Ardisia crenata carbon glycosyl transferase AcCGT64 gene and application thereof in preparation of bergenin

Abstract

The invention relates to an ardisia crenata carbon glycosyl transferase AcCGT64 gene and application of the ardisia crenata carbon glycosyl transferase AcCGT64 gene in preparation of bergenin, and belongs to the technical field of biology. The nucleotide sequence of the ardisia crenata carbon glycosyl transferase AcCGT64 gene is as shown in SEQ ID NO.1, and the total length of the sequence is 1353bp; the amino acid sequence of the encoded protein is shown as SEQ ID NO.2, and 450 amino acid residues are encoded. The ardisia crenata carbon glycosyl transferase AcCGT64 gene disclosed by the invention can be used as a biosynthesis regulation gene of bergenin, is applied to preparation of bergenin, and is remarkable in application prospect and easy to popularize and apply.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a cinnabar root carbonyl transferase AcCGT64 gene and an application thereof in preparing bergenin. Background Art

[0002] Ardisia crenata Sims is a plant of the genus Ardisia in the family Primulaceae. This genus is distributed in China, India, Myanmar, the Malay Peninsula, Indonesia and Japan; in China, it is distributed from southeastern Tibet to Taiwan, Hubei to Hainan Island and other regions. There are many medicinal ingredients in the cinnabar root plant, and bergenin is one of the main active ingredients. Bergenin has attracted wide attention due to its various pharmacological activities, such as antitussive, anti-inflammatory, antianxiety, antioxidant, antimalarial, anticancer, treatment of diabetes, anti-hepatotoxicity, immunomodulation and neuroprotection. Unlike morphine-like central inhibitory cough suppressants, bergenin has a selective inhibitory effect on the cough center and has no inhibitory effect on other nerve centers. It has the characteristics of small toxic side effects, few adverse reactions, and no drug resistance after continuous use. Bergenin has been made into a variety of dosage forms and is widely used in China to treat respiratory diseases, such as compound bergenin tablets and bergenin capsules.

[0003] The precursor of bergenin biosynthesis is 2-glucose-4-methoxygallic acid, which is generated by intramolecular dehydration, rearrangement and ring closure; 2-glucose-4-methoxygallic acid is generated by 4-methoxygallic acid connecting glucose at the 2-position with uridine diphosphate glucose (UDP-glucose) as the glycosyl donor under the catalysis of carbon glycosyltransferase (CGT); 4-methoxygallic acid is catalyzed by gallic acid oxygen methyltransferase (OMT) with gallic acid as substrate and S-adenosyl-methionine (SAM) as the methyl donor; gallic acid is catalyzed by shikimate dehydrogenases (SDH) with NADP+ as the proton donor.

[0004] With the increasing number of chronic bronchitis diseases, the gap in raw materials for bergenin is increasing. At present, the raw materials for bergenin on the market are mainly obtained by extraction, and the annual demand for dry medicinal materials for extracting bergenin has reached more than 2,000 tons. The extensive mining in recent decades has caused a sharp decline in wild resource reserves and is on the verge of exhaustion. At the same time, the high altitude, many mountainous areas and cold climate conditions in the origin make it difficult to cultivate artificially on a large scale, further expanding the gap in the market for bergenin raw materials and causing prices to rise year by year. In recent years, with the rapid development of the field of synthetic biology, the use of synthetic biology technology to produce natural drug monomers can effectively solve the above problems. However, to understand 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 studying the biosynthetic pathways of plant metabolites. Currently, the synthesis pathway of 4-methoxygallic acid catalyzing the glycosylation reaction at the C-2 position to form 2-glucose-4-methoxygallic acid has been clarified, but the function of the carbon glycosyltransferase responsible for glycosylation has been less verified, which has affected the advancement of the biosynthesis of bergenin. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a cinnabar root carbon glycosyltransferase AcCGT64 gene, which can be used as a biosynthesis regulatory gene of bergenin and can be used in the preparation of bergenin.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a cinnabar root carbon glycosyltransferase AcCGT64 gene, the nucleic acid sequence of the cinnabar root carbon glycosyltransferase AcCGT64 gene is shown in SEQ ID NO.1, and the full length of the sequence is 1353 bp.

[0008]

[0009] The second aspect of the present invention provides a protein encoded by the above-mentioned cinnabar root carbon glycosyltransferase AcCGT64 gene, the amino acid sequence of the protein is shown in SEQ ID NO.2, encoding 450 amino acid residues.

[0010] MSNSGNQEPRPHIALLPSAGMGHLNPLLRLTATLAARGCHVTVITPTPTFTAAESEHMDEFFSTYPNISRLKFQIVGDEPPSEFDPPVFLRWQSINRSGHILRPLLTALSPPI TAIFSDFVAGLCICPIADDLKIPNYFVVTSSARFFSVWAFLAYTNHIDDEILIPGLSPFHKSTIPQPFFNPKQLFTKLATSNAPLLSKAKGILLNTFDYFEPDTITAIRKGTAL TDLPQVFPIGPLKDYKFGKGNYPTWLDDQEEGSVVYVSFGSRTAVSRDQIWELGDGGLERSGFPFLWVLKTSKEDDVELRDLLGDTFFEKTGKKGIVVKGWADQEAIMAHPAIGG FVSHCGWNSVTEATRYGVPILAWPQHGDQRLNAEVLEKAGMGVWERDWGWQGERLIKGEEIGEKIEKMMTDEKLKTQAKKIRDEAKKALEANGSSEKALSGLITTVKKN; (SEQ ID NO.2)

[0011] The third aspect of the present invention provides a recombinant plasmid containing the above-mentioned cinnabarinaceous root carbon glycosyltransferase AcCGT64 gene.

[0012] Preferably, the recombinant plasmid is obtained by homologous recombination of the above-mentioned cinnabarinaceous root carbon glycosyltransferase AcCGT64 gene with the pET28a vector and is named pET28a-AcCGT64.

[0013] The fourth aspect of the present invention provides a transgenic engineering bacterium, which contains the recombinant plasmid, or the genome of the genetic engineering bacterium is integrated with the exogenous cinnabarinaceous root carbonyl transferase AcCGT64 gene.

[0014] Preferably, the genetically modified engineered bacteria is the Escherichia coli BL21 (DE3) strain.

[0015] The fifth aspect of the present invention provides an application of a cinnabar root carbonyl transferase AcCGT64 gene in the preparation of bergenin.

[0016] Preferably, 4-methoxygallic acid and glycosyl donor uridine diphosphate glucose (UDP-glucose) are used as raw materials, and a glycosylation reaction is carried out at the C-2 position of 4-methoxygallic acid under the catalysis of cinnabar root carbon glycosyltransferase encoded by the above-mentioned cinnabar root carbon glycosyltransferase AcCGT64 gene to produce bergenin.

[0017] The present invention obtains the target protein after in vitro expression through the recombinant plasmid, and directly generates bergenin by further catalyzing the substrate 4-methoxygallic acid.

[0018] The cinnabar root carbon glycosyltransferase AcCGT64 gene of the present invention is identified from the cinnabar root plant through transcriptome sequencing and bioinformatics technology, and after a large number of experiments and screening; RNA of the cinnabar root rhizome is extracted using RNA reagent, and reverse transcribed into cDNA and then PCR amplified. The amplification primers of the cinnabar root carbon glycosyltransferase AcCGT64 gene are as follows:

[0019] 5'F:ATGTCTAACTCCGGTAACCAAG; (SEQ ID NO.3)

[0020] 3'R: CTAATTTTTCTTCACCGTAGTAATT; (SEQ ID NO.4)

[0021] In addition, when homologous recombination is performed with the vector pET28a, the AcCGT64 gene needs to be amplified and recovered using primers with homologous walls. The primers with homologous walls are as follows:

[0022] Upstream homology arm primer:

[0023] 5'F: cagcaaatgggtcgcggatccATGTCTAACTCCGGTAACCAAG; (SEQ ID NO.5)

[0024] Downstream homology arm primer:

[0025] 3'R: acggagctcgaattcggatccCTAATTTTTCTTCACCGTAGTAATT; (SEQ ID NO.6)

[0026] The glycosyltransferase AcCGT64 gene isolated and identified from the root of Cinnabaris rubra can be used as an important marker gene for molecular-assisted breeding of Cinnabaris rubra, and can also be used as an important candidate gene for the production of bergenin in the construction of yeast chassis cells.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The present invention provides a cinnabar root carbonyl transferase AcCGT64 gene, which can be used as a biosynthesis regulatory gene for bergenin and can be used in the preparation of bergenin.

[0029] (2) With the rapid development of bioinformatics technology, the mining of key enzyme genes in the biosynthetic pathway of bergenin has been greatly promoted. The biosynthetic regulatory gene of bergenin in the present invention, namely the cinnabar root carbon glycosyltransferase AcCGT64 gene, was identified and successfully verified for the first time, opening up a new biosynthetic method for producing bergenin. The present invention obtains the target product by enzyme catalysis in vitro through heterologous expression of Escherichia coli proteins, adopts in vitro biosynthesis, and carries out directional production, which has the advantages of less by-products, etc.

[0030] (3) The present invention also provides a recombinant plasmid, a genetically engineered bacterium and a recombinant protein containing the cinnabar root carbon glycosyltransferase AcCGT64 gene, which lays a foundation for synthesizing bergenin in large quantities through bioengineering methods and further for constructing a cell factory that produces bergenin.

[0031] (4) The in vitro biosynthesis of bergenin is highly controllable, which can reduce the demand for raw material planting, produce a single product, facilitate the separation and purification of bergenin in the later stage, and reduce the difficulty of chemical synthesis and the complexity of the synthesis path. The cinnabar root carbon glycosyltransferase AcCGT64 gene, as a key gene for the biosynthesis of bergenin, can also be used for breeding research of plants rich in bergenin such as Ardisia japonica. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the synthetic route derived for bergenin;

[0033] Figure 2 Schematic diagram of the construction of the recombinant expression plasmid Pet28a-AcCGT64;

[0034] Figure 3 The results of electrophoresis detection of the recombinant gene of cinnabar root carbonyl transferase AcCGT64.

[0035] Mar, 1 is the positive single colony detection result;

[0036] Figure 4 This is the SDS-PAGE protein electrophoresis detection diagram of the cinnabar root carbonyl transferase AcCGT64. Among them, M is the protein molecular weight standard; lanes 1, 2, 3, and 4 are the flow-through eluent, precipitation eluent, 20mmol / L imidazole eluent, and 50mmol / L imidazole eluent respectively; lanes 5-11 are all proteins in 250mmol / L imidazole eluent;

[0037] Figure 5HPLC detection of the glycosylation of 4-methoxygallic acid by carbon glycosyltransferase AcCGT64. The horizontal axis is time, unit min, and the vertical axis is the response value, unit mAU; among them, CK: the enzyme activity reaction result of the control group (4-methoxygallic acid + uridine diphosphate glucose + inactivated cinnabar root carbon glycosyltransferase AcCGT64) enzyme inactivation; standard: 4-methoxygallic acid standard + bergenin standard; AcCGT64: the enzyme activity reaction result of the experimental group (4-methoxygallic acid + uridine diphosphate glucose + cinnabar root carbon glycosyltransferase AcCGT64);

[0038] Figure 6 is the mass spectrometry analysis (LC / MS / MS) spectrum of the standard, wherein A is the standard 4-methoxy

[0039] The retention time of bergenic acid is 20.04 minutes; B is the retention time of the standard substance bergenin is 19.43 minutes;

[0040] Figure 7 Mass spectrometry (LC / MS / MS) of the reaction products to verify enzyme activity, where A is the substrate 4-methoxy

[0041] The retention time of A is 20.04 minutes for methyl gallic acid; B is the retention time of the reaction product bergenin 19.41 minutes;

[0042] Figure 8 The fragment ion pattern of the standard substance bergenin (theoretical molecular weight 328) (LC / MS / MS);

[0043] Fig. 9 The fragment ion pattern of the reaction product bergenin (theoretical molecular weight 328) (LC / MS / MS). DETAILED DESCRIPTION

[0044] The present invention is further described in detail below in conjunction with embodiments.

[0045] Those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the art or the product specifications are used. If the manufacturer of the materials or equipment used is not specified, they are all conventional products that can be purchased.

[0046] Example 1

[0047] Based on the Unigene basic functional annotation information of the cinnabar root transcriptome, CGT candidate genes were screened in the sequencing annotation results. At the same time, the carbon glycosyltransferase (CGT) identified in plants was analyzed by sequence local BLAST, and then the screening results were sorted and analyzed. Finally, a carbon glycosyltransferase (CGT) 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 detection, the target candidate carbon glycosyltransferase AcCGT64 gene (which can catalyze the glycosylation reaction of 4-methoxygallic acid to produce bergenin) was finally identified. Figure 1 The operation steps of each stage of the synthesis of bergenin are as follows (the reagents, raw materials, instruments and equipment used in the following implementation are all commercially available):

[0048] (1) Preparation of cDNA template

[0049] Fresh samples of the roots of Cinnabarinum were taken, sliced, and then quickly frozen in liquid nitrogen for RNA extraction. Total RNA was extracted using the HiPure Plant RNA Mini Kit from Magen (Guangzhou Meiji Biotechnology Co., Ltd.). RNA was extracted according to the operating procedures of the kit. After passing the test, the RNA was reverse transcribed into cDNA using the TAKARA reverse transcription kit and stored at -20°C for later use.

[0050] (2) Gene amplification and recovery

[0051] Primers for amplifying the cinnabar root carbon glycosyltransferase AcCGT64 gene were designed using primer design software (CE Design) v1.04. The primers consisted of primer F (SEQ ID NO.3) and primer R (SEQ ID NO.4). The KOD high-fidelity enzyme was used according to the instructions for use of the high-fidelity KOD enzyme, and the gene was amplified using the Bergenia cDNA as a template. The PCR reaction program was: 94°C, 5min; 94°C, 30S, 58°C, 90S, 72°C, 1min, 35 cycles; 72°C, 7min. After the PCR was completed, the gel was run, and the target band was recovered after confirming that the amplification was successful. The EasyPure Quick Gel Extraction Kit of Beijing Quanshijin Biotechnology Co., Ltd. was used for gene gel extraction and recovery to recover the target gene. After recovery, the recovery concentration was measured on a NanoReady ultra-micro UV-visible spectrophotometer, and finally stored in a -20°C refrigerator for later use. The cinnabar root carbon glycosyltransferase AcCGT64 gene fragment was obtained, and its nucleic acid sequence was sequenced as shown in SEQ ID NO.1.

[0052] 5'F:ATGTCTAACTCCGGTAACCAAG; (SEQ ID NO.3)

[0053] 3'R: CTAATTTTTCTTCACCGTAGTAATT; (SEQ ID NO.4)

[0054] In addition, when the cinnabar root carbon glycosyltransferase AcCGT64 gene fragment with a vector homology arm undergoes homologous recombination with the vector pET28a (the homology arm is Escherichia coli pET28a), the cinnabar root carbon glycosyltransferase AcCGT64 gene needs to be amplified and recovered using primers with homology walls (i.e., homology arm primers). The homology arm primers are composed of an upstream homology arm primer (shown in SEQ ID NO.5 in the sequence table) and a downstream homology arm primer (shown in SEQ ID NO.6 in the sequence table). Using the cinnabar root carbon glycosyltransferase AcCGT64 gene as a template, follow the instructions for use of the high-fidelity KOD enzyme and perform PCR amplification again to obtain the cinnabar root carbon glycosyltransferase AcCGT64 gene fragment with a vector homology arm.

[0055] Upstream homology arm primer:

[0056] 5'F:cagcaaatgggtcgcggatccATGTCTAACTCCGGTAACCAAG. (SEQ ID NO.5)

[0057] Downstream homology arm primer:

[0058] acggagctcgaattcggatccCTAATTTTTCTTCACCGTAGTAATT. (SEQ ID NO. 6).

[0059] The lowercase letters in the above upstream homology arm primer (SEQ ID NO.5) and downstream homology arm primer (SEQ ID NO.6) represent the pET28a homology arms, and the uppercase letters represent the primer sequences for amplifying the cinnabarinaceous root carbonyl transferase AcCGT64 gene.

[0060] (3) Construction and identification of gene recombination vectors

[0061] A schematic diagram of homologous recombination is shown in Figure 2. First, the vector pET28a was linearized, and the linearized vector was obtained by single enzyme digestion with BamH I enzyme. During homologous recombination, the assembly was carried out according to the operating instructions of the homologous recombination enzyme, and then the concentrations of the pET28a homologous arms inserted with the vector homologous arms, the capital letters represent the amplified cinnabar root carbon glycosyltransferase AcCGT64 gene fragment and the pET28a vector were calculated, and the amount of each component was calculated according to the recombination instructions; finally, each component was added to the PCR reaction tube on ice, and the pET28a homologous arms, the capital letters represent the amplified cinnabar root carbon glycosyltransferase AcCGT64 gene and the pET28a vector were homologously recombined to obtain a recombinant plasmid, named pET28a-AcCGT64. After assembly, the results were tested and sent to the company for sequencing. The electrophoresis test results after assembly are shown in Figure 3 , indicating that the assembly is successful. Reassemble the operation according to the following process:

[0062] Table 1 Candidate gene recombination reaction system

[0063]

[0064] Among them, X = (0.02×pET28a base pair number) ng / linearized pET28a concentration ng / μL; Y = (0.02×pET28a base pair number) ng / cinnabar root carbon glycosyltransferase AcCGT64 recovery concentration ng / μL, and the inserted gene fragment is the cinnabar root carbon glycosyltransferase AcCGT64 gene fragment inserted with the vector homology arm.

[0065] (4) SDS-PAGE protein electrophoresis detection

[0066] After a small protein expression test, the protein induction conditions of AcCGT64 were determined to be: 18°C, 0.1mM IPTG, 220r / min, induction for 12h; then shake vigorously, collect bacteria, break the cell wall, obtain protein supernatant after high-speed centrifugation (12000r / min), and then use SDS-PAGE protein electrophoresis and detection. The test results are shown in Figure 4 , Figure 4 This indicates that AcCGT64 protein can be eluted and purified in 250 mmol / L imidazole elution buffer.

[0067] (5) Enzyme activity reaction

[0068] The enzyme activity of the cinnabar root carbon glycosyltransferase AcCGT64 gene was determined by glycosylation reaction to synthesize bergenin in a 1.5mL centrifuge tube. The mixture in the experimental sample reaction system contained: 2 microliters of 100mM uridine diphosphate glucose, 2 microliters of 100mM 4-methoxygallic acid, 40μg of purified cinnabar root carbon glycosyltransferase AcCGT64 protein, and 50mM Tris-HCl buffer (pH 8.0) was added to a total volume of 100μL, and the total volume of the reaction system was 100μL. After incubation at 32℃ for 2 hours, an equal volume of 1M hydrochloric acid was terminated, and the supernatant was taken after brief centrifugation (12000r / min). Finally, the reaction product was detected by HPLC and LC-MS / MS analysis.

[0069] Control group (CK) reaction system: 2 μL of 100 mM 4-methoxygallic acid, 2 μL of 100 mM UDP-glucose, 40 μg of inactivated purified cinnabar root carbon glycosyltransferase AcCGT64 protein, 50 mM Tris-HCl buffer (pH 8.0) was added to a total volume of 100 μL, and the total volume of the reaction system was 100 μL.

[0070] Standard products: 50 μl of 10 mM 4-methoxygallic acid, 50 μl of 10 mM bergenin.

[0071] (6) Product testing

[0072] HPLC detection conditions are as follows:

[0073] The instrument used for HPLC detection is Agilent 1290 ultra-high performance liquid chromatograph. The chromatographic column is XBridge ShieldRP18 (4.6mm×250mm, 5μm), column temperature: 30°C; the mobile phase for the determination of bergenin 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; 35-38min, 90%-90% B; 38-45min, 90%-100% B; elution time: 45min; injection volume: 10 microliters; flow rate: 0.6ml / min; detection wavelength 270nm. The test results are shown in Figure 5 , indicating that the experimental samples produced bergenin under the catalysis of cinnabar root carbonyl transferase AcCGT64.

[0074] LC-MS / MS detection conditions are as follows:

[0075] 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 used was negative ion mode, voltage: 3500 V; fragmentation voltage: 135 V; cone voltage: 60 V; radio frequency voltage: 750 V, scanning range: 100-1000 m / z, scanning mode: SRM. Chromatographic conditions: the chromatographic column is XBridge Shield RP18 (4.6mm, × 250mm, 5μm), the column temperature is 32°C, the mobile phase for determining bergenin 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; 35-38min, 90%-90% B; 38-45min, 90%-100% B; elution time: 45min; injection volume: 10 μL; flow rate: 0.6ml / min; detection wavelength is 270nm.

[0076] Test results are shown in Figure 6 to Figure 9 From the results, it can be seen that the peak time and characteristic fragmentation ions of the product are consistent with those of the standard substance bergenin, confirming that the reaction product is bergenin. Finally, it was concluded that the carbon glycosyltransferase AcCGT64 has the ability to catalyze the glycosylation of 4-oxymethyl gallic acid to produce bergenin.

[0077] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A Cinnabarinaceous Root Carbonyl Glycosyltransferase AcCGT64 A gene characterized by Cinnabarinum root carbonyl transferase AcCGT64 The nucleic acid sequence of the gene is shown in SEQ ID NO.

1.

2. The cinnabar root carbonyl transferase according to claim 1 AcCGT64 The gene encodes a protein characterized in that The amino acid sequence of the encoded protein is shown in SEQ ID NO.

2.

3. A composition containing the cinnabar root carbonyl transferase according to claim 1. AcCGT64 Recombinant plasmid of gene.

4. The method according to claim 3 containing cinnabarinum root carbonyl transferase AcCGT64 A recombinant plasmid of a gene, characterized in that Cinnabarinum sibiricum Citrate Transferase AcCGT64 The gene was homologously recombined with the pET28a vector to obtain pET28a - AcCGT64 Heavy Plasmid .

5. A genetically modified bacteria, characterized in that: The recombinant plasmid of claim 3 or 4, or the genome of the genetically engineered bacteria is integrated with the exogenous cinnabar root carbonyl transferase of claim 1 AcCGT64 Gene.

6. The genetically modified bacteria according to claim 5, characterized in that: The transgenic engineering bacteria are Escherichia coli BL21 (DE3) strains.

7. The cinnabar root carbonyl transferase according to claim 1 AcCGT64 Application of genes in the preparation of bergenin.

8. The cinnabar root carbonyl transferase according to claim 7 AcCGT64 The application of the gene in the preparation of bergenin is characterized in that: Using 4-methoxygallic acid as substrate and UDP-glucose as glycosyl donor, the above-mentioned cinnabar root carbonyl glycosyltransferase AqCGT51 Under the catalysis of the genetically encoded cinnabar root carbonyl glycosyltransferase, a glycosylation reaction occurs at the C-2 position of 4-methoxygallic acid to produce bergenin.

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