Application of a Dendrobium Methyltransferase in the Synthesis of Regulatory Gastrointestinal Activity Flavonoids

Abstract

The present invention discloses the application of the methyltransferase encoded by the DhOMT8 gene of Dendrobium huoshanense in the biosynthesis of regulating gastrointestinal active flavonoids, belonging to the field of biotechnology. The nucleotide sequence of the DhOMT8 gene encodes a nucleotide sequence of an amino acid sequence shown in SEQ ID NO.2. In vitro, the methylated flavonoids are obtained by an enzymatic catalytic biosynthesis method using the DhOMT8 protein, providing a new method for synthesizing the gastrointestinal active flavonoids.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to the application of Dendrobium methyltransferase in the synthesis of flavonoids that regulate gastrointestinal activity. Background Art

[0002] Dendrobium has a long history of medicinal use. It was listed as a top-grade herb in "Shennong's Herbal Classic", which recorded that it "is mainly used for treating internal injuries, dispelling rheumatism, lowering qi, tonifying the five internal organs for emaciation and weakness, strengthening yin, and taking it for a long time can strengthen the stomach and intestines". After more than two thousand years of development, there are currently more than 30 species of Dendrobium used for medicine in China, and the commonly used ones include Dendrobium officinale, Dendrobium huoshanense, Dendrobium nobile, Dendrobium chrysotoxum, Dendrobium fimbriatum, Dendrobium devonianum, etc. Dendrobium "benefits the stomach and promotes the production of body fluid, nourishes yin and clears heat", and its "stomach-benefiting" effect is definite, and it is a commonly used "gastrointestinal drug" in current clinical practice. There are many discussions in ancient books about its "strengthening the stomach and intestines", "regulating the stomach qi", "clearing stomach heat", "invigorating the spleen and promoting digestion", etc., and the relevant pharmacological effects have been reported in many studies (Yan Meiqiu, Chen Suhong, Lv Guiyuan. Pharmacological research and application progress of Dendrobium related to "strengthening the stomach and intestines" [J]. Chinese Traditional and Herbal Drugs, 2016, 47(21): 3918-3924.). Flavonoid O-methyltransferases are involved in the biosynthesis and metabolism of flavonoids in plants. These enzymes can catalyze the reaction in which the hydroxyl group (-OH) on the flavonoid skeleton is replaced by a methoxy group (-OCH3), thereby generating methoxylated flavonoid derivatives. O-methyltransferases change the chemical structure of flavonoids by introducing methyl groups at the hydroxyl positions of flavonoids, and thus affect their biological activities. Methylated flavonoids usually have higher liposolubility, are more likely to cross cell membranes, and increase their bioavailability and stability. Some methylated flavonoids have the effect of regulating gastrointestinal activity (Huang Xiaoyan, Zou Menglong, Chen Yalu, et al. Mechanism of Astragalus membranaceus in treating ulcerative colitis based on network pharmacology and molecular docking analysis [J]. Traditional Chinese Drug Research & Clinical Pharmacology, 2021, 32(6): 815-824.). In addition, methylated flavonoids also exhibit enhanced antioxidant and anti-inflammatory effects. Therefore, the study of flavonoid O-methyltransferases helps to deeply understand the biosynthesis mechanism of these compounds and provides a theoretical basis and potential biotechnological means for new drug development.

[0003] Research shows that the chemical constituents of Dendrobium huoshanense contain a rich class of methylated flavonoids, which have the ability to regulate gastrointestinal activity. Its methyltransferase can catalyze the methylation of the oxygen atom of the hydroxyl group on flavonoid compounds. The synthesis methods of methylated flavonoid compounds mainly include chemical synthesis and enzymatic synthesis. The chemical synthesis method has harsh reaction conditions, involves organic reagents, has a low yield and is prone to produce by-products harmful to the environment and human body, so it is not the best choice for synthesizing methylated flavonoid compounds. In contrast, the enzymatic synthesis method is more suitable. So far, no literature reports on the methyltransferase of Dendrobium huoshanense have been found. Therefore, it is of great significance to identify and develop an enzyme with the function of synthesizing methylated flavonoid compounds and apply it in the synthesis of flavonoid compounds that regulate gastrointestinal activity. Summary of the Invention

[0004] The present invention mainly aims at the above technical problems, and provides a methyltransferase gene that catalyzes the generation of flavonoid compounds that regulate gastrointestinal activity, as well as a method for biosynthesizing flavonoid compounds that regulate gastrointestinal activity, so as to solve the deficiencies of the existing chemical synthesis of flavonoid compounds that regulate gastrointestinal activity.

[0005] Specifically, the present invention provides the following technical solutions:

[0006] On the one hand, the present invention provides a methyltransferase, the amino acid sequence of which contains the sequence shown in SEQ ID NO.2.

[0007] On the other hand, the present invention provides a methyltransferase gene, the sequence of which is a nucleotide sequence encoding an amino acid sequence containing the sequence shown in SEQ ID NO.2,

[0008] Or based on the principle of complementary pairing, the methyltransferase gene provided by the present invention can be a sequence that is fully complementary to the nucleotide sequence encoding an amino acid sequence containing the sequence shown in SEQ ID NO.2.

[0009] In a preferred embodiment, the nucleotide sequence of the above methyltransferase gene contains the sequence shown in SEQ ID NO:1, or a nucleotide sequence that is completely complementary to the sequence shown in SEQ ID NO.1.

[0010] As is well known to those skilled in the art, the gene sequence can also contain introns, promoters and various regulatory elements. Therefore, the nucleotide sequence of the above methyltransferase gene can also contain introns, promoters and various regulatory elements.

[0011] On the other hand, the present invention provides an application of a methyltransferase gene in the synthesis of flavonoids that regulate gastrointestinal activity, wherein the nucleic acid sequence of the methyltransferase gene is a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO.2, or a sequence that is fully complementary paired with the nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO.2.

[0012] In a preferred embodiment, the above methyltransferase gene sequence is a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO.2, or a sequence that is fully complementary paired with the nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO.2.

[0013] In a preferred embodiment, the nucleotide sequence of the above methyltransferase gene is as shown in SEQ ID NO:1, or a nucleotide sequence that is fully complementary paired with the sequence shown in SEQ ID NO.1.

[0014] On the other hand, the present invention provides an application of the aforementioned methyltransferase in the synthesis of flavonoids that regulate gastrointestinal activity.

[0015] On the other hand, the present invention provides a method for synthesizing flavonoids that regulate gastrointestinal activity, the method comprising the following steps:

[0016] 1) Obtain the aforementioned methyltransferase;

[0017] 2) Use the methyltransferase in step 1) to catalytically synthesize flavonoids that regulate gastrointestinal activity in an enzyme activity reaction system.

[0018] In a preferred embodiment, the above methyltransferase is obtained by prokaryotic expression.

[0019] In a preferred embodiment, the above methyltransferase is obtained by biosynthesis.

[0020] In a preferred embodiment, the enzyme activity reaction system contains the above methyltransferase, SAM, a substrate for synthesizing flavonoids that regulate gastrointestinal activity, and a buffer.

[0021] On the other hand, the present invention provides a method for synthesizing flavonoids that regulate gastrointestinal activity, characterized in that the method comprises the following steps:

[0022] 1) Obtain a methyltransferase containing an amino acid sequence as shown in SEQ ID NO.2;

[0023] 2) Use the above methyltransferase to catalytically synthesize flavonoids that regulate gastrointestinal activity in an enzyme activity reaction system.

[0024] In a preferred embodiment, the above methyltransferase is obtained by prokaryotic expression.

[0025] In a preferred embodiment, the above methyltransferase is obtained by chemical synthesis.

[0026] In a preferred embodiment, the enzyme activity reaction system contains the above methyltransferase, SAM, the substrate for synthesizing flavonoids with regulatory activity on gastrointestinal tract, and a buffer.

[0027] In a preferred embodiment, the enzyme activity reaction system further contains divalent cations.

[0028] In a preferred embodiment, the reaction temperature of the enzyme activity reaction system is 4 - 70 °C.

[0029] In a preferred embodiment, the pH of the enzyme activity reaction system is 7.0 - 10.0.

[0030] In a preferred embodiment, the substrate for synthesizing flavonoids with regulatory activity on gastrointestinal tract is isorhamnetin.

[0031] Compared with the prior art, the present invention has the following advantages:

[0032] 1) Compared with the prior art, the present invention uses an enzyme-catalyzed biosynthesis method to obtain flavonoids with regulatory activity on gastrointestinal tract, which has the advantages of simpler steps, less pollution, and more single products compared with the chemical synthesis method.

[0033] 2) The present invention provides a new methyltransferase, which has a relatively wide temperature range suitable for reaction (4 - 70 °C).

[0034] 3) The methyltransferase in the present invention does not depend on metal ions.

[0035] 4) The methyltransferase of the present invention reaches a relatively high reaction rate in a short time, can completely convert the substrate into the product, and obtains a pure product. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The method and its beneficial effects of the present invention will be described in detail below in conjunction with the drawings and specific embodiments.

[0037] Figure 1 Results of the cloned gene fragment. A: Gene cloning, M is the 2000bp DNA Marker, and lane 1 is DhOMT8.

[0038] Figure 2SDS-PAGE gel electrophoresis pattern of pET-28a(+)-DhOMT8 fusion protein. The protein Marker (15-180 kDa) is on the left, and the target protein conditions are on the right (1, 2, and 3 are empty vector, crude enzyme of DhOMT8, and purified protein of DhOMT8 respectively).

[0039] Figure 3 Results of the reaction of DhOMT8 with the substrate. A: Liquid phase diagram of the reaction of DhOMT8 with quercetin; B: Liquid phase diagram of the standard product; C: MS peak diagram of the catalytic product.

[0040] Figure 4 Results of the detection of the enzymatic kinetic parameters for the conversion of quercetin to isorhamnetin catalyzed by DhOMT8. Detailed implementation manners

[0041] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without any creative efforts shall fall within the protection scope of the present invention.

[0042] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs.

[0043] Example 1

[0044] Cloning of the target methyltransferase gene

[0045] 1. Total RNA was extracted from Dendrobium huoshanense using a total RNA extraction kit. The total RNA extracted above was taken, and the experimental operations were carried out according to the instructions of the Nanjing Novizan IIQ RT SuperMix reverse transcription kit to obtain ideal cDNA. Using this as a template, polymerase chain reaction (PCR) was performed to amplify the full-length fragment of the DhOMT8 gene. The specific primers were designed according to the transcriptome data of this experiment, as shown in Table 1: IIQ RT SuperMix reverse transcription kit to obtain ideal cDNA. Using this as a template, polymerase chain reaction (PCR) was performed to amplify the full-length fragment of the DhOMT8 gene. The specific primers were designed according to the transcriptome data of this experiment, as shown in Table 1:

[0046] Table 1 Primers sequence

[0047]

[0048] Using the cDNA of Dendrobium huoshanense extracted in the above experiments as the PCR template respectively, the target fragment was amplified using the primers DhOMT8-F and DhOMT8-R. The polymerase used in PCR was Nanjing Novoprotein 2×Phanta Max Master Mix. Prepare a 25 μL PCR reaction system according to Table 10.

[0049] Table 2 25 μL PCR reaction system for amplifying the target gene (25 μL)

[0050]

[0051]

[0052] Place the prepared PCR reaction system in a PCR instrument and perform the amplification of the target gene according to the PCR reaction program in Table 11.

[0053] Table 3 PCR reaction program for amplifying the gene

[0054]

[0055] After the reaction, use 1% agarose gel electrophoresis to detect the product size, and the electrophoresis diagram is shown in Figure A.

[0056] 2. Connect the amplified product to the T vector (pET-28a, Nanjing Novoprotein Biotechnology Co., Ltd.), and transform it into Escherichia coli DH5α. Select single colonies and culture them in LB medium, and perform colony PCR verification. The colony PCR reaction system is as follows: 12.5 μL Green Taq Mix (P131, Nanjing Novoprotein Biotechnology Co., Ltd.), 1 μL each of the upstream and downstream primers, 2 μL of the template (bacterial solution), and make up to 25 μL with dd H2O. Send the samples with correct colony PCR results for sequencing.

[0057] The nucleotide sequence obtained by sequencing is as shown in SEQ ID NO.1. This gene sequence contains 1077 nucleotides and encodes 358 amino acids (shown in SEQ ID NO.2).

[0058] >SEQ ID NO.1

[0059] ATGGGCAGTTACAATGCCACCGCCGATGGAAGTATCGACGACGAGGCCGCCTGCCTCTA

[0060] CGCGTTGCAGCTCGTCAGCTTCTCCATCCTCCCCATGACCCTCAAGGCCGCCATCGAACT

[0061] CAAACTCCTCGAAACCATCTCCCTCGCCGGCCCCGGCGCTCAACTCTCTCCGTCAGAAC

[0062] TTGCCTCTCGCCTCCCCTTCATCACAAACCCCCAAGCCCCCGTCATGCTCGACCGCATCC

[0063] TCCGCCTTCTGGCCAGCTACTCCATCCTCACTTGCTCTATCTCCCCATCCGGCGAGCGCC

[0064] GCTATGGCGCCGCACCGGTTTGTAAATTCCTCACGCCCAATTCCGATGGAGTTTCTATGG

[0065] CTCCGCTTGCTCTAATGAATCAGGATAAGGTGTTGATGGAGAGCTGGTACTATCTAAAGG

[0066] ATGCGGTGATCGAAGGCGGTATTCCGTTCAATAAGGCGTATGGGATGACGGCGTTTGAGT

[0067] ACCATGGAACGGATCCAAGGTTCAATAAGGTGTTTAACGATGGGATGTCAGGCCATTCA

[0068] ACTATCATTACGAATAAATTGTTGGAGATTTATGAGGGTTTTGATGGGTTGGGTTCGCTGG

[0069] TGGATGTGGGAGGCGGAGTGGGGGCGACGTTGGGAAAGATTACGGCCAAGTATACCGG

[0070] GATTAGAGGAATTAATTTTGATCTCCCGCATGTAATCTCCGAGGCTCCTCCTCTTCCAGGA

[0071] GTGGAGCATGTGGGCGGAGATATGTTCGAGAGTGTTCCCACCGCTGATGCCATTTTCATG

[0072] AAGTGGATTCTTCATGATTGGAGCGACGATCATTGCCTCAAATTGTTGAAGAATTGTTGG

[0073] AAGGCATTGCCTGAAAATGGGAAAGTGATTGTGGCCGAGTGCATTCTTCCGGTGGAGCC

[0074] CGAACAAACTGTGGCAGCAAAAGGTGTCTTTCATGTCGACCTCATCATGTTGGCTCATA

[0075] ACCCTGGAGGAAAAGAGAGAACGGAGAATGAATTTAAATCATTGGCAAAGGAGGCTGG

[0076] CTTCTCTGATTTCAAATCCATCTACATATTTTCAGGCTGTTGGGTTATGGAATTCATCAAGT

[0077] AG

[0078] >SEQ ID NO.2

[0079] MGSYNATADGSIDDEAACLYALQLVSFSILPMTLKAAIELKLLETISLAGPGAQLSPSELASR

[0080] LPFITNPQAPVMLDRILRLLASYSILTCSISPSGERRYGAAPVCKFLTPNSDGVSMAPLALMN

[0081] QDKVLMESWYYLKDAVIEGGIPFNKAYGMTAFEYHGTDPRFNKVFNDGMSGHSTIITNKLL

[0082] EIYEGFDGLGSLVDVGGGVGATLGKITAKYTGIRGINFDLPHVISEAPPLPGVEHVGGDMFE

[0083] SVPTADAIFMKWILHDWSDDHCLKLLKNCWKALPENGKVIVAECILPVEPEQTVAAKGVF

[0084] HVDLIMLAHNPGGKERTENEFKSLAKEAGFSDFKSIYIFSGCWVMEFIK

[0085] Expression and Detection of the Target Protein in Example 2

[0086] Enlarge the culture of the target bacterial solution with correct sequencing and extract the recombinant plasmid pET-28a-DhOMT8 from it. Transform the recombinant plasmid into competent Escherichia coli BL21(DE3) cells, spread on plates, and pick out clones into LB medium. Culture at 37°C and 200 rpm for 16 h to obtain the mother solution. Inoculate the mother solution into LB medium containing 50 μg / mL KaN at a ratio of 1:100, and culture at 37°C until OD 600 = 0.6 - 0.8 (using sterile LB medium as a control), then add IPTG with a final concentration of 0.5 mM, and finally culture at 16°C and 200 rpm for 18 - 20 h.

[0087] Centrifuge to collect the induced bacterial solution, resuspend the cells with pre-cooled Tris-HCl (50 mM) buffer at pH = 9.0, centrifuge again, and collect the cells again. Repeat once to remove the residual medium. Finally, add 15 mL of the above Tris-HCl buffer to each gram of wet cells. The obtained cell suspension can be frozen overnight in a -20°C refrigerator and then broken at low temperature on an ultrasonic cell disruptor with a power of 150 W, working for 3 s and intermittent for 3 s. Stop the disruption when the cell suspension becomes clear. Finally, centrifuge the disrupted solution at 4°C and 14,000 rpm for 4 min, and the supernatant is the crude enzyme solution, which is stored in a -20°C refrigerator.

[0088] According to the His-Tag on the fusion protein, purify the target protein using a nickel affinity chromatography column, elute the target protein with different concentrations of imidazole solution, and finally detect the molecular weight and purity of the target protein by 10% SDS-PAGE electrophoresis. As Figure 2 shown, the target protein appears near 43 kDa (using 1, 2, and 3 as empty vector, crude enzyme, and purified protein respectively), indicating that the protein is successfully expressed in the supernatant, and the purified protein can be used for subsequent experiments.

[0089] Example 3 In Vitro Enzyme Activity Detection

[0090] 1. Enzyme Activity Reaction System: 10 μg of pure enzyme, 0.5 mM quercetin, 5 mM SAM methyl donor, 14 mM β-mercaptoethanol, and make up to 200 μL with 50 mM, pH = 9.0 Tris-HCl buffer. After the reaction solution is incubated in a water bath at 45°C for 45 min, add double the volume of pre-cooled methanol, vortex and mix well, and centrifuge at 4°C and 14,000 rpm for 4 min. Filter the supernatant and detect it by high performance liquid chromatography.

[0091] 2. HPLC Conditions

[0092] Phase A: 0.1% formic acid aqueous solution; Phase B: acetonitrile

[0093] Elution gradient: 0 - 5 min: 5% - 20% B; 5 - 8 min: 20% - 22% B; 8 - 17 min: 22% - 25% B; 17 - 23 min: 25% - 35% B; 23 - 25 min: 35% - 50% B; 25 - 32 min: 50% - 95% B; 32 - 37 min: 95% B; 37 - 38 min: 95% - 5% B; 38 - 42 min: 5% B.

[0094] 3. LC-MS conditions:

[0095] The detection mode is positive ion mode, capillary voltage 3500 V, sheath gas temperature 280 °C, flow rate 11 L·min -1 , drying gas temperature 320 °C, flow rate 10 L·min -1 , nozzle voltage 155 V, flow rate 0.250 mL·min -1 .

[0096] 4. Detection results:

[0097] The results are shown in Figure 3 , when using quercetin as the substrate, for DhOMT8, 1 product peak can be observed in the reaction. By comparing with the standard product control and based on the retention time and mass-to-charge ratio m / z, this peak is determined to be the product isorhamnetin.

[0098] Example 4

[0099] Detection of enzymatic kinetic parameters

[0100] Using quercetin as the substrate and SAM as the methyl donor, the effects of pH, temperature, metal ions, and reaction time on the catalytic activity were investigated.

[0101] 1. Temperature: Using quercetin as the methyl acceptor and SAM as the methyl donor, the variation law of enzyme activity at different reaction temperatures (4 °C, 30 °C, 40 °C, 50 °C, 60 °C, and 70 °C) was investigated. The reaction system contained 10 μg of pure protein, 0.5 mM of substrate, 5 mM of SAM, 14 mM of β-mercaptoethanol, and was made up to 200 μL with 50 mM, pH = 9.0 Tris-HCl buffer. The reaction solution was reacted at each temperature for 45 min. After the reaction, twice the volume of pre-cooled methanol was immediately added to the reaction system to terminate the reaction. After centrifugation, the supernatant was taken and filtered for HPLC detection. Each reaction was set up with 3 parallel experiments. The conversion rate of the substrate was estimated based on the peak area ratio in the chromatogram.

[0102] 2. pH: Different pH buffers at 50 mM were investigated: 7.0 - 8.0, Na2HPO4 - NaH2PO4 Buffer; 8.0 - 9.0, Tris - HCl Buffer; 9.0 - 10.0, Na2CO3 - NaHCO3 Buffer for their effects on enzyme activity. The reaction system was as follows: In a 200 μL reaction system, it contained 14 mM β - mercaptoethanol, 5 mM SAM, 10 μg protein, 0.5 mM compound quercetin, and was supplemented to the specified volume with the corresponding buffer. The reaction solution was reacted at 45 °C for 40 min. Immediately after the reaction, twice the volume of pre - cooled methanol was added. After centrifugation, the supernatant was filtered and analyzed by HPLC. To ensure the reliability of the data, three replicate samples were set up for each group of reactions. Finally, the conversion rate was calculated through the peak area of the chromatogram.

[0103] 3. Metal ions: The receptor molecule of the reaction solution was compound quercetin, and the donor was SAM. Different divalent metal ions were investigated: Co 2+ , Zn 2+ , Ba 2+ , Ca 2+ , Mg 2+ and EDTA for their effects on enzyme activity. In a 200 μL reaction system, it contained 14 mM β - mercaptoethanol, 5 mM SAM donor, 10 μg protein, 0.5 mM substrate, 5 mM divalent metal ion, and was supplemented to the specified volume with 50 mM, pH = 9.0 Tris - HCl buffer. The reaction solution was reacted at 30 °C for 4 min. Immediately after the reaction, twice the volume of pre - cooled methanol was added and centrifuged at 14000 rpm at 4 °C for 4 min. The supernatant was filtered and analyzed by HPLC. Three replicate samples were set up for each group of reactions. Finally, the conversion rate was calculated through the peak area of the chromatogram.

[0104] 4. Reaction time: A 200 μL Tris - HCl (50 mM, pH = 9.0) reaction system contained 14 mM β - mercaptoethanol, 5 mM SAM, 10 μg purified protein, 0.5 mM substrate quercetin, and was reacted in a 50 °C water bath for 3 min, 5 min, 8 min, 10 min, 20 min, 30 min, 40 min, 60 min respectively. After the reaction, twice the volume of pre - cooled methanol was quickly added to each reaction solution to terminate the reaction, and centrifuged at 14000 rpm at 4 °C for 4 min. The supernatant was filtered and detected by HPLC, and the conversion rate of the substrate was calculated. To ensure the accuracy of the data, three parallel experiments were set up for each group. The substrate conversion rate was calculated according to the ratio of the peak areas in the HPLC chromatogram.

[0105] 6. Detection results:

[0106] Enzymatic kinetic studies showed that ( Figure 4) DhOMT8 has the highest enzyme activity at 50 °C and has a good conversion rate in the range of 4 - 70 °C. The optimal pH of the reaction solution is 9.0, suitable for reaction in the pH range of 7.0 - 9.0, and its catalytic activity in Tris-HCl buffer is higher than that in Na2CO3-NaHCO3 buffer. This enzyme does not depend on metal ions. The relative conversion efficiency can reach over 70% in 10 min and the relative conversion rate can reach 100% in 30 min.

[0107] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to the above embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Use of a methyltransferase in the synthesis of regulatory gastrointestinal active flavonoids, characterized in that, The amino acid sequence of the methyltransferase is as shown in SEQ ID NO.2, the flavonoid compound regulating gastrointestinal activity is isorhamnetin, and the synthesis of the flavonoid compound regulating gastrointestinal activity uses quercetin as a substrate and S-adenosylmethionine as a methyl donor to synthesize isorhamnetin.

2. Use of a methyltransferase gene in the synthesis of regulatory gastrointestinal active flavonoids, characterized in that, The gene sequence of the methyltransferase is a nucleotide sequence encoding the amino acid sequence as shown in SEQ ID NO.2, or a sequence that is fully complementary paired with the nucleotide sequence encoding the amino acid sequence as shown in SEQ ID NO.

2. The flavonoid compound regulating gastrointestinal activity is isorhamnetin, and the synthesis of the flavonoid compound regulating gastrointestinal activity uses quercetin as a substrate and S-adenosylmethionine as a methyl donor to synthesize isorhamnetin.

3. A method for synthesizing a flavonoid compound regulating gastrointestinal activity, the flavonoid compound regulating gastrointestinal activity is isorhamnetin, and the synthesis of the flavonoid compound regulating gastrointestinal activity uses quercetin as a substrate and S-adenosylmethionine as a methyl donor to synthesize isorhamnetin. The method comprises the following steps: 1) Obtain a methyltransferase with an amino acid sequence as shown in SEQ ID NO.2; 2) Use the methyltransferase in step 1) to catalytically synthesize a flavonoid compound regulating gastrointestinal activity in an enzymatic reaction system.

4. The method according to claim 3, characterized in that In step 1), the methyltransferase is obtained by prokaryotic expression or by chemical synthesis.

5. The method for synthesizing a flavonoid compound with gastrointestinal activity regulation according to claim 3, characterized in that, In step 2), the enzymatic reaction system contains the methyltransferase described in step 1), S-adenosylmethionine (SAM), and a substrate for synthesizing a flavonoid compound regulating gastrointestinal activity.

6. A method for synthesizing a flavonoid compound with gastrointestinal activity regulation as claimed in claim 5, characterized in that, In step (2), the enzyme activity reaction system also contains Ba 2+ , Ca 2+ , Mg 2+ or EDTA.

7. A method for synthesizing a flavonoid compound for regulating gastrointestinal activity according to claim 5, characterized in that, In step 2), the reaction temperature of the enzymatic reaction system is 4 - 70 °C.

8. A method for synthesizing a gastrointestinal activity-regulating flavonoid compound as claimed in claim 5, characterized in that, In step 2), the pH of the enzymatic reaction system is 7.0 - 10.0.

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