CN113462668A

CN113462668A - Oleanolic acid glucuronyl transferase and coding gene and application thereof

Info

Publication number: CN113462668A
Application number: CN202110699993.6A
Authority: CN
Inventors: 杨生超; 唐卿雁; 张广辉; 范伟; 陈庚
Original assignee: Yunnan Agricultural University
Current assignee: Yunnan Agricultural University
Priority date: 2018-09-19
Filing date: 2018-09-19
Publication date: 2021-10-01
Also published as: CN113249355A; CN109266626B; CN109266626A; CN113249354A

Abstract

The invention relates to oleanolic acid glucuronyl transferase and a coding gene and application thereof, belonging to the field of plant genetic engineering and biotechnology. The amino acid sequence of the oleanolic acid glucuronyl transferase is shown in SEQ ID NO. 3. The nucleotide sequence is shown as SEQ ID NO. 7. The oleanolic acid glucuronic acid transferase can specifically take uridine diphosphate glucuronic acid as a glycosyl donor to catalyze the glycosylation reaction of the C-3 position of oleanolic acid, so that oleanolic acid 3-O-beta-glucuronic acid is generated. The transferase is an enzyme in an upstream step in the synthesis process of oleanane-type saponin, and has important significance for analyzing the synthesis way of the oleanane-type saponin.

Description

Oleanolic acid glucuronyl transferase and coding gene and application thereof

Technical Field

The invention belongs to the field of plant genetic engineering and biotechnology, and particularly relates to oleanolic acid glucuronyl transferase and a coding gene and application thereof.

Background

Ginger-shaped pseudo-ginseng (Panax zingiberensis C.Y.Wu et K.M.Feng) is a perennial herb of Panax of Araliaceae, and is distributed in southeast Yunnan, middle of Nipol, high altitude area without lead, Myanmar east branch, whispering, etc. The rhizome and meat of the rhizomes of the zingiber officinale roscoe are like ginger blocks, belong to endangered species, and due to over development, wild resources are extremely rare at present. Rhizoma Zingiberis recens-shaped Notoginseng radix has effects of removing blood stasis, relieving pain, and stopping bleeding, and can be used for treating traumatic injury, fracture, hematemesis, functional metrorrhagia, and traumatic hemorrhage.

The saponin is the main active component of rhizoma Zingiberis recens Notoginseng radix, and mainly contains dammarane type saponin and oleanane type saponin, such as ginsenoside R_OChikusetsusaponin IV and chikususaponin IVa. The oleanane-type saponin has pharmacological effects of resisting tumor, reducing blood lipid, reducing blood glucose, and protecting liver. Oleanane-type saponins are converted from oleanolic acid 3-O- β -glucuronic acid, which is catalyzed by an unknown glucuronic acid transferase (OAGT).

Although oleanane-type saponins are also abundant in other Panax species and their transcriptome information has been reported, glycosyltransferases involved in oleanane-type saponin synthesis have not been identified. Therefore, how to overcome the defects of the prior art is a problem which needs to be solved in the field of plant genetic engineering and biotechnology at present.

Disclosure of Invention

The invention aims to solve the defects of the prior art and provides oleanolic acid glucuronyl transferase, a coding gene and application thereof.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the amino acid sequence of the oleanolic acid glucuronyl transferase is shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO. 4.

The invention also protects the gene for coding the oleanolic acid glucuronyl transferase.

The method specifically comprises the following steps: the coding sequence of the gene is a nucleotide sequence shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO. 8.

Another purpose of the invention is to provide a recombinant vector containing the oleanolic acid glucuronic acid transferase gene, which is constructed by directly connecting the genes shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.8 with different expression vectors. Meanwhile, the invention protects the recombinant gene engineering bacteria obtained by the transformation of the recombinant vector.

The invention also relates to the application of the oleanolic acid glucuronyl transferase in synthesizing oleanane type saponin.

The specific application method comprises the following steps: oleanolic acid glucuronyl transferase is used as a catalyst, and uridine diphosphate glucuronic acid is used as a substrate to catalyze oleanolic acid to generate oleanolic acid 3-O-beta-glucuronic acid.

The applicant of the present invention found that notoginseng root is an ideal material for researching oleanane-type saponin synthesis, and in the present invention, Illumina HiSeq is used^TM2000 sequencing platform establishes transcriptome database of Panax notoginseng, and functionally identifies key steps in biosynthesis of oleanane-type saponin, i.e., glucuronyl transferase encoding enzyme gene catalyzing oleanolic acid to form oleanolic acid 3-O-beta-glucuronic acid.

The invention firstly detects the enzyme activity of 4 screened genes (3 are from panax notoginseng, and 1 is from another panax plant panax japonicus containing oleanane type saponin) for coding oleanolic acid glucuronic acid transferase, and the oleanolic acid glucuronic acid transferase is found to specifically transfer glucuronic acid to the C-3 position of oleanolic acid to form oleanolic acid 3-O-beta-glucuronic acid. The invention provides valuable genetic information for the research of the zingiber officinale roscoe, provides candidate genes for the biosynthesis of the oleanane type ginsenoside and lays a foundation for further gene function identification.

Compared with the prior art, the invention has the beneficial effects that:

1. the oleanolic acid glycosyltransferase can specifically catalyze the glycosylation reaction at the C3 position of oleanolic acid by taking uridine diphosphate glucuronate (UDP-GlcA) as a glycosyl donor.

2. The invention provides valuable genetic information for the research of the zingiber officinale roscoe, provides candidate genes for the biosynthesis of the oleanane type ginsenoside and lays a foundation for further gene function identification.

3. The cloned oleanolic acid glycosyltransferase is an enzyme in a key step in the saponin synthesis process, and has important significance for analyzing the synthesis path of oleanane type saponin, disclosing the molecular mechanism of ginsenoside synthesis in panax plants, further laying a foundation for establishing a medicinal plant triterpene saponin heterologous synthesis system, promoting the biosynthesis of triterpene saponin in the panax plants and improving the yield of the triterpene saponin.

Drawings

FIG. 1 is a graph of UGT cluster analysis involving ginsenoside biosynthesis. Wherein, 6 unigene marked triangles have higher homology with BvUGT73C 10-UGT 73C 13. It was reported that Arabidopsis thaliana UGT73C 10-UGT 73C13 can catalyze the 3-O-glycosylation of oleanane-type saponins, oleanolic acid, and hederagenin (Augustin et al, 2012), and thus it is presumed that these 6 genes may have the effect of catalyzing the 3-O-glycosylation of oleanolic acid.

The numbering of each sequence in the NCBI database is as follows: ginseng, UGTPg101(AKQ76389), UGTPg1(AIE12479), UGTPg100(AKQ76388), PgUGT94Q2(AGR44632), UGTPg29(AKA44579), UGTPg45(AKA44586), PgUGT74AE2(AGR 44631);

american ginseng, Pq3-O-UGT2(ALE 15280);

alfalfa MtUGT71G1(AAW56092), MtUGT73F3(ACT34898), MtUGT73K1(AAW 56091);

rice, OsUGT707A3(BAC83989), OsUGT706C1(BAB68090), OsUGT706D1(BAB68093), OsUGT709A4(BAC80066), OsRUGT-10(BAD 69345);

crocus sativus, CsUGT707B1(CCG85331), CsGT45(ACM66950), CsUGT84A22(ALO19890), CsUGT78A14(ALO 19888);

cowherb seed, SvUGT74M1(ABK 76266);

soybean, GmUGT73F4(BAM29363), GmUGT73F2(BAM29362), GmSGT2(BAI99584), GmSGT3(BAI 99585);

solanum muricatum, SaGT4A (BAD 89042);

arabidopsis, AtUGT73C5(OAP09184), AtUGT73C6(OAP 07438);

arabidopsis thaliana, BvUGT73C10(AFN26666), BvUGT73C11(AFN26667), BvUGT73C12(AFN26668), BvUGT73C13(AFN 26669).

FIG. 2 is a diagram of pCzn1-jzsqUnigene0023775(RC) sequencing validation aligned to the expected sequence.

FIG. 3 shows the result of enzyme cleavage identification of the recombinant plasmid pCzn1-jzsqUnigene0023775(RC), wherein the Marker (Marker): 200,500,800,1200,2000,3000,4500, respectively; m: marking; 1, plasmid before enzyme digestion; 2, cutting the plasmid.

FIG. 4 is a map of a recombinant expression vector; wherein, MCS: a multiple cloning site; TEE: a translation enhancing element; cspA: cold shock proteins; lac: a lactose operon; amp: ampicillin.

FIG. 5 is an SDS-PAGE analysis of protein expression identification; wherein, M is the protein molecular mass standard; 1, no induction; 2, after induction; 3, inducing the crushed supernatant; and 4, precipitating after induced crushing.

FIG. 6 is an SDS-PAGE analysis of protein purification; m is protein molecular mass standard; 1, treating a sample after crushing; 2, flowing out; and 3, eluting.

FIG. 7 is a protein Western Blot identification and analysis diagram; wherein, M is the protein molecular mass standard; 1, purifying the sample.

FIG. 8 shows a ginsenoside biosynthetic pathway predicted from a prior report;

wherein, AACT, acetyl-CoA acetyltransferase; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme a; HMGS, 3-hydroxy-3-methylglutaryl coenzyme A synthetase; HMGR, 3-hydroxy-3-methylglutaryl coenzyme A reductase; MVK, mevalonate kinase; PMK, phosphomevalonate kinase; MVD, mevalonate diphosphate decarboxylase; FPP, farnesyl pyrophosphate; DMAPP, dimethylpropylene diphosphate; DXP, 1-deoxy-D-xylulose 5-phosphate; MEP, methylerythritol phosphate; FPS, farnesyl pyrophosphate synthetase; SS, squalene synthetase; SE, squalene epoxidase; DDS, dammarenediol synthetase; beta-AS, beta-amyrin synthase; CAS, cycloartenol synthase; LAS, lanosterol synthase; PPDS, protopanaxadiol synthase; PPTS, protopanaxatriol synthase; CYP, cytochrome P450; glc, β -D-glucopyranosyl; ara (f), a-L-arabinofuranyl; GlcA, β -D-glucopyranoside; OAGT, oleanolic acid glucuronyl transferase; GlcT-n represents uridine diphosphate saccharide: glycosyltransferase (GlcT) catalyzes the first glycosylation reaction on the nth carbon atom; GlcT-n (2) indicates catalysis of a second glycosylation reaction at the nth carbon atom; UGRdGT, uridine diphosphate glucose: ginsenoside Rd glycosyltransferase.

FIG. 9 shows the key step in the biosynthesis of oleanane-type saponins, i.e., the enzyme glucuronic acid transferase catalyzes oleanolic acid to form oleanolic acid 3-O- β -glucuronic acid.

Fig. 10 shows that 3 unigene highly expressed in the root of zingiber officinale are selected from 6 unigene obtained by clustering analysis and subjected to enzyme activity test, note that: in addition, 1 unigene is derived from panax japonicus, and as can be seen from figure 1, the gene has higher homology with 3 unigenes of panax notoginseng.

FIG. 11 is a graph showing HPLC analysis of Oleanolic Acid Glucuronyl Transferase (OAGT) on the glycosylation at C-3 position of oleanolic acid;

in vitro enzyme activity assays of 4 selected genes encoding glucuronic acid transferase (3 from Panax notoginseng (Burk.) F.H.Chen and 1 from Panax japonicus) were performed using denatured purified oleanolic acid glucuronic acid transferase protein as a control, and it was found that these four recombinant proteins could not specifically transfer glucuronic acid to C-3 position of oleanolic acid to produce oleanolic acid 3-O-. beta. -glucuronic acid, although uridine diphosphate glucose (UDP-Glc) was not used as a sugar donor. In the enzyme activity verification test, ginger-like pseudo-ginseng unigene0023775, unigene0022968 and unigene0004252 are respectively named as PzGAT1, PzGAT2 and PzGAT3, and panax japonicus unigene0053366 is named as PjGAT. In the figure, 1: oleanolic acid control (authetic OA); 2: oleanolic acid 3-O-. beta. -glucuronic acid (alcoholic OAGA); control: comparison; "+" indicates that two substances were involved in the reaction system, such as PzGAT1+ UDP-GlcA, and that the recombinant proteins PzGAT1 and uridine diphosphate glucuronate (UDP-GlcA) were added to the reaction system.

FIG. 12 is a schematic diagram of liquid chromatography-mass spectrometry (LC-MS) of an enzyme activity reaction system of a protein encoded by an oleanolic acid glucuronic acid transferase gene in the process of oleanolic acid glycosylation;

a. and (3) a total ion flow diagram of recombinant protease activity detection. Wherein the oleanolic acid is a substrate oleanolic acid standard product, and the 3-O-beta-glucuronic acid is a product oleanolic acid 3-O-beta-glucuronic acid standard product.

b. Mass spectrum of oleanolic acid uronic acid product in negative ion mode, wherein the upper right figure is oleanolic acid 3-O-beta-glucuronic acid, 631.3850 is [ M-H ] -, 455.3529 is [ M-Gla-H ] - (wherein Gla is glucuronide and H is proton). The lower right figure is oleanolic acid, 455.3529 is [ M-H ] -. The characteristic fragment ions of the saponin components in the ESI negative ion mode are mainly [ M-H ] -, and in the process of secondary mass spectrometry, the saponin is easy to break glycosidic bonds to generate deglycosylated ions.

Detailed Description

The present invention will be described in further detail with reference to examples.

It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The materials or equipment used are not indicated by manufacturers, and all are conventional products available by purchase.

The percentage numbers are percentages by mass unless otherwise indicated.

In order to find oleanolic acid glycosyltransferase (OGAT) genes, genes related to glycosylation are searched in transcriptome data of panax notoginseng and panax beaded, the genes are clustered with glycosyltransferase genes with known functions in other species to construct an evolutionary tree (figure 1), 4 unigene genes which are predicted to have the functions of glycosylation at C-3 site and have high expression at root are found as candidate genes of OGAT to be subjected to enzyme activity detection, the genes comprise unigene0023775, unigene0022968 and unigene0004252 from panax notoginseng and unigene0053366 which are named PzGAT1, PzGAT2, PzGAT3 and PjGAT respectively, the amino acid sequences sequentially correspond to SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, and the nucleotide sequences sequentially correspond to SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO. 8.

The process of protein recombination is illustrated by taking PzGAT1 as an example. The protein recombination process of PzGAT2, PzGAT3 and PjGAT is the same.

Designing a full-length splicing primer by adopting a PCR-based Accurate Synthesis method PAS (polymerase chain reaction), synthesizing a gene ginger-shaped pseudo-ginseng Unigene0023775(RC), which is abbreviated as jzsqUnigene0023775(RC) in the following, and connecting a carrier pCzn1 by enzyme digestion connection; the obtained recombinant plasmid pCzn1-jzsqUnigene0023775(RC) was transferred into TOP10 clone strain and Arctic-Express^TMAnd (3) expressing the strain.

Isopropyl thiogalactoside (IPTG) was used to induce expression of the protein of interest jzsqUnigene0023775 (RC). Optimizing expression condition, adjusting induction condition to 37 deg.C, analyzing target protein mainly in inclusion body form. And (3) re-dissolving the target protein jzsqUnigene0023775(RC) by a renaturation mode, and performing Ni column affinity purification to obtain the target protein.

The specific experimental method and results are as follows:

sequencing validation of pCzn1-jzsqUnigene0023775(RC)

The obtained recombinant plasmid pCzn1-jzsqUnigene0023775(RC) is transferred into a TOP10 clone strain, and the specific transfer method comprises the following steps: (1) adding 1 μ L of recombinant plasmid into 100 μ L of TOP10 competent bacteria, and placing on ice for 20 min; (2) thermally shocking at 42 deg.C for 90sec, and rapidly placing in ice for 5 min; adding 600 μ L LB culture solution; (3) after shaking at 37 ℃ for 1h at 220r/min, the whole was centrifuged and plated on LB plates containing 50. mu.g/mL of ampicillin, and cultured overnight at 37 ℃ in an inverted manner. And (4) picking positive clones for sequencing. The sequencing result is aligned with the expected sequence, and the intercepted alignment sequence is shown in figure 2.

2. Plasmid restriction enzyme identification

2.1 enzyme digestion System

Wherein, the plasmid is: jzsqUnigene0023775

The results of enzyme cleavage identification are shown in FIG. 3. The digested plasmid OD260/OD280: 1.82.

3. Prokaryotic protein expression

3.1 expression identification of prokaryotic proteins

The theoretical molecular weight of the PzGAT1 protein is about 56.857KD (containing histidine tag (His-tag)), and the amino acid sequence is translated as follows:

3.1.1 pCzn1-jzsqUnigene0023775(RC) vector was transformed into E.coli expression system (Arctic Express)

(1) 1. mu.l of pCzn1-jzsqUnigene0023775(RC) plasmid was added to 100. mu.l of competent bacteria (Arctic Express) and placed on ice for 20 min;

(2) thermally shocking at 42 deg.C for 90s, and rapidly placing in ice for 5 min; adding 600 mul LB culture liquid;

(3) after shaking at 37 ℃ for 1h at 220r/min, the whole was centrifuged and plated on LB plates containing 50. mu.g/ml ampicillin (Amp), and the plates were cultured overnight at 37 ℃ in an inverted manner.

3.1.2 IPTG Induction of expression of pCzn1-jzsqUnigene0023775(RC) vector fusion protein

(1) Picking up a single clone on a transformation plate (Amp + LB) and inoculating the single clone in a test tube containing 3ml of LB culture solution of 50 mu g/ml Amp, shaking at 37 ℃ and 220r/min overnight;

(2) inoculating into 30ml LB culture solution containing 50 μ g/ml ampicillin (Amp) at a volume ratio of 1:100 the next day, and shaking at 37 deg.C and 220r/min until thallus OD600 is 0.6-0.8;

(3) taking out 1ml of culture, centrifuging at 10000r/mim for 2min at room temperature, discarding the supernatant, and resuspending the thallus precipitate with 100 μ l of 1 × loading buffer;

(4) adding isopropyl thiogalactoside (IPTG) to the rest culture to a final concentration of 0.5mM, shaking at 37 deg.C and 220r/min for 4h, and inducing fusion protein expression;

(5) 1ml of the culture was removed, centrifuged at 10000 r/mm at room temperature for 2min, the supernatant was discarded, and the pellet was resuspended in 100. mu.l of 1 Xloading buffer. Centrifuging the rest culture at 4000r/mim for 10min, discarding the supernatant, and resuspending the thallus precipitate with phosphate buffer solution with pH 7.4; after the resuspension liquid is subjected to ultrasonic crushing, supernatant and precipitation liquid are respectively taken and added into a sample loading buffer solution for resuspension.

(6) SDS-PAGE gel concentration 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis was performed, and Coomassie brilliant blue staining was observed, and the results are shown in FIG. 5.

3.1.3 analysis of expression identification results

Protein expression was induced by isopropyl thiogalactoside (IPTG), and the target protein was mainly present in the precipitate as analyzed by SDS-PAGE gel electrophoresis at a gel concentration of 12%, as shown in FIG. 5.

3.2 renaturation of Inclusion body proteins

(1) Resuspending the bacterial pellet in 20ml of lysis solution [20mM Tris-HCl (Tris-hydroxymethyl-aminomethane hydrochloride solution) containing 1mM PMSF (phenylmethylsulfonyl fluoride) and protease inhibitor mixture ], pH8.0], and carrying out ultrasonic disruption with ultrasonic power of 400W in an ultrasonic mode: working for 4s, and pausing for 8s for 20 min;

(2) centrifuging the cell lysate subjected to ultrasonic disruption at 4 ℃ and 10000r/mim for 20min, and collecting precipitates (inclusion bodies);

(3) the inclusion bodies were washed 3 times with an inclusion body wash [20mM Tris (Tris hydroxymethyl aminomethane), 1mM EDTA, 2M urea, 1M NaCl, 1% Triton X-100 (polyethylene glycol octylphenyl ether), pH8.0 ];

(4) the inclusion bodies were solubilized with 30ml of solubilization buffer [20mM Tris (Tris hydroxymethyl aminomethane), 5mM DTT (dimercaptothreitol), 8M urea, pH8.0] and left overnight at 4 ℃; centrifuging at room temperature at 10000r/mim for 15 min;

(5) gradually adding dropwise (1ml/min) the supernatant obtained by the previous step into 120ml buffer solution containing 20mM Tris-HCl (Tris hydrochloride solution) and 0.15M NaCl (sodium chloride) and having a pH of 8.0, gradually diluting in a gradient manner (1:4), slowly stirring, and dialyzing the protein solution in 2L buffer solution containing 20mM Tris-HCl (Tris hydrochloride solution) and 0.15M NaCl (sodium chloride) and having a pH of 8.0 overnight.

3.3 Nickel (Ni) column affinity purification and results analysis of fusion proteins

3.3.1 Ni column purification

(1) Loading the supernatant obtained after the centrifugation of the dialysate into a Ni-IDA Binding-Buffer (Ni-iminodiacetic acid Binding Buffer) pre-balanced Ni-IDA-Sepharose (Ni-iminodiacetic acid-Sepharose) CL-6B affinity chromatographic column at the flow rate of 0.5ml/min by using a low-pressure chromatographic system;

(2) flushing with Ni-IDA Binding-Buffer (nickel-iminodiacetic acid Binding Buffer) at a flow rate of 0.5ml/min until the effluent OD280 value reaches the baseline;

(3) washing with Ni-IDA Washing-Buffer [20mM Tris-HCl, 20mM imidazole, 0.15M NaCl, pH8.0] at a flow rate of 1ml/min until the effluent OD280 value reaches baseline;

(4) eluting the target protein with Ni-IDA Elution-Buffer (nickel-iminodiacetic acid Elution Buffer) [20mM Tris-HCl, 250mM imidazole, 0.15M NaCl, pH8.0] at the flow rate of 1ml/min, and collecting the effluent;

(5) the above-collected protein solution was added to a dialysis bag, and dialysis was performed overnight using 2L of a solution containing 20mM Tris-HCl (Tris-hydroxymethyl-aminomethane hydrochloride solution), 0.15M NaCl (sodium chloride), pH 8.0;

(6) the purified protein after dialysis was analyzed by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) with a gel concentration of 12%, and the results are shown in FIG. 4.

3.3.2 analysis of purification results

The inclusion bodies are subjected to renaturation, the target protein is re-dissolved, the target protein is obtained through nickel column affinity purification, and 12% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) analysis is carried out, and the result is shown in FIG. 6, and the corresponding target protein is purified.

3.4 Western Blot (Western Blot) and analysis of results

3.4.1 Western Blot (Western Blot) procedure

(1) The purified dialyzed sample was sampled at 5. mu.l

(2) After the sample loading is finished, the polyacrylamide gel runs out the laminated gel at 90V, and then the voltage is increased to 200V until the electrophoresis is finished.

(3) After electrophoresis, the gel was removed and the membrane was transferred at constant pressure of 100V for 1.5h and constant current of 250mA

(4) After the electrotransfer was completed, the membrane was removed and washed with PBST (phosphate buffered saline containing Tween-20) for 5min 4 times.

(5) The membrane was placed in a 5% (w/v) skim milk powder blocking solution and blocked at 37 ℃ for 1 h.

(6) Primary antibody was diluted with blocking solution and membranes were incubated overnight at 4 ℃ in primary antibody dilution.

(7) The next day, the membrane was removed and washed with PBST (phosphate buffered saline containing Tween-20) for 5min 4 times,

(8) the secondary antibody was diluted with blocking solution containing 5% (w/v) milk. The membrane was reacted in a secondary antibody at 37 ℃ for 1 h.

(9) After the reaction, the membrane was taken out and placed in a clean box to wash the membrane for 4 times, 5min each time.

(10) ECL (electrochemiluminescence) development, exposure. The primary antibody was Anti His mAb ((mouse) Anti His-tag mAb) and the 1:1000 secondary antibody was HRP-goat Anti-rabbit IgG1: 5000. The dilution ratio is shown in Table 1, for example.

TABLE 1

Numbering	First antibody name	Dilution ratio	Name of secondary antibody	Dilution ratio
						1	His	1:1000	Sheep anti rabbit	1:5000

3.4.2 Western Blot (Western Blot) analysis of results

And (3) verifying by western blot, wherein the purified protein is the expressed target protein.

The recombinant protein obtained by the steps is used in a subsequent enzyme activity verification test.

Predicting a possible pathway of saponin synthesis (figure 8), performing an enzyme activity reaction test on the glycosyltransferase by taking a compound in the pathway as a substrate, performing the enzyme activity reaction by respectively taking uridine diphosphate glucose (UDP-glucose) and uridine diphosphate glucuronic acid (UDP-glucuronide) as glycosyl donors and oleanolic acid as glycosyl acceptors, and detecting whether a product is generated by HPLC after the reaction is finished.

Enzyme activity detection conditions: 200 μ L of the mixture contained 25mM TAPS-HCl (pH 8.6), 1mM DTT, 1mM of the receptor substrate oleanolic acid, 5mM UDP-glucose or 5mM UDP-glucuronic acid and 50 μ g of purified recombinase, and the purified marker protein histone was used as a control after denaturation by heating in boiling water for 20 min. After incubating the enzyme reaction system at 30 ℃ for 30min, the reaction was stopped by adding 200. mu.L of methanol. Finally, product identification and detection is performed by High Performance Liquid Chromatography (HPLC) methods.

HPLC determination conditions: the high performance liquid chromatography is completed by an Agilent 1260 series high performance liquid chromatograph. The chromatographic column is Agilent ZORBAX SB-C18(250mm multiplied by 4.6mm,5 μm), the mobile phase is 0.1% phosphoric acid water solution (A) -acetonitrile (B) by mass concentration, and the gradient elution is as follows: 0-10min, 20% B; 10-18min, 50% B; 18-26min, 100% B. Flow rate 1.0 mL/min^-1The detection wavelength is 203nm, the column temperature is 25 ℃, and the injection volume is 10 mu L.

Ultra-high performance liquid chromatography-quadrupole time of flight mass spectrometry (UPLC-QTOF-MS) analysis conditions: the instrument is an IUPLC-QTOF-MS/MS instrument of Waters company in the United states; column was ACQUITY UPLC BEH C18(2.1X100mm, 1.7 μm), mobile phase: a (0.1% formic acid in water by mass), B (acetonitrile), gradient elution: 0-1.5 min, 20% B; 1.5-2.0 min, 35% B; 2.0-4.0 min, 50% B; 4.0-6.0 min, 65% B; 6.0-7.5 min, 85% B; 7.5-8.0 min, 95% B; 8.0-10.0 min, 20% B. Flow rate: 0.5mL/min^-1(ii) a Column temperature: the analysis time is 10min at 35 ℃, the sample injection amount is 1.0 mu L, and an automatic sample injection mode is adopted.

Mass spectrum parameters: the ion scanning mode is ESI negative ion scanning; the scanning range is 100-1200 Da; the ion source temperature is 100 ℃; temperature of the desolventizing gas: 400 ℃; flow rate of desolventizing gas: 800 L.h^-1(ii) a Capillary voltage: 2.5kV (negative ions); taper hole voltage: 40V; low energy collision energy: 6V; high energy collision energy: 30-50V.

As shown in FIG. 11, the results of the reaction of 4 genes PzGAT1, PzGAT2, PzGAT3 and PjGAT recombinant proteins encoding glucuronyl transferase with oleanolic acid as a glycosyl acceptor and uridine diphosphate glucuronate (UDP-GlcA) as a glycosyl donor were examined to show that the product oleanolic acid 3-O-. beta. -glucuronic acid was produced, whereas the reaction with uridine diphosphate glucose (UDP-Glc) as a glycosyl donor was not produced, as compared with the control. The result shows that the oleanolic acid glycosyltransferase acts on the glycosylation modification of oleanolic acid in the saponin synthesis process, so that oleanolic acid is glycosylated at the C3 position to finally form oleanolic acid 3-O-beta-glucuronic acid.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Sequence listing

SEQ ID NO.1

SEQ ID NO.2

SEQ ID NO.3

SEQ ID NO.4

SEQ ID NO.5

SEQ ID NO.6

SEQ ID NO.7

SEQ ID NO.8

Sequence listing

<110> Yunnan university of agriculture

<120> oleanolic acid glucuronyl transferase, and coding gene and application thereof

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 492

<212> PRT

<213> Artificial sequence ()

<400> 1

Met Ser Lys Met Gln Asn Gln Leu His Phe Val Leu Val Pro Leu Leu

1 5 10 15

Ala Gln Gly His Met Ile Pro Met Ile Asp Met Ala Arg Leu Leu Ala

20 25 30

Gln His Gly Val Val Val Ser Leu Val Thr Thr Pro His Asn Ala Ser

35 40 45

Arg Phe Ala Ser Thr Ile His Arg Ala Arg Asp Ser Gly Leu Lys Ile

50 55 60

Gln Leu Ile Gln Ile Pro Phe Pro Trp Gln Glu Val Gly Leu Pro Pro

65 70 75 80

Gly Cys Glu Asn Leu Asp Ser Leu Pro Ser Arg Asp Leu Ile Gly Asn

85 90 95

Phe Phe Ser Ala Leu Asn Lys Leu Gln Gln Pro Leu Glu Gln His Leu

100 105 110

Gln Glu Leu Met Pro Pro Pro Asn Cys Ile Ile Ser Asp Lys Tyr Leu

115 120 125

Ser Trp Thr Thr Lys Thr Ala Gln Lys Phe His Val Pro Arg Leu Val

130 135 140

Phe His Gly Met Cys Cys Phe Ser Leu Leu Ser Ser His Asn Ile Arg

145 150 155 160

Leu Tyr Asn Ala His Leu Ser Val Thr Ser Asp Ser Gln Pro Phe Val

165 170 175

Val Pro Gly Met Pro Gln Arg Val Glu Ile Thr Lys Ala Gln Leu Pro

180 185 190

Gly Ala Phe Val Thr Leu Pro Asp Leu Asp Asp Ile Arg Asp Gln Met

195 200 205

Arg Glu Ala Glu Ser Ser Ala Tyr Gly Val Val Val Asn Ser Phe Ser

210 215 220

Glu Leu Glu Gln Gly Cys Ser Glu Glu Tyr Lys Lys Ala Ile Ala Lys

225 230 235 240

Lys Val Trp Cys Ile Gly Pro Val Ser Leu Cys Asn Lys Asp Asn Leu

245 250 255

Asp Lys Phe Glu Arg Gly Asn Lys Ala Ser Ile Asp Glu Thr His Cys

260 265 270

Thr Glu Trp Leu Asp Ser Met Lys Pro Lys Ser Val Ile Tyr Ala Cys

275 280 285

Leu Gly Ser Gln Cys Arg Leu Val Pro Ala Gln Leu Met Glu Leu Gly

290 295 300

Leu Ala Leu Glu Ser Ser Lys His Pro Phe Ile Trp Val Ile Lys Glu

305 310 315 320

Gly Glu Arg Phe Gln Glu Leu Glu Lys Trp Leu Val Glu Glu Glu Phe

325 330 335

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

340 345 350

Gln Val Leu Ile Leu Ser His Pro Ala Ile Lys Ala Phe Leu Thr His

355 360 365

Cys Gly Trp Asn Ser Thr Ile Glu Gly Val Cys Ser Gly Val Pro Met

370 375 380

Ile Thr Trp Pro Met Phe Ala Glu Gln Phe Phe Asn Glu Lys Leu Ile

385 390 395 400

Val Asp Ile Leu Arg Ile Gly Ile Lys Val Gly Val Gln Val Ser Val

405 410 415

Arg Trp Gly Glu Glu Glu Lys Ile Gly Val Leu Val Lys Arg Glu Gln

420 425 430

Ile Gln Lys Ala Ile Glu Thr Ile Met Asn Gly Gly Gly Glu Glu Gly

435 440 445

Gly Ile Arg Lys Arg Ala Thr Lys Leu Ser Lys Val Gly Ala Arg Ala

450 455 460

Met Glu Asp Gly Gly Ser Ser His Phe Asn Ile Ser Leu Leu Ile Gln

465 470 475 480

Asp Ile Trp Lys Gln Lys Asn Asn Gln Glu Lys Leu

485 490

<210> 2

<211> 486

<212> PRT

<213> Artificial sequence ()

<400> 2

Met Asp Ser Pro Ser Asp Gln Leu His Ile Val Met Ile Pro Leu Met

1 5 10 15

Cys Pro Gly His Leu Ile Pro Met Val Asp Met Ala Lys Leu Leu Ala

20 25 30

Gln Arg Ala Val Thr Val Thr Ile Val Ala Thr Pro Arg Asn Ala Ile

35 40 45

Arg Phe Gly Ala Val Ile Gly Arg Ala Ile Glu Ser Gly Leu Pro Ile

50 55 60

Arg Leu Leu Glu Val Arg Phe Pro Ala Leu Glu Ala Gly Leu Pro Glu

65 70 75 80

Gly Cys Glu Ser Val Asp Asp Leu Pro Ser Leu Ala Met Ser Ile Asn

85 90 95

Phe Phe Ala Ala Thr Lys Met Leu Gln Glu Pro Val Glu Lys Met Leu

100 105 110

Lys Asp Ile Lys Pro Ser Pro Ser Cys Ile Leu Ser Asp Lys His Val

115 120 125

Phe Trp Thr Ser Asp Thr Ala Lys Lys Leu Gln Ile Pro Trp Ile Met

130 135 140

Phe Asp Gly Met Ser Cys Phe Thr Gln Leu Cys Thr Glu Asn Ile Tyr

145 150 155 160

Asn Ser Lys Val His Glu Ser Val Ser Ser Glu Ser Glu Ser Phe Val

165 170 175

Val Arg Gly Leu Pro Asp His Ile Glu Phe Thr Lys Ala Gln Leu Pro

180 185 190

Gly Leu Phe Asn Pro Gly Ser Val Pro Ala Ile Asp Glu Ile Arg Glu

195 200 205

Gln Val Arg Ala Thr Glu Val Gly Ala Tyr Gly Val Val Ile Asn Ser

210 215 220

Phe Glu Glu Leu Glu Gln Asp Tyr Val Asp Glu Phe Lys Lys Val Arg

225 230 235 240

Arg Asp Lys Val Trp Cys Val Gly Pro Leu Ser Leu Asn Asn Glu Asn

245 250 255

Met Leu Asp Lys Ala Gln Arg Gly His Asn Asn Ala Ser Ile Asp Gly

260 265 270

Asn Lys Cys Leu Gln Trp Leu Asp Asn Trp Ala Asn Gly Ser Val Ile

275 280 285

Tyr Ala Cys Leu Gly Ser Ile Ser Ser Leu Thr Cys Thr Gln Leu Met

290 295 300

Glu Leu Ala Leu Gly Leu Glu Ala Ser Glu Arg Pro Phe Val Trp Val

305 310 315 320

Val Arg Ala Gly Gly Lys Gln Lys Glu Ile Glu Lys Trp Ile Leu Glu

325 330 335

Glu Gly Phe Glu Glu Ser Thr Lys Gly Arg Gly Leu Leu Ile Arg Gly

340 345 350

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

355 360 365

Val Thr His Cys Gly Trp Asn Ser Thr Ile Glu Gly Ile Cys Ser Gly

370 375 380

Val Pro Met Ile Thr Trp Pro Leu Phe Ala Glu Gln Phe Phe Asn Glu

385 390 395 400

Lys Leu Val Val Gln Val Leu Glu Thr Gly Val Ser Val Gly Ala Lys

405 410 415

Glu Val Met Pro Leu Gly Glu Glu Glu Lys Phe Gly Val Lys Val Arg

420 425 430

Ser Lys Asn Val Lys Glu Ala Ile Glu Cys Ile Met Leu Glu Gly Lys

435 440 445

Glu Gly Gln Glu Arg Arg Lys Arg Ala Arg Glu Leu Ala Lys Glu Ala

450 455 460

Val Arg Ala Val Glu Glu Gly Gly Ser Ser His Leu Asn Ile Thr Leu

465 470 475 480

Leu Ile Glu Asp Ile Met

485

<210> 3

<211> 497

<212> PRT

<213> Artificial sequence ()

<400> 3

Met Ala Ser Ser Ser Ser Ser Gln Leu Gln Gln Leu His Phe Val Leu

1 5 10 15

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

20 25 30

Arg Leu Phe Ala Gln His Gly Val Ile Val Thr Ile Val Ser Thr Pro

35 40 45

Leu Asn Thr Lys Arg Phe Lys Thr Ile Val Asp Arg Ala Ile Asp Ser

50 55 60

Gly Leu Gln Ile Arg Ile Ile Asp Leu Tyr Phe Pro Ala Ala Glu Ala

65 70 75 80

Cys Leu Pro Arg Gly Cys Glu Asn Met Asp Ser Ile Ser Arg Asn Leu

85 90 95

Ile Lys Asn Phe Phe Met Ala Ser Ser Met Leu Gln Gln Pro Phe Asp

100 105 110

Gln Leu Phe Asp Gln Leu Ser Pro Arg Pro Ser Cys Ile Ile Ser Gly

115 120 125

Lys Asn Gln Ala Trp Thr Val Glu Thr Ala Arg Lys Phe Asn Ile Pro

130 135 140

Arg Leu Phe Phe Asp Gly Met Gly Cys Phe Ser Phe Ser Cys Thr His

145 150 155 160

Asn Leu Lys Met Ser Glu Glu Phe Gln Arg Val Thr Ser Lys Phe Glu

165 170 175

Thr Phe Leu Val Pro Gly Leu Pro His Glu Ile Glu Leu Thr Lys Ala

180 185 190

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

195 200 205

Val Arg Asn Lys Met Thr Ala Ala Glu Ser Ile Ala Asp Gly Ile Ile

210 215 220

Val Asn Ser Phe Glu Glu Leu Glu Pro Glu Tyr Val Glu Met Tyr Thr

225 230 235 240

Lys Val Lys Gly Gly Asn Ile Trp Cys Ile Gly Pro Val Ser Ala Ser

245 250 255

Asn Lys Leu Ile Leu Asp Lys Ala Glu Arg Gly Ser Phe Ala Pro Thr

260 265 270

Glu Asn Glu Ile Gln Cys Leu Glu Trp Leu Asp Leu Gln Glu Pro Asn

275 280 285

Ser Val Val Tyr Ala Cys Leu Gly Ser Ile Ser Gly Leu Thr Ala Ser

290 295 300

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

305 310 315 320

Ile Trp Val Ile Arg Gly Gly Glu Arg Ser Lys Glu Leu Glu Arg Trp

325 330 335

Ile Lys Gln Glu Arg Phe Glu Glu Arg Thr Lys Gly Arg Gly Leu Leu

340 345 350

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

355 360 365

Gly Gly Phe Leu Thr His Cys Gly Trp Asn Ser Thr Leu Glu Gly Val

370 375 380

Ser Ala Gly Lys Pro Ile Ile Ala Cys Pro Leu Phe Ala Glu Gln Phe

385 390 395 400

Tyr Asn Glu Lys Leu Val Val Lys Val Leu Gly Thr Gly Ala Ser Val

405 410 415

Gly Val Glu Ala Ala Val Thr Trp Gly Met Glu Asp Gln Phe Gly Leu

420 425 430

Val Met Lys Arg Glu Asn Val Glu Lys Ala Ile Gln Glu Val Met Asp

435 440 445

Lys Gly Val Glu Ala Glu Glu Arg Arg Lys Arg Ala Arg Glu Phe Gly

450 455 460

Asp Met Ala Lys Arg Ala Ile Glu Glu Gly Gly Ser Ser Tyr Leu Asn

465 470 475 480

Ile Arg Ser Leu Ile Gln His Val Lys Glu Lys Asn Glu Leu Lys His

485 490 495

Ala

<210> 4

<211> 498

<212> PRT

<213> Artificial sequence ()

<400> 4

Met Ser Gln Ser Pro Ala Met Ser Lys Met Gln Asn Gln Leu His Phe

1 5 10 15

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

20 25 30

Met Ala Arg Leu Leu Ala Gln His Gly Val Val Val Ser Leu Val Thr

35 40 45

Thr Pro His Asn Ala Ser Arg Phe Ala Ser Thr Ile His Arg Ala Arg

50 55 60

Asp Ser Gly Leu Lys Ile Gln Leu Ile Gln Ile Pro Phe Pro Cys Gln

65 70 75 80

Glu Val Gly Leu Pro Pro Gly Cys Glu Asn Leu Asp Ser Val Pro Ser

85 90 95

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

100 105 110

Pro Leu Glu Gln His Leu Gln Glu Leu Met Pro Pro Pro Ser Cys Ile

115 120 125

Ile Ser Asp Lys Tyr Leu Ser Trp Thr Thr Lys Thr Ala Glu Lys Ile

130 135 140

His Val Pro Arg Leu Val Phe His Gly Met Cys Cys Phe Ser Leu Leu

145 150 155 160

Ser Ser His Asn Ile Arg Leu Tyr Asn Ala His Leu Ser Val Thr Ser

165 170 175

Asp Ser Gln Pro Phe Val Val Pro Gly Met Pro Gln Arg Val Glu Ile

180 185 190

Thr Lys Ala Gln Leu Pro Gly Ala Phe Val Thr Leu Pro Gly Leu Asp

195 200 205

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

210 215 220

Val Val Asn Ser Phe Ser Glu Leu Glu Gln Gly Cys Ser Glu Glu Tyr

225 230 235 240

Lys Lys Ala Ile Ala Lys Lys Val Trp Cys Ile Gly Pro Val Ser Leu

245 250 255

Cys Asn Lys Asp Asn Leu Asp Lys Phe Glu Arg Gly Asn Lys Ala Ser

260 265 270

Ile Asp Glu Thr Leu Cys Thr Glu Trp Leu Asp Ser Met Lys Pro Lys

275 280 285

Ser Val Ile Tyr Ala Cys Leu Gly Ser Gln Cys Arg Leu Val Pro Ala

290 295 300

Gln Leu Met Glu Leu Gly Leu Ala Leu Glu Ser Ser Lys His Pro Phe

305 310 315 320

Ile Trp Val Ile Lys Glu Gly Glu Arg Phe Gln Glu Leu Glu Lys Trp

325 330 335

Leu Val Glu Glu Glu Phe Glu Glu Arg Ile Lys Arg Arg Gly Leu Leu

340 345 350

Ile Lys Gly Trp Ala Pro Gln Val Leu Ile Leu Ser His Pro Ala Ile

355 360 365

Lys Ala Phe Leu Thr His Cys Gly Trp Asn Ser Thr Ile Glu Gly Val

370 375 380

Cys Ser Gly Val Pro Met Ile Thr Trp Pro Met Phe Ala Glu Gln Phe

385 390 395 400

Phe Asn Glu Lys Leu Ile Val Asp Ile Leu Arg Ile Gly Ile Lys Val

405 410 415

Gly Val Gln Val Ser Val Arg Trp Gly Glu Glu Glu Lys Ile Gly Val

420 425 430

Leu Val Lys Arg Glu Gln Ile Gln Lys Ala Ile Glu Thr Ile Met Asn

435 440 445

Gly Gly Gly Glu Glu Gly Gly Ile Arg Lys Arg Ala Thr Lys Leu Ser

450 455 460

Lys Val Gly Ala Arg Ala Met Glu Asp Gly Gly Ser Ser His Phe Asn

465 470 475 480

Ile Ser Leu Leu Ile Gln Asp Ile Trp Lys Gln Lys Asn Asn Gln Glu

485 490 495

Lys Leu

<210> 5

<211> 1476

<212> DNA

<213> Artificial sequence ()

<400> 5

atgtccaaaa tgcaaaacca gcttcacttt gtgctagtac cattactggc tcaaggccac 60

atgatcccaa tgatagacat ggccagacta ctcgcgcaac atggcgtcgt agtaagcttg 120

gtcaccaccc ctcacaacgc ctccagattc gcctcaacaa ttcaccgcgc aagagattct 180

gggcttaaaa ttcaactcat acaaattcca ttcccctggc aagaagtagg gcttccacca 240

ggatgtgaaa atctcgacag cctgccttcc cgagacctta taggaaactt ttttagtgct 300

cttaataaat tacaacagcc tctagaacag cacctgcaag aactaatgcc ccctccaaac 360

tgcataatct cggacaagta cctttcgtgg acaacaaaaa cagcccaaaa attccatgtt 420

ccaagattag tttttcatgg gatgtgctgt ttttcactac taagttccca taatataagg 480

ctttataatg ctcacttatc tgtcacttca gactcccaac cttttgtggt gccaggaatg 540

ccccaaaggg ttgaaataac caaagcccag ctaccaggag catttgttac attgccagat 600

ttggatgata ttcgtgatca aatgcgcgag gccgaatcaa gcgcttatgg ggttgtggtg 660

aacagcttca gtgagcttga gcaggggtgt tccgaagagt acaaaaaggc cattgcgaaa 720

aaagtttggt gcattggacc ggtttctcta tgtaacaagg ataatttaga caaatttgag 780

agaggaaaca aagcttcaat cgatgagaca cactgcaccg aatggcttga ttcaatgaaa 840

ccgaaatctg taatctacgc ttgtttgggt agccaatgca ggcttgtacc agcacaactt 900

atggaacttg ggttggcatt agaatcatca aaacatccat ttatttgggt gattaaagaa 960

ggggaaaggt ttcaagaatt ggagaagtgg ttagtagagg aggaatttga agagagaatc 1020

aaaaggaggg ggcttttgat caaggggtgg gctccacaag ttctcattct ttctcacccg 1080

gcaatcaagg cttttttaac tcattgcggg tggaattcaa ccattgaagg agtttgctct 1140

ggtgtgccaa tgataacatg gccgatgttt gctgagcaat ttttcaatga aaaattaata 1200

gttgatattt taaggattgg gatcaaagtc ggggttcaag tctctgttag atggggagaa 1260

gaagaaaaga ttggagtttt ggttaagagg gagcaaattc agaaagctat agaaacaata 1320

atgaatgggg gaggagaaga aggaggtata agaaagaggg cgacaaagct ttcaaaagtt 1380

ggcgcaaggg caatggaaga tggagggtcc tctcatttca acatatcact gttgattcaa 1440

gatatctgga aacagaagaa caatcaagaa aagttg 1476

<210> 6

<211> 1458

<212> DNA

<213> Artificial sequence ()

<400> 6

atggattcac cgtcagacca gcttcacatt gttatgatac ccttaatgtg cccaggccac 60

cttattccca tggtggacat ggccaaattg ctagcacaac gtgccgtgac cgtcaccatt 120

gtcgccacac cccgtaatgc catccgtttc ggtgcagtca tcggacgtgc catcgaatct 180

ggcctgccga ttcgactcct cgaagtccgt tttccggcct tggaggccgg cttgccggag 240

ggatgcgaaa gcgtggacga tttaccctct ttggctatgt ctataaattt ttttgctgca 300

accaaaatgc tacaagagcc ggtggaaaaa atgttaaaag atataaagcc tagtccaagc 360

tgcatactat ctgacaagca tgttttctgg acgtctgata ctgccaaaaa actccaaatt 420

ccgtggatta tgtttgatgg aatgagttgc ttcacgcaat tatgtacgga aaatatatac 480

aattccaagg tgcacgaaag tgtgtcgtcc gagtcagagt cctttgttgt gcgtggtctg 540

cctgatcata tcgagttcac taaagcccag ctgcccggat tattcaatcc ggggtcagta 600

cctgcgattg atgagatccg tgaacaggta agagctactg aagtaggagc atatggggtt 660

gtgattaata gttttgagga gttggaacaa gattatgttg atgaatttaa aaaagtccga 720

agagataaag tttggtgtgt tggtccgtta tcactcaaca acgagaacat gttggacaag 780

gctcagagag gacataataa tgcgtcgatt gatggaaaca agtgcttaca atggcttgac 840

aattgggcaa atggctccgt catttatgcc tgccttggaa gcataagcag ccttacttgt 900

acacaactca tggagcttgc tctgggctta gaagcatcgg aacgtccgtt tgtatgggtg 960

gtaagggcag ggggcaaaca aaaagagatc gagaaatgga tattggaaga gggattcgag 1020

gaaagtacga aagggagagg gcttttgatc cggggttggg cgccacaagt gcttatattg 1080

tcacaccctg caattggagg attcgtaacg cattgtggtt ggaattcgac tattgaaggg 1140

atttgtagcg gcgttcccat gatcacttgg cctttgtttg ccgagcaatt tttcaatgag 1200

aagttggtag tgcaggtact tgagactggc gtgagtgttg gggctaaaga agttatgcct 1260

ttgggtgagg aagagaagtt cggggtgaaa gtgaggagta agaatgtgaa agaggctata 1320

gagtgcataa tgcttgaagg aaaagaaggt caagagagaa gaaaaagagc tagggagctt 1380

gcaaaagagg ctgtcagggc agttgaagag ggaggatctt ctcaccttaa tatcactctt 1440

ttaattgagg acattatg 1458

<210> 7

<211> 1494

<212> DNA

<213> Artificial sequence ()

<400> 7

atggcctcct cctcctcctc acagcttcaa cagctccact ttgttctcat acccctaatg 60

tcccctggcc accttatgcc tattgtagac atggccagat tatttgccca acatggagtc 120

attgtcacaa tagtcagtac tcccctcaac actaaaagat tcaaaactat tgtggatcgc 180

gcaattgatt caggccttca aatccgaatt attgaccttt attttcctgc agctgaagcc 240

tgtttgcctc ggggatgcga aaacatggat tccatttcac ggaatttgat caagaatttc 300

tttatggcat ctagcatgtt acaacaacca tttgaccaac tatttgatca actaagcccc 360

cgcccgagct gcataatttc gggcaaaaat caagcatgga cagttgagac tgcccggaag 420

tttaacattc caagactttt tttcgatggg atgggctgtt tttctttttc atgtacacat 480

aatttaaaga tgtccgagga atttcagcgt gtcacctcca aatttgagac ctttttggtg 540

cctggtttgc cacatgaaat tgagttgact aaagcccagc taccggagtc tctgaatcca 600

ggcgggtcag gagatctgat tgatgttcgt aacaagatga ccgcagccga gtcgatagcg 660

gatgggatta tagtcaattc attcgaggaa ttagaacccg aatatgtgga aatgtataca 720

aaggtgaaag gtggtaacat ttggtgcatt ggccctgttt cggcttctaa caagttgatc 780

ttggacaagg ctgaaagagg cagtttcgcc cctacagaaa atgagattca atgcttggag 840

tggcttgatt tacaagagcc aaactcagtt gtttatgctt gtttaggcag catcagtggc 900

ctaacagctt cacaactcgt ggagctcggg ttaggcttgg aagcatcaaa aaggccgttt 960

atttgggtga taagaggagg ggagagatca aaagagctgg agagatggat taaacaagag 1020

cgattcgaag agaggactaa agggagggga cttttggtac gaggctgggc gcctcagtta 1080

ctaatcctgt cccattcgtc caccggaggc ttcttaacgc actgcggttg gaattcaacg 1140

ctcgaaggtg tgtccgcggg taagcccata attgcttgtc ctttgtttgc cgagcaattt 1200

tataatgaga agttggtggt caaagtattg ggaactgggg cgagtgtggg agttgaggct 1260

gctgtgacat ggggaatgga agatcagttt gggttggtaa tgaagagaga gaatgtagaa 1320

aaggcaatac aagaggtaat ggataaagga gtagaagcag aagaaagaag aaaaagagca 1380

agagagtttg gggatatggc aaaaagggcg atagaagaag gaggatcttc ttaccttaac 1440

attagaagtt taatccaaca tgtcaaggaa aagaatgaac ttaagcatgc atga 1494

<210> 8

<211> 1494

<212> DNA

<213> Artificial sequence ()

<400> 8

atgtcccaaa gtccagcaat gtccaaaatg caaaaccagc ttcactttgt gctagtccca 60

ttactggctc aaggccacat gatcccaatg atagacatgg ccaggctact cgcgcaacat 120

ggcgtcgtag taagcttggt caccacccct cacaacgcct ccagattcgc ctcaacaatt 180

caccgcgcaa gagattctgg gcttaaaatt caactcatac aaattccatt cccctgccaa 240

gaagtagggc ttccaccagg atgtgaaaat ctcgacagcg tgccttcccg agaccttata 300

ggaaactttt ttagtgctct taataaatta caacagcctc tagaacagca cctgcaagaa 360

ctaatgcccc ctccaagctg cataatctcg gacaagtacc tttcgtggac aacaaaaaca 420

gccgaaaaaa tccatgttcc aagattagtt tttcatggga tgtgctgttt ttcactacta 480

agttcccata atataaggct ttataatgcc cacttatctg tcacttcaga ctcccaacct 540

tttgtggtgc caggaatgcc ccaaagggtt gaaataacca aagcccagct accaggagca 600

tttgttacat tgccaggttt ggatgatatt cgtgatcaaa tgcgcgaggc cgaatcaagc 660

gcttatgggg ttgtggtgaa cagcttcagt gagcttgagc aggggtgttc cgaagagtac 720

aaaaaggcca ttgcgaaaaa agtttggtgc attggaccag tttctctatg taacaaggat 780

aatttagaca aatttgagag aggaaacaaa gcttcaatcg atgagacact ctgcaccgaa 840

tggcttgatt caatgaaacc gaaatctgta atctacgctt gtttgggtag ccaatgcagg 900

cttgtaccag cacaacttat ggaacttggg ttggcattag aatcatcaaa acatccattt 960

atttgggtga ttaaagaagg ggaaaggttt caagaattgg agaagtggtt agtagaggag 1020

gaatttgaag agagaatcaa aaggaggggg cttctgatca aggggtgggc tccacaagtt 1080

ctcattcttt ctcacccggc aatcaaggct tttttaactc attgcgggtg gaattcaacc 1140

attgaaggag tttgctctgg tgtgccaatg ataacatggc cgatgtttgc tgagcaattt 1200

ttcaatgaaa aattaatagt tgatatttta aggattggga tcaaagttgg ggttcaggtc 1260

tctgttagat ggggagaaga agaaaagatt ggagtcttgg ttaaaaggga gcaaattcag 1320

aaagctatag aaacaataat gaatggggga ggagaagaag gaggtataag aaagagggcg 1380

acaaagcttt caaaagttgg cgcaagggca atggaagatg gagggtcctc tcatttcaac 1440

atatcactct tgattcaaga tatctggaaa cagaagaaca atcaagaaaa gtta 1494

Claims

1. The oleanolic acid glucuronyl transferase is characterized in that the amino acid sequence of the oleanolic acid glucuronyl transferase is shown as SEQ ID NO. 3.

2. A gene encoding the oleanolic acid glucuronyltransferase of claim 1.

3. The gene of claim 2, wherein the coding sequence of the gene is the nucleotide sequence shown in SEQ ID No. 7.

4. A recombinant vector comprising the gene according to claim 2 or 3.

5. A recombinant genetically engineered bacterium transformed with the recombinant vector of claim 4.

6. The use of oleanolic acid glucuronic acid transferase of claim 1 in the synthesis of oleanane-type saponin, wherein oleanolic acid glucuronic acid transferase is used as a catalyst, and uridine diphosphate glucuronic acid is used as a substrate to catalyze oleanolic acid to produce oleanolic acid 3-O- β -glucuronic acid.