integrated; the expression of the HMGR gene and the ERG9 gene of the Saccharomyces cerevisiae is enhanced; the Saccharomyces cerevisiae includes Saccharomyces cerevisiae BY4742; The recombinant Agrobacterium includes Agrobacterium integrated with the gene of claim 2, as well as CAS gene, HMGR gene, CYP94D109 gene, C5-SD2 gene, CPI-5 gene, CYP51 gene, SMO1-3 gene, SMO2-2 gene, SSR1-3 gene, CYP90B71 gene, CYP90G6 gene, CYP94N8 gene, CYP72A616 gene, CYP90G4 gene, CYP90B27 gene, UGT80A41 gene and UGT73CE1 gene; the Agrobacterium includes Agrobacterium LBA4404; The accession number of the HMGR gene in NCBI is OP243233.1; The accession number of the ERG9 gene in NCBI is WNM97123.1; The accession number of the DHCR7 gene in NCBI is NP_958487.2; The accession number of the DHCR24 gene in NCBI is NP_001008645.1; The accession number of the RHM gene in NCBI is CAD92667.1; The accession number of the CYP90B27 gene in NCBI is KX904822.1; The accession number of CYP90G4 in NCBI is QDS03628.1; The accession number of CYP94D109 in NCBI is QDS03630.1; The accession number of UGT80A41 in NCBI is WIL59759.1; The accession number of UGT73CE1 in NCBI is WIL59760.1; The accession number of the CAS gene in NCBI is MN368727.1; The accession number of the C5-SD2 gene in NCBI is OP243261.1; The accession number of the CPI-5 gene in NCBI is OP243252.1; The accession number of the CYP51 gene in NCBI is OP243253.1; The accession number of the SMO1-3 gene in NCBI is OP243242.1 The accession number of the SMO2-2 gene in NCBI is OP243245.1; The accession number of the SSR1-3 gene in NCBI is OP243236.1; The accession number of the CYP90B71 gene in NCBI is QPZ88854.1; The accession number of the CYP90G6 gene in NCBI is QPZ88855.1; The accession number of the CYP94N8 gene in NCBI is QPZ88856.1; The accession number of the CYP72A616 gene in NCBI is MK636705.1 The nucleotide sequence of the cas9 gene is shown in SEQ ID NO.

11.

5. Use of the glycosyltransferase UGT73DY2 according to claim 1 or the gene according to claim 2 or the recombinant vector, expression cassette or recombinant bacteria according to claim 3 in synthesizing steroidal saponin compounds, characterized in that: The steroidal saponin compounds include Paris polyphylla saponin compounds and Ophiopogon japonicus saponin compounds.

6. The use according to claim 5, characterized in that: The Paris polyphylla saponin compounds include diosgenin-3-O-α-L-rhamnose-(1→4)-[α-L-rhamnose-pyranosyl]-(1→2)-β-D-glucoside and diosgenin-3-O-α-L-rhamnose-(1→4)-[α-L-rhamnose-pyranosyl]-(1→2)-β-D-glucoside; The ophiopogon japonicus saponin compound includes ophiopogon japonicus saponin-3-O-α-L-rhamnopyranosyl-(1→4)-[α-L-rhamnopyranosyl]-(1→2)-β-D-glucoside; The chemical structural formula of the diosgenin-3-O-α-L-rhamnose-(1→4)-[α-L-rhamnose pyranosyl]-(1→2)-β-D-glucoside is shown in Formula I, the chemical structural formula of the diosgenin-3-O-α-L-rhamnose-(1→4)-[α-L-rhamnose pyranosyl]-(1→2)-β-D-glucoside is shown in Formula II, and the chemical structural formula of the ophiopogon japonicus saponin compound including ophiopogon japonicus saponin-3-O-α-L-rhamnose-(1→4)-[α-L-rhamnose pyranosyl]-(1→2)-β-D-glucoside is shown in Formula III; 7. A method for synthesizing steroidal saponin compounds, characterized in that: The steps include: Using Paris polyphylla saponin or Ophiopogon japonicus saponin-3-O-α-L-rhamnose-(1→2)-β-D-glucoside as substrate and UDP-rhamnose as sugar donor, the reaction is catalyzed by the glycosyltransferase UGT73DY2 described in claim 1; Or: ferment the recombinant Saccharomyces cerevisiae described in claim 4 in YPDA medium.

8. The method according to claim 7, characterized in that: When the substrate is Paris saponin V, the catalytic reaction generates diosgenin-3-O-α-L-rhamnose-(1→4)-[α-L-rhamnose pyranosyl]-(1→2)-β-D-glucoside; when the substrate is Paris saponin VI, the catalytic reaction generates diosgenin-3-O-α-L-rhamnose-(1→4)-[α-L-rhamnose pyranosyl]-(1→2)-β-D-glucoside; when the substrate is Ophiopogonin-3-O-α-L-rhamnose-(1→2)-β-D-glucoside, the catalytic reaction generates Ophiopogonin-3-O-α-L-rhamnose-(1→4)-[α-L-rhamnose pyranosyl]-(1→2)-β-D-glucoside.

9. Use of the glycosyltransferase UGT73DY2 according to claim 1 or the gene according to claim 2 or the recombinant vector, expression cassette or recombinant bacteria according to claim 3 in preparing a transgenic organism having the function of biosynthesizing Paris polyphylla saponin compounds.

10. The use according to claim 9, characterized in that: The genetically modified organisms include genetically modified tobacco; The transgenic tobacco is Nicotiana benthamiana whose genome is integrated with the gene of claim 2, as well as CAS gene, HMGR gene, CYP94D109 gene, C5-SD2 gene, CPI-5 gene, CYP51 gene, SMO1-3 gene, SMO2-2 gene, SSR1-3 gene, CYP90B71 gene, CYP90G6 gene, CYP94N8 gene, CYP72A616 gene, CYP90G4 gene, CYP90B27 gene, UGT80A41 gene and UGT73CE1 gene; The Paris polyphylla saponin compound includes diosgenin-3-O-α-L-rhamnopyranose-(1→4)-[α-L-rhamnopyranose]-(1→2)-β-D-glucoside.

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