CN113736758B - A kind of BpOMT1 gene of petracea oxymethyltransferase and its application in the preparation of 4-methoxygallic acid - Google Patents

Abstract

The invention discloses a rock cabbage oxygen methyltransferase BpOMT1 gene and its application in the preparation of 4-methoxygallic acid. The sequence of the rock cabbage oxygen methyltransferase BpOMT1 gene is shown in SEQ ID NO:1. Using gallic acid and S-adenosyl-methionine as raw materials, 4-methoxygallic acid is generated under the catalysis of BpOMT1, which is of great significance for the study of the regulation of the biosynthesis of petracenin.

Description

A kind of BpOMT1 gene of rock cabbage oxygen methyltransferase and its application in the preparation of 4-methoxygallic acid

technical field

The invention belongs to the field of biological technology, and in particular relates to a gene of rock cabbage oxygen methyltransferase BpOMT1 and its application in the preparation of 4-methoxygallic acid.

Background technique

Rock cabbage (Bergenia purpurascens) is a plant of the genus Bergenia in the saxifrage family. There are 10 species of this genus in Asia, except for 3 species produced in East Asia, northern South Asia and southeastern Central Asia, they are mainly distributed in Shaanxi (Qinling), Xinjiang, Sichuan, Yunnan and Tibet in China. The whole plant of the genus of the genus Bructus contains bagenin, especially the content of 8.2% of the material extracted from the rhizome of the genus Bacillus. Bergenin has various effects such as antitussive, anti-inflammatory, anti-anxiety, anti-oxidation, anti-malarial, anti-cancer, treatment of diabetes, anti-hepatotoxicity, immune regulation and neuroprotection (Shihao Xiang,. BergeninExerts Hepatoprotective Effects by Inhibiting the Release of Inflammatory Factors, Apoptosis and Autophagy via the PPAR-γ Pathway. Drug Des Devel Ther. 2020; 14:129-143.). Scholars at home and abroad have also found that it is different from morphine-like antitussives that inhibit the central nervous system. Petgenin has a selective inhibitory effect on the cough center and has no inhibitory effect on other nerve centers. It has less toxic and side effects, fewer adverse reactions, and continuous use does not produce drug resistance. Sex and other characteristics (Wang Jin, Human and animal pharmacokinetics of petracenin and its effect on IGABA. Shandong University, 2010.).

Brutin is an isocoumarin compound, and the biosynthetic precursor of Brutin is 2-glucose-4-methoxygallic acid via rearrangement after intramolecular dehydration or in the unknown dehydratase (dehydratase) Under the action of closed-loop generation (Gan B.Bajracharya.Diversity,pharmacology and synthesis of bergenin and its derivatives:potential materials for therapeutic usages.Fitoterapia.2015;101:133-52.;); 2-glucose-4-methoxygalloc The acid is generated from 4-methoxygallic acid under the catalysis of carbon glycosyltransferase (CGT, C-glucosyltransferase) using uridine diphosphate glucose (UDP-glucose) as the sugar donor to connect glucose at the 2-position; 4- Methoxygallic acid (4-O-Methylgallic acid, 4-O-ME-GA) is derived from gallic acid (Gallic acid, GA), and is converted to S by gallic acid oxygen methyltransferase (OMT, O-methyltransferase) -Adenosyl-methionine (SAM, S-adenosyL-methionine) is catalyzed by a methyl donor. Therefore, the biosynthesis of 4-methoxygallic acid is of great significance for the study of the regulation of petracenin biosynthesis.

In recent years, due to disorderly excavation and other reasons, the wild resources of rock cabbage medicinal materials are decreasing day by day. Carrying out the research on the biosynthetic pathway of petracenin can provide a basis for artificial cultivation measures and increase the content of medicinal components in petracenin, and relieve the pressure on wild resources. At the same time, the planting period of the raw materials for the extraction of petogenin is long, and the requirements for planting plots and planting techniques are relatively high. Therefore, how to efficiently obtain these useful secondary metabolites has always been a problem that scientific researchers think about and study. For high value-added natural products, the establishment of homologous or heterologous expression systems to efficiently produce medicinal active ingredients using modern biotechnology has been widely considered to be an important technical means to solve the shortage of medicinal resources in the future. However, in order to find out the biosynthetic pathways of these active ingredients, it is necessary to identify the key genes related to these pathways, and discovering these catalytic enzyme genes has become a key link in the study of the biosynthetic pathways of plant metabolites. At present, the synthetic pathway of gallic acid to catalyze the methylation reaction of the C-4 hydroxyl group to form 4-methoxygallic acid is not clear, and the function of the oxymethyltransferase responsible for methylation has not been verified, which affects rock The advancement of the work on the biosynthesis of brassicin.

Contents of the invention

In order to solve the technical problem of how to obtain a large amount of high-purity 4-methoxygallic acid biosynthesis, the present invention provides a BpOMT1 gene of rock cabbage oxygen methyltransferase, which can be used as a biosynthesis regulation gene of 4-methoxygallic acid and Applied to the preparation of 4-methoxy gallic acid.

The invention provides a rock cabbage oxygen methyltransferase BpOMT1 gene, the nucleic acid sequence of which is shown in SEQ ID NO: 1, and the full length of the sequence is 1077bp.

The present invention also provides a rock cabbage oxygen methyltransferase BpOMT1, which is encoded by the rock cabbage oxygen methyltransferase BpOMT1 gene and encodes 358 amino acid residues, the amino acid series of which are shown in SEQ ID NO: 2 Show.

The invention also provides a recombinant plasmid containing the BpOMT1 gene of the rock cabbage oxygen methyltransferase.

Preferably, the recombinant plasmid is obtained by homologous recombination of the above-mentioned Pecticola oxymethyltransferase BpOMT1 gene and the pET28a vector, and is named pET28a-BpOMT1.

The present invention also provides a genetically modified engineered bacterium, the genetically engineered bacterium contains the above-mentioned recombinant plasmid, or the genome of the genetically engineered bacterium is integrated with the exogenous BpOMT1 gene.

Preferably, the genetically engineered bacteria is Escherichia coli BL21 (DE3) strain.

The present invention also provides the application of the rock cabbage oxygen methyltransferase BpOMT1 in the preparation of 4-methoxygallic acid.

As a preference, in the application of the rock cabbage oxygen methyltransferase BpOMT1 in the preparation of 4-methoxy gallic acid, gallic acid and methyl donor S-adenosyl-methionine are used as raw materials, in Under the catalysis of the rock cabbage oxygen methyltransferase (amino acid series as shown in SEQ ID NO: 2) encoded by the above-mentioned rock cabbage oxygen methyltransferase BpOMT1 gene, at the C-4 of gallic acid (Gallic acid) A methylation reaction is carried out on the hydroxyl group at the position to generate 4-methoxygallic acid (4-O-Methylgallic acid, 4-O-ME-GA).

The present invention also provides a primer for cloning the BpOMT1 gene of the rock cabbage oxygen methyltransferase, the primer is composed of primer F and primer R, and the nucleotide sequence of the primer F is shown in SEQ ID NO: 3 , the nucleotide sequence of the primer R is shown in SEQ ID NO: 4.

The present invention obtains the target protein (Babca oxymethyltransferase BpOMT1) after in vitro expression through the recombinant plasmid, and further catalyzes the substrate gallic acid to directly generate 4-methoxy gallic acid.

The rock cabbage oxygen methyltransferase BpOMT1 gene of the present invention is identified from the rhizome of rock cabbage through transcriptome sequencing and bioinformatics technology after a large number of experiments; Extract the RNA from the rhizome of rock cabbage, reverse transcribe it into cDNA, and then amplify it by PCR.

Compared with prior art, the beneficial effect of the present invention:

(1) With the rapid development of bioinformatics technology, the mining of key enzyme genes in the biosynthesis pathway of 4-methoxygallic acid has been greatly promoted. The biosynthesis regulation gene of 4-methoxygallic acid in the present invention, that is, the oxygen methyltransferase BpOMT1 gene, is identified and successfully verified for the first time, and a new biosynthetic method for producing 4-methoxygallic acid has been developed. The invention obtains the target product through heterologous expression and in vitro enzyme catalysis, adopts in vitro biosynthesis for directional production, and has the advantages of less by-products and the like.

(2) The present invention provides recombinant plasmids, genetically engineered bacteria and recombinant proteins containing the BpOMT1 gene of B. chinensis, for the large-scale synthesis of 4-methoxygallic acid by bioengineering methods, and further for the biosynthesis of B. Laying the foundation for regulatory research.

(3) Through in vitro biosynthesis of 4-methoxygallic acid, the controllability is strong, and the demand for raw material planting can be reduced, and the production product is single, which is convenient for the separation and purification of 4-methoxygallic acid in the later stage; it can also reduce chemical Difficult synthesis, complex synthesis path and other issues. The rock cabbage oxygen methyltransferase BpOMT1 gene is a key gene for the biosynthesis of 4-methoxygallic acid, and can also be studied by plant breeding of rock cabbage and the like.

(4) The BpOMT1 gene isolated and identified from B. chinensis in the present invention can be used as an important marker gene for molecular assisted breeding of B. chinensis, and can also be used as a marker gene for the production of 4-methoxy in yeast chassis cell construction. An important candidate gene for gallic acid.

In the sequence listing, SEQ ID NO: 1 shows the nucleotide sequence of the BpOMT1 gene of the rock cabbage oxygen methyltransferase.

In the sequence listing, SEQ ID NO: 2 shows the amino acid series of BpOMT1 of the rock cabbage oxygen methyltransferase BpOMT1.

SEQ ID NO: 3 in the sequence listing is the nucleotide sequence of primer F.

Shown in SEQ ID NO: 4 in the sequence listing is the nucleotide sequence of primer R.

SEQ ID NO: 5 in the sequence listing is the nucleotide sequence of the upstream homology arm primer.

SEQ ID NO: 6 in the sequence listing is the nucleotide sequence of the downstream homology arm primer.

Description of drawings

Figure 1 is a schematic diagram of the deduced synthetic route of 4-methoxygallic acid.

Fig. 2 is a schematic diagram of the construction of the recombinant expression plasmid pET28a-BpOMT1 (used to express the BpOMT1 gene of B. cabbage oxygen methyltransferase).

Fig. 3 is the result of electrophoresis detection after the recombination of the BpOMT1 gene of the rock cabbage oxygen methyltransferase. Among them, M is the nucleic acid marker, and 1, 2, and 3 are the detection results of three positive single colonies.

Fig. 4 is the SDS-PAGE protein electrophoresis detection diagram of BpOMT1 of B. cabbage oxygen methyltransferase. Among them, M: protein molecular mass standard; lanes 1, 2, 3, 4, 5, 6, and 7 are precipitation, flow-through eluent, 20mmol/L imidazole eluent, 30mmol/L imidazole eluent, 50mmol/L imidazole elution solution, 100mmol/L imidazole elution solution, 250mmol/L imidazole elution solution under protein.

Fig. 5 is HPLC detection of the methylation effect of oxygen methyltransferase BpOMT1 on the C-4 hydroxyl group of gallic acid (Gallic acid). Figure 5A: The estimated path of methylation on the hydroxyl group at the C-4 position of gallic acid (Gallic acid, GA); Figure 5B: The result of the enzymatic reaction of the rock cabbage oxygen methyltransferase BpOMT1, the abscissa is time, unit min; the ordinate is the response value, and the unit is mAU; among them, CK: the enzyme activity of the control group (gallic acid + S-adenosyl-methionine + inactivated rock cabbage oxygen methyltransferase BpOMT1) enzyme inactivation Reaction result; standard substance: gallic acid standard substance + 4-methoxygallic acid standard substance; BpOMT1: the enzyme of the experimental group (gallic acid + S-adenosyl-methionine + rock cabbage oxygen methyltransferase BpOMT1) live reaction results.

Fig. 6 is the mass spectrometry analysis (LC/MS/MS) collection of illustrative plates of standard substance, and wherein, Fig. 6-A is total ion chromatogram;

Figure 6-B shows the retention time of the standard gallic acid, 15.96 minutes; Figure 6-C, the retention time of the standard 4-methoxygallic acid, 23.26 minutes.

Fig. 7 is the mass spectrometry analysis (LC/MS/MS) collection of illustrative plates of enzyme activity verification reaction product, and wherein, Fig. 7-A is total ion chromatogram; Fig. 7-B is the retention time 15.45 minutes of substrate gallic acid; Fig. 7- C is the retention time of the reaction product 4-methoxygallic acid, 23.86 minutes.

Figure 8 is a fragment ion diagram (theoretical molecular weight 184) (LC/MS/MS) of the standard product 4-methoxygallic acid.

Fig. 9 is a fragment ion diagram (theoretical molecular weight 184) (LC/MS/MS) of the reaction product 4-methoxygallic acid.

Detailed ways

Example 1

Based on the basic function annotation information of the transcriptome Unigene of P. chinensis, the OMT candidate genes were screened in the sequencing annotation results. At the same time, the oxygen methyltransferase (OMT) identified in the plant was analyzed by sequence-local BLAST, and then the screening results were sorted out. Finally, an oxygen methyltransferase (OMT) gene was found. After a series of work such as cDNA preparation, candidate gene amplification and recovery, homologous recombination, protein expression, in vitro enzyme activity reaction, and HPLC and LC/MS/MS detection, it was finally identified that it can catalyze gallic acid (Gallic acid). acid) The methylation reaction on the hydroxyl group at the C-4 position generates the target candidate oxygen methyltransferase BpOMT1 gene of 4-methoxygallic acid ( FIG. 1 ). The steps of each stage of 4-methoxygallic acid synthesis are as follows (the following reagents, raw materials, instruments and equipment, etc. are all commercially available):

(1) Preparation of cDNA template

Fresh samples of rock cabbage rhizomes were taken, sliced and then quick-frozen in liquid nitrogen for total RNA extraction. The total RNA was extracted using the HiPure Plant RNA Mini Kit kit from Magen (Guangzhou Meiji Biotechnology Co., Ltd.), and the total RNA was extracted according to the operation steps of the kit. Reverse transcribed into cDNA and stored at -20°C for future use.

(2) Gene amplification and recovery

Utilize primer design software (CE Design) v1.04, design the primer of amplifying rock cabbage oxygen methyltransferase BpOMT1 gene, described primer is by primer F (SEQ ID NO: 3) and primer R (SEQ ID NO: 4) The composition is operated according to the instruction manual of the high-fidelity KOD enzyme, and the KOD high-fidelity enzyme is used to perform gene amplification using the cDNA of rock cabbage as a template. The PCR reaction program was: 94°C, 5min; 94°C, 30S, 58°C, 50S, 72°C, 1min, 35 cycles; 72°C, 7min. After the PCR, run the gel to confirm the success of the amplification and recover the target band. For gene extraction and gel extraction, the EasyPureQuick Gel Extraction Kit kit from Beijing Quanshijin Biotechnology Co., Ltd. was used for target gene recovery. After recovery, measure its recovery concentration on a NanoReady ultra-micro-ultraviolet-visible spectrophotometer, and finally store it in a refrigerator at -20°C for future use, and obtain the BpOMT1 gene fragment of the rock cabbage oxygen methyltransferase. After sequencing, its nucleic acid sequence is shown as SEQ ID NO: 1 shown.

In addition, when carrying out homologous recombination of the BpOMT1 gene fragment with the vector homology arm and the vector pET28a (the homology arm is Escherichia coli pET28a), the BpOMT1 gene with the same The primer (being the homology arm primer) of source wall is amplified and reclaimed, and described homology arm primer is by upstream homology arm primer (shown in SEQ ID NO: 5 in sequence listing) and downstream homology arm primer (sequence listing The composition shown in SEQ ID NO: 6), using the BpOMT1 gene of Bacillus oxymethyltransferase as a template, operated according to the instruction manual of the high-fidelity KOD enzyme, and performed PCR amplification again to obtain BpOMT1 with carrier homology arms Gene fragment of base transferase BpOMT1.

Upstream homology arm primer: atgggtcgcggatcc ATGGCTCCACAAAATGAAGCAG (SEQ ID NO: 5).

Downstream homology arm primers:

acggagctcgaattcggatccCTACTTCAAGAATTCCATGATATAAGAATTGAAAGC (shown in SEQ ID NO: 6).

The lowercase letters in the above-mentioned upstream homology arm primer (SEQ ID NO: 5) and downstream homology arm primer (shown in SEQ ID NO: 6) represent pET28a homology arms, and capital letters represent the amplified rock cabbage oxygen methyltransferase BpOMT1 Gene primer sequence.

(3) Construction and identification of gene recombination vector

See Figure 2 for a schematic diagram of homologous recombination. First, the vector pET28a was linearized, and the linearized vector was obtained by single enzyme digestion with BamH I enzyme. In the case of homologous recombination, assemble according to the operating instructions of the homologous recombination enzyme, and then calculate the dosage of each component according to the concentration of the BpOMT1 gene fragment inserted into the homologous arm of the carrier and the pET28a vector, and follow the recombination instructions ; Finally, each component was added to a PCR reaction tube on ice, and the gene of B. cabbage oxygen methyltransferase BpOMT1 was homologously recombined with the pET28a vector to obtain a recombinant plasmid, which was named pET28a-BpOMT1. After assembly, the results were detected and sent to the company for sequencing. The electrophoresis detection results after assembly are shown in Figure 3, indicating that the assembly was successful. Reorganize operations as follows:

Table 1 Candidate Gene Recombination Reaction System

Among them, X=(0.02×pET28a base log) ng/concentration of linearized pET28a ng/μL; Y=(0.02×pET28a base log) ng/recovery concentration of rock cabbage oxygen methyltransferase BpOMT1 ng/μL , the inserted gene fragment is a gene fragment inserted into the BpOMT1 gene fragment of the cabbage oxygen methyltransferase with the homology arm of the vector.

(4) SDS-PAGE protein electrophoresis detection

After a small protein expression test, the protein induction conditions of BpOMT1 were determined to be: 17°C, 0.1mM IPTG, 220r/min, induced for 12h; then shaken, collected bacteria, broken the wall, and centrifuged at a high speed (12000r/min) Obtain the protein supernatant, and then use SDS-PAGE protein electrophoresis and detection. The detection results are shown in Figure 4, which shows that the BpOMT1 protein can be eluted and purified in the 250mmol/L imidazole eluent.

(5) Enzyme activity reaction

The enzymatic activity of BpOMT1 was determined by methylation reaction to synthesize 4-methoxygallic acid in a 1.5mL centrifuge tube. The mixture in the experimental sample reaction system contains: 2 microliters of 100mM S-adenosyl-methionine, 2 microliters of 100mM gallic acid, 40 micrograms of purified rock cabbage oxygen methyltransferase BpOMT1, adding 50mM Tris-HCl buffer (pH 8.0 ) to a total volume of 100 μL, the total volume of the reaction system is 100 μL. After incubating at 31° C. for 2 hours, an equal volume of 1M hydrochloric acid was used to terminate the reaction, centrifuged briefly (12000 r/min), and the supernatant was taken. Finally, the reaction product was detected by HPLC and LC-MS/MS analysis.

Control group (CK) reaction system: 2 microliters of 100mM gallic acid, 2 microliters of 100mM S-adenosyl-methionine, 40 micrograms of inactivated purified rock cabbage oxygen methyltransferase BpOMT1, adding 50mM Tris-HCl buffer solution (pH 8.0) to a total volume of 100 μL, and the total volume of the reaction system is 100 μL.

Standard products: 50 microliters of 10mM gallic acid standard substance, 50 microliters of 10mM 4-methoxygallic acid standard substance.

(6) Product detection

HPLC detection conditions are as follows:

The instrument used for HPLC detection is Agilent 1290 ultra-high performance liquid chromatography. The chromatographic column is XBridge ShieldRP18 (4.6mm×250mm, 5μm), column temperature: 30°C; the mobile phase for the determination of 4-methoxygallic acid is 0.01% v/v formic acid aqueous solution (A)-acetonitrile (B), gradient elution : 0～8min, 1%～5%B; 8～13min, 5%～10%B; 13～20min, 10%～20%B; 20～25min, 20%～45%B; 25～35min, 45% %～90%B; 35～40min, 90%～90%B; Elution time: 40min; Injection volume: 10 microliters; Flow rate: 0.6ml/min; Detection wavelength: 230nm. The test results are shown in Fig. 5, which shows that the experimental sample produced 4-methoxygallic acid under the catalysis of BpOMT1.

LC-MS/MS detection conditions are as follows:

In order to further confirm the reaction products detected by HPLC, Agilent 1290UPLC/6540Q-TOF liquid chromatography-mass spectrometry (LC/MS/MS) was used for detection: mass spectrometry conditions: the ion source is in negative ion mode, voltage: 3500V; Fragmentation voltage: 135V; cone voltage: 60V; RF voltage: 750V, scanning range: 100-1000m/z, scanning method: SRM. Chromatographic conditions: chromatographic column is XBridge Shield RP18 (4.6mm, × 250mm, 5μm), column temperature: 30°C, mobile phase for determination of 4-methoxygallic acid is 0.01% v/v formic acid aqueous solution (A) - acetonitrile (B ), gradient elution: 0～8min, 1%～5%B; 8～13min, 5%～10%B; 13～20min, 10%～20%B; 20～25min, 20%～45%B; 25～35min, 45%～90%B; 35～40min, 90%～90%B; Elution time: 40min; Injection volume: 10 microliters; Flow rate: 0.6ml/min; Detection wavelength: 230nm.

The test results are shown in Figures 6 to 9. It can be seen from the results that the peak elution time and characteristic fragment ions of the product are consistent with the standard 4-methoxygallic acid, confirming that the reaction product is 4-methoxygallate acid. Finally, it was concluded that the oxymethyltransferase BpOMT1 had the ability to catalyze the methylation of the oxygen on the C4 position of gallic acid to generate 4-methoxygallic acid.

sequence listing

<110> Yunnan Agricultural University

<120> A BpOMT1 Gene of Oxygen Methyltransferase and Its Application in the Preparation of 4-Methoxygallic Acid

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<170> SIPOSequenceListing 1.0

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Ser Ala Ile Glu Leu Asp Leu Leu Glu Ile Met Ala Lys Ser Gly Pro

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Gly Ala Tyr Met Ser Pro Ile Asp Ile Ala Ser Gln Leu Pro Thr Asn

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Asn Pro Asp Ala Pro Val Met Leu Asp Arg Ile Leu Arg Leu Leu Ala

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Cys Tyr Ser Val Leu Thr Cys Ser Val Arg Asn Leu Pro Asp Gly Arg

85 90 95

Val Glu Arg Leu Tyr Gly Leu Ala Pro Val Cys Lys Tyr Leu Thr Lys

100 105 110

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

115 120 125

Lys Ile Leu Met Glu Ser Trp Tyr His Leu Lys Asp Ala Val Leu Asp

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Gly Gly Ile Pro Phe Asn Lys Ala Tyr Gly Met Ser Ala Phe Glu Tyr

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His Gly Thr Asp Pro Arg Phe Asn Lys Val Phe Asn Arg Gly Met Ser

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Asp His Ser Thr Ile Thr Met Lys Lys Ile Leu Glu Thr Tyr Lys Gly

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Phe Glu Gly Leu Thr Ser Val Val Asp Val Gly Gly Gly Thr Gly Ala

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Thr Leu Asn Met Ile Leu Ser Lys Tyr Pro Asn Ile Arg Gly Ile Asn

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Phe Asp Leu Pro His Val Ile Glu Asp Ala Pro Ser Tyr Pro Gly Val

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Glu His Val Gly Gly Asp Met Phe Val Ser Val Pro Lys Gly Asp Ala

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Ile Phe Met Lys Trp Ile Cys His Asp Trp Ser Asp Glu His Cys Leu

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<213> Rock cabbage (Bergenia purpurascens)

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<213> Rock cabbage (Bergenia purpurascens)

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<213> Rock cabbage (Bergenia purpurascens)

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<213> Rock cabbage (Bergenia purpurascens)

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Claims (9)

1. a rock cabbage oxygen methyltransferase BpOMT1 gene, its nucleotide sequence is as shown in SEQ ID NO: 1. 2. A rock cabbage oxygen methyltransferase BpOMT1, the amino acid series of which is shown in SEQ ID NO: 2. 3. A recombinant plasmid containing the BpOMT1 gene of the rock cabbage oxygen methyltransferase according to claim 1. 4. The recombinant plasmid according to claim 3, characterized in that, the recombinant plasmid is obtained by homologous recombination of the rock cabbage oxygen methyltransferase BpOMT1 gene described in claim 1 and the pET28a vector, named pET28a-BpOMT1 . 5. A genetically modified engineered bacterium, characterized in that it contains the recombinant plasmid according to claim 3 or 4, or the genome of the genetically modified engineered bacterium is integrated with the oxymethyl transfer of the rock cabbage described in claim 1 Enzyme BpOMT1 gene. 6. The genetically modified engineered bacterium according to claim 5, characterized in that, the genetically modified engineered bacterium is Escherichia coli BL21 (DE3) strain. 7. The application of the rock cabbage oxygen methyltransferase BpOMT1 described in claim 2 in the preparation of 4-methoxygallic acid. 8. application according to claim 7, is characterized in that, with gallic acid and S-adenosyl-methionine as raw material, under the catalysis of rock cabbage oxygen methyltransferase BpOMT1 described in claim 2, This produces 4-methoxygallic acid. 9. a kind of primer of cloning claim 1 BpOMT1 gene of rock cabbage oxygen methyltransferase, is characterized in that, described primer is made up of primer F and primer R, and the nucleotide sequence of described primer F is as SEQ ID As shown in NO: 3, the nucleotide sequence of the primer R is shown in SEQ ID NO: 4.

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