CN114480323A

CN114480323A - Oat glycosyltransferase AsUGT73E1 and application thereof in synthesis of steroid saponin

Info

Publication number: CN114480323A
Application number: CN202011251664.7A
Authority: CN
Inventors: 薛哲勇; 宋伟; 苏同兵
Original assignee: Northeast Forestry University
Current assignee: Northeast Forestry University
Priority date: 2020-11-11
Filing date: 2020-11-11
Publication date: 2022-05-13
Anticipated expiration: 2040-11-11
Also published as: CN114480323B

Abstract

The invention relates to a saponin metabolic pathway, in particular to oat glycosyltransferase AsUGT73E1 and application thereof in synthesis of steroid saponin. The glycosyltransferase provided by the invention is a protein as described in a1 or a 2: a1. protein with amino acid sequence as shown in SEQ ID No. 1; a2. 1 through substitution and/or deletion and/or addition of one or more amino acid residues to form the protein with glycosyltransferase activity. The glycosyltransferase can take trillin or pennogenin-3-O-glucoside as a substrate and introduce rhamnosyl to generate the paris polyphylla saponin V or paris polyphylla saponin VI.

Description

Oat glycosyltransferase AsUGT73E1 and application thereof in synthesis of steroid saponin

Technical Field

The invention relates to a saponin metabolic pathway, in particular to oat glycosyltransferase AsUGT73E1 and application thereof in synthesis of steroid saponin.

Background

Rhizoma paridis is a generic name of plants in genus of rhizoma paridis of family Liliaceae, can be used as main raw material of Chinese patent medicines such as Yunnan Baiyao, GONGXUENING and pyretic toxicity removing, and has high medicinal and economic values. Steroid saponin is the main chemical component of Paris polyphylla, and more than 160 kinds of steroid saponin including Paris polyphylla saponin I, II, III, V, VI and VII are separated and identifiedHas wide physical activity. The anti-tumor effect is the main effect of the paris polyphylla saponin I, and the paris polyphylla saponin I induces autophagy and cell cycle arrest (He et al.2019) by inhibiting PDK1/Akt/mTOR signal pathways in human gastric cancer HGC-27 cells and down-regulating cyclin B1. Paris saponin VI induces apoptosis and autophagy in non-small cell lung cancer through the ROS-triggered mTOR signaling pathway (Teng et al.2019). The paris polyphylla saponin VII can promote mitochondria to generate ROS and activate MAPK and PTEN/p53 pathways, and jointly induce HepG2 human hepatoma cell apoptosis (Zhang et al.2016). Paris saponin VII is also effective in antibacterial and anti-inflammatory aspects, can remarkably inhibit the growth of cladosporium cladosporioides, candida and propionibacterium acnes, and can be used as an effective substitute for synthetic drugs (Deng et al.2008; Qin et al.2012). Diosgenin (diosgenin) can enhance the activity of lipoprotein lipase, hepatic lipase, superoxide dismutase, glutathione peroxidase and nitric oxide synthase of hyperlipidemic mice, improve lipid distribution, and reduce blood lipid (Gong et al 2010). Paris saponin III has excellent anthelmintic activity and can kill dactylogyrus (EC) parasitizing at gill part of goldfish₅₀＝18.06mg l^-1) And has low toxicity to goldfish (Wang et al.2010). In the research of the new corona pneumonia, the saponin molecules are found to have potential anti-new corona virus activity. Through docking screening, it was speculated that Paris Saponin I could bind to 2019-nCoV major protease (M protease) and prevent viral replication (Yan et al.2020).

The research on the paris polyphylla saponin at the present stage mainly focuses on medicine and clinic, so that the understanding of the metabolic pathway, especially the downstream biosynthesis process is quite lacking. 2,3-Oxidosqualene (2,3-Oxidosqualene) is a common precursor for sterol and triterpene synthesis, and the sterol or triterpene skeleton is generated under the catalysis of 2,3-Oxidosqualene cyclase (2,3-Oxidosqualene cyclases, OSCs). Most pentacyclic triterpene synthases are capable of catalyzing 2, 3-oxidation of squalene to form dammarane-type cations in a "chain-chain" conformation, which subsequently undergo further rearrangement to produce pentacyclic triterpenes such as α -amyrin, β -amyrin and lupeol (lupeol) (Xue et al 2018). Cycloartenol synthase (cycloartenol synthase) catalyzes 2, 3-oxidation of squalene to form a pre-sterol cation in a "chain-boat-chain" conformation, which is then converted to cycloartenol, and synthesizes cholesterol through a series of reactions. The cholesterol content in plants, although generally low, is an important constituent of phytosterols (phytostanols) and is also the direct precursor of steroid saponins (C a rdenas et al 2015). Steroid sapogenins require hydroxylation of cholesterol side chain for synthesis, and are mainly modified by cytochrome P450 enzyme (CYP). For example, CYP90Bs evolved sterol polyhydroxylase activity through gene replication in Paris polyphylla (Paris polyphylla), and PpCYP90G4 catalyzed hydroxylation of cholesterol C16 and C22 with E-ring closure. 16,22(S) -dihydroxy cholesterol is further hydroxylated at C26 under the action of enzymes such as PpCYP94D108 and the like to form diosgenin (Christ et al 2019), while P450 enzyme at C17 of pennogenin is still to be resolved. Finally, diosgenin or pennogenin is glycosylated under the action of glycosyltransferase (UGT) to form rhizoma paridis saponin with multiple biological activities. At present, researches on glycosylation modification of steroid sapogenin are rarely reported.

The paris polyphylla saponin is basically derived from plant extraction, but due to over development, paris polyphylla resources are exhausted. The use of synthetic biology to construct heterologous biosynthetic pathways is increasingly becoming an effective way to obtain natural active ingredients. However, the genome of the paris plant is huge, the types of saponins are numerous, and the metabolic pathway of the paris saponin is difficult to analyze through a gene coexpression network. Oats (Avena sativa L.) are the only saponin-rich plants in grasses (Vincken et al 2007), and two different types of saponins can be synthesized. One is the triterpenoid saponin, avenacins (avenacins), synthesized in roots and root tips, and the other is the steroid saponin, avenacosides, which accumulate in leaves and grains. The oat is an annual herbaceous plant, has a short growth cycle, can complete one growth cycle in 3-4 months under a long-day condition, and can be used as an ideal mode material for researching a synthetic approach of steroid saponins. With the discovery of the triterpene saponin avencins metabolic gene cluster, the synthesis pathway of the oat triterpene saponin is completely analyzed through years of research (Louvau et al 2018; Orme et al 2019).

Disclosure of Invention

In order to analyze the metabolic pathway of the paris polyphylla saponin and realize the artificial synthesis of the paris polyphylla saponin, oat is taken as a model material, and two oat glycosyltransferases are obtained and are named as AsUGT73E5 and AsUGT73E1 respectively. To verify the function of glycosyltransferase, we designed AsUGT73E5, PCR primer AsUGT73E5-ORF-F/R, AsUGT73E1-ORF-F/R of coding region of AsUGT73E 1. PCR amplification is carried out by taking reverse transcribed oat seedling cDNA as a template, and AsUGT73E5 and AsUGT73E1 genes are obtained. The open reading frame of the AsUGT73E1 gene contains 1473 basic groups, the nucleotide sequence is shown as SEQ ID NO. 2, and the coded amino acid sequence is shown as SEQ ID NO. 1. The open reading frame of the AsUGT73E5 gene contains 1548 bases, the nucleotide sequence is shown as SEQ ID NO. 4, and the coded amino acid sequence is shown as SEQ ID NO. 3.

Then, we performed protein expression and purification. The Open Reading Frames (ORF) of the genes AsUGT73E5 and AsUGT73E1 are cloned into a prokaryotic expression vector pGEX-6p-1 by homologous recombination, and the expression competence Rosetta is transformed (DE 3). The cells were cultured in Amp-resistant LB liquid medium until OD600 became 0.6, and then IPTG was added thereto to induce overnight at low temperature. After the cells were sonicated, the proteins in the supernatant were purified by glutaminone Beads, and the fractions were collected and analyzed by SDS-PAGE (FIG. 2). Compared with the control sample without induction, the recombinant protein shows a remarkable band around 80 KDa. The AsUGT73E5 and AsUGT73E1 proteins respectively comprise 515 and 490 amino acids, and the molecular weights after fusion with GST tags are 82.21KDa and 79.79KDa respectively.

Next, we performed functional validation of the glycosyltransferases AsUGT73E5 and AsUGT73E1 with diosgenin, pennogenin and neutigogenin, respectively, with the following results:

HPLC detection is carried out on a product obtained by reacting the AsUGT73E5 protein with diosgenin and UDP-Glc (figure 3), and compared with an unloaded reaction, a new peak (product 1) appears in the product of the AsUGT73E5 at 22.3min, and the peak appearance time is the same as that of a trillin (trillin) standard product. Recovering the sample, adding AsUGT73E1 protein and UDP-Rha to continue reacting, and generating a new product peak (product 2) at 20.7min, wherein the time is consistent with the standard product of the paris polyphylla saponin V. 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, and are consistent with the molecular weights of trillin (576.3) and paris polyphylla saponin V (722.4), which indicates that diosgenin generates paris polyphylla saponin V after continuous catalysis of AsUGT73E5 and AsUGT73E 1.

After HPLC detection of the product of the reaction of AsUGT73E5 protein with pennogenin and UDP-Glc (FIG. 5), a new peak (product 3) appeared at 18.8min in the product of AsUGT73E5 compared with the unloaded reaction. Recovering a sample, adding AsUGT73E1 protein and UDP-Rha to continue reacting, and generating a new product peak (product 4) within 17.1min, wherein the time is consistent with that of a paris polyphylla saponin VI standard product. 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, and are consistent with the molecular weights of pennogenin-3-O-glucoside (592.3) and paris polyphylla saponin VI (738.4), which indicates that the pennogenin generates paris polyphylla saponin VI after continuous catalysis of AsUGT73E5 and AsUGT73E 1.

After HPLC detection of the product of the reaction of AsUGT73E5 protein with Numerigenin and UDP-Glc (FIG. 8), a new peak (product 5) appeared at 16.7min in the product of AsUGT73E5 compared with the unloaded reaction. The sample was recovered and the reaction was continued by adding AsUGT73E1 protein and UDP-Rha, and a second product peak (product 6) appeared at 15.6 min. TOF Positive ion Scan mode detection As shown in FIG. 9,

products

5 and 6 have molecular weights of 593.37(M + H), respectively⁺) And 739.43(M + H)⁺) The corresponding glycosylation product is generated after the neutiagenin is continuously catalyzed by AsUGT73E5 and AsUGT73E 1.

Based on the above studies, the present invention provides a glycosyltransferase which is a protein described in the following a1 or a 2:

a1. protein with amino acid sequence as shown in SEQ ID No. 1;

a2. 1 through substitution and/or deletion and/or addition of one or more amino acid residues to form the protein with glycosyltransferase activity.

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

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

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

In some embodiments, the Vector is a cloning Vector, contains the gene encoding the glycosyltransferase and elements required for plasmid replication, for example, a pClone007 Blunt Simple Vector into which the encoding gene is inserted. In other embodiments, the vector is an expression vector comprising a gene encoding the glycosyltransferase and an element that enables successful expression of the protein, e.g., a pGEX-6p-1 vector into which the encoding gene is inserted.

In some embodiments, the recombinant bacterium is a recombinant bacterium containing a cloning vector, e.g., e.coli DH5 α, and the gene encoding the glycosyltransferase is replicated by culturing the recombinant bacterium. In other embodiments, the recombinant bacterium is a recombinant bacterium comprising an expression vector, which is cultured under suitable conditions, e.g., with the addition of an appropriate amount of IPTG, and expression of the glycosyltransferase is induced at 16 ℃.

The invention also provides a preparation method of the glycosyltransferase, which comprises the following steps: constructing an expression vector of the encoding gene of the glycosyltransferase, introducing the expression vector into an expression host bacterium to obtain a recombinant bacterium, culturing the recombinant bacterium, inducing protein expression, collecting thalli, and extracting and purifying protein.

The use of said glycosyltransferase in glycosyltransfer reactions also belongs to the scope of protection of the present invention.

The application of the glycosyltransferase in the synthesis of steroid saponin also belongs to the protection scope of the invention. The synthesis of steroid saponins includes in vitro synthesis and in vivo synthesis, for example, synthesis of steroid saponins in microorganisms (e.g., yeast) or plants.

In some embodiments of the invention, in the synthesis of the steroid saponin, trillin or pennogenin-3-O-glucoside is used as a substrate, and rhamnosyl is introduced under the action of the glycosyltransferase to generate the paris saponin V or the paris saponin VI.

The invention also provides a method for synthesizing the paris polyphylla saponin V, which comprises the following steps: and (3) reacting the glycosyltransferase with trillin and UDP-Rha to generate the paris polyphylla saponin V.

The invention also provides a method for synthesizing the paris polyphylla saponin VI, which comprises the following steps: the glycosyltransferase is used for reacting with pennogenin-3-O-glucoside and UDP-Rha to generate the paris polyphylla saponin VI.

The glycosyltransferase provided by the invention is helpful for researching the synthetic route of the plant steroid saponin, and provides gene resources for obtaining a large amount of the paris polyphylla saponin or other saponins.

Drawings

FIG. 1 shows electrophoretograms of amplification products of AsUGT73E5 and AsUGT73E1 genes. M is DL 2000; 1 is AsUGT73E5 gene band; and 2 is an AsUGT73E1 gene band.

FIG. 2 shows an SDS-PAGE electrophoresis chart of prokaryotic expression of AsUGT73E5 and AsUGT73E1 proteins. M: page-ruler protein molecular markers; 1: not inducing; 2: IPTG-induced whole cell protein; 3: cell supernatants after IPTG induction; 4: cell precipitation after IPTG induction; 5: purifying the supernatant GST column; 6: and (4) carrying out ultrafiltration concentration on the purified protein.

FIG. 3 chromatograms of glycosylation products of reaction of AsUGT73E5, AsUGT73E1 with diosgenin. The abscissa is retention time (min) and the ordinate is electrical signal (mAU). pGEX-No-load: the cell containing pGEX-6P-1 empty vector is subjected to protein induction expression and then extracted and purified protein is used as a blank control; pGEX-AsUGT73E 5: the AsUGT73E5 protein is extracted and purified after the cells containing the pGEX-AsUGT73E5 vector are subjected to protein induction expression; and the cells containing the pGEX-AsUGT73E1 vector are subjected to protein induction expression, and then extracted and purified AsUGT73E1 protein.

FIG. 4 Mass Spectroscopy of the glycosylation products of the reaction of AsUGT73E5, AsUGT73E1 with diosgenin. The abscissa is mass to charge ratio and the ordinate is ion intensity.

FIG. 5 chromatograms of glycosylation products of AsUGT73E5, AsUGT73E1 reacted with pennogenin. The abscissa is retention time (min) and the ordinate is electrical signal (mAU). pGEX-No-load: the cell containing pGEX-6P-1 empty vector is subjected to protein induction expression and then extracted and purified protein is used as a blank control; pGEX-AsUGT73E 5: the AsUGT73E5 protein is extracted and purified after the cells containing the pGEX-AsUGT73E5 vector are subjected to protein induction expression; and the cells containing the pGEX-AsUGT73E1 vector are subjected to protein induction expression, and then extracted and purified AsUGT73E1 protein.

FIG. 6 Mass Spectrometry analysis of glycosylation products of AsUGT73E5, AsUGT73E1 reacted with pennogenin. The abscissa is mass to charge ratio and the ordinate is ion intensity.

Figure 7. the process of catalytic glycosylation of steroid sapogenins by assegt 73E5 and assegt 73E 1.

FIG. 8 chromatogram of glycosylation products of the reaction of AsUGT73E5, AsUGT73E1 with neutiagenin. The abscissa is retention time (min) and the ordinate is electrical signal (mAU). pGEX-No-load: extracting and purifying protein after protein induction expression of cells containing pGEX-6P-1 empty vectors to serve as blank control; pGEX-AsUGT73E 5: the AsUGT73E5 protein is extracted and purified after the cells containing the pGEX-AsUGT73E5 vector are subjected to protein induction expression; and the cells containing pGEX-AsUGT73E1 vectors are subjected to protein induction expression, and then extracted and purified AsUGT73E1 protein.

FIG. 9 Mass Spectrometry analysis of glycosylation products of AsUGT73E5, AsUGT73E1 reacted with neutigogenin. The abscissa is mass-to-charge ratio and the ordinate is ionic strength.

Detailed Description

The present invention is further described below in conjunction with the following examples, which are to be understood as being merely illustrative and explanatory of the invention and not limiting the scope of the invention in any way.

Experimental materials:

the plant material used in the following experiments was diploid oats (Avena strigosa, S75), and known varieties are described in non-patent document Papadopoulou et al 1999. The above biological materials are also stored in the laboratory and the applicant states that they can be released to the public for verification experiments within twenty years from the filing date.

Escherichia coli (Escherichia coli) DH5 alpha and Rosetta (DE3) were purchased from Shanghai-only Biotechnology Ltd. The cloning Vector pClone007 Blunt Simple Vector was purchased from Biotechnology Ltd of New Engineers, Kyoto. The prokaryotic expression vector pGEX-6P-1 is stored in the laboratory and can also be obtained commercially.

PCR primers:

the main reagents are as follows:

diosgenin: CAS number: 512-04-9, molecular formula: c₂₇H₄₂O₃The english name diosgenin, purchased from kyoto pily science development limited, cat No. BP 0504.

Pennogenin: CAS number: 507-89-1, molecular formula: c₂₇H₄₂O₄English name pennogenin. The pennogenin used in the experiment is obtained by enzymolysis, separation and purification of the paris polyphylla saponin VI, and the enzymolysis, separation and purification method refers to Li, W., Wang, Z., Gu, J., Chen, L., Hou, W., Jin, Y.P.,&wang, Y.P. (2015), Bioconversion of angioside Rd to angioside M1 by y strain hormone hydrolysis and its enhancement effect on insulin secretion in vitro, die Pharmazie,70: 340-. Pennogenin is also commercially available.

The extraction method comprises the following steps of (1) neurargigenin: CAS number: 6811-35-4, molecular formula: c₂₇H₄₂O₄English name nutagenin. The nuarigenin used in the experiment is obtained by enzymolysis, separation and purification of an avenacoside (avenacosides) extract, and the method for extracting the avenacoside (avenacosides) refers to Yang, J.L., Wang, P.s., Wu, W.B., ZHao, Y.T., Idehen, E.s.,& Sang,S.M.(2016).Steroidal saponins in oat bran.Journal of Agricultural&food Chemistry, 64(7), 1549-1556, the methods for enzymatic separation and purification refer to Li, W, Wang, Z, Gu, J, Chen, L, Hou, W, Jin, Y.P.,&Wang,Y.P.(2015).Bioconversion of ginsenoside Rd to ginsenoside 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, formula: c₁₅H₂₂N₂Na₂O₁₇P₂Purchased from Kulai Bokoku technologies, Beijing, under the brand name CU11611-500 mg.

UDP-Rhamnose (UDP-Rha): CAS number: 1526988-33-9, formula: c₁₅H₂₂N₂Na₂O₁₆P₂The English name UDP 5' -diphospho-a-L-rhamnose, available from Suzhou Han enzymes Biotechnology Ltd.

Trillin: CAS number: 14144-06-0, formula: c₃₃H₅₂O₈Purchased from Doudu Pury science development, Inc., cat # BP 1124.

Rhizoma paridis saponin V: CAS number: 19057-67-1, formula: c₃₉H₆₂O₁₂Purchased from Doudu Pury science and technology development Inc., cat number BP 1151.

And (3) paris polyphylla saponin VI: CAS number: 55916-51-3, formula: c₃₉H₆₂O₁₃Purchased from Doudu Pury science development Inc., cat number BP 1131.

Chromatographic methanol: purchased from merck, usa under serial number 1.06007.4008.

Unless otherwise specified, the reagents used in the following examples are conventional in the art, and are either commercially available or formulated according to conventional methods in the art; the experimental methods and conditions used are all conventional in the art, and reference can be made to relevant experimental manuals, well-known literature or manufacturer instructions. Unless defined otherwise, 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

We used oats as model material and found two glycosyltransferase genes, which were named AsUGT73E5 and AsUGT73E 1. The Open Reading Frame (ORF) of the AsUGT73E1 gene contains 1473 bases, the nucleotide sequence of the ORF is shown as SEQ ID NO. 2, and the glycosyltransferase with the coded amino acid sequence shown as SEQ ID NO. 1 is obtained. The Open Reading Frame (ORF) of the AsUGT73E5 gene contains 1548 bases, the nucleotide sequence is shown as SEQ ID NO. 4, and the coding amino acid sequence is shown as glycosyltransferase as SEQ ID NO. 3. By transcriptome-to-target metabolite correlation analysis (Pearson correlation), we found that expression of these two glycosyltransferases in 10 tissues of oat was highly synergistic with cholesterol synthesis and therefore likely involved in steroid saponin synthesis. In order to verify the functions of the two glycosyltransferases in steroid saponin synthesis, gene cloning and expression are carried out.

2. Cloning of genes

2.1 extraction of Total RNA from oat seedlings

The RNA extraction was carried out using the RNAprep Pure Plant Kit (cat # DP441) from Tiangen, according to the Kit instructions, and the following steps were carried out:

(1) rapidly grinding 50-100mg of oat leaf into powder in liquid nitrogen, adding 450 μ L of RL (adding beta-mercaptoethanol before use), and mixing by vortexing and shaking vigorously;

(2) transferring the solution to a filter column CS, centrifuging at 12,000rpm for 2-5min, and sucking supernatant in a collecting tube to an RNase-Free centrifuge tube;

(3) adding 0.5 times of anhydrous ethanol, mixing, transferring the obtained solution and precipitate into adsorption column CR3, centrifuging at 12,000rpm for 30-60sec, pouring off waste liquid, and placing adsorption column CR3 back into the collecting tube;

(4) adding 350 μ L deproteinizing solution RW1 into adsorption column CR3, centrifuging at 12,000rpm for 30-60sec, pouring off waste liquid, and placing adsorption column CR3 back into the collection tube;

(5) preparing DNase I working solution: putting 10 mu L of DNase I stock solution into a new RNase-Free centrifuge tube, adding 70 mu L of RDD buffer solution, and gently and uniformly mixing;

(6) adding 80 μ L DNase I working solution into the center of the adsorption column CR3, and standing at room temperature for 15 min;

(7) adding 350 μ L deproteinized solution RW1 into adsorption column CR3, centrifuging at 12,000rpm for 30-60sec, pouring off waste liquid, and placing adsorption column CR3 back into the collection tube;

(8) adding 500 μ L of rinsing solution RW (ethanol before use) into adsorption column CR3, standing at room temperature for 2min, centrifuging at 12,000rpm for 30-60sec, pouring off waste liquid in the collection tube, and placing adsorption column CR3 back into the collection tube, and repeating once;

(9) centrifuging at 12,000rpm for 2min, pouring off waste liquid, placing adsorption column CR3 at room temperature for several minutes, and air drying the residual rinsing liquid completely;

(10) placing the adsorption column CR3 into a new RNase-Free centrifuge tube, and dripping 30-100 μ L RNase-Free ddH into the middle part of the adsorption membrane₂O, standing at room temperature for 2min, and centrifuging at 12,000rpm for 2min to obtain an RNA solution.

2.2 Synthesis of cDNA

The SuperScript III Rreverse Transcriptase kit (Invitrogen, cat # 18080085) was used and the procedures were performed according to the kit instructions:

(1) denaturation of RNA template

Heating at 65 deg.C for 5min, rapidly cooling on ice, and standing on ice for 2 min.

(2) First Strand cDNA Synthesis

And (5) centrifuging for a short time and mixing uniformly. Reacting at 55 deg.C for 60min, heating at 70 deg.C for 15min to terminate the reaction, and storing the product at-20 deg.C.

2.3 amplification of the Gene of interest

Designing a glycosyltransferase gene AsUGT73E5, an AsUGT73E1 coding region primer AsUGT73E5-ORF-F/R, AsUGT73E 1-ORF-F/R. The cDNA of oat seedlings diluted by 5 times is used as a template for PCR amplification, primers AsUGT73E5-ORF-F/R, AsUGT73E1-ORF-F/R and 2 x Phanta Max Master Mix high fidelity enzyme (vazyme, the cargo number is P515-02) are respectively used for PCR amplification of a target gene, and the reaction system is as follows:

reaction conditions are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30sec, annealing at primer Tm (AsUGT73E 5-ORF-F/R: 60 ℃, AsUGT73E 1-ORF-F/R: 60 ℃) for 30sec, extension at 72 ℃ for 1min, for 33 cycles; extension was complete at 72 ℃ for 7 min. After the reaction is finished, the PCR product is detected by 1% agarose gel electrophoresis.

As shown in FIG. 1, the gene bands were all 1500bp in size, with 1 being AsUGT73E5 and 2 being AsUGT73E 1.

2.4DNA gel recovery

The target gene fragment was recovered using Gel Extraction Kit (Omega, cat # D2500-02) Kit, and the procedures were performed according to the Kit instructions, as follows:

(1) the agarose gel containing the band of interest was cut in a UV dicer, an equal volume of Binding Buffer/Binding Buffer was taken and the mixture incubated at 55 ℃ for 7min until the gel was completely thawed.

(2) Sucking 700 μ L of the mixed solution, transferring into a DNA adsorption column with a 2mL collecting tube, standing for 1min, centrifuging at 10,000g for 1min, and discarding the filtrate.

(3) The adsorption column was placed back in the collection tube, and 700. mu.L of anhydrous ethanol diluted SPW Wash Buffer was added, 10,000g was centrifuged for 1min, and the filtrate was discarded. And repeating the steps once.

(4) The filtrate was discarded, and the empty adsorption column was returned to the centrifuge tube and centrifuged at 12,000g for 2 min.

(5) The air adsorption column was placed in a sterilized 1.5mL centrifuge tube, the tube lid was opened and allowed to stand for 1min, 30. mu.L of sterile water (preheated at 60 ℃) was added to the center of the adsorption membrane, and allowed to stand at room temperature for 1 min. The DNA was eluted by centrifugation at 12,000g for 1 min.

2.5 cloning vector ligation

The gene of interest was cloned into pClone007 Blunt Simple Vector (Beijing Okagaku Biopsis, cat # TSV-007BS) in the following reaction system:

reacting at room temperature for 5 min.

2.6 transformation of E.coli

(1) Taking 100 mu L of the competent cell DH5 alpha (Shanghai Diego) melted in ice bath, adding the target DNA, gently mixing uniformly, and then placing for 30min in ice bath;

(2) carrying out water bath heat shock at 42 ℃ for 60s, and quickly transferring the centrifuge tube into an ice bath for 2 min;

(3) adding 200 mu L of nonresistant sterile LB culture solution into a centrifuge tube, uniformly mixing, and culturing for 1h at 37 ℃ in a shaking table at 180rpm to recover bacteria;

(4) absorbing the competent cells transformed in the last step, adding the competent cells to an LB agar culture medium containing ampicillin (Amp, screening concentration of 100mg/L) resistance, uniformly spreading the cells, blow-drying the liquid on the surface of the culture medium, and inverting the plate at 37 ℃ for overnight culture;

(5) several single colonies were picked and added to 500. mu.L LB liquid medium containing Amp resistance (100mg/L), cultured at 37 ℃ for 4h at 180rpm, and the bacteria liquid was identified by PCR with primers M13-F/R, and the positive clones were sequenced by Biotech Limited, Borneo, Senui, to obtain cloning vectors pClon 007-AsUGT73E5 and pClon 007-AsUGT73E1 with correct sequences.

2.7 plasmid extraction

After the preservation of the correctly sequenced samples, plasmids were extracted using E.Z.N.A.plasmid Mini Kit I Kit (omega, cat # D6942-02) according to the instructions, following the procedure:

(1) taking 5mL of bacterial liquid (12-16h) cultured overnight at 37 ℃, centrifuging 10,000g for 1min, and removing supernatant;

(2) adding 250 mu L of Solution I (added with RNase A) into a centrifuge tube, blowing and stirring uniformly;

(3) adding 250 μ L Solution II, reversing the upper part and the lower part for 4-6 times, mixing, standing for 2min to fully crack the thallus (the total time is less than 5 min);

(4) add 350. mu.L Solution III, immediately reverse 6-8 times, let the Solution mix thoroughly, at which point a large amount of white flocculent precipitate appears. Centrifuging at 13,000g for 10 min;

(5) placing the adsorption column in a collection tube, sucking centrifuged supernatant, adding into the adsorption column, centrifuging at 10,000g for 1min, and removing filtrate;

(6) 700. mu.L of DNA Wash Buffer was added to the column, 10,000g was centrifuged for 1min, and the filtrate was discarded. Repeating the steps once;

(7) placing the empty adsorption column into the collection tube, centrifuging for 2min at 13,000g, transferring the adsorption column into a new 1.5mL centrifuge tube, opening the tube cover to dry the adsorption column for 1min, and volatilizing residual rinsing liquid in the adsorption column;

(8) adding 50 μ L of sterile water preheated to 55 deg.C into the center of the membrane of the adsorption column, standing for 2min, and centrifuging at 13,000g for 1 min. The adsorption column was discarded and the plasmid was stored at-20 ℃ until use.

3. Protein expression

3.1 prokaryotic expression vector construction

The pClone007 vector (pClone007-AsUGT73E5 and pClone007-AsUGT73E1) containing the target gene AsUGT73E5 and AsUGT73E1 open reading frames is used as a template, a recombinant primer AsUGT73E5-pGEXF/R, AsUGT73E1-pGEXF/R is designed for PCR amplification, and the reaction system and the reaction conditions are the same as those in 2.3, so that the gene fragment for constructing the expression vector is obtained.

pGEX-6p-1 is used as a prokaryotic expression vector. pGEX-6p-1 vector was linearized with EcoRI (Thermo, cat # FD0274) and SalI (Thermo, cat # FD0644) rapid endonucleases as follows:

the reaction was terminated after 1 hour at 37 ℃.

And detecting the PCR amplification product and the linearized pGEX-6p-1 vector by agarose gel electrophoresis and then recovering the PCR amplification product and the linearized pGEX-6p-1 vector. Homologous recombination of the recovered gene fragment and linearized pGEX-6p-1 was performed using the Cloneexpress II One Step Cloning Kit (vazyme, cat # C112-02) according to the instructions, and ORFs of genes AsUGT73E5 and AsUGT73E1 were cloned into prokaryotic expression vector pGEX-6p-1, respectively, as follows:

reacting at 37 deg.C for 30min, cooling to 4 deg.C or cooling on ice to obtain reaction product, and transforming Escherichia coli DH5 alpha by the same transformation method as 2.6. After overnight culture, picking single colony, adding into 500 μ L LB liquid medium containing Amp resistance (100mg/L), culturing at 37 deg.C and 180rpm for 4h, carrying out PCR identification on bacterial liquid, identifying primer as pGEX-F/R, and sequencing positive clone by Borneo Biotechnology Limited. After the samples with correct sequencing result are preserved, plasmids pGEX-AsUGT73E5/pGEX-AsUGT73E1 are extracted, and the plasmid extraction method is the same as the above 2.7. Coli Rosseta (DE3) was transformed with the extracted plasmid to express competence (Shanghai unique organism) while pGEX-6p-1 empty vector transformation was set as a control, and the transformation procedure was as described above for 2.6.

3.2 protein inducible expression

Inoculating Rosseta (DE3) bacterial liquid containing pGEX-AsUGT73E5/pGEX-AsUGT73E1/pGEX-6p-1 empty vector into 1L LB liquid culture medium containing Amp resistance (100mg/L) respectively according to the volume ratio of 1:100, and culturing at 37 ℃ with a shaker at 200rpm until OD₆₀₀0.6 mM IPTG was added and induced overnight at 16 ℃ on a shaker at 160 rpm. The cells were collected by centrifugation at 4,000rpm at 4 ℃ and 10mL of a precooled PBS solution (concentration 0.01M, pH 7.4, formulation: 8.0g NaCl, 0.2g KCl, Na₂HPO₄ 1.44g，KH₂PO₄0.24g, adding distilled water to 1L), resuspending, and ultrasonically crushing on ice until the solution is translucent. Centrifuging at 12,000rpm for 15min at 4 deg.C, collecting supernatant and precipitate, and detecting by SDS-PAGE electrophoresis.

3.3 protein purification

Equilibration/eluent (formulation identical for equilibration and eluent) and eluent were prepared and 1mM DTT was added prior to use. Equilibration/wash (1L): 140mM NaCl, 2.7mM KCl, 10mM Na₂HPO₄，1.8mM KH₂PO₄pH 7.4. Eluent (1L): 50mM Tris-HCl,10mM reduced glutathione, pH 8.0.

(1) Glutathieone Beads (Changzhou heaven and earth people and biology, the product number: SA008010) is loaded into a proper chromatographic column and is balanced by a balancing liquid with 5 times of the column volume, so that the filler is in a buffer system the same as that of the target protein to play a role in protecting the protein;

(2) adding the sample into the well-balanced glutaminone Beads, ensuring that the target protein is fully contacted with the glutaminone Beads, improving the recovery rate of the target protein, and collecting effluent;

(3) washing with a washing solution with 10 times of column volume to remove non-specifically adsorbed hybrid protein, and collecting the washing solution;

(4) collecting the eluate, i.e. the target protein component, by using 5 column volumes of the eluate;

(5) balancing the filler by using 3 times of column volume of balancing liquid and 5 times of column volume of deionized water in sequence;

(6) the purified protein solution was added to millipore 15mL ultrafiltration tube (10KD), the sample was concentrated by centrifugation at 4,000rpm at 4 ℃ to 500. mu.L, and 15mL of PBS phosphate buffer (concentration 0.01M, formulation: NaCl 8.0g, KCl 0.2g, Na)₂HPO₄ 1.44g，KH₂PO₄0.24g, adjusting pH to 7.4, adding distilled water to 1L), and concentrating to 500 μ L. Repeating the steps once;

(7) sucking purified protein, diluting, adding glycerol to final concentration of 10%, and storing at-80 deg.C.

SDS-PAGE detects the purified protein. The results are shown in FIG. 2, where 1 is the whole cell protein without induction; 2 is whole cell protein after IPTG induction; 3 is cell supernatant after IPTG induction; 4 is cell precipitation after IPTG induction; 5 is protein purified by a supernatant GST column; 6 is the purified protein after ultrafiltration concentration. Compared with the control sample without induction, the recombinant protein shows a remarkable band around 80 KDa. The AsUGT73E5 and AsUGT73E1 proteins respectively comprise 515 and 490 amino acids, and the molecular weights after fusion with GST tags are 82.21KDa and 79.79KDa respectively.

Example 2 functional characterization of the glycosyltransferases AsUGT73E5 and AsUGT73E1

1. Enzyme activity detection

(1) AsUGT73E5 catalyzed glycosylation reaction

Accurately weighing 1mM steroid sapogenin (diosgenin/pennogenin/Nuo-alpha-apogenin), 1mM UDP-Glucose (UDP-Glc), 50 μ L purified glycosyltransferase AsUGT73E5, and dissolving in PBS phosphate buffer (concentration 0.01M, formulation: NaCl 8.0g, KCl 0.2g, Na₂HPO₄ 1.44g，KH₂PO₄0.24g, pH adjusted to 8.0, distilled water added to 1L) to make the final volume 300. mu.L. Reacting at 37 deg.C for 2 hr, adding methanol with equal volume to stop enzyme activity, vacuum drying the product, and dissolving in 500 μ L chromatographic AAlcohol is tested.

(2) AsUGT73E1 catalyzed glycosylation reaction

The glycosylation reaction in (1) was repeated, the product was concentrated and dried to be used as a substrate in its entirety, and 1mM UDP-Rhamnose (UDP-Rha), 50. mu.L of the purified glycosyltransferase AsUGT73E1, dissolved in PBS phosphate buffer (pH 8.0), was added to make the final volume 300. mu.L. After reacting for 2h at 37 ℃, adding methanol with the same volume to stop the enzyme activity, decompressing and spin-drying the product, and dissolving the product in 500 mu L of chromatographic methanol to be tested.

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

(1) Liquid chromatography

The experiment used a Thermo Ultimate 3000 liquid chromatograph, Thermo Hypersil GOLD C₁₈HPLC detection was carried out using a liquid chromatography column (250 mm. times.4.6 mm, 5 μm). The mobile phase is water (A) and acetonitrile (B). Elution gradient: 0-6 min, 20-30% of B; 30-60% of B for 6-15 min; 15-21 min, 60% -100% B; 21-30 min, 100% B; 30-35 min, 100-20% of B. Flow rate 1mL/min, column temperature 30 ℃, sample injection amount 10 μ L, detection wavelength: 210 nm.

(2) Mass spectrometric detection

This experiment was detected using an AB SCIEX TripleTOF 6600 ultra high resolution mass spectrometer. Positive ion data acquisition mode, conditions are: the capillary voltage is 3.6kV, the taper hole voltage is 35kV, the ion source temperature is 105 ℃, the desolvation gas temperature is 340 ℃, the reverse taper hole airflow is 55L/h, the desolvation gas is 650L/h, and the extraction taper hole is 4V. Mass to charge ratio data scan range: 50-1500 m/z.

Results and analysis

And drying a product obtained by reacting the AsUGT73E5 protein with diosgenin and UDP-Glc by using a vacuum concentrator, adding 500 mu L of chromatographic methanol for dissolving, filtering by using a 0.22 mu m filter membrane, and carrying out HPLC detection. As shown in FIG. 3, the product of AsUGT73E5 showed a new peak (product 1) at 22.3min compared to the unloaded reaction, which was the same time as the trillin (trillin) standard. Recovering the sample, adding AsUGT73E1 protein and UDP-Rha to continue reacting, and generating a new product peak (product 2) at 20.7min, wherein the time is consistent with the standard product of the paris polyphylla saponin V. TOF Positive ion Scan mode detection see FIG. 4, product 1 andthe product 2 has a molecular weight of 577.38(M + H)⁺) And 723.43(M + H)⁺) Consistent with the molecular weights of trillin (576.3) and yamogenin V (722.4), it was shown that yamogenin produced yamogenin V after continuous catalysis by AsUGT73E5 and AsUGT73E 1.

After HPLC detection of the product of the reaction of AsUGT73E5 protein with pennogenin and UDP-Glc (FIG. 5), a new peak (product 3) appeared at 18.8min in the product of AsUGT73E5 compared with the unloaded reaction. Recovering a sample, adding AsUGT73E1 protein and UDP-Rha to continue to react, and generating a new product peak (product 4) within 17.1min, wherein the time is consistent with the standard product of the paris saponin VI. TOF Positive ion Scan mode detection As shown in FIG. 6,

products

3 and 4 have molecular weights of 593.37(M + H), respectively⁺) And 739.43(M + H)⁺) The molecular weight of the derivative is consistent with that of the pennogenin-3-O-glucoside (592.3) and the paris polyphylla saponin VI (738.4), which shows that the pennogenin generates the paris polyphylla saponin VI after being continuously catalyzed by AsUGT73E5 and AsUGT73E 1. The process of catalyzing steroid sapogenin glycosylation by the AsUGT73E5 and AsUGT73E1 proteins is shown in FIG. 7.

And drying a product obtained by reacting the AsUGT73E5 protein with the Nurseothiagenin and the UDP-Glc by using a vacuum concentrator, adding 500 mu L of chromatographic methanol for dissolving, filtering by using a 0.22 mu m filter membrane, and carrying out HPLC detection. As shown in FIG. 8, the product of AsUGT73E5 showed a new peak at 16.7min (product 5) compared to the unloaded reaction. The sample was recovered and the reaction was continued by adding AsUGT73E1 protein and UDP-Rha, and a second product peak (product 6) appeared at 15.6 min. TOF Positive ion Scan mode detection As shown in FIG. 9,

products

5 and 6 have molecular weights of 593.37(M + H), respectively⁺) And 739.43(M + H)⁺) The corresponding glycosylation product is generated after the neutiagenin is continuously catalyzed by AsUGT73E5 and AsUGT73E 1. Reference documents:

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 steroid biosynthesis 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.Planta Medica,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 human vascular endothelial cells against hydrogen peroxide-induced apoptosis.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-deficient plants.Proceedings of the National Academy of Sciences,96(22),12923-12928.

Qin,X.J.,Sun,D.J.,Ni,W.,Chen,C.X.,Hua,Y.,He,L.,&Liu,H.Y.(2012).Steroidal saponins with antimicrobial activity from stems and leaves of Paris polyphylla var.yunnanensis. Steroids,77(12),1242-1248.

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.induces apoptotic 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.

Wang,G.X.,Han,J.,Zhao,L.W.,Jiang,D.X.,Liu,Y.T.,&Liu,X.L.(2010).Anthelmintic activity of steroidal saponins from Paris Polyphylla.Phytomedicine,17(14),1102-1105.

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 product specificity of 2,3-oxidosqualene cyclase in Oryza species.New Phytologist,218(3):1076-1088.

Zhang,C.,Jia,X.J.,Bao,J.L.,Chen,S.H.,Wang,K.,Zhang,Y.L.,…&He,C.W.(2016). Polyphyllin VII induces apoptosis in HepG2 cells through ROS-mediated mitochondrial dysfunction and MAPK pathways.BMC Complementary and Alternative Medicine,16(58).

sequence listing

<110> northeast university of forestry

<120> oat glycosyltransferase AsUGT73E1 and application thereof in steroid saponin synthesis

<130> P200850-DBL

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 490

<212> PRT

<213> oat (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> oat (Avena sativa L.)

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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> oat (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> oat (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 cgagggggtg 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 (Artificial Sequence)

<400> 5

atggcggatc tacacttcct 20

<210> 6

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ttaattagct tgggacagta ga 22

<210> 7

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atggttgcca gccgtgtga 19

<210> 8

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ctagctaggc acctgctct 19

<210> 9

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gcccctggga tccccggaat tcatggcgga tctacacttc ct 42

<210> 10

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

cgatgcggcc gctcgagtcg acttaattag cttgggacag taga 44

<210> 11

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gcccctggga tccccggaat tcatggttgc cagccgtgtg a 41

<210> 12

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

cgatgcggcc gctcgagtcg acctagctag gcacctgctc t 41

<210> 13

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tgtaaaacga cggccagt 18

<210> 14

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

caggaaacag ctatgacc 18

<210> 15

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

cagcaagtat atagcatggc c 21

<210> 16

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

ggagctgcat gtgtcagagg 20

Claims

1.A glycosyltransferase which is a protein according to a1 or a 2:

a1. protein with amino acid sequence as 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 as SEQ ID NO. 2.

4. An expression cassette, vector or recombinant bacterium comprising the gene of claim 2 or 3.

5. The method for producing the glycosyltransferase of claim 1, comprising the steps of: constructing an expression vector of the gene of claim 2 or 3, introducing the expression vector into an expression host bacterium to obtain a recombinant bacterium, culturing the recombinant bacterium and inducing protein expression, collecting the bacterium, and extracting and purifying the protein.

6. Use of the glycosyltransferase of claim 1 in a glycosyltransfer reaction.

7. Use of the glycosyltransferase of claim 1 in steroid saponin synthesis.

8. Use according to claim 7, characterized in that: in the synthesis of the steroid saponin, trillin or pennogenin-3-O-glucoside is used as a substrate, rhamnosyl is introduced under the action of glycosyltransferase, and the paris polyphylla saponin V or paris polyphylla saponin VI is generated.

9.A method for synthesizing rhizoma paridis saponin V comprises the following steps: reacting glycosyltransferase of claim 1 with trillin, UDP-Rha, to produce polyphyllin V.

10. A method for synthesizing paris polyphylla saponin VI comprises the following steps: reacting the glycosyltransferase of claim 1 with pennogenin-3-O-glucoside, UDP-Rha to produce Paris Saponin VI.