CN111411099A - Snow bile acetyltransferase and its encoding gene and its application in the preparation of snow bile A - Google Patents

Abstract

The invention discloses a gallbladder acetyltransferase, its encoding gene and its application in preparing gallbladder A. The gallbladder acetyltransferase has an amino acid sequence as shown in SEQ ID No. 1. The present invention identifies and obtains the key enzyme in the synthetic pathway of seglanin A for the first time, that is, seglanyl acetyltransferase; the segmentol acetyltransferase can convert the hydroxyl group at the C-25 position of seglantoin into acetyl group, thereby obtaining cholestyramine; based on this, the present invention constructs a recombinant vector containing the acetyltransferase encoding gene of leucanthemum, and further constructs a genetically engineered bacterium, and utilizes the transgenic engineering bacterium to express acetyltransferase of leucanthemum in vitro The enzyme, after further catalyzing the substrate sebacillin, can directly obtain sever bilirubin, without the need to extract sever bilirubin after planting selenium plants; it provides an important way for the large and rapid preparation of serobilis. .

Description

Snow bile acetyltransferase and its encoding gene and its application in the preparation of snow bile A

technical field

The invention belongs to the technical field of biotechnology, and in particular relates to a snow bile acetyltransferase, an encoding gene thereof and an application in the preparation of snow bile A.

Background technique

Hemsleya chinensis Cogn (Hemsleya chinensis Cogn) is a plant of the Cucurbitaceae Hewsleva Cogn. There are 31 species of Hemsleya genus in the world. Except for 2 species produced in India and Vietnam, the others are mainly distributed in Yunnan, China, China. Southwest regions such as Sichuan and Guizhou.

Studies have found that cucurbitacin and other triterpenoid saponins are rich in the tubers of the genus Scutellariae, especially Scutellarin extracted from this genus plant (the mixture of Scutellarin A and Scetobilis B, which is a mixture of Scutellarin and Saccharin). It has been developed into "Snow Biliformin Tablets", which exerts various effects such as clearing heat and detoxifying, antibacterial and anti-inflammatory, and is commonly used in clinical treatment of bacillary dysentery, enteritis, bronchitis and acute tonsillitis and other diseases.

In addition, domestic and foreign scholars have also found that Cucurbitacin IIa has obvious cytotoxic effects. Cucurbitacin can destroy the actin cytoskeleton by inhibiting the downstream survivin of JAK2/STAT3, and guide cells to generate PARP mediated apoptosis, thereby inhibiting the proliferation of tumor cells, which makes snow bilirubin a new class of anticancer drugs.

At present, snow bilirubin is mainly extracted from the tubers of the genus Snow biliary. In recent years, due to the disorderly excavation by humans, the wild resources of snow gallbladder medicinal materials are decreasing day by day. In order to relieve the pressure of wild resources, people have gradually carried out artificial cultivation of the genus Pyrex, but the planting cycle of P. The work was not going well.

How to efficiently obtain these useful secondary metabolites has always been a problem that researchers think and study. For high value-added natural products, using modern biotechnology to establish homologous or heterologous expression systems to efficiently produce medicinal active ingredients has been widely regarded as an important technical means to solve the shortage of medicinal resources in the future. However, to understand the biosynthetic pathways of these active ingredients, it is necessary to identify the key genes related to these pathways, and the discovery of these catalytic enzyme genes has become a key link in the study of the biosynthetic pathways of plant metabolites.

It is generally believed in the academic community that cerebilin A is obtained by acetylation of the hydroxyl group at the C25 position of cerebilin. The function of acetyltransferase has not been verified, which affects the progress of the biosynthesis of snow bilirubin.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a cucurbitacyltransferase and its encoding gene and application in the preparation of cucurbitacin A, the cucurbitacin acetyltransferase can convert Cucurbitacin IIb (Cucurbitacin IIb) in vitro The hydroxyl group at the C-25 position is converted into an acetyl group to obtain cerebilin A, which provides an important way for the mass preparation of cerebilin A.

For realizing the above-mentioned purpose of the invention, the technical scheme of the present invention is as follows:

The snow gall acetyltransferase of the present invention has the amino acid sequence shown in SEQ ID No.1.

The present invention identifies and obtains the key enzyme in the synthesis pathway of seglanin A for the first time: seglanyl acetyltransferase, which can convert the hydroxyl group at the C-25 position of sables into acetyl base, thereby obtaining seleuronan.

Further, the present invention identifies and obtains the encoding gene of snow gall acetyltransferase in snow gall for the first time, and the encoding gene has the nucleotide sequence shown in SEQ ID No. 2.

In order to improve the yield of stevia, and reduce the dependence of the production of stevia on the genus stevia, the present invention provides a recombinant vector containing the encoding gene of the above steat acetyltransferase; and, expressing the above steat acetyl Transgenic engineering bacteria of transferase.

In the present invention, there are two ways in which the encoding gene of snow bile acetyltransferase exists in the transgenic engineering bacteria, one of which: the above-mentioned encoding gene containing snow bile acetyl transferase is included in the transgenic engineering bacteria The second is: the genome of the transgenic engineering bacteria is integrated with the encoding gene of the snow bile acetyltransferase.

The present invention also provides the application of the above-mentioned transgenic engineering bacteria expressing sebile acetyltransferase in the preparation of seglanin A.

Preferably, the application includes: using the transgenic engineering bacteria to produce scaphole acetyltransferase, and then using the scapile acetyltransferase to prepare scapolin A.

The present invention also provides the application of the above-mentioned sequoia acetyltransferase in the preparation of sequoia A.

Preferably, the application includes: using sebacin and an acetyl donor as raw materials, using the selenium acetyltransferase as a catalyst, and reacting at 28-35° C. for 20-40 min to obtain selephane white.

In the invention, the transgenic engineering bacteria can be used to express the gallbladder acetyltransferase in vitro, and the substrate gallbladder can be further catalyzed to directly obtain the gallbladder, without the need to extract the gallbladder after planting the gallbladder plant. . The present invention also provides the application of the above-mentioned gene encoding snow gall acetyltransferase as a molecular marker for assisted breeding of snow gall.

Given that the gene encoding snow gall acetyltransferase is a key gene for plant synthesis of gallbladder A, the gene encoding gall gall acetyltransferase can be used as an important marker gene for molecularly assisted breeding of gall bladder, and can also be used as a yeast chassis cell construct. An important candidate gene for the production of snow bilirubin.

Compared with the prior art, the beneficial effects of the present invention are embodied in:

The present invention identifies and obtains the key enzyme in the synthesis pathway of seglanin A for the first time: seglanyl acetyltransferase, which can convert the hydroxyl group at the C-25 position of sables into acetyl Based on this, the present invention constructs a recombinant vector containing the encoding gene of cholestasis, and further constructs a genetically engineered bacterium, and uses the transgenic engineering bacterium to express cholestylin in vitro , after further catalyzing the substrate bilirubin, directly obtaining bilirubin, without the need to extract bilirubin after planting the genus bilmobilis; it provides an important way for the preparation of bilbilate in large quantities and quickly.

Description of drawings

Fig. 1 is the synthetic schematic diagram that the sable acetyltransferase of the present invention catalyzes sable bile to generate sable cholin A;

Wherein, Cucurbitacin IIb represents cerebilin B, Cucurbitacin IIa represents cerebilin A, and HCAT1 represents cerebilin acetyltransferase of the present invention, the same below;

Fig. 2 is the electrophoresis detection result figure of the recombinant plasmid pET32a-HCAT1 containing the gene encoding snow bile acetyltransferase of the present invention;

M represents Marker protein standard, the same below;

Fig. 3 is the structural representation of the recombinant plasmid pET32a-HCAT1 containing the gene encoding snow bile acetyltransferase of the present invention;

Fig. 4 is the result of protein electrophoresis detection of snow bile acetyltransferase HCAT1 prepared by the present invention;

Fig. 5 is the Western blot detection result diagram of snow bile acetyltransferase HCAT1 prepared by the present invention;

Fig. 6 is the HPLC detection result diagram of the enzymatic activity reaction of snow bile acetyltransferase HCAT1 of the present invention;

Among them, Control means blank control, and standard means standard product;

Fig. 7 is the characteristic ion peak figure of snow bilirubin;

Among them, Mass-to-Charge (m/z) represents the mass-to-charge ratio (m/z), the same below;

Fig. 8 is the characteristic ion peak figure of snow bilirubin;

Fig. 9 is the result diagram of the enzymatic activity reaction FC-MS detection of snow bile acetyltransferase HCAT1 of the present invention;

Among them, Substrate represents the reaction substrate, Product represents the reaction product, and Retention time (min) represents the peak time (minutes).

Detailed ways

The technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

In this example, based on the basic functional annotation information of the snow gall transcriptome Unigene, the candidate genes of BAHD type acetyltransferase (BAHD-AT) were screened in the sequencing annotation results. Genes, mainly using CsACT identified in cucumber (Cucumis sativus) and CmACT identified in melon (Cucumis melo) as clues, through local BLAST analysis of the sequence, and then sorting and analyzing the screening results, and finally found 2 in the snow gall transcriptome. BAHD-AT gene. After a series of work such as cDNA preparation, candidate gene amplification and recovery, homologous recombination, protein expression, in vitro enzymatic reaction, and HPLC and LC/MS detection, a series of work was finally identified that can catalyze Cucurbitacin (Cucurbitacin). IIb) Acetylation of the hydroxyl group at the C-25 position to generate the target candidate acetyltransferase HCAT1 gene of sevbilin A (Fig. 1).

Specifically include the following steps:

(1) Preparation of cDNA template

Fresh samples of snow gall tubers were taken, sliced and snap-frozen in liquid nitrogen, and RNA extraction was performed with the HiPure Plant RNA Mini Kit kit from Magen (Guangzhou Meiji Biotechnology Co., Ltd.). After the extracted RNA was qualified, the RNA was reverse transcribed into cDNA using TAKARA reverse transcription kit, and stored at -20°C for future use.

(2) Gene amplification and recovery

Use primer design software (CE Design) v1.04 to design amplification primers for the acetyltransferase HCAT1 gene:

Upstream primer: 5'-ATGGAGATGCCATTGAAAGTG-3' (SEQ ID No. 3);

Downstream primer: 5'-CTAAACTTGAATGACATTAGGATTTATGG-3' (SEQ ID No. 4);

In order to facilitate the homologous recombination of the acetyltransferase HCAT1 gene obtained by amplification with the vector pET32a, it is necessary to add a homology arm to the primer (the homology arm is Escherichia coli pET32a); namely:

Upstream primer: 5'-ccatggctgatatcggatccATGGAGATGCCATTGAAAGTG-3' (SEQ ID No. 5);

Downstream primers:

5'-tgtcgacggagctcgaattcCTAAACTTGAATGACATTAGGATTTATGG-3' (SEQ ID No. 6);

Gene amplification was then performed using KOD high-fidelity enzyme.

The PCR reaction system was: 94°C, 5min; 94°C, 30s, 62°C, 50S, 72°C, 1min, 35 cycles; 72°C, 7min.

After PCR, run the gel to confirm that the amplification is successful and then recover the target band. Gene Gel Extraction The target gene was recovered using the EasyPure Quick Gel Extraction Kit from Beijing Quanshijin Biotechnology Co., Ltd. After recovery, the recovered concentration was measured on a NanoReady ultra-trace UV-Vis spectrophotometer, and finally stored in a -20°C refrigerator for later use.

(3) Construction and identification of gene recombination vector

First, use BamH I enzyme to perform single digestion to obtain a linearized pET32a vector; then perform homologous recombination according to the following reaction system, and add each component to a PCR reaction tube on ice:

Wherein X=(0.02×pET32a base pairs)ng/Linearized pET32a concentration ng/μL; Y=(0.02×pET32a base pairs)ng/HCAT1 recovery concentration ng/μL;

After the recombination was completed, the results were detected and sent to the company for sequencing. The electrophoresis detection results after the assembly were shown in Figure 2, indicating that the assembly was successful; the structural schematic diagram of the obtained recombinant plasmid pET32a-HCAT1 is shown in Figure 3.

(4) SDS-PAGE protein electrophoresis and western blot detection

After the protein expression test, it was determined that the induction conditions of acetyltransferase HCAT1 were: 17 °C, 0.1 mM IPTG, 220 r/min, induction for 12 h; then shaken vigorously, collected bacteria, broken the wall, and obtained the protein after high-speed centrifugation The supernatant was then electrophoresed and detected by SDS-PAGE. The test results are shown in Figure 4.

To further confirm the target protein, immunoblotting was performed. Because the N-terminus of acetyltransferase HCAT1 carries a His-tag, a high-purity mouse-derived monoclonal antibody His-Tag antibody (GenStar, Shenzhen, China) should be used, and the antibody should be blocked and incubated according to the requirements of WB detection.

First, dilute the primary antibody at a dilution of 1:2000 in PBST and 5% w/v skim milk, inoculate the membrane loaded with transfer protein, incubate for 1 h at room temperature or overnight at 4 °C, recover the primary antibody mixture, and then add Incubate in PBST buffer for 20 min (40 rpm) on a shaker at room temperature, and then use rabbit anti-mouse immunoglobulin G-horseradish peroxidase (IgG-HRP)-conjugated secondary antibody (GenStar, Shenzhen, China) at 1 : 5000 dilution with PBST and 5% w/v skim milk, shake at room temperature for 1 h; finally, discard the secondary antibody mixture, shake with 20 mL of 1×PBST for 10 min, repeat 3 times, and pass StarSignal high-sensitivity chemiluminescence The luminescent substrate provided by the detection kit (GenStar, Shenzhen, China), and then the membrane was exposed to X-ray film for visual luminescent detection of the conjugated secondary antibody. The detection results are shown in Figure 5, indicating that the supernatant protein of the target gene was obtained.

(5) Enzyme activity reaction

The activity of the acetyltransferase HCAT1 was determined by synthesizing cucurbitacin IIa (ie, icicle A) through an acetylation reaction in a 1.5 mL centrifuge tube.

The reaction system contained: 40 mM acetyl CoA, 400 μM substrate cebilin, 50 mM sodium phosphate buffer (pH 7.5), and 40 μg purified acetyltransferase HcAT1, and the total volume of the reaction system was 100 μL.

After incubation at 31° C. for 30 min, the reaction mixture was extracted three times with an equal volume of ethyl acetate, followed by brief centrifugation, and the supernatant was collected and concentrated. The product was dissolved in methanol and finally analyzed by HPLC and LC-MS for reaction product detection.

(6) Product detection

HPLC detection conditions are as follows:

The instrument used for HPLC detection was an Agilent 1290 ultra-high performance liquid chromatograph. The chromatographic column is Phenomenex Kinetex C18 analytical column (4.6×100mm, 2.6μm), and the mobile phase for the determination of selebilins (including selebilins A and B) is 0.2% phosphoric acid aqueous solution (A)-acetonitrile (B), gradient Elution: 0～15min, 25%～33%B; 15～20min, 33%～40%B; 20～24min, 40%～60%B; 24～28min, 60%～90%B; detection wavelength 212nm . The detection results are shown in Figure 6, indicating that there is snow bilirubin in the reaction product.

LC-MS detection conditions are as follows:

In order to further confirm the reaction product detected by HPLC, Agilent 1290 UPLC/6540 Q-TOF liquid chromatography mass spectrometer (LC/MS) was used for detection, and the detection method was as follows:

Mass spectrometry conditions: The ion source adopts positive ion mode, voltage: 3500V; fragmentation voltage: 135V; cone voltage: 60V; radio frequency voltage: 750V, scanning range: 100-1000m/z;

Chromatographic conditions: the chromatographic column is Phenomenex Kinetex C18 analytical column (4.6×100mm, 2.6μm), the mobile phase for the determination of snow bilirubin is 0.2% phosphoric acid aqueous solution (A)-acetonitrile (B), gradient elution: 0～15min, 25 %～33%B; 15～20min, 33%～40%B; 20～24min, 40%～60%B; 24～28min, 60%～90%B; detection wavelength 212nm. The test results are shown in Figure 7, Figure 8 and Figure 9.

It can be seen from the results in Figure 9 that the peak times of the substrate Cucurbitacin IIb and the reaction product Cucurbitacin IIa in the reaction solution are significantly different from those of the Cucurbitacin standard and Cucurbitacin IIa. The peak time of the standard product is consistent; it is further confirmed that the generated product is cerebilin A.

sequence listing

<110> Yunnan Agricultural University

<120> Snow bile acetyltransferase and its encoding gene and its application in the preparation of snow bile A

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Val Gly Gly Gly Asp Cys Glu Asp Ala Asn Glu Arg Ser Arg Ser Leu

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Lys Val Ser Leu Ser Asp Glu Leu Thr Arg Phe Tyr Pro Leu Ala Gly

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Arg Val Lys Glu Asp Asn Glu Ser Ile Phe Cys Asn Asp Glu Gly Ala

85 90 95

Ile Tyr Val Glu Ala Lys Ala Asn Cys Leu Leu Ser Asp Phe Leu Asn

100 105 110

Gln Leu Glu Ile Asp Ser Leu Asn Asp Phe Leu Pro Phe Asp Ser Ala

115 120 125

Lys Gly Cys Ile Leu Leu Val Gln Ile Thr Ser Phe Glu Cys Gly Gly

130 135 140

Met Ala Ile Gly Leu Leu Met Ser His Lys Ile Ser Asp Ala Ser Ser

145 150 155 160

Ile Ser Ala Phe Ile Lys Ser Trp Thr Ala Thr Ser Arg Gly Cys Lys

165 170 175

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

180 185 190

Pro Pro Gln Asp Phe Pro Ile Ser Thr Pro Thr Ser Asp Ser Gly Ile

195 200 205

His Ala Lys Gly Val Thr Lys Arg Leu Val Phe Glu Ala Ser Lys Ile

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Ile Glu Leu Lys Ala Lys Ala Thr Ser Ala Thr Val Lys Gln Pro Thr

225 230 235 240

Arg Val Glu Ala Val Thr Gly Leu Ile Trp Lys Cys Ala Ile Ala Ala

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Claims (10)

1. A hemsleya amabilis acetyl transferase is characterized by having an amino acid sequence shown as SEQ ID No. 1.

2. The gene encoding cucurbitaceyltransferase of claim 1.

3. The gene encoding cucurbitacin acetyltransferase of claim 2, having a nucleotide sequence shown as SEQ id No. 2.

4. A recombinant vector comprising a gene encoding the cucurbitaceyltransferase of claim 2 or 3.

5. A genetically engineered bacterium expressing the cucurbitacin acetyltransferase of claim 1.

6. The genetically engineered bacterium expressing cucurbitaceyltransferase of claim 5, comprising the recombinant vector containing the gene encoding cucurbitaceyltransferase of claim 4;

alternatively, the gene encoding the cucurbitacin acetyltransferase of claim 2 or 3 is integrated into the genome.

7. The use of the genetically engineered bacterium of claim 5 or 6 expressing cucurbitaceyltransferase in the preparation of cucurbitacin.

8. The use of the cucurbitacin acetyltransferase of claim 1 in the preparation of cucurbitacin.

9. The use of cucurbitacin acetyltransferase of claim 8 in the preparation of cucurbitacin, comprising:

reacting at 28-35 deg.C for 20-40min with hemsleyadin and acetyl donor as raw materials and hemsleyadin acetyltransferase as catalyst to obtain cucurbitacin.

10. The use of the gene encoding cucurbitacin acetyltransferase of claim 2 or 3 as a molecular marker for assisted breeding of cucurbitacin.

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