Glycosyl transferase gene for biosynthesis of polygonatum cyrtonema steroid saponin and application of glycosyl transferase gene

Abstract

The invention discloses a glycosyl transferase gene for biosynthesis of polygonatum cyrtonema steroid saponin and application of the glycosyl transferase gene. The glycosyl transferase gene directly related to steroid saponin synthesis in the UGT85A subfamily is cloned and functionally verified from polygonatum cyrtonema for the first time, and gene resources of steroid saponin biosynthesis are enriched; the activity of the PcUGT85As gene for catalyzing the glycosylation of the sitenogenin is defined, and the key enzyme action of the PcUGT85As gene is proved; a key target spot is provided for realizing metabolic regulation and synthetic biological production of the polygonatum cyrtonema active saponin; the gene can be applied to microbial cell factory construction, transgenic plant breeding or biological manufacturing of high-yield saponin, and has a good application prospect and industrial value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant biotechnology, and relates to a glycosyltransferase gene for biosynthesis of polyphylla saponins and application thereof. BACKGROUND

[0002] Polygonatum cyrtonema Hua is a traditional food and medicine plant in China, and its rhizome has been recorded in the Pharmacopoeia of the People's Republic of China, which has the effects of nourishing yin and lung, tonifying qi and spleen, etc. Modern pharmacological studies have shown that P. cyrtonema is rich in various active secondary metabolites, among which steroidal saponins are important characteristic components, which have anti-inflammatory, anti-tumor, hypoglycemic, immune-enhancing, and cardiovascular protection activities, and thus have become a research hotspot in the development and utilization of plant medicines.

[0003] Steroidal saponins are synthesized step by step by multiple enzymes in plants, and glycosylation modification is a key step in the formation of steroidal saponin structure, which has important influence on its physicochemical properties, biological activity and stability. Previous studies have found that UDP-glycosyltransferase (UGT) plays a key role in the glycosylation of steroidal saponins, but the research on the glycosylation enzyme genes of P. cyrtonema steroidal saponins, especially the jatrophatrino-type steroidal saponins, is still relatively weak, and the related functional genes have not been clearly analyzed, which hinders the comprehensive elucidation of the synthesis mechanism of steroidal saponins and the in-depth development of metabolic engineering.

[0004] In previous studies, the UGT85A subfamily was reported to catalyze the glycosylation modification of triterpenes, flavonoids and phenylpropanoids, but its role in the synthesis of steroidal saponins still lacks direct evidence. Especially in P. cyrtonema, although the UGT85A-type genes were observed to have some relevance to the synthesis of steroidal saponins through metabolomic and transcriptomic data, their functions have not been systematically verified.

[0005] Based on the secondary metabolome database of P. cyrtonema constructed in the previous stage, the research team of the present application identified as many as 94 kinds of steroidal saponins, among which jatrophatrino-type steroidal saponins were the main type, accounting for as high as 28.72%. By using multi-omics methods such as targeted metabolome and whole genome association analysis (mGWAS), weighted gene co-expression network analysis (WGCNA), and joint analysis of metabolome and transcriptome, three tandem repeat UGT85A subfamily genes significantly related to the synthesis of jatrophatrino-type steroidal saponins were further screened out, which were PcUGT85A129, PcUGT85A130 and PcUGT85A131, respectively.

[0006] The function verification of the above genes is carried out through a Polyphylla Przewalskii hairy root transformation system, and the results show that the three can significantly improve the accumulation of jatamansi-type steroidal saponin-3-O-glucosyl-rhamnoseyl-glucosyl-glucoside (referred to as HJkp0414) and other jatamansi-type steroidal saponins in Polyphylla Przewalskii hairy roots, and it is speculated that they may catalyze the glycosyltransfer reaction of the 3-O site of steroidal saponins.

[0007] The present application first clearly proposes that PcUGT85A129, PcUGT85A130 and PcUGT85A131 can be used as key functional genes for the glycosylation reaction of jatamansi-type steroidal saponins in Polyphylla Przewalskii, which provides a theoretical basis and technical support for in-depth revealing of the biosynthesis network of steroidal saponins, carrying out precise molecular breeding and constructing a steroidal saponin metabolic engineering production system. SUMMARY

[0008] The present application aims to provide a glycosyltransferase gene for the biosynthesis of steroidal saponins in Polyphylla Przewalskii and its application, and to clarify its function in the glycosylation modification process of steroidal saponins, thereby providing a basis for the biosynthesis mechanism research and metabolic engineering of steroidal saponins in Polyphylla Przewalskii.

[0009] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0010] On the one hand, the present application provides a glycosyltransferase gene for the biosynthesis of steroidal saponins in Polyphylla Przewalskii, which is any one or a combination of the following:

[0011] PcUGT85A129 gene, the nucleotide sequence of the PcUGT85A129 gene is shown as SEQ ID NO: 1;

[0012] PcUGT85A130 gene, the nucleotide sequence of the PcUGT85A130 gene is shown as SEQ ID NO: 2;

[0013] PcUGT85A131 gene, the nucleotide sequence of the PcUGT85A131 gene is shown as SEQ ID NO: 3.

[0014] On the other hand, the present application also provides a recombinant expression vector, which contains the above-mentioned gene and its promoter and terminator, and is fused with green fluorescent protein sfGFP for expression.

[0015] On the other hand, the present application also provides a method for constructing the above-mentioned recombinant expression vector, comprising the following steps:

[0016] (1) designing specific primers containing the PcUGT85A129, PcUGT85A130 or PcUGT85A131 gene;

[0017] (2) using a homologous recombination method to fuse the above-mentioned gene with sfGFP and connect it to the pEarleyGate101 vector;

[0018] (3) transforming into E. coli for amplification and screening positive clones.

[0019] In another aspect, the present application also provides a Polyphylla Przewalskii hairy root high-efficiency transformation system, which is transformed with the above-mentioned recombinant expression vector, and the transformation system is a Polyphylla Przewalskii hairy root transformation system.

[0020] In another aspect, the present application also provides a transgenic Polyphylla Przewalskii hairy root expressing a glycosyltransferase gene, the content of steroidal saponins of which is significantly higher than that of wild-type Polyphylla Przewalskii.

[0021] According to the present application, the glycosylation site of the steroidal saponins of the transgenic Polyphylla Przewalskii hairy root is C3-OH.

[0022] In another aspect, the present application also provides a method for obtaining a transgenic Polyphylla Przewalskii by a hairy root transformation method mediated by Agrobacterium, which comprises the following steps:

[0023] (1) constructing a recombinant vector containing the above-mentioned glycosyltransferase gene;

[0024] (2) transforming the above-mentioned recombinant vector into Agrobacterium rhizogenes;

[0025] (3) infecting the roots of Polyphylla Przewalskii with the above-mentioned Agrobacterium to induce the formation of hairy roots;

[0026] (4) screening and verifying the expression of the target gene and the change in the content of steroidal saponins in the transgenic plant.

[0027] In another aspect, the present application also provides a method for preparing a Polyphylla Przewalskii steroidal saponin product, which comprises using a transgenic Polyphylla Przewalskii hairy root to synthesize saponins, and obtaining the target steroidal saponins by extraction and purification.

[0028] In another aspect, the present application also provides the application of a glycosyltransferase gene in the biosynthesis of Polyphylla Przewalskii steroidal saponins, and the preparation of a modified plant containing the glycosyltransferase gene, the expression of which is increased in the modified plant relative to wild-type plants; wherein the glycosyltransferase gene is any one or a combination of the following:

[0029] PcUGT85A129 gene, the nucleotide sequence of which is shown in SEQ ID NO: 1;

[0030] PcUGT85A130 gene, the nucleotide sequence of the PcUGT85A130 gene is shown as SEQ ID NO: 2;

[0031] PcUGT85A131 gene, the nucleotide sequence of the PcUGT85A131 gene is shown as SEQ ID NO: 3.

[0032] According to the application, the glycosyltransferase gene can promote the synthesis of the oleanolic saponin-3-O-glucosyl-rhamnoseyl-glucosyl-glucoside.

[0033] The application successfully identifies three candidate UDP-glycosyltransferase genes: PcUGT85A129, PcUGT85A130 and PcUGT85A131 through systematic mining of key glycosylation enzyme genes in the synthesis of oleanolic saponins in Polygonatum cyrtonema Hua. The full-length cDNA sequences of the above genes are obtained by using molecular cloning technology, and expression vectors are constructed to realize heterologous expression in the Polygonatum cyrtonema Hua hairy root induction system. The RT-qPCR analysis results show that the expression amount of the above genes in the three kinds of transgenic hairy roots is significantly up-regulated, and the expression level of PcUGT85A131 is increased by about 20 times compared with the wild type. Further metabolic analysis shows that the total content of oleanolic saponins in the hairy roots expressing PcUGT85A129, PcUGT85A130 and PcUGT85A131 increases significantly, indicating that the three have the function of enhancing the accumulation of oleanolic saponins. In combination with the expression amount and the change of metabolic products, it is speculated that PcUGT85A129, PcUGT85A130 and PcUGT85A131 are involved in the glycosylation modification process of the 3-O position of saponins, and are key functional genes in the biosynthesis pathway of oleanolic saponins.

[0034] Compared with the prior art, the application first clones and functionally verifies the glycosyltransferase genes in the UGT85A subfamily directly related to the synthesis of saponins from Polygonatum cyrtonema Hua, enriches the gene resources of saponin biosynthesis; it is clear that the PcUGT85As gene has the activity of catalyzing the glycosylation of oleanolic sapogenin, and confirms its key enzyme effect; it provides a key target for realizing the metabolic regulation and synthetic biology production of active saponins of Polygonatum cyrtonema Hua; the gene can be applied to the construction of microbial cell factories, transgenic plant breeding or biological manufacturing of high-yield saponins, and has good application prospect and industrial value. BRIEF DESCRIPTION OF DRAWINGS

[0035] The drawings described herein are used to provide further understanding of the application, constitute a part of the application, the illustrative embodiments of the application and the description thereof are used to explain the application, and do not constitute improper limitation on the application. In the drawings: Figure 1The total RNA electrophoresis result diagram of Polygonatum cyrtonema Hua of the present application.

[0036] Figure 2 The pEarleyGate 101 vector of the present application PcUGT85A129 / 130 / 131 Gene PCR amplification result.

[0037] Figure 3 The pEarleyGate 101 vector of the present application

[0038] Figure 4 The pEarleyGate 101 vector of the present application PcUGT85A129 / 130 / 131 Gene and sfGFP Gene together PCR amplification electrophoretogram.

[0039] Figure 5 The pEarleyGate 101-PcUGT85A129 / 130 / 131-sfGFP vector schematic diagram of the present application.

[0040] Figure 6 The bacteria detection result after the pEarleyGate 101-PcUGT85A129 / 130 / 131-sfGFP vector of the present application is transformed into K599 Agrobacterium.

[0041] Figure 7 The pEarleyGate 101-PcUGT85A129 / 130 / 131-sfGFP vector schematic diagram of the present application. PcUGT85A129 / 130 / 131-sfGFP Fluorescence and overexpression level of the gene in the hairy roots of Polygonatum cyrtonema Hua; A: expression of the PcUGT85A129 / 130 / 131-sfGFP fusion protein in the hairy roots of Polygonatum cyrtonema Hua, the magnification of the bright field (Bright) picture and the GFP fluorescence picture is 2.5 times, and the magnification of the GFP 10x fluorescence picture is 10 times; B, C, D: overexpression level of the PcUGT85A129 / 130 / 131 gene, the Polygonatum cyrtonema Hua Actin gene is used as an internal reference gene, P <0.05, P <0.01, P <0.001.

[0042] Figure 8 A is the relative content diagram of HJkp0414 in the transgenic hairy roots of the present application, and 8B is the secondary mass spectrum diagram of HJkp0414.

[0043] Figure 9 The relative content diagram of the staticeno type steroidal saponin of the present application. DETAILED DESCRIPTION

[0044] The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0045] The materials, reagents and the like used in the following examples can be obtained from commercial channels unless otherwise specified.

[0046] In order to make the purposes, technical solutions and advantages of the present application clearer and more apparent, the present application will be further described in detail below in combination with the drawings and specific examples. It should be understood that the specific examples described herein are only used to explain the present application and are not used to limit the present application.

[0047] 1. Experimental materials and reagents

[0048] 1.1 Plant materials

[0049] The Polygonatum cyrtonema Hua seedlings used in the experiments of the present application were taken from the laboratory of the present team (temperature 22℃, light time 16h·d-1).

[0050] 1.2 Chemical reagents

[0051] The main reagents used in the experiments of the present application are shown in Table 1.1 below.

[0052] Table 1.1 Main reagents required for experiments

[0053]

[0054] 1.3 Instruments and equipment

[0055] Lanyan constant temperature metal bath; AB104-N electronic analytical balance; PCR instrument; Thermo nanodrop 2000 spectrophotometer; Sigma table type high-speed refrigerated centrifuge; D-37520 type high-speed centrifuge; DK-S24 type electric heating constant temperature water bath; light absorption enzyme label SpectraMax 190; DHG-9070HA precision forced convection drying oven; GENSYS10S UV-Vis ultraviolet spectrophotometer; ZEISS laser confocal microscope; LUYOR-3415RG-LUYOR-3415 dual-wavelength fluorescent protein excitation light source; ALPHA1-2LD Plus freeze dryer, etc.

[0056] 1.4 Main solution formula

[0057] 1.4.2 Preparation of P. cyrtonema transformation expression buffer

[0058]

[0059] The MES solution is more prone to bacterial contamination, and the mother liquor needs to be prepared again after bacterial contamination. Acetyl-syringone is dissolved in N-N-dimethylformamide and then is divided into small portions, and the buffer must be prepared and used immediately.

[0060] 2. Experimental methods

[0061] 2.1 Extraction of total RNA from P. cyrtonema

[0062] 2.1.1 Preparation of experimental supplies

[0063] Grinding supplies were sterilized by high-temperature drying at 180°C. The young rhizomes T41_7 of Polygonatum cyrtonema with the highest content of steroidal saponin HJkp0414 were selected as the RNA extraction material and divided into multiple replicate groups.

[0064] 2.1.2 Extraction of total RNA from Polygonatum cyrtonema

[0065] RNA was extracted using TaKaRa MiniBEST Plant RNA Extraction Kit, and the detailed steps are as follows: https: / / www.takarabiomed.com.cn / ProductShow.aspx?m=20141220151825763053&productID=20141226153536140120.

[0066] 2.2 Detection of total RNA from Polygonatum cyrtonema

[0067] 5 μL of Polygonatum cyrtonema RNA was added to 1 μL of TaKaRa 6×RNA loading buffer, and agarose gel electrophoresis was performed at 115 V for 15 min. The spectrophotometer was used to detect the RNA concentration and A230 / A260, A260 / A280.

[0068] 2.3 Acquisition of PcUGT85A genes

[0069] 2.3.1 Design of primers for full-length PcUGT85A genes

[0070] Primer 6.0 software was used to design primers for the full-length CDS region of Polygonatum cyrtonema PcUGT85A129, PcUGT85A130 and PcUGT85A131 genes (Table 2.1).

[0071] Table 2.1 Primer sequences for cloning PcUGT85A129, PcUGT85A130 and PcUGT85A131

[0072]

[0073] 2.3.2 Synthesis of first-strand cDNA

[0074] cDNA was obtained using the PrimeScript TM RT reagent Kit with gDNA Eraser, and the detailed steps are as follows: http: / / www.baoxinbio.com.cn / product / 67929.html .

[0075] Table 2.2 Reaction system for removing genomic DNA

[0076]

[0077] Table 2.3 RNA reverse transcription reaction system

[0078]

[0079] Amplification of PcUGT85A gene fragment.

[0080] Table 2.4 PCR amplification reaction system

[0081]

[0082]

[0083] PCR reaction program: pre-denaturation: 98℃ 2min; 30 cycles (denaturation: 98℃ 10s; annealing: temperature is the average value of upstream and downstream primers 10s; extension: 72℃ 2min); final extension: 72℃ 5min; preservation: 16℃ 2h.

[0084] 2.3.4 Agarose gel electrophoresis

[0085] The electrophoresis condition is 145V for 15min.

[0086] 2.3.5 PCR product gel recovery

[0087] FlaPure Gel Purification Kit kit is used, and the detailed steps are as follows: https: / / genesand.com / page68?product_id=65&_l=zh_CN .

[0088] 2.3.6 Connection of vector and target gene

[0089] The connection system is as shown in Table 2.5. Gently mix, react at room temperature for 5min. After the reaction is completed, place on ice.

[0090] Table 2.5 Connection system

[0091]

[0092] 2.3.7 Escherichia coli transformation

[0093] The transformed only biological dh5DH5α, details are as follows: https: / / www.weidibio.com / display.php?id= 581 .

[0094] 2.3.8 Screening of positive clones

[0095] Table 2.6 PCR verification reaction system

[0096]

[0097] Single colonies on the long bacteria plate were picked into LB liquid containing corresponding antibiotics, placed in a 37°C shaker at 180 rpm for amplification, and then verified by PCR (Table 2.6) and sequencing. The PCR reaction program was as follows: pre-denaturation at 98°C for 2 min; denaturation at 98°C for 10 s; annealing at 50°C for 10 s; extension at 72°C for 1 min, for a total of 34 cycles; extension at 72°C for 10 min.

[0098] 2.3.9 Extraction of positive clone plasmid

[0099] The TaKaRa MiniBEST Plasmid Purification Kit was used, and the detailed steps were as follows: https: / / www.takarabiomed.com.cn / ProductShow.aspx?m=20141215114017640217&productID= 20141226133731220739

[0100] 2.4 Construction of pEarleyGate 101-PcUGT85A129 / 130 / 131-sfGFP overexpression vector

[0101] The pEarleyGate 101-PcUGT85A129 / 130 / 131-sfGFP overexpression vector was constructed by homologous recombination. The enzyme digestion site on the pEarleyGate 101 vector was SpeI and XholI, and the PcUGT85A gene was connected to the sfGFP gene by a small fragment linker. The primers required for constructing the pEarleyGate 101-PcUGT85A-sfGFP overexpression vector were designed using SnapGene (Table 2.7). To obtain the linearized vector, the plant expression vector pEarleyGate 101-sfGFP was digested with the restriction endonuclease SpeI.

[0102] Table 2.7 Primer sequences for pEarleyGate 101-PcUGT85A129 / 130 / 131-sfGFP overexpression vector

[0103]

[0104] The enzyme digestion reaction system was incubated at 37°C for 1 h, and then the reaction was terminated by heating at 80°C for 30 s. Subsequently, agarose gel electrophoresis was performed, followed by gel recovery, and then the QIAquick Gel Extraction Kit was used. - Basic Seamless Cloning and Assembly Kit kit, 50°C for 20 min. Next step is E. coli transformation, positive clone screening and positive plasmid extraction, followed by Agrobacterium transformation, the Agrobacterium used for transformation is K599 Chemically Competent Cell, see details in: https: / / www.weidibio.com / display.php?id=320 . Finally, single colony from plate is picked into LB liquid containing Rif (20 pg / mL) and Kan (50 pg / mL), 28°C, 180 rpm incubation. PCR verification and glycerol preservation.

[0105] 2.5 Polyphylla rhizome hairy root transformation

[0106] (1) K599 positive bacteria liquid culture to OD600 about 0.6-0.8;

[0107] (2) Preparation of Polyphylla transformation expression buffer;

[0108] (3) 3000 rpm centrifugation for 10 min of the above bacteria liquid, pour off the supernatant, add buffer to resuspend the precipitate, continue 3000 rpm centrifugation for 10 min, pour off the supernatant, repeat twice;

[0109] (4) Add buffer to resuspend the precipitate again, so that the final Agrobacterium buffer OD600 is between 0.3-0.5, dark treatment for 2h;

[0110] (5) Polyphylla is taken out from the soil, washed, and try to keep most of the roots of Polyphylla during washing. Use a knife to make incisions on the surface of the Polyphylla rhizome, focus on the tender white area for incision, and at the same time, use needle pricking method to make small wounds on the surface of the Polyphylla rhizome. Soak the incised Polyphylla in the prepared infection liquid for 30 min. After treatment, put the Polyphylla back into the soil, loosen the soil appropriately and water appropriately to ensure the vitality of the Polyphylla;

[0111] (6) Incubate in a culture room at 22°C with 8h light and 16h dark for 60-120 days.

[0112] 2.6 Identification of transgenic Polyphylla hairy roots

[0113] 2.6.1 Fluorescence observation

[0114] (1) Take the Polyphylla out of the soil, wash it, and pay attention to prevent damaging the Polyphylla hairy roots and try to ensure the integrity of the Polyphylla root system;

[0115] (2) Use a dual-wavelength fluorescent protein excitation light source (440-460 nm) to irradiate the Polyphylla hairy roots in a dark room, and use a yellow filter to observe and take pictures.

[0116] 2.6.2 Quantitative Real-time PCR (qRT-PCR) analysis Specific primers for PcUGT85A129 / 130 / 131-sfGFP gene were designed using Primer Premier 6.0, see Table 2.8:

[0117] Table 2.8 qRT-PCR primer table

[0118]

[0119] qRT-PCR reaction was performed using TaKaRa kit, PCR reaction program was shown in Table 2.9:

[0120] Table 2.9 qRT-PCR reaction program

[0121]

[0122] 2.7 Steroidal saponin content determination of transgenic P. multiflorum hairy roots

[0123] 2.7.1 Sample preparation

[0124] PcUGT85A129, PcUGT85A130 and PcUGT85A131 overexpression hairy roots with green excitation fluorescence after fluorescent lamp irradiation were cut, and then were quickly frozen in liquid nitrogen for 2 min and vacuum freeze-dried for 63 h.

[0125] 2.7.2 Sample extraction

[0126] The freeze-dried hairy root sample was ground into powder by a grinding instrument (MM 400, Retsch) (33 Hz, 1.5 min). 30 mg of hairy root sample powder was added into 1500 μL of 70% methanol water internal standard extraction solution pre-cooled at 20°C (if less than 30 mg, 1500 μL of extraction agent was added per 30 mg of sample). The internal standard extraction solution was prepared by dissolving 1 mg of standard into 1 mL of 70% methanol water to prepare 1000 μg / mL standard stock solution, and then the 1000 μg / mL stock solution was further diluted with 70% methanol to prepare 250 μg / mL internal standard solution, and then vortexed to mix.

[0127] Centrifuged at 12 000 rpm for 3 min, and the supernatant was filtered by a microporous filter (0.22 μm) into a sample bottle.

[0128] 2.7.3 Chromatography mass spectrometry collection conditions

[0129] The data collection instrument system mainly includes an ultra performance liquid chromatography (UPLC) (Exion LC TM AD, https: / / sciex.com.cn / ) and tandem mass spectrometry (MS / MS).

[0130] The liquid phase conditions mainly include:

[0131] (1) chromatographic column: Agilent SB-C18 1.8 μm, 2.1 mm*100 mm;

[0132] (2) mobile phase: A phase is ultrapure water (0.1% formic acid is added), B phase is acetonitrile (0.1% formic acid is added);

[0133] (3) elution gradient: 0.00 min B phase ratio is 5%, B phase ratio is linearly increased to 95% within 9.00 min, and maintained at 95% for 1 min, 10.00-11.10 min, B phase ratio is reduced to 5%, and balanced at 5% for 14 min;

[0134] (4) flow rate 0.35 mL / min; column temperature 40℃; injection volume 2 μL.

[0135] The effluent is alternately connected to an ESI triple quadrupole linear ion trap (QTRAP)-MS.

[0136] The mass spectrometry conditions mainly include: electrospray ionization (ESI) temperature 500℃; ion spray voltage (IS) 5500V (positive ion mode) / -4500V (negative ion mode); ion source gas I (GSI), gas II (GSII) and curtain gas (CUR) are set to 50, 60 and 25 psi respectively, and the collision-induced ionization parameter is set to high. QQQ scanning uses MRM mode, and the collision gas (nitrogen) is set to medium. Through further optimization of declustering potential (DP) and collision energy (CE), the DP and CE of each MRM ion pair are completed. According to the metabolites eluted in each period, a specific group of MRM ion pairs is monitored in each period.

[0137] 3. Experimental results

[0138] 3.1 Extraction of total RNA of Polygonatum cyrtonema Hua

[0139] RNA agarose gel electrophoresis results showed that the 28S and 18S bands in lane 2 were bright, and the 5S band was clearly visible. The RNA concentration was measured to be 125.80 ng / μL, with an A260 / 280 of 2.06 and an A260 / 230 of 1.78. Therefore, the RNA quality was good and it could be used for subsequent reverse transcription.

[0140] 3.2 Cloning of the PcUGT85A129 / 130 / 131 gene

[0141] The target fragments of the PcUGT85A129, PcUGT85A130, and PcUGT85A131 genes were amplified. Figure 1 The fragment sizes were 1443bp, 1428bp, and 1458bp, respectively, consistent with the theoretical sizes, indicating that the PCR amplification products were correct. The amplified genes were ligated into a T-vector and transformed into competent *E. coli* for bacterial detection and sequencing verification. The bacterial detection bands were correct, and the sequencing results showed that the sequences were identical to those in the *Polygonatum cyrtonema* genome, indicating that the *PcUGT85A129*, *PcUGT85A130*, and *PcUGT85A131* genes had been successfully cloned.

[0142] Construction of the 3.3pEarleyGate 101-PcUGT85A129 / 130 / 131-sfGFP overexpression vector

[0143] 3.3.1 Obtaining the Linear Vector and Target Gene

[0144] The pEarleyGate 101-sfGFP vector, prepared in advance in the laboratory, was digested with TaKaRa EcoRI restriction endonuclease to obtain a linear pEarleyGate 101-sfGFP vector. Electrophoresis after gel recovery confirmed the linearity of the vector. Figure 2 As shown in the figure, the results indicate that the pEarleyGate101-sfGFP vector was correctly digested and banded.

[0145] PCR amplification of the PcUGT85A129, PcUGT85A130, PcUGT85A131, and sfGFP gene fragments yielded PcUGT85A129, PcUGT85A130, PcUGT85A131, and sfGFP gene fragments with homologous arms. These fragments were then recovered from the gel and used to construct vectors. Figure 3 As shown in the figure, the results indicate that the target gene band was successfully amplified.

[0146] 3.3.2 Vector Construction and Microbial Detection

[0147] The pEarleyGate 101-PcUGT85A129 / 130 / 131-sfGFP overexpression vector was successfully constructed. A 6×His tag was attached to the C-terminus of PcUGT85A129, PcUGT85A130, and PcUGT85A131, and the Superfolder GFP (sfGFP) green fluorescent protein gene was attached to the N-terminus. Figure 4 The bacterial detection results of *Agrobacterium rhizogenes* K599 transformed with pEarleyGate 101-PcUGT85A129 / 130 / 131-sfGFP are as follows: Figure 5 As shown in the figure, the results indicate that the pEarleyGate 101-PcUGT85A129 / 130 / 131-sfGFP vector was successfully transformed into K599 Agrobacterium rhizogenes.

[0148] 3.4 Detection of hairy root transformation and expression level of Polygonatum multiflorum

[0149] pEarleyGate 101-PcUGT85A129 / 130 / 131-sfGFP was transformed into K599 Agrobacterium rhizogenes, successfully obtaining PcUGT85A129, PcUGT85A130, and PcUGT85A131 overexpressing Polygonatum odoratum hairy roots. Polygonatum odoratum hairy roots transformed with pEarleyGate101-sfGFP vector served as a negative control. After 60 days of culture in the native environment, a green fluorescent signal of sfGFP was detected in the Polygonatum odoratum hairy root cells under handheld fluorescent light in a dark room. Figure 6 This indicates that the PcUGT85A129, PcUGT85A130, and PcUGT85A131 genes have been successfully transferred into the target plant and are all normally expressed in the hairy root cells of Polygonatum cyrtonema. Green fluorescent hairy roots were cut, flash-frozen in liquid nitrogen, and RNA was extracted and reverse-transcribed into cDNA for further real-time quantitative PCR. Using the Actin gene of Polygonatum cyrtonema as an internal control, the expression levels of the transferred genes were analyzed. The results showed that the expression levels of PcUGT85A129, PcUGT85A130, and PcUGT85A131 genes were significantly increased in the hairy roots of Polygonatum cyrtonema transformed with the overexpression vector. Among them, PcUGT85A131 had the highest expression level, with an expression level more than 20-fold higher than that of wild-type overexpressed hairy roots of Polygonatum cyrtonema. Figure 6 ).

[0150] Analysis of steroidal saponin content in the hairy roots of Polygonatum multiflorum overexpressed by 3.5PcUGT85A129 / 130 / 131

[0151] The present application is based on the secondary metabolism database of Polygonatum cyrtonema Hua, and it is found that the stanozolol type of steroidal saponin is the main type of steroidal saponin in Polygonatum cyrtonema Hua. Through resequencing and metabolome data of 162 Polygonatum cyrtonema Hua materials Genome-Wide Association Study (GWAS), three UGT85 family genes PCUGT85A129, PCUGT85AI30 and PcUGT85A131 significantly associated with the stanozolol type of steroidal saponin (stanozolol type of steroidal saponin-3-O-glucosyl-rhamnose-glucosyl-glucoside (HJkp0414)) are mined. After the three UGT85 family genes are transferred into the hairy roots of Polygonatum cyrtonema Hua, the metabolites of the hairy roots of Polygonatum cyrtonema Hua overexpressing the genes PcUGT85A129, PcUGT85A130 and PcUGT85A131 are subjected to qualitative and quantitative mass spectrometry analysis, and 245 steroidal saponin compounds are detected, of which 27 are stanozolol type of steroidal saponin, and it is also found that HJkp0414 exists Figure 8 A and Figure 8 B).

[0152] The content change of the main type of steroidal saponin of Polygonatum cyrtonema Hua, the stanozolol type of steroidal saponin, is analyzed Figure 9A total of 27 types of steroidal saponins were detected. In the hairy roots of Polygonatum multiflorum overexpressed by PcUGT85A129, the content of 25 types (92.59%) of steroidal saponins was higher than that of the wild type. In the hairy roots of Polygonatum multiflorum overexpressed by PcUGT85A130, the content of 23 types (85.19%) of steroidal saponins was higher than that of the wild type. In the hairy roots of Polygonatum multiflorum overexpressed by PcUGT85A131, the content of 19 types (70.37%) of steroidal saponins was higher than that of the wild type. Among them, PcUGT85A129 overexpression of five types of steroidal saponins in the hairy roots of Polygonatum odoratum include: HJkp0404 (sparganin 3-O-glucosyl-xylosyl-glucosyl-xylososide), HJkp0435 (sparganin 3-O-glucosyl-glucosyl-glucososide), HJkp0412 (sparganin 3-O-rhamnosyl-glucosyl-glucosyl-xylososide), and HJkn0378 ((25S)-Spirostan-5-en-12-one-3-O-β-D-glucopyranosyl-(1→2)-O-[β-D-glucopyranosyl-(1→3)]-O-β-Dglucop The contents of yranosyl-(1→4)-β-D-galactopyranoside and HJkp0378 (Yunnan Polygonatum saponin D) increased by more than 4 times, with HJkn0378 showing the highest increase at 817.23 times, followed by HJkp0435 at 37.89 times. In the hairy roots of Polygonatum multiflorum overexpressed by PcUGT85A130, the contents of two steroidal saponins, HJkp0439 and HJkn0378, showed the highest increases, both exceeding 6 times. In the hairy roots of Polygonatum multiflorum overexpressed by PcUGT85A131, the contents of three steroidal saponins, HJkp0404, Zjhp082657 and HJkn0378, increased by more than 4 times, with the highest increase being 9.24 times for HJkn0378. Notably, the contents of HJkp0414 all increased significantly. Figure 8 A) The HJkp0414 content in the hairy roots of *Polygonatum cyrtonema* overexpressed with PcUGT85A130 increased by 3.40-fold; followed by a 2.33-fold increase in the hairy roots of *Polygonatum cyrtonema* overexpressed with PcUGT85A129; and a 1.50-fold increase in the hairy roots of *Polygonatum cyrtonema* overexpressed with PcUGT85A131. Simultaneously, the glycosylation sites of the phentermine-type steroidal saponins were all located at C3-OH, suggesting that PcUGT85A129 / 130 / 131 participate in the 3-O-glycosylation process of phentermine-type steroidal saponins.

[0153] The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the technical principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A glycosyltransferase gene for the biosynthesis of polyphyllin steroidal saponins, characterized in that, The gene is any one or a combination of the following: a PcUGT85A129 gene, the nucleotide sequence of which is shown as SEQ ID NO: 1; a PcUGT85A130 gene, the nucleotide sequence of which is shown as SEQ ID NO: 2; a PcUGT85A131 gene, the nucleotide sequence of which is shown as SEQ ID NO:

3.

2. A recombinant expression vector, characterized in that, The vector contains the gene and its promoter and terminator according to claim 1, and is fused with green fluorescent protein sfGFP for expression.

3. A method of constructing the recombinant expression vector of claim 2, wherein, The method comprises the following steps: (1) designing specific primers containing the PcUGT85A129, PcUGT85A130 or PcUGT85A131 gene; (2) using a homologous recombination method to fuse the gene with sfGFP and connect it to the pEarleyGate101 vector; (3) transforming into E. coli for amplification and screening positive clones.

4. A hairy root high-efficiency transformation system of Polygonatum cyathopetalum, characterized in that, The transformation system is transformed with the recombinant expression vector according to claim 2, and the transformation system is a Polyphylla rubescens hairy root transformation system.

5. A transgenic hairy root of Polygonum multiflorum expressing the gene of claim 1, wherein, The content of steroidal saponins in the transgenic Polyphylla rubescens hairy roots is significantly increased compared with that in wild-type Polyphylla rubescens.

6. The transgenic P. multiflorum hairy roots of claim 5, wherein, The glycosylation site of the steroidal saponins in the transgenic Polyphylla rubescens hairy roots is C3-OH.

7. A method for obtaining the transgenic Polygonatum cyrtonema of claim 5 by a hairy root transformation method mediated by Agrobacterium, characterized in that, The method comprises the following steps: (1) constructing a recombinant vector containing the glycosyltransferase gene; (2) transforming Agrobacterium rhizogenes with the recombinant vector; (3) infecting the roots of Polyphylla rubescens with the Agrobacterium to induce the formation of hairy roots; (4) screening and verifying the expression of the target gene and the change in the content of steroidal saponins in the transgenic plants.

8. A method for preparing a polyphyllin steroidal saponin product, characterized by, The method comprises using the transgenic Polyphylla rubescens hairy roots according to claim 5 for saponin synthesis, and obtaining the target steroidal saponins by extraction and purification.

9. The use of the glycosyltransferase gene according to claim 1 in the biosynthesis of polyphyllin steroidal saponins, characterized in that, A modified plant containing a glycosyltransferase gene is prepared, and the expression of the glycosyltransferase gene in the modified plant is increased relative to that in a wild-type plant; wherein the glycosyltransferase gene is any one or a combination of the following: a PcUGT85A129 gene, the nucleotide sequence of which is shown as SEQ ID NO: 1; a PcUGT85A130 gene, the nucleotide sequence of which is shown as SEQ ID NO: 2; a PcUGT85A131 gene, the nucleotide sequence of which is shown as SEQ ID NO:

3.

10. The use according to claim 9, wherein the glycosyltransferase gene can promote the synthesis of a sitosterol-type steroidal saponin-3-O-glucosyl-rhamnosyl-glucosyl-glucoside.

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