CN114480323B - Oat glycosyltransferase AsUGT73E1 and its application in the synthesis of steroidal saponins - Google Patents

Abstract

The present invention relates to saponin metabolism pathways, specifically to oat glycosyltransferase AsUGT73E1 and its application in the synthesis of steroidal saponins. The glycosyltransferase provided by the present invention is a protein as described in a1 or a2 as follows: a1. A protein whose amino acid sequence is shown in SEQ ID NO: 1; a2. The amino acid sequence shown in SEQ ID NO: 1 is passed through one or A protein with glycosyltransferase activity formed by the substitution and/or deletion and/or addition of several amino acid residues. The glycosyltransferase can use trillium or metanosogenin-3-O-glucoside as a substrate, introduce rhamnosyl group, and generate chrysanthemum saponin V or chrysanthemum saponin VI.

Description

Oat glycosyltransferase AsUGT73E1 and its application in the synthesis of steroidal saponins

Technical field

The present invention relates to saponin metabolism pathways, specifically to oat glycosyltransferase AsUGT73E1 and its application in the synthesis of steroidal saponins.

Background technique

Chonglou is the general name for plants of the genus Chonglou in the Liliaceae family. It can be used as the main raw material of Chinese patent medicines such as Yunnan Baiyao, Gongxuening and Reduqing, and has extremely high medicinal and economic value. Steroidal saponins are the main chemical components of Chonglou plants. Currently, more than 160 species have been isolated and identified, mainly including Chonglou saponins (polyphyllin) Ⅰ, Ⅱ, Ⅲ, Ⅴ (diosgenate saponins) and Ⅵ and Ⅶ (bianosaponins). ), has a wide range of pharmacological activities. Anti-tumor is the main effect of Chonglou saponin. Chonglou saponin I induces autophagy and cell cycle arrest by inhibiting the PDK1/Akt/mTOR signaling pathway and downregulating cyclin B1 in human gastric cancer HGC-27 cells (Heet al. 2019) . Chonglousaponin VI induces apoptosis and autophagy through the ROS-triggered mTOR signaling pathway in non-small cell lung cancer (Teng et al. 2019). Chonglousaponin VII can promote mitochondrial production of ROS and activate the MAPK and PTEN/p53 pathways, jointly inducing apoptosis in HepG2 human liver cancer cells (Zhang et al. 2016). Chonglou saponin VII is also very effective in antibacterial and anti-inflammatory, and can significantly inhibit the growth of Cladosporium cladosporum, Candida and Propionibacterium acnes, and can be used as an effective substitute for synthetic drugs (Deng et al. 2008; Qin et al. .2012). Diosgenin can enhance the activities of lipoprotein lipase, hepatic lipase, superoxide dismutase, glutathione peroxidase and nitric oxide synthase in mice with hyperlipidemia, and improve lipid distribution. , which has the effect of lowering blood lipids (Gong et al. 2010). Chonglou saponin III has excellent anthelmintic activity and can kill anthromycin parasites on the gills of goldfish (EC ₅₀ = 18.06 mg l ^-1 ), and is low-toxic to goldfish (Wang et al. 2010). In research on COVID-19, it has been found that saponin molecules have potential anti-COVID-19 activity. Through docking screening, it is speculated that Chonglousaponin I can bind to the 2019-nCoV main protease (M protease) to prevent virus replication (Yan et al. 2020).

At this stage, research on Chonglou saponins is mainly focused on medicine and clinical practice, resulting in a lack of understanding of its metabolic pathways, especially the downstream biosynthetic processes. 2,3-oxidosqualene is a common precursor for the synthesis of sterols and triterpenes, and is produced under the catalysis of 2,3-Oxidosqualene cyclases (OSCs). Sterol or triterpenoid skeleton. Most pentacyclic triterpene synthases can catalyze 2,3-oxysqualene to form a dammarane cation in the "chair-chair-chair" conformation, which is then further rearranged to produce α-amyrin, β-amyrin and lupeol. (lupeol) and other pentacyclic triterpenes (Xue et al. 2018). Cyclartenol synthase catalyzes 2,3-oxysqualene to form a prosterol cation in the "chair-boat-chair" conformation, which is then converted into cycloartenol and synthesizes cholesterol through a series of reactions. Although the content of cholesterol in plants is generally low, it is an important component of phytosterols and the direct precursor of steroidal saponins (Cárdenas et al. 2015). The synthesis of steroidal sapogenins requires the hydroxylation of the cholesterol side chain, which is mainly modified by cytochrome P450 monooxygenase (CYP). For example, it was found in Paris polyphylla that CYP90Bs evolved sterol polyhydroxylase activity through gene duplication, and PpCYP90G4 can catalyze the hydroxylation of cholesterol C16 and C22 with E-ring closure. 16,22(S)-dihydroxycholesterol is further hydroxylated at the C26 position under the action of enzymes such as PpCYP94D108 to form diosgenin (Christ et al. 2019), while the P450 enzyme at the C17 position of pennogenin remains to be analyzed. Finally, diosgenin or metanosogenin undergoes glycosylation under the action of glycosyltransferase (UDP-glucosyltransferase, UGT) to form diosgenin with a variety of biological activities. At present, there are few reports on the glycosylation modification of steroidal sapogenins.

Chonglou saponins are basically extracted from plants. However, over-exploitation has led to the depletion of Chonglou resources. The use of synthetic biology to construct heterologous biosynthetic pathways has gradually become an effective way to obtain natural active ingredients. However, the genome of Chrysanthemum plants is huge and there are many types of saponins, making it difficult to analyze the metabolic pathways of Chrysanthemum saponins through gene co-expression networks. Oats (Avena sativa L.) is the only plant rich in saponins among the grasses (Vincken et al. 2007) and can synthesize two different types of saponins. One type is avencins, a triterpenoid saponin synthesized in roots and root tips, and the other is avenacosides, a steroidal saponin that accumulates in leaves and grains. Oat is an annual herb with a short growth cycle. It can complete a growth cycle in 3 to 4 months under long daylight conditions. It can be used as an ideal model material for studying the synthesis pathway of steroidal saponins. With the discovery of the triterpene saponin avencins metabolic gene cluster, the synthesis pathway of oat triterpene saponins has been relatively completely analyzed after years of research (Louveau et al. 2018; Orme et al. 2019).

Contents of the invention

In order to analyze the metabolic pathway of Chonglou saponin and realize the artificial synthesis of Chonglou saponin, we used oat as a model material and obtained two oat glycosyltransferases, named AsUGT73E5 and AsUGT73E1 respectively. To verify the function of glycosyltransferase, we designed PCR primers AsUGT73E5-ORF-F/R and AsUGT73E1-ORF-F/R for the coding regions of glycosyltransferase genes AsUGT73E5 and AsUGT73E1. The reverse-transcribed oat seedling cDNA was used as a template for PCR amplification, and the AsUGT73E5 and AsUGT73E1 genes were obtained. The open reading frame of the AsUGT73E1 gene contains 1473 bases, its nucleotide sequence is shown in SEQ ID NO:2, and its encoded amino acid sequence is shown in SEQ ID NO:1. The open reading frame of the AsUGT73E5 gene contains 1548 bases, its nucleotide sequence is shown in SEQ ID NO:4, and its encoded amino acid sequence is shown in SEQ ID NO:3.

Then, we performed protein expression and purification. The open reading frames (ORFs) of genes AsUGT73E5 and AsUGT73E1 were cloned into the prokaryotic expression vector pGEX-6p-1 using homologous recombination, and then transformed into expression-competent Rosetta (DE3). Cultivate in Amp-resistant LB liquid medium until OD600=0.6, add IPTG for low-temperature induction overnight. After the bacterial cells were disrupted by sonication, the proteins in the supernatant were purified using Glutathione Beads, and each component was collected for SDS-PAGE analysis (Figure 2). Compared with the uninduced control sample, the recombinant protein showed obvious bands near 80KDa. AsUGT73E5 and AsUGT73E1 proteins contain 515 and 490 amino acids respectively, and their molecular weights after fusion with the GST tag are 82.21KDa and 79.79KDa respectively.

Next, we used diosgenin, metanosagenin and nuaartigenin to functionally verify the glycosyltransferases AsUGT73E5 and AsUGT73E1 respectively. The results are as follows:

The reaction product of AsUGT73E5 protein with diosgenin and UDP-Glc was detected by HPLC (Figure 3). Compared with the unloaded reaction, the product of AsUGT73E5 showed a new peak (product 1) at 22.3 minutes, which was similar to trillium. (trillin) standards peak at the same time. The sample was recovered, AsUGT73E1 protein and UDP-Rha were added to continue the reaction. A new product peak (product 2) appeared at 20.7 minutes, and the time was consistent with the Chonglousaponin V standard. The TOF positive ion scanning mode detection is shown in Figure 4. The molecular weights of product 1 and product 2 are 577.38 (M+H+) and 723.43 (M+H+) respectively, which are similar to trillium (576.3) and chrysanthemum saponin V (722.4). The molecular weights of diosgenin are consistent, indicating that diosgenin is catalyzed by AsUGT73E5 and AsUGT73E1 to generate diosgenin V.

After HPLC detection of the reaction product of AsUGT73E5 protein with metanogenin and UDP-Glc (Figure 5), compared with the unloaded reaction, the product of AsUGT73E5 showed a new peak (product 3) at 18.8 minutes. The sample was recovered, AsUGT73E1 protein and UDP-Rha were added to continue the reaction. A new product peak (product 4) appeared at 17.1 minutes, and the time was consistent with the Chonglousaponin VI standard. TOF positive ion scanning mode detection is shown in Figure 6. The molecular weights of product 3 and product 4 are 593.37 (M+H+) and 739.43 (M+H+) respectively, which are similar to those of metanosogenin-3-O-glucoside (592.3) and The molecular weight of chrysanthemum saponin VI (738.4) is consistent, indicating that the metanosaponin is continuously catalyzed by AsUGT73E5 and AsUGT73E1 to generate chrysanthemum saponin VI.

After HPLC detection of the reaction product of AsUGT73E5 protein with neuaartinin and UDP-Glc (Figure 8), compared with the unloaded reaction, a new peak of AsUGT73E5 product appeared at 16.7 minutes (product 5). The sample was recovered, AsUGT73E1 protein and UDP-Rha were added to continue the reaction, and the second product peak (product 6) appeared at 15.6 minutes. The TOF positive ion scanning mode detection is shown in Figure 9. The molecular weights of product 5 and product 6 are 593.37 (M+H ⁺ ) and 739.43 (M+H ⁺ ) respectively, indicating that neuaartinogen has been continuously catalyzed by AsUGT73E5 and AsUGT73E1. The corresponding glycosylation products are generated.

Based on the above research, the present invention provides a glycosyltransferase, which is a protein as described in a1 or a2 below:

a1. The protein whose amino acid sequence is shown in SEQ ID NO:1;

a2. A protein with glycosyltransferase activity formed by substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence shown in SEQ ID NO:1.

The gene encoding the glycosyltransferase also belongs to the protection scope of the present invention.

In some embodiments of the invention, the nucleotide sequence of the gene is shown in SEQ ID NO: 2.

Expression cassettes, vectors or recombinant bacteria containing the genes also belong to the protection scope of the present invention.

In some embodiments, the vector is a cloning vector, comprising a gene encoding the glycosyltransferase and elements required for plasmid replication, for example, pClone007 Blunt Simple Vector with the encoding gene inserted. In other embodiments, the vector is an expression vector, comprising a gene encoding the glycosyltransferase and elements capable of successfully expressing the protein, for example, a pGEX-6p-1 vector in which the encoding gene is inserted.

In some embodiments, the recombinant bacterium is a recombinant bacterium containing a cloning vector, such as E. coli DH5α, and the gene encoding the glycosyltransferase is replicated by culturing the recombinant bacterium. In other embodiments, the recombinant bacterium is a recombinant bacterium containing an expression vector, and the recombinant bacterium is cultured under appropriate conditions, for example, adding an appropriate amount of IPTG and inducing the expression of the glycosyltransferase at 16°C.

The present invention also provides a method for preparing the glycosyltransferase, which includes the following steps: constructing an expression vector for the encoding gene of the glycosyltransferase, introducing the expression vector into an expression host bacterium, obtaining recombinant bacteria, cultivating the recombinant bacteria and inducing Protein expression, collecting bacterial cells, extracting and purifying proteins.

The use of the glycosyltransferase in glycosyl transfer reactions also falls within the protection scope of the present invention.

The use of the glycosyltransferase in the synthesis of steroidal saponins also falls within the protection scope of the present invention. The synthesis of steroidal saponins includes in vitro synthesis and in vivo synthesis, for example, steroidal saponins are synthesized in microorganisms (such as yeast) or plants.

In some embodiments of the present invention, during the synthesis of the steroidal saponins, trillium or metanosogenin-3-O-glucoside is used as a substrate, and is introduced into mice under the action of the glycosyltransferase. Liposyl, generates Chonglousaponin V or Chonglousaponin VI.

The present invention also provides a method for synthesizing saponin V, which includes the following steps: utilizing the glycosyltransferase to react with trillium and UDP-Rha to generate saponin V.

The present invention also provides a method for synthesizing saponin VI, which includes the following steps: utilizing the glycosyltransferase to react with metanosapogenin-3-O-glucoside and UDP-Rha to generate saponin VI.

The glycosyltransferase provided by the present invention will help us explore the synthesis pathway of plant steroidal saponins, and at the same time, it also provides genetic resources for obtaining a large amount of Chonglou saponin or other saponins.

Description of the drawings

Figure 1. Electropherogram of AsUGT73E5 and AsUGT73E1 gene amplification products. M is DL 2000; 1 is AsUGT73E5 gene band; 2 is AsUGT73E1 gene band.

Figure 2. SDS-PAGE electrophoresis diagram of prokaryotic expression of AsUGT73E5 and AsUGT73E1 proteins. M: Page-ruler protein molecular marker; 1: not induced; 2: whole cell protein after IPTG induction; 3: cell supernatant after IPTG induction; 4: cell pellet after IPTG induction; 5: supernatant GST column purification; 6: Purified proteins are concentrated by ultrafiltration.

Figure 3. Chromatogram of the glycosylation products of the reaction between AsUGT73E5, AsUGT73E1 and diosgenin. The abscissa is the retention time (min), and the ordinate is the electrical signal (mAU). pGEX-empty: The protein extracted and purified from the cells containing the pGEX-6P-1 empty vector after protein induction and expression, as a blank control; pGEX-AsUGT73E5: The protein extracted and purified from the cells containing the pGEX-AsUGT73E5 vector after protein induction and expression. AsUGT73E5 protein; AsUGT73E1 protein extracted and purified after protein induction and expression in cells containing pGEX-AsUGT73E1 vector.

Figure 4. Mass spectrometry analysis of the glycosylation products of the reaction between AsUGT73E5, AsUGT73E1 and diosgenin. The abscissa is the mass-to-charge ratio, and the ordinate is the ionic strength.

Figure 5. Chromatogram of the glycosylation products of the reaction between AsUGT73E5, AsUGT73E1 and metanosaponogenin. The abscissa is the retention time (min), and the ordinate is the electrical signal (mAU). pGEX-empty: The protein extracted and purified from the cells containing the pGEX-6P-1 empty vector after protein induction and expression, as a blank control; pGEX-AsUGT73E5: The protein extracted and purified from the cells containing the pGEX-AsUGT73E5 vector after protein induction and expression. AsUGT73E5 protein; AsUGT73E1 protein extracted and purified after protein induction and expression in cells containing pGEX-AsUGT73E1 vector.

Figure 6. Mass spectrometry analysis of the glycosylation products of the reaction between AsUGT73E5, AsUGT73E1 and metanosaponogenin. The abscissa is the mass-to-charge ratio, and the ordinate is the ionic strength.

Figure 7. AsUGT73E5 and AsUGT73E1 catalyze the glycosylation process of steroidal sapogenins.

Figure 8. Chromatogram of the glycosylation products of the reaction between AsUGT73E5, AsUGT73E1 and nuaartidin. The abscissa is the retention time (min), and the ordinate is the electrical signal (mAU). pGEX-empty: The protein extracted and purified from the cells containing the pGEX-6P-1 empty vector after protein induction and expression, as a blank control; pGEX-AsUGT73E5: The protein extracted and purified from the cells containing the pGEX-AsUGT73E5 vector after protein induction and expression. AsUGT73E5 protein; AsUGT73E1 protein extracted and purified after protein induction and expression in cells containing pGEX-AsUGT73E1 vector.

Figure 9. Mass spectrometry analysis of the glycosylation products of the reaction between AsUGT73E5, AsUGT73E1 and nuaartinogen. The abscissa is the mass-to-charge ratio, and the ordinate is the ionic strength.

Detailed ways

The present invention will be further described below in conjunction with the examples. It should be understood that the following examples are only used to explain and illustrate the present invention and do not limit the scope of the present invention in any way.

Experimental Materials:

The plant material used in the following experiments was diploid oat (Avena strigosa, S75), a known species, which was recorded in the non-patent document Papadopoulou et al. 1999. The above biological materials are also preserved in this laboratory, and the applicant declares that they can be released to the public for verification experiments within twenty years from the date of application.

Escherichia coli DH5α and Rosetta (DE3) were purchased from Shanghai Weidi Biotechnology Co., Ltd. The cloning vector pClone007 Blunt Simple Vector was purchased from Beijing Qingke Xinye Biotechnology Co., Ltd. The prokaryotic expression vector pGEX-6P-1 is preserved in the laboratory and is also commercially available.

PCR primers:

Main reagents:

Diosgenin: CAS number: 512-04-9, molecular formula: C ₂₇ H ₄₂ O ₃ , English name diosgenin, purchased from Chengdu Prefa Technology Development Co., Ltd., product number BP0504.

Pennogenin: CAS number: 507-89-1, molecular formula: C ₂₇ H ₄₂ O ₄ , English name pennogenin. The metanogenin used in the experiment was obtained by enzymatic separation and purification of Chonglou saponin VI. For the enzymatic separation and purification method, please refer to Li, W., Wang, Z., Gu, J., Chen, L., Hou, W., The method described in Jin, YP, & Wang, YP (2015). Bioconversion of ginsenosideRd to ginsenoside M1 by snailase hydrolysis and its enhancement effect oninsulin secretion in vitro. Die Pharmazie, 70:340–346. Metanogenin is also commercially available.

Nuatigenin: CAS number: 6811-35-4, molecular formula: C ₂₇ H ₄₂ O ₄ , English name nuatigenin. The neoartigenin used in the experiment was obtained by enzymatic separation and purification of avenacosides extract. For the extraction method of avenacosides, please refer to Yang, JL, Wang, P., Wu, WB, Zhao, YT, Idehen. ,E.,&Sang,SM(2016).Steroidal saponins in oat bran.Journal of Agricultural&FoodChemistry, 64(7):1549-1556., For enzymatic separation and purification methods, please refer to Li,W.,Wang,Z.,Gu,J .,Chen,L.,Hou,W.,Jin,YP,&Wang,YP(2015).Bioconversion of ginsenoside Rd toginsenoside M1 by snailase hydrolysis and its enhancement effect on insulin secretion in vitro.Die Pharmazie,70:340–346. .

UDP-Glucose (UDP-Glc): CAS number: 28053-08-9, molecular formula: C ₁₅ H ₂₂ N ₂ Na ₂ O ₁₇ P ₂ , purchased from Beijing Coolab Technology Co., Ltd., product number CU11611-500mg.

UDP-Rhamnose (UDP-Rha): CAS number: 1526988-33-9, molecular formula: C ₁₅ H ₂₂ N ₂ Na ₂ O ₁₆ P ₂ , English name UDP 5'-diphospho-aL-rhamnose, purchased from Suzhou Hanzyme Biotechnology Co., Ltd.

Trillium: CAS number: 14144-06-0, molecular formula: C ₃₃ H ₅₂ O ₈ , purchased from Chengdu Prefa Technology Development Co., Ltd., product number BP1124.

Chonglou saponin V: CAS number: 19057-67-1, molecular formula: C ₃₉ H ₆₂ O ₁₂ , purchased from Chengdu Prefa Technology Development Co., Ltd., product number BP1151.

Chonglou saponin VI: CAS number: 55916-51-3, molecular formula: C ₃₉ H ₆₂ O ₁₃ , purchased from Chengdu Prefa Technology Development Co., Ltd., product number BP1131.

Chromatographic methanol: purchased from Merck, USA, product number 1.06007.4008.

Unless otherwise specified, the reagents used in the following examples are all conventional reagents in the field, and can be purchased commercially or prepared according to conventional methods in the field; the experimental methods and conditions used are all conventional experimental methods and conditions in the field. Please refer to relevant experimental manuals, publicly known literature or manufacturer's instructions. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Example 1. Discovery, cloning and expression of glycosyltransferase genes AsUGT73E5 and AsUGT73E1

1. Gene discovery

Using oat as a model material, we discovered two glycosyltransferase genes and named them AsUGT73E5 and AsUGT73E1. The open reading frame (ORF) of the AsUGT73E1 gene contains 1473 bases, its nucleotide sequence is shown in SEQ ID NO: 2, and it encodes a glycosyltransferase whose amino acid sequence is shown in SEQ ID NO: 1. The open reading frame (ORF) of the AsUGT73E5 gene contains 1548 bases, its nucleotide sequence is shown in SEQ ID NO: 4, and it encodes a glycosyltransferase whose amino acid sequence is shown in SEQ ID NO: 3. Through transcriptome and target metabolite correlation analysis (Pearson correlation), we found that the expression of these two glycosyltransferases in 10 oat tissues is highly coordinated with cholesterol synthesis, and therefore is likely to be involved in the synthesis of steroidal saponins. In order to verify the functions of these two glycosyltransferases in the synthesis of steroidal saponins, we performed gene cloning and expression.

2. Gene cloning

2.1 Extraction of total RNA from oat seedlings

RNA was extracted using the RNAprep Pure Plant Kit (Cat. No.: DP441) from Tiangen Biotechnology Company. Follow the instructions for use with the kit. The steps are as follows:

(1) 50-100mg oat leaves are quickly ground into powder in liquid nitrogen, add 450μL RL (add β-mercaptoethanol before use), vortex vigorously and mix;

(2) Transfer the solution to the filter column CS, centrifuge at 12,000 rpm for 2-5 minutes, and transfer the supernatant in the collection tube to an RNase-free centrifuge tube;

(3) Add 0.5 times the volume of supernatant absolute ethanol and mix evenly. Transfer the resulting solution and precipitate to the adsorption column CR3, centrifuge at 12,000 rpm for 30-60 seconds, discard the waste liquid, and put the adsorption column CR3 back into the collection tube;

(4) Add 350 μL of protein-removing solution RW1 to the adsorption column CR3, centrifuge at 12,000 rpm for 30-60 seconds, discard the waste liquid, and put the adsorption column CR3 back into the collection tube;

(5) Preparation of DNase I working solution: Put 10 μL of DNase I storage solution into a new RNase-Free centrifuge tube, add 70 μL of RDD buffer, and mix gently;

(6) Add 80 μL of DNase I working solution to the center of the adsorption column CR3 and leave it at room temperature for 15 minutes;

(7) Add 350 μL of protein-removing solution RW1 to the adsorption column CR3, centrifuge at 12,000 rpm for 30-60 seconds, discard the waste liquid, and put the adsorption column CR3 back into the collection tube;

(8) Add 500 μL of rinse solution RW to the adsorption column CR3 (add ethanol before use), let it stand at room temperature for 2 minutes, centrifuge at 12,000 rpm for 30-60 seconds, pour out the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube. ,repeat;

(9) Centrifuge at 12,000 rpm for 2 minutes, discard the waste liquid, place the adsorption column CR3 at room temperature for a few minutes, and dry the remaining rinse liquid thoroughly;

(10) Place the adsorption column CR3 into a new RNase-Free centrifuge tube, drop 30-100 μL RNase-Free ddH ₂ O into the middle of the adsorption membrane, leave it at room temperature for 2 minutes, and centrifuge at 12,000 rpm for 2 minutes to obtain an RNA solution. .

2.2 Synthesis of cDNA

Apply SuperScript III Rreverse Transcriptase kit (Invitrogen, Cat. No.: 18080085) and operate according to the kit instructions:

(1) RNA template denaturation

Heat at 65°C for 5 minutes, quickly cool on ice, and let stand on ice for 2 minutes.

(2) First-strand cDNA synthesis

Centrifuge briefly to mix. React at 55°C for 60 minutes, heat at 70°C for 15 minutes to terminate the reaction, and store the product at -20°C.

2.3 Target gene amplification

Design the glycosyltransferase gene AsUGT73E5 and AsUGT73E1 coding region primers AsUGT73E5-ORF-F/R and AsUGT73E1-ORF-F/R. PCR amplification was performed using 5-fold diluted oat seedling cDNA as a template, using primers AsUGT73E5-ORF-F/R, AsUGT73E1-ORF-F/R and 2×Phanta Max Master Mix high-fidelity enzyme (vazyme, Cat. No.: P515 -02) Perform PCR amplification of the target gene. The reaction system is as follows:

Reaction conditions: Pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 30 seconds, annealing for 30 seconds according to the primer Tm value (AsUGT73E5-ORF-F/R: 60°C, AsUGT73E1-ORF-F/R: 60°C), and extension at 72°C for 1 minute. 33 cycles; complete extension at 72°C for 7 minutes. After the reaction, the PCR products were detected by 1% agarose gel electrophoresis.

The results are shown in Figure 1, 1 is AsUGT73E5, 2 is AsUGT73E1, and the gene band sizes are both about 1500bp.

2.4DNA gel recovery

Use the Gel Extraction Kit (Omega, Cat. No.: D2500-02) to recover the target gene fragment, and follow the kit instructions, as follows:

(1) Cut the agarose gel containing the target band in a UV gel cutter, take an equal volume of binding buffer/Binding Buffer, and incubate the mixture at 55°C for 7 minutes until the gel is completely melted.

(2) Take 700 μL of the mixed solution, transfer it to a DNA adsorption column with a 2 mL collection tube, let it stand for 1 min, centrifuge at 10,000 g for 1 min, and discard the filtrate.

(3) Place the adsorption column back into the collection tube, add 700 μL SPW Wash Buffer diluted with absolute ethanol, centrifuge at 10,000g for 1 min, and discard the filtrate. repeat.

(4) Discard the filtrate, place the empty adsorption column back into the centrifuge tube, and centrifuge at 12,000g for 2 minutes.

(5) Place the empty adsorption column in a sterilized 1.5 mL centrifuge tube, open the tube lid and let it sit for 1 min. Add 30 μL of sterile water (preheated at 60°C) in the center of the adsorption membrane and let it stand at room temperature for 1 min. Centrifuge at 12,000g for 1 minute to elute DNA.

2.5 Cloning vector connection

Clone the target gene into pClone007 Blunt Simple Vector (Beijing Qingke Biotech, Cat. No.: TSV-007BS). The reaction system is as follows:

React at room temperature for 5 minutes.

2.6 Transformation of E. coli

(1) Take 100 μL of competent cells DH5α (Shanghai VideBio) melted in ice bath, add the target DNA, mix gently and then place in ice bath for 30 minutes;

(2) Heat shock in a 42°C water bath for 60 seconds, then quickly transfer the centrifuge tube to an ice bath for 2 minutes;

(3) Add 200 μL of non-resistant sterile LB culture solution to the centrifuge tube, mix well and incubate in a 37°C shaker at 180 rpm for 1 hour to resuscitate the bacteria;

(4) Add the competent cells transformed in the previous step to the LB agar medium containing ampicillin (Amp, screening concentration 100 mg/L) resistance, spread the cells evenly, blow dry the liquid on the surface of the medium, and place the plate upside down Incubate overnight at 37°C;

(5) Pick a number of single colonies, add them to 500 μL LB liquid culture medium containing Amp resistance (100 mg/L), and culture them at 37°C and 180 rpm for 4 hours. The bacterial liquid is identified by PCR. The identification primer is M13-F/R. Positive clones are sent Ruibo Xingke Biotechnology Co., Ltd. conducted sequencing and obtained the cloning vectors pClone007-AsUGT73E5 and pClone007-AsUGT73E1 with correct sequences.

2.7 Plasmid extraction

After the correctly sequenced samples are preserved, use the E.Z.N.A. Plasmid Mini Kit I (omega, Cat. No.: D6942-02) to extract the plasmid according to the instruction manual. The steps are as follows:

(1) Take 5mL of bacterial liquid cultured overnight at 37°C (12-16h), centrifuge at 10,000g for 1 minute, and discard the supernatant;

(2) Add 250 μL Solution I (RNase A has been added) to the centrifuge tube and mix by pipetting;

(3) Add 250 μL Solution II, mix by inverting 4-6 times, and let stand for 2 minutes to fully lyse the bacteria (the total time is less than 5 minutes);

(4) Add 350 μL Solution III and immediately turn it upside down 6-8 times to mix the solution thoroughly. At this time, a large amount of white flocculent precipitate will appear. Centrifuge at 13,000g for 10 minutes;

(5) Place the adsorption column in the collection tube, add the centrifuged supernatant to the adsorption column, centrifuge at 10,000g for 1 minute, and discard the filtrate;

(6) Add 700 μL of DNA Wash Buffer to the adsorption column, centrifuge at 10,000 g for 1 min, and discard the filtrate. repeat;

(7) Put the empty adsorption column back into the collection tube, centrifuge at 13,000g for 2 minutes, transfer the adsorption column to a new 1.5mL centrifuge tube, open the tube cover and dry the adsorption column for 1 minute, and evaporate the remaining rinse liquid in the adsorption column;

(8) Add 50 μL of sterile water preheated to 55°C to the center of the membrane of the adsorption column, let stand for 2 minutes, and centrifuge at 13,000g for 1 minute. Discard the adsorption column and store the plasmid at -20°C until use.

3. Protein expression

3.1 Construction of prokaryotic expression vector

Using the pClone007 vector (pClone007-AsUGT73E5 and pClone007-AsUGT73E1) containing the open reading frames of the target genes AsUGT73E5 and AsUGT73E1 as the template, design the recombination primers AsUGT73E5-pGEXF/R and AsUGT73E1-pGEXF/R for PCR amplification. The reaction system and reaction conditions are the same. 2.3 above, obtain the gene fragment used to construct the expression vector.

pGEX-6p-1 was used as the prokaryotic expression vector. The pGEX-6p-1 vector was linearized with EcoRⅠ (Thermo, Cat. No.: FD0274) and SalⅠ (Thermo, Cat. No.: FD0644) fast endonucleases. The system is as follows:

The reaction was terminated after 1 hour at 37°C.

The PCR amplification product and linearized pGEX-6p-1 vector were detected by agarose gel electrophoresis and recovered. Use ClonExpress II One Step Cloning Kit (vazyme, Cat. No.: C112-02) according to the instructions to perform homologous recombination on the recovered gene fragment and linearized pGEX-6p-1, and clone the ORFs of genes AsUGT73E5 and AsUGT73E1 into prokaryotic expression respectively. In the vector pGEX-6p-1, the reaction system is as follows:

React at 37°C for 30 minutes, then lower to 4°C or cool on ice. The reaction product obtained is transformed into E. coli DH5α. The transformation method is the same as 2.6 above. After overnight culture, single colonies were picked, added to 500 μL LB liquid medium containing Amp resistance (100 mg/L), and cultured at 37°C 180 rpm for 4 hours. The bacterial liquid was identified by PCR. The identification primer was pGEX-F/R. Positive clones were sent Sequencing was performed by Ruibo Xingke Biotechnology Co., Ltd. For samples with correct sequencing results, the plasmid pGEX-AsUGT73E5/pGEX-AsUGT73E1 is extracted after culture preservation. The plasmid extraction method is the same as 2.7 above. Use the extracted plasmid to transform Escherichia coli Rossetta (DE3) expression competent form (Shanghai Vetech), and set up pGEX-6p-1 empty vector transformation as a control. The transformation method is the same as 2.6 above.

3.2 Protein induced expression

The Rossetta (DE3) bacterial solution containing pGEX-AsUGT73E5/pGEX-AsUGT73E1/pGEX-6p-1 empty vector was inoculated into 1L LB liquid medium containing Amp resistance (100mg/L) at a volume ratio of 1:100. , culture at 37°C with a shaker at 200 rpm until OD ₆₀₀ = 0.6, add 0.2mM IPTG, and induce overnight with a shaker at 16°C at 160 rpm. Collect the bacterial cells by centrifugation at 4°C and 4,000 rpm, add 10 mL of pre-cooled PBS solution (concentration 0.01M, pH 7.4, formula: NaCl 8.0g, KCl 0.2g, Na ₂ HPO ₄ 1.44g, KH ₂ PO ₄ 0.24g, add Distilled water to 1L), resuspend and sonicate on ice until the solution becomes translucent. Centrifuge at 12,000 rpm for 15 min at 4°C, collect the supernatant and precipitate, and detect by SDS-PAGE electrophoresis.

3.3 Protein purification

Prepare the equilibrium/wash solution (the formula of the balance solution and the wash solution are the same) and eluent, and add 1mMDTT before use. Equilibration/wash solution (1L): 140mM NaCl, 2.7mM KCl, 10mM Na ₂ HPO ₄ , 1.8mM KH ₂ PO ₄ , pH 7.4. Eluent (1L): 50mM Tris-HCl, 10mM reduced glutathione, pH 8.0.

(1) Load Glutathione Beads (Changzhou Tiandi Renhe Biology, Cat. No.: SA008010) into a suitable chromatography column, balance it with 5 times the column volume of equilibrium solution, so that the filler is in the same buffer system as the target protein. The role of protective proteins;

(2) Add the sample to the balanced Glutathione Beads to ensure full contact between the target protein and GlutathioneBeads, improve the recovery rate of the target protein, and collect the effluent;

(3) Wash with 10 times the column volume of impurity solution to remove non-specifically adsorbed impurity proteins and collect the impurity solution;

(4) Use 5 times the column volume of eluent to collect the eluate, which is the target protein component;

(5) Use 3 times the column volume of equilibrium solution and 5 times the column volume of deionized water to balance the packing;

(6) Add the purified protein solution to a millipore 15mL ultrafiltration tube (10KD), centrifuge at 4°C, 4,000rpm to concentrate the sample to 500μL, and add 15mL PBS phosphate buffer (concentration 0.01M, formula: NaCl 8.0g, KCl 0.2g , Na ₂ HPO ₄ 1.44g, KH ₂ PO ₄ 0.24g, adjust pH to 7.4, add distilled water to 1L), and continue to concentrate to 500 μL. repeat;

(7) Aspirate the purified protein, dilute it and add glycerol to a final concentration of 10%, and store it at -80°C.

Purified protein was detected by SDS-PAGE. The results are shown in Figure 2. 1 is the whole cell protein without induction; 2 is the whole cell protein after IPTG induction; 3 is the cell supernatant after IPTG induction; 4 is the cell pellet after IPTG induction; 5 is the supernatant. Protein purified by GST column; 6 is the purified protein after ultrafiltration and concentration. Compared with the uninduced control sample, the recombinant protein showed obvious bands near 80KDa. AsUGT73E5 and AsUGT73E1 proteins contain 515 and 490 amino acids respectively, and their molecular weights after fusion with the GST tag are 82.21KDa and 79.79KDa respectively.

Example 2. Functional identification of glycosyltransferases AsUGT73E5 and AsUGT73E1

1. Enzyme activity detection

(1) Glycosylation reaction catalyzed by AsUGT73E5

Accurately weigh 1mM steroidal sapogenin (diosgenin/patenogenin/neaartinogenin), 1mM UDP-Glucose (UDP-Glc), 50μL purified glycosyltransferase AsUGT73E5, and dissolve in PBS phosphate buffer solution (concentration 0.01M, formula: NaCl 8.0g, KCl 0.2g, Na ₂ HPO ₄ 1.44g, KH ₂ PO ₄ 0.24g, adjust pH 8.0, add distilled water to 1L), so that the final volume reaches 300 μL. After reacting at 37°C for 2 hours, an equal volume of methanol was added to terminate the enzyme activity. The product was spin-dried under reduced pressure and then dissolved in 500 μL chromatographic methanol for testing.

(2) Glycosylation reaction catalyzed by AsUGT73E1

Repeat the glycosylation reaction in (1), concentrate and dry the product and use it as a substrate. Add 1mM UDP-Rhamnose (UDP-Rha), 50μL of purified glycosyltransferase AsUGT73E1, and dissolve it in PBS phosphate buffer ( pH 8.0), bringing the final volume to 300 μL. After reacting at 37°C for 2 hours, an equal volume of methanol was added to terminate the enzyme activity. The product was spin-dried under reduced pressure and then dissolved in 500 μL chromatographic methanol for testing.

2. Identification of enzyme products by HPLC and LC-Q-TOF

(1) Liquid chromatography

This experiment used a Thermo UltiMate 3000 liquid chromatograph and a Thermo Hypersil GOLD C ₁₈ liquid chromatography column (250mm×4.6mm, 5μm) for HPLC detection. The mobile phases are water (A) and acetonitrile (B). Elution gradient: 0~6min, 20%~30%B; 6~15min, 30%~60%B; 15~21min, 60%~100%B; 21~30min, 100%B; 30~35min, 100 %～20%B. The flow rate is 1mL/min, the column temperature is 30°C, the injection volume is 10μL, and the detection wavelength is 210nm.

(2) Mass spectrometry detection

This experiment used the AB SCIEX TripleTOF 6600 ultra-high resolution mass spectrometer for detection. Positive ion data acquisition mode, the conditions are: capillary voltage 3.6kV, cone voltage 35kV, ion source temperature 105℃, desolvation gas temperature 340℃, reverse cone gas flow 55L/h, desolvation gas 650L/h, extraction cone hole 4V. Mass-to-charge ratio data scanning range: 50-1500m/z.

results and analysis

The product of the reaction between AsUGT73E5 protein, diosgenin, and UDP-Glc was dried with a vacuum concentrator, dissolved in 500 μL of chromatographic methanol, and filtered with a 0.22 μm filter for HPLC detection. As shown in Figure 3, compared with the unloaded reaction, the product of AsUGT73E5 has a new peak (product 1) at 22.3 minutes, which is the same peak time as the trillin standard. The sample was recovered, AsUGT73E1 protein and UDP-Rha were added to continue the reaction. A new product peak (product 2) appeared at 20.7 minutes, and the time was consistent with the Chonglousaponin V standard. The TOF positive ion scanning mode detection is shown in Figure 4. The molecular weights of product 1 and product 2 are 577.38 (M+H ⁺ ) and 723.43 (M+H ⁺ ) respectively, which are similar to trillium (576.3) and chrysanthemum saponin V ( 722.4), indicating that diosgenin was continuously catalyzed by AsUGT73E5 and AsUGT73E1 to generate diosgenin V.

After HPLC detection of the reaction product of AsUGT73E5 protein with metanogenin and UDP-Glc (Figure 5), compared with the unloaded reaction, the product of AsUGT73E5 showed a new peak (product 3) at 18.8 minutes. The sample was recovered, AsUGT73E1 protein and UDP-Rha were added to continue the reaction. A new product peak (product 4) appeared at 17.1 minutes, and the time was consistent with the Chonglousaponin VI standard. TOF positive ion scanning mode detection is shown in Figure 6. The molecular weights of product 3 and product 4 are 593.37 (M+H ⁺ ) and 739.43 (M+H ⁺ ) respectively, which is similar to that of metanosogenin-3-O-glucoside (592.3 ) is consistent with the molecular weight of chrysanthemum saponin VI (738.4), indicating that metanosaponin generates chrysanthemum saponin VI after continuous catalysis by AsUGT73E5 and AsUGT73E1. The process of glycosylation of steroidal sapogenins catalyzed by AsUGT73E5 and AsUGT73E1 proteins is shown in Figure 7.

The product of the reaction between AsUGT73E5 protein, neuaartinin, and UDP-Glc was dried with a vacuum concentrator, dissolved in 500 μL of chromatographic methanol, filtered with a 0.22 μm filter, and then subjected to HPLC detection. As shown in Figure 8, compared with the unloaded reaction, the product of AsUGT73E5 showed a new peak (product 5) at 16.7 minutes. The sample was recovered, AsUGT73E1 protein and UDP-Rha were added to continue the reaction, and the second product peak (product 6) appeared at 15.6 minutes. The TOF positive ion scanning mode detection is shown in Figure 9. The molecular weights of product 5 and product 6 are 593.37 (M+H ⁺ ) and 739.43 (M+H ⁺ ) respectively, indicating that neuaartinogen has been continuously catalyzed by AsUGT73E5 and AsUGT73E1. The corresponding glycosylation products are generated. references:

Cárdenas, P.D., Sonawane, P.D., Heinig, U., Bocobza, S.E., Burdman, S., & Aharoni, A. (2015). The bitter side of the nightshades: Genomics drives discovery in Solanaceae steroidal alkaloid metabolism. Phytochemistry, 113, 24 -32.

Christ, B., Xu, C.C., Xu, M.L., Li, F.S., Wada, N., Mitchell, A.J.,… & Weng, J.K. (2019). Repeated evolution of cytochrome P450-mediated spiroketal steroidbiosynthesis in plants. Nature Communications, 10 ,3206.

Deng,D.,Lauren,D.R.,Cooney,J.M.,Jensen,D.J.,Wurms,K.V.,Upritchard,J.E.,…&Li,M.Z. (2008).Antifungal saponins from Paris polyphylla Smith.PlantaMedica,74(11),1397-1402 .

Gong,G.H.,Qin,Y.,Huang,W.,Zhou,S.,Wu,X.H.,Yang,X.H,…&Li.D.(2010).Protective effects of diosgenin in the hyperlipidemic rat model and in humanvascular endothelial cells against hydrogen peroxide-induced poptosis. Chemico-Biological Interactions, 184(3), 366-375. Journal of agricultural and food chemistry, 64(7), 1549-1556.

He,J.L.,Yu,S.,Guo,C.J.,Tan,L.,Song,X.M.,Wang,M.,…&Peng,C.(2019).Polyphyllin I induces autophagy and cell cycle arrest via inhibiting PDK1/Akt/ mTOR signal and downregulating cyclin B1 in human gastric carcinoma HGC-27 cells. Biomedicine&Pharmacotherapy,117,109189.

Louveau,T.,Orme,A.,Pfalzgraf,H.,Stephenson,M.J.,Melton,R.,Saalbach,G.,...&Langdon,T. (2018).Analysis of two new arabinosyltransferases belonging to the carbohydrate-active enzyme(CAZY)glycosyl transferase family1 provides insights into disease resistance and sugar donor specificity. The Plant Cell, 30(12), 3038-3057.

Orme,A.,Louveau,T.,Stephenson,M.J.,Appelhagen,I.,Melton,R.,Cheema,J.,…&Osbourn, A.(2019).A noncanonical vacuolar sugar transferase required for biosynthesis of antimicrobial defense compounds in oat.Proceedings of the National Academy of Sciences, 201914652.

Papadopoulou,K.,Melton,R.E.,Leggett,M.,Daniels,M.J.,&Osbourn,A.E.(1999). Compromised disease resistance in saponin-deficientplants.Proceedings of the National Academy of Sciences,96(22),12923-12928.

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Teng,J.F.,Qin,D.L.,Mei,Q.B.,Qiu,W.Q.,Pan,R.,Xiong,R.,…&Wu.,A.G.(2019). Polyphyllin VI, a saponin from Trillium tschonoskii Maxim.inducesapoptotic and autophagic cell death via the ROS triggered mTOR signaling pathway in non-small cell lung cancer. Pharmacological Research,147,104396.

Vincken,J.P.,Heng,L.,de Groot,A.,&Gruppen,H.(2007).Saponins,classification and occurrence in the plant kingdom.Phytochemistry,68(3),275-297.

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Xue,Z.Y.,Tan,Z.W.,Huang,A.C.,Zhou,Y.,Sun,J.C.,Wang,X.N.,…&Qi,X.Q.(2018). Identification of key amino acid residues determining productspecificity of 2,3-oxidosqualene cyclase in Oryza species.New Phytologist,218(3):1076-1088.

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sequence list

<110> Northeast Forestry University

<120> Oat glycosyltransferase AsUGT73E1 and its application in the synthesis of steroidal saponins

<130> P200850-DBL

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 490

<212> PRT

<213> Oats (Avena sativa L.)

<400> 1

Met Val Ala Ser Arg Val Lys Lys Leu Arg Val Leu Leu Ile Pro Phe

1 5 10 15

Phe Ala Thr Ser His Ile Glu Pro Tyr Thr Glu Leu Ala Ile Arg Leu

20 25 30

Ala Gly Ala Lys Pro Asp Tyr Ala Val Glu Pro Thr Ile Ala Val Thr

35 40 45

Pro Ala Asn Val Pro Ile Val Gln Ser Leu Leu Glu Arg Arg Gly Gln

50 55 60

Gln Gly Arg Ile Lys Ile Ala Thr Tyr Pro Phe Pro Ala Val Glu Gly

65 70 75 80

Leu Pro Ala Gly Val Glu Asn Leu Gly Lys Val Ala Ala Ala Asp Ala

85 90 95

Trp Arg Ile Asp Ala Ala Ala Ile Ser Asp Thr Leu Met Arg Pro Ala

100 105 110

Gln Glu Ala Leu Val Arg Ala Gln Ser Pro Asp Ala Met Val Ala Asp

115 120 125

Pro His Phe Ser Trp Gln Ala Gly Ile Ala Ala Asp Leu Gly Val Pro

130 135 140

Leu Val Ser Phe Ser Val Val Gly Ala Phe Ser Gly Leu Val Met Gly

145 150 155 160

Lys Leu Met Ala Tyr Gly Ala Val Glu Asp Gly Glu Asp Ala Val Thr

165 170 175

Ile Pro Gln Phe Pro Leu Pro Glu Ile Arg Ile Pro Val Thr Glu Leu

180 185 190

Pro Glu Phe Leu Arg Thr His Leu Leu Glu Arg Asp Gly Lys Asp Val

195 200 205

Asp Ser Ile Gly Lys Val Ser Val Gly Gln Asn Phe Gly Leu Ala Ile

210 215 220

Asn Thr Ala Ser His Leu Glu Gln Gln Tyr Cys Glu Met His Thr Ser

225 230 235 240

Gly Gly Gln Ile Lys Arg Ala Tyr Phe Val Gly Pro Leu Ser Leu Gly

245 250 255

Ala Glu Ala Val Ala Pro Gly Gly Gly Gly Gly Glu Thr Gln Ala Pro

260 265 270

Pro Cys Ile Arg Trp Leu Asp Ser Lys Pro Asp Arg Ser Val Val Tyr

275 280 285

Leu Cys Phe Gly Ser Leu Thr His Val Ser Asp Ala Gln Leu Asp Glu

290 295 300

Leu Ala Leu Gly Leu Glu Ala Ser Gly Lys Ala Phe Leu Trp Val Val

305 310 315 320

Arg Ala Ala Glu Ala Trp Arg Pro Pro Ala Gly Trp Ala Glu Arg Val

325 330 335

Gln Asp Arg Gly Met Leu Leu Thr Ala Trp Ala Pro Gln Thr Ala Ile

340 345 350

Leu Gly His Arg Ala Val Gly Ala Phe Val Thr His Cys Gly Trp Asn

355 360 365

Ser Val Leu Glu Ala Val Ala Ala Gly Leu Pro Val Leu Thr Trp Pro

370 375 380

Met Val Phe Glu Gln Phe Ile Thr Glu Arg Leu Val Thr Glu Val Met

385 390 395 400

Gly Ile Gly Glu Arg Phe Trp Pro Glu Gly Ala Gly Arg Arg Ser Thr

405 410 415

Arg Tyr Glu Glu His Gly Leu Val Pro Ala Glu Asp Val Ala Arg Ala

420 425 430

Val Thr Thr Phe Met Cys Pro Gly Gly Ala Gly Asp Ala Lys Arg Gln

435 440 445

Arg Ala Met Glu Leu Ala Ala Glu Ser Arg Ala Ala Met Ala Glu Gly

450 455 460

Gly Ser Ser His Arg Asp Leu Cys Arg Leu Val Asp Asp Leu Val Ala

465 470 475 480

Ala Lys Leu Glu Arg Glu Gln Val Pro Ser

485 490

<210> 2

<211> 1473

<212> DNA

<213> Oats (Avena sativa L.)

<400> 2

atggttgcca gccgtgtgaa gaagctgcgt gtcctgctca ttcccttctt cgcgacaagc 60

cacatcgagc cctacaccga gctcgccatc cgcctcgccg gcgccaagcc ggactacgcc 120

gtggagccaa caattgcggt gacgccggcg aacgtcccaa tcgtccagtc cttgctggag 180

cgacgcggac agcaggggcg catcaagatc gcgacgtacc cgttcccggc cgtggagggc 240

ctcccggcgg gcgtggagaa cctgggcaag gtcgcggcgg ccgacgcctg gcgcatcgac 300

gcggccgcca tcagcgacac cctgatgcgg cccgcgcagg aggcgctggt gagggcgcag 360

tcccccgacg ccatggtcgc cgacccgcac ttctcctggc aggccggcat cgccgccgat 420

ctgggcgtgc cgctggtgtc gttcagcgtg gtgggcgcct tctcggggct cgtcatgggc 480

aaactcatgg cctacggcgc cgtcgaggac ggcgaagacg ccgttacgat ccctcagttt 540

ccccttccgg agatacggat accggtgacc gagctgccgg agttcctgag gacccacctg 600

ctcgagcgtg acgggaagga cgtcgatagc atcggcaaag tttcggtggg acagaatttc 660

ggcctcgcca tcaacacggc gtcgcacctg gagcagcagt actgcgagat gcacaccagc 720

ggcggccaaa tcaagcgagc ctacttcgtg gggcccctct cgctgggagc cgaagcagtt 780

gcccccggcg gcggcggcgg cgagacacag gcgccgccgt gcatccgttg gctggactcg 840

aagccggacc ggtcggtggt gtacctgtgc ttcgggagcc tgacccacgt ctcggacgcg 900

cagctggacg agctggctct cgggctggag gcgtccggga aggcgttcct gtgggtggtg 960

agggcggcgg aggcgtggcg gccgccggcg gggtgggcgg agcgcgtgca ggacaggggg 1020

atgctcctga ccgcctgggc cccgcagacc gccatcctgg gccaccgcgc cgtgggcgcc 1080

ttcgtgacgc actgcgggtg gaactcggtg ctggaggcgg tggcggcggg gctgccggtg 1140

ctgacgtggc cgatggtgtt cgagcagttc atcacggaga ggctggtgac ggaggtgatg 1200

gggatcgggg agcggttctg gccggagggc gccggacggc ggagcaccag gtacgaagag 1260

cacgggctgg tcccggcgga ggacgtggcg cgggcggtga caacgttcat gtgccccgga 1320

ggagcagggg acgccaagag gcagagggcg atggagctcg ccgccgagtc tcgtgcggcc 1380

atggcggaag gaggctcgtc gcaccgtgat ctgtgccgcc tcgttgacga tctcgtcgca 1440

gctaagctag agagagagca ggtgcctagc tag 1473

<210> 3

<211> 515

<212> PRT

<213> Oats (Avena sativa L.)

<400> 3

Met Ala Asp Leu His Phe Leu Val Val Pro Leu Ala Ala Gln Gly His

1 5 10 15

Ile Ile Pro Met Val Asp Val Ala Arg Leu Leu Ala Ala Arg Gly Ser

20 25 30

Arg Val Thr Val Val Thr Thr Pro Val Asn Ala Ala Arg Asn Arg Ala

35 40 45

Ala Val Asp Gly Ala Arg Lys Ala Gly Leu Ala Val Glu Leu Leu Glu

50 55 60

Leu Pro Phe Pro Ser Ala Gln Leu Gly Leu Pro Glu Gly Leu Glu Ala

65 70 75 80

Val Asp Gln Leu Asn Gly Gln Pro Pro Glu Ile Ser Ile Gly Leu Phe

85 90 95

Lys Ala Ile Trp Thr Leu Ala Gly Pro Leu Glu Glu Tyr Leu Arg Ala

100 105 110

Leu Pro Arg Leu Pro Asp Cys Leu Val Ala Asp Leu Cys Asn Pro Trp

115 120 125

Thr Ala Pro Val Cys Glu Arg Leu Gly Ile Pro Arg Leu Val Met His

130 135 140

Cys Pro Ser Ala Tyr Phe Gln Leu Ala Val His Arg Leu Asn Glu His

145 150 155 160

Gly Val Tyr Gly Gly Gly Val Glu Asp Tyr Asp Pro Thr Pro Ile Glu

165 170 175

Val Pro Gly Phe Pro Val Arg Ala Phe Gly Ser Lys Thr Thr Met Arg

180 185 190

Gly Phe Phe Gln Tyr Pro Gly Val Glu Gln Glu His Leu Glu Ala Leu

195 200 205

His Ala Glu Ala Thr Ala Asp Gly Leu Leu Phe Asn Ser Phe Arg Ala

210 215 220

Ile Glu Ala Asp Phe Leu Asp Ala Tyr Ala Ala Ala Leu Gly Lys Thr

225 230 235 240

Thr Trp Ala Val Gly Pro Thr Ala Leu Val Asn Asp Thr Thr Thr Thr

245 250 255

Thr Ala Ser Ser Arg Ser Ser Thr Ile Val Ser Trp Leu Asp Ala Arg

260 265 270

Pro Pro Asp Ser Val Leu Tyr Val Ser Phe Gly Ser Ile Ser Leu Leu

275 280 285

Ser Ala Lys Gln Leu Ala Lys Leu Ala Asp Gly Leu Glu Ala Ser Gly

290 295 300

Arg Pro Phe Val Trp Ala Ile Lys Glu Asp Lys Ala Asp Ala Ala Val

305 310 315 320

Arg Ser Gln Leu Asp Glu Glu Gly Gly Phe Glu Ala Arg Val Lys Asp

325 330 335

Arg Gly Leu Leu Val Arg Gly Trp Ala Pro Gln Val Ala Ile Leu Ser

340 345 350

His Pro Ala Val Gly Gly Phe Leu Thr His Cys Gly Trp Asn Ser Thr

355 360 365

Leu Glu Ala Leu Ser His Gly Val Pro Ala Leu Thr Trp Pro Thr Asn

370 375 380

Ala Asp Gln Phe Cys Ser Glu Gln Val Ile Val Asp Val Leu Asp Val

385 390 395 400

Gly Val Arg Ser Gly Val Lys Ile Pro Ala Leu Tyr Val Pro Pro Glu

405 410 415

Ala Glu Gly Val Gln Val Glu Ser Gly Asp Val Glu Arg Ala Ile Val

420 425 430

Glu Leu Met Asp Gly Gly Pro Glu Gly Ala Ala Arg Arg Ala Arg Ala

435 440 445

Arg Lys Ile Ala Val Glu Ala Lys Ala Ala Met Glu Glu Gly Gly Thr

450 455 460

Ser His Ser Asp Leu Thr Asp Met Ile Arg His Val Ser Glu Leu Ser

465 470 475 480

Arg Lys Lys Arg Leu Gln Leu Glu Thr Ala Asp Ala Thr Cys Glu Glu

485 490 495

Ala Thr Arg Ala Ala Asp Asn Ala Ala Ala Val Leu Pro Leu Leu Ser

500 505 510

Gln Ala Asn

515

<210> 4

<211> 1548

<212> DNA

<213> Oats (Avena sativa L.)

<400> 4

atggcggatc tacacttcct ggtcgtgccg ctggcggcgc agggccacat catccccatg 60

gtggacgtgg cgcgcctcct cgccgcgcgt ggctcgcggg tcaccgtcgt caccacgccc 120

gtcaacgccg cgcgcaaccg ggccgccgtg gacggcgcca ggaaggcggg cctcgccgtc 180

gagctcctgg agctcccgtt ccccagcgcg cagctcggcc tgcctgaggg cctggaggcc 240

gtcgaccagc tgaacgggca gccacctgaa atctccatcg gcctcttcaa ggccatctgg 300

accctggccg gaccgctgga ggagtacctc cgcgcgctgc cgcgcctgcc ggactgcctc 360

gtcgccgact tgtgcaaccc ttggacggcg ccggtctgcg agcgcctcgg catcccgagg 420

ctggtgatgc actgcccgtc cgcctacttc cagctcgccg tgcaccgcct gaacgagcac 480

ggcgtgtacg gcggaggcgt cgaggactac gaccccacgc ctatcgaggt gccgggcttc 540

cccgtgcgcg ccttcgggag caagaccacc atgcggggct tcttccagta ccccggcgtc 600

gagcaggagc accttgaagc gctccacgcc gaggccaccg ccgacggcct gctcttcaac 660

agcttccgcg ccatcgaggc cgacttcctc gacgcctacg cggcggcgct cggcaagacg 720

acgtgggccg tcgggccgac cgccttggtg aacgacacca ccaccaccac cgcctcctcg 780

aggtcgagca ccatcgtgtc gtggctcgac gcccggccgc cggactccgt gctgtacgtc 840

agcttcggca gcatctccct gctgtcggcg aagcagctgg cgaagctcgc ggacgggctg 900

gaggcgtcgg ggcggccgtt cgtgtgggcg atcaaggagg acaaggcgga cgcggcggtg 960

cggtcgcagc tggacgagga gggagggttc gaggcgcggg tcaaggacag gggcctcctg 1020

gtgcgcgggt gggcgccgca ggtggccatc ctctcgcacc cggcggtggg cggcttcctc 1080

acgcactgcg gctggaacag cacgctggag gccctctcac acggcgtgcc ggcgctgacg 1140

tggcccacca acgccgacca gttctgcagc gagcaggtga tcgtggacgt cctcgacgtc 1200

ggcgtcaggt ctggcgtcaa gatcccggcc ctgtacgtgc ccccggaggc cgaggggggtg 1260

caggtggaga gcggcgacgt ggagagggcg atcgtggagc tgatggacgg cgggccggag 1320

ggagcggcga ggagggccag ggcaaggaag attgccgtgg aggccaaggc ggccatggag 1380

gaaggcggga cgtcgcactc cgacctaacg gacatgatcc gccatgtctc ggagctgtcc 1440

aggaagaaga ggctccagct cgagacagcc gacgcgacct gtgaagaagc aacaagagca 1500

gcagacaacg ctgccgcagt actgcctcta ctgtcccaag ctaattaa 1548

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 5

atggcggatc tacacttcct 20

<210> 6

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 6

ttaattagct tgggacagta ga 22

<210> 7

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 7

atggttgcca gccgtgtga 19

<210> 8

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 8

ctagctaggc acctgctct 19

<210> 9

<211> 42

<212> DNA

<213> Artificial Sequence

<400> 9

gcccctggga tccccggaat tcatggcgga tctacacttc ct 42

<210> 10

<211> 44

<212> DNA

<213> Artificial Sequence

<400> 10

cgatgcggcc gctcgagtcg acttaattag cttggggacag taga 44

<210> 11

<211> 41

<212> DNA

<213> Artificial Sequence

<400> 11

gcccctggga tccccggaat tcatggttgc cagccgtgtg a 41

<210> 12

<211> 41

<212> DNA

<213> Artificial Sequence

<400> 12

cgatgcggcc gctcgagtcg acctagctag gcacctgctc t 41

<210> 13

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 13

tgtaaaacga cggccagt 18

<210> 14

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 14

caggaaacagctatgacc 18

<210> 15

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 15

cagcaagtat atagcatggc c 21

<210> 16

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 16

ggagctgcat gtgtcagagg 20

Claims (8)

1. A glycosyltransferase, the amino acid sequence of which is shown in SEQ ID NO: 1. 2. A gene encoding the glycosyltransferase of claim 1. 3. The gene according to claim 2, characterized in that: the nucleotide sequence of the gene is shown in SEQ ID NO: 2. 4. Expression cassette, vector or recombinant bacteria containing the gene of claim 2 or 3. 5. The preparation method of the glycosyltransferase according to claim 1, comprising the following steps: constructing an expression vector for the gene described in claim 2 or 3, introducing the expression vector into the expression host bacteria, obtaining recombinant bacteria, cultivating the recombinant bacteria and Protein expression is induced, bacterial cells are collected, and proteins are extracted and purified. 6. The use of the glycosyltransferase according to claim 1 in the synthesis of steroidal saponins, characterized in that: in the synthesis of the steroidal saponins, trillium or metanosogenin-3-O-glucoside is used. As a substrate, the rhamnosyl group is introduced under the action of the glycosyltransferase to generate chrysanthemum saponin V or chrysanthemum saponin VI. 7. A method for synthesizing chrysanthemum saponin V, comprising the following steps: utilizing the glycosyltransferase of claim 1 to react with trillium and UDP-Rha to generate chrysanthemum saponin V. 8. A method for synthesizing saponin VI, comprising the following steps: utilizing the glycosyltransferase of claim 1 to react with metanosogenin-3-O-glucoside and UDP-Rha to generate saponin VI. .