Tartary buckwheat flavone methylation catalyzed by an oxygen methyltransferase, genes, primer sets and applications

Abstract

The application provides a kind of catalytic bitter buckwheat oxymethyl flavone synthesis oxymethyl transferase, gene, primer set and application, belong to bitter buckwheat oxymethyl flavone technical field.The application is based on the genome and transcriptome data analysis of bitter buckwheat, and the OMT gene catalyzing oxymethyl flavone synthesis is mined, and the in vitro enzyme activity expression system is used to verify FtOMT19, FtOMT20, FtOMT24 All can catalyze oxymethyl flavone formation, and have multiple modification sites, which not only provides important gene elements for the biosynthesis of bitter buckwheat oxymethyl flavone compounds, but also provides key gene sites for bitter buckwheat molecular breeding, and provides a theoretical basis for other flavone methylation modification.

Description

Technical Field

[0001] This invention belongs to the field of buckwheat oxymethyl flavonoid technology, and particularly relates to a class of oxymethyltransferases, genes, primer sets and applications that catalyze the methylation of baicalin, 7,8-dihydroxyflavone, myricetin, quercetin, quercetin or rutin. Background Technology

[0002] Oxymethyltransferase (OMT) O O-methyltransferases (OMTs) are a class of active proteins that catalyze the transfer of the methyl group from S-adenosyl-L-methionine (SAM) to the hydroxyl group of the acceptor molecule, thereby forming an oxygen methyl group (-OCH3). Based on substrate specificity and catalytic mechanism, plant OMTs are mainly divided into two categories: caffeoyl-CoA oxygen methyltransferases (CCoAOMTs) and caffeate oxygen methyltransferases (COMTs). Methylation modification can significantly improve the lipophilicity, metabolic stability, and bioavailability of flavonoids, playing an important role in enhancing their pharmacological activity and improving transmembrane transport efficiency. In recent years, researchers have successfully identified OMT genes involved in flavonoid methylation modification in various plants. For example, in citrus... CsOMT Catalytic multi-site methylation of flavonoids; Arabidopsis thaliana AtOMT1 OMTs are involved in the methylation modification of flavonols such as quercetin. However, the OMT family exhibits high substrate specificity and site selectivity, with significant differences in catalytic spectra among OMTs from different species. Therefore, the discovery and functional identification of key OMT genes have become a core step in elucidating the biosynthetic pathway of oxymethylflavonoids in plants.

[0003] Buckwheat ( Fagopyrum tataricum (L.) Gaertn. is a medicinal and edible plant recorded in the Compendium of Materia Medica. Its main active ingredients are oxymethylflavonoids, such as isorhamnetin, syringin, and narcissin. These components have significant pharmacological activities, including antioxidant, anti-inflammatory, anticancer, and cardiovascular protective effects. Currently, buckwheat has been developed into various functional foods such as buckwheat tea, buckwheat wine, and buckwheat vinegar. Clinically, it is mainly used to assist in lowering blood sugar and blood lipids, as well as in the prevention of chronic diseases, with significant efficacy. Oxymethylflavonoids have diverse structures, and their chemical synthesis is cumbersome due to the need for regioselective methylation reactions, resulting in low yield, high cost, and heavy environmental burden. Currently, the main source of oxymethylflavonoids relies on direct extraction from buckwheat. This highly resource-dependent approach severely restricts the production and development of pharmaceuticals and health products with oxymethylflavonoids as functional components. To date, no research has been reported on the OMT gene in buckwheat that specifically catalyzes the synthesis of oxymethylflavonoids. Therefore, it is urgent to analyze the key OMT gene in the biosynthetic pathway of oxymethylflavonoids in tartary buckwheat and to expand the sources of oxymethylflavonoids with new technologies and methods to alleviate the current shortage of resources. Summary of the Invention

[0004] To address the aforementioned problems in the prior art, this invention provides a class of oxygen methyltransferases, genes, primer sets, and applications for catalyzing the methylation of flavonoids in tartary buckwheat, particularly a class of oxygen methyltransferases, genes, primer sets, and applications for catalyzing the methylation of baicalin, 7,8-dihydroxyflavone, myricetin, quercetin, quercetin glycoside, or rutin. This invention, through systematic research on oxygen methyltransferases, provides a key catalytic module for the synthetic biology research of tartary buckwheat oxygen methyl flavonoids; simultaneously, it provides important gene elements for the breeding and quality evaluation of new tartary buckwheat varieties; furthermore, it provides an important reference for in-depth research on the methylation modification mechanism of flavonoid compounds.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a class of oxymethyltransferases that catalyze the synthesis of oxymethylflavonoids from tartary buckwheat, comprising: FtOMT19 FtOMT20 or FtOMT24 The FtOMT19 The amino acid sequence is shown in SEQ ID NO. 1; FtOMT20 The amino acid sequence is shown in SEQ ID NO. 2; FtOMT24 The amino acid sequence is shown in SEQ ID NO. 3.

[0006] The present invention also provides a gene encoding the said oxygen methyltransferase, wherein FtOMT19 The nucleotide sequence is shown in SEQ ID NO. 4. FtOMT20 The nucleotide sequence is shown in SEQ ID NO. 5. FtOMT24 The nucleotide sequence is shown in SEQ ID NO. 6.

[0007] The present invention also provides a primer set for amplifying the said gene, including an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 7. FtOMT19 -F and downstream primers with nucleotide sequences as shown in SEQ ID NO. 8 FtOMT19 -R; or Including upstream primers with nucleotide sequences as shown in SEQ ID NO. 9 FtOMT20 -F and the downstream primer with the nucleotide sequence shown in SEQ ID NO. 10 FtOMT20 -R; or Including the upstream primer with a nucleotide sequence as shown in SEQ ID NO. 11. FtOMT24 -F and the downstream primer with the nucleotide sequence shown in SEQ ID NO. 12 FtOMT24 -R.

[0008] The present invention also provides the application of the oxygen methyltransferase in catalyzing the methylation of tartary buckwheat flavonoids.

[0009] The present invention also provides the application of the oxygen methyltransferase in catalyzing the methylation of baicalin, 7,8-dihydroxyflavone, myricetin, quercetin, quercetin or rutin.

[0010] Furthermore, the baicalin is catalyzed to generate 6-oxomethylbaicalin and 7-oxomethylbaicalin; The 7,8-dihydroxyflavone was catalyzed to generate 8-hydroxy-7-oxomethylflavone and 7-hydroxy-8-oxomethylflavone; The myricetin is catalyzed to generate syringin, 3',4'-dioxymethylmyricetin, and 3',4',5'-trioxymethylmyricetin; The quercetin catalyzes the formation of isorhamnetin and 3',4'-dioxymethylquercetin; The quercetin is catalyzed to produce 3'-oxymethylquercetin and 3',4'-dioxymethylquercetin; The rutin catalyzes the formation of narcissin.

[0011] The present invention also provides the application of the oxymethyltransferase in the synthesis of oxymethylflavonoids.

[0012] This invention also provides the application of the aforementioned oxymethyltransferase in molecular marker-assisted breeding of tartary buckwheat.

[0013] The present invention also provides the application of the aforementioned oxymethyltransferase in the breeding of new varieties of tartary buckwheat.

[0014] The beneficial effects of this invention compared to the prior art are as follows: This invention provides an oxygen methyltransferase (OMT) derived from tartary buckwheat, its encoding gene, and its applications. The OMT gene has a specific nucleotide sequence, and the encoded OMT protein has a specific amino acid sequence. This invention utilizes integrated whole-genome resequencing, transcriptomics, spatial transcriptomics, metabolomics, and spatial metabolomics data analysis to screen and clone the OMT gene from tartary buckwheat, construct a prokaryotic expression vector, and obtain the recombinant protein. In vitro enzymatic reaction results show that this OMT can use various flavonoid compounds, including quercetin, quercetin, rutin, myricetin, baicalin, and 7,8-dihydroxyflavone, as substrates. This invention is the first to reveal the OMT gene in tartary buckwheat involved in the biosynthesis of oxygen methylflavone, providing a highly efficient enzymatic catalytic element for the green biomanufacturing of oxygen methylflavone, helping to reduce dependence on natural tartary buckwheat resources, and promoting the development and application of related functional foods and pharmaceuticals.

[0015] This invention, based on tartary buckwheat genome data, systematically integrated whole-genome resequencing and metabolomics analysis of 220 tartary buckwheat germplasm resources. Combined with transcriptome, spatial transcriptome, metabolome, and spatial metabolome data from the representative variety Y063, it successfully screened out the OMT gene, which is related to the biosynthesis of oxymethylflavone and is specifically highly expressed in the fruit. This multi-omics joint analysis strategy significantly improved the accuracy and specificity of key gene screening, filling the gap in existing technologies regarding the systematic discovery of tartary buckwheat OMT genes. Furthermore, this invention established an E. coli prokaryotic expression platform for verifying the catalytic function of OMT, providing a reliable technical means for rapidly and efficiently identifying the substrate specificity and catalytic activity of OMT genes. The establishment of this platform not only reduces dependence on the complex environment within the plant but also lays an experimental foundation for subsequent enzyme engineering and functional modification. This invention aims to discover the key OMT enzyme involved in the formation of oxymethylflavone in tartary buckwheat. The obtained functional gene elements effectively open up the biosynthetic pathway of oxymethylflavone, providing core catalytic elements for its synthetic biology research. Meanwhile, this invention provides a theoretical basis and technical support for the efficient biosynthesis of oxymethylflavonoids and for carrying out marker-assisted breeding, and has significant scientific research value and industrial application prospects. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is the biosynthetic pathway for oxymethylflavonoids; Figure 2 For phylogenetic tree and expression level analysis; Figure 3 Electrophoresis diagrams for gene cloning and vector construction are shown. Figure A is an agarose gel electrophoresis diagram of gene cloning, Figure B is an agarose gel electrophoresis diagram of pET32a recombinant vector ligation, and Figure C is an electrophoresis diagram of PCR products from BL21 Escherichia coli transformed with the recombinant vector. 1, 2, and 3 represent... FtOMT19 , FtOMT20 , FtOMT24 ; Figure 4The images show LC-MS diagrams obtained after functional identification of the target gene in *E. coli*. Figure A shows the products obtained using quercetin (Que) as a substrate: isorhamnetin (Iso) and 3',4'-dioxymethylquercetin (3',4'-DMQue). Figure B shows the products obtained using quercetin (Que-R) as a substrate: 3'-oxymethylquercetin (Iso-R) and 3',4'-dioxymethylquercetin (3',4'-DMQue-R). Figure C shows the product obtained using rutin (Rut) as a substrate: narcissin (Nar). Figure D shows the product obtained using myricetin (Myr) as a substrate. The products are Syr, 3',4'-dioxomethylmyricetin (3',4'-DMMyr), and 3',4',5'-trioxomethylmyricetin (3',4',5'-TMMyr). Figure E uses baicalein as a substrate, and the products are 6-oxomethylbaicalein (6-MBai) and 7-oxomethylbaicalein (7-MBai). Figure F uses 7,8-dihydroxyflavone (7,8-DHF) as a substrate, and the products are 8-hydroxy-7-oxomethylflavone (8,7-MF) and 7-hydroxy-8-oxomethylflavone (7,8-MF). Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] This invention provides a class of oxymethyltransferases that catalyze the methylation of baicalin, 7,8-dihydroxyflavone, myricetin, quercetin, quercetin, or rutin, comprising... FtOMT19 , FtOMT20 or FtOMT24 The FtOMT19 The amino acid sequence is shown in SEQ ID NO. 1. FtOMT20 The amino acid sequence is shown in SEQ ID NO. 2. FtOMT24 The amino acid sequence is shown in SEQ ID NO. 3.

[0024] In this invention, the FtOMT19 The amino acid sequence is MAEEQKKASTNEGEQQTRHQEIGHKSLLQSDALYQYILETSVYPREPEVMKELRDITAKHPWNIMTTSADEGQFLNMIVKLINAKNTMEIGVYTGYSLLATALALPDDGKILAMDINRENYELG LPVIQKAGVAHKIEFKEGPALPVLDQMLADEKNLGSYDFIFVDADKDNYWNYHKRLIDLIKVGGLIGYDNTLWNGSVVAADDAPMRKYVKYYRDFVLEFNKSIVADPRVEICQLPVGDGITLCRRIA (SEQ IDNO. 1); The FtOMT20The amino acid sequence is MPPLEEAFQSNLSISGTDEEETMAHVAQIVDSIAFPMTMQAAIELGLLEIISSAGPDSRLSASEVAAKLPSENPQAPDMVDRILRLLSAFSVISCSVIGDAGERKRVYGLTPVSKYFVKDEDGVSLGPLLRLLQDKVFLESWYKLKDAVLEGGIAFNKAHGMNAFEYPGVDQRFNEVFNVA MFNHTSIMMKKILQSYKGFENINQLVDVGGGLGHNLKIILSKYPNIKGINYDLPHVTKHGIPHPGMEHVGGDMFEGVPCAEAIFMKWILHDWGDEYCLTLLKNCYKALPDTGKVVVVESVVSEVPETTTAAKAFCEMDLIMMTQNPGGKERSKQEFLDLAKEAGFAGIRFECFVASLWVMEFYK (SEQ ID NO. 2); The FtOMT24 The amino acid sequence is MASEAPAGNGETTQTFRHQEVGHKSLLQSDELYQYILETSVYPNEPKCMKELRDVTANHPWNIMTTSADEGQFLNMLIKLINAKNTMEIGVYTGYSLLATALALPEDGKILAMDINRENYELG LPIIQKAGVAHKIDFKEGPALPVLDQMIQEGKYHGTFDFIFVDADKDNYLNYHKRLIDLVKVGGVIGYDNTLWNGSVVAPPNAQLRKYVRYYRDFVLELNKTLPADPRIEICQLPVGDGITLCRRIS (SEQ ID NO. 3).

[0025] The present invention also provides a gene encoding the said oxygen methyltransferase, wherein FtOMT19 The nucleotide sequence is shown in SEQ ID NO. 4. FtOMT20 The nucleotide sequence is shown in SEQ ID NO. 5. FtOMT24 The nucleotide sequence is shown in SEQ ID NO. 6.

[0026] In this invention, the FtOMT19The nucleotide sequence is ATGGCAGAAGAGCAGAAGAAAGCTTCAACGAACGAAGGTGAGCAGCAAACTAGGCATCAAGAGATCGGCCATAAGAGCCTTCTTCAAAGCGATGCTCTCTATCAGTACATCTTGGAGACGAGTGTTTACCCCAGAGAGCCCGAGGTCATGAAAGAGCTCCGCGATATCACAGCCAAACATCCATGGAACATAATGACGACATCAGCGGATGAAGGGCAGTTCTTGAACATGATAGTGAAGCTGATCAACGCCAAGAACACAATGGAGATCGGAGTTTATACCGGTTACTCTCTTCTTGCTACCGCTCTTGCCCTCCCTGATGACGGAAAGATTTTGGCAATGGACATAAACAGAGAGAACTATGAACTGGGATTGCCTGTCATCCAGAAAGCAGGGGTTGCACATAAAATTGAATTCAAAGAAGGCCCTGCTTTGCCTGTTCTTGATCAGATGCTTGCAGATGAGAAGAATCTGGGATCGTATGATTTCATATTTGTGGATGCTGACAAGGACAATTACTGGAATTACCATAAGAGGTTGATCGATCTGATCAAAGTGGGAGGGCTGATCGGCTACGACAACACCCTATGGAACGGCTCTGTGGTCGCAGCGGATGATGCGCCAATGAGGAAATACGTCAAGTACTATAGAGACTTTGTTCTCGAGTTCAACAAGTCCATCGTGGCTGATCCTAGGGTCGAGATTTGCCAGCTCCCTGTGGGTGATGGAATTACCCTGTGCCGCCGTATCGCTTGA (SEQ ID NO. 4); The FtOMT20 The FtOMT24 The nucleotide sequence is (SEQ ID NO. 6).

[0027] The present invention also provides a primer set for amplifying the said gene, including an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 7. FtOMT19 -F and downstream primers with nucleotide sequences as shown in SEQ ID NO. 8 FtOMT19 -R; or Including upstream primers with nucleotide sequences as shown in SEQ ID NO. 9 FtOMT20-F and the downstream primer with the nucleotide sequence shown in SEQ ID NO. 10 FtOMT20 -R; or Including the upstream primer with a nucleotide sequence as shown in SEQ ID NO. 11. FtOMT24 -F and the downstream primer with the nucleotide sequence shown in SEQ ID NO. 12 FtOMT24 -R.

[0028] In this invention, the FtOMT19 The nucleotide sequence of -F is ATGGCAGAAGAGCAGAAGAAAG (SEQ ID NO. 7), which... FtOMT19 The nucleotide sequence of -R is TCAGCGATACGGCGGCACA (SEQ ID NO. 8). The FtOMT20 The nucleotide sequence of -F is ATGCCTCCGTTGGAAGAAGCTT (SEQ ID NO. 9), which... FtOMT20 The nucleotide sequence of -R is TCACTTATAGAACTCCATGACC (SEQ ID NO. 10). The FtOMT24 The nucleotide sequence of -F is ATGGCAAGTGAAGCTCCCG (SEQ ID NO. 11), which... FtOMT24 The nucleotide sequence of -R is TCAGCTGATACGGCGGCACA (SEQ ID NO. 12).

[0029] The present invention also provides the application of the oxygen methyltransferase in catalyzing the methylation of tartary buckwheat flavonoids.

[0030] The present invention also provides the application of the oxygen methyltransferase in catalyzing the methylation of baicalin, 7,8-dihydroxyflavone, myricetin, quercetin, quercetin or rutin; The baicalin is catalyzed to produce 6-oxomethylbaicalin and 7-oxomethylbaicalin; The 7,8-dihydroxyflavone was catalyzed to generate 8-hydroxy-7-oxomethylflavone and 7-hydroxy-8-oxomethylflavone; The myricetin is catalyzed to generate syringin, 3',4'-dioxymethylmyricetin, and 3',4',5'-trioxymethylmyricetin; The quercetin catalyzes the formation of isorhamnetin and 3',4'-dioxymethylquercetin; The quercetin is catalyzed to produce 3'-oxymethylquercetin and 3',4'-dioxymethylquercetin; The rutin catalyzes the formation of narcissin.

[0031] The present invention also provides the application of the oxymethyltransferase in the synthesis of oxymethylflavonoids.

[0032] This invention also provides the application of the aforementioned oxymethyltransferase in molecular marker-assisted breeding of tartary buckwheat.

[0033] The present invention also provides the application of the aforementioned oxymethyltransferase in the breeding of new varieties of tartary buckwheat.

[0034] Example 1: Gene Cloning OMT gene mining: Hidden Markov Model (HMM) files (PF01596, PF00891, Methyltransf.HMM) of conserved OMT domains were downloaded from the Pfam database (https: / / pfam.xfam.org / ). Gene searches were performed on the tartary buckwheat genome (https: / / www.mbkbase.org / Pinku1 / ) using HMMER 3.0, identifying 38 tartary buckwheat OMT genes. FtOMT19 , FtOMT20 , FtOMT24 Three genes were screened based on multi-omics data, with corresponding sequence numbers C_AA168263.1, C_AA168264.1, and C_AA168265.1 (National Center for Biotechnology Information GeneBase database). Combining transcriptome data from green and mature buckwheat fruits, it was found that all three OMT genes were highly expressed in the fruits (e.g., ...). Figure 2 (As shown).

[0035] OMT gene cloning: Primers were designed using Snapgene software in accordance with primer design principles. The designed primers are shown in Table 1.

[0036] Table 1 Primers used for gene cloning

[0037] From May to September 2023, all tartary buckwheat materials were planted in an experimental field in Beizhai Village, Qiaozhen Town, Huairou District, Beijing (116°32'57''E, 40°20'13''N, altitude 54 m). Green fruits of the 'Jinqiao No. 2' variety were collected, and total RNA was extracted from the green fruits using the FastPure Universal Plant Total RNA Isolation Kit (purchased from Nanjing Novizan Biotechnology Co., Ltd., product number RC411-01). After RNA quality testing, TransScript was used to extract the RNA. II. One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (purchased from Beijing TransGen Biotech Co., Ltd., product number AH311-03) was used to reverse transcribe cDNA. Using cDNA as a template, PCR amplification was performed using 2×KeyPo master Mix high-fidelity enzyme (purchased from Nanjing Novizan Biotechnology Co., Ltd., product number PK511-03). The total volume of the PCR system was 25 μL, containing 1 μL of forward primer (10 μM), 1 μL of reverse primer (10 μM), 1 μL of template, 13 μL of 2×KeyPo master Mix, and 9 μL of ddH2O. The PCR reaction program was: 98℃, 30 s; 98℃, 10 s, 54℃, 5 s, 68℃, 30 s, 32 cycles; 72℃, 60 s.

[0038] The PCR amplification products were detected by 1% agarose gel electrophoresis (results are shown below). Figure 3 As shown in A), the target fragment was recovered using the FastPureGel DNA Extraction Mini Kit (purchased from Nanjing Novizan Biotechnology Co., Ltd., product number DC301). The recovered fragment was ligated into the cloning vector pEASY-Blunt (purchased from Beijing TransGen Biotech Co., Ltd., product number CB101-01). The total ligation volume was 5 μL: 1 μL pEASY-Blunt vector and 4 μL purified product. The ligation reaction was carried out at 25℃ for 30 min. The ligation product was transformed into E. coli competent cells DH5α (purchased from Shanghai Weidi Biotechnology Co., Ltd., product number DL1001M) using the heat shock method. Single colonies were picked and colony PCR was performed. The total volume of the reaction system was 25 μL: containing 13 μL 2×Taq PCR Mix, 1 μL template, 1 μL forward primer, 1 μL reverse primer and 9 μL water. The PCR reaction program was: 94℃, 3 min; 94℃, 30 s, 55℃, 30 s, 72℃, 90 s, 32 cycles; 72℃, 5 min. Plasmids were extracted from positive strains using the FastPure Plasmid Mini Kit (purchased from Nanjing Novizan Biotechnology Co., Ltd., product number DC201). Positive clones were selected based on PCR results and sequenced by Beijing Nuosai Genome Research Center Co., Ltd. The sequencing results were compared with the genome sequence, and the nucleotide sequence was found to be 100% similar to the original data.

[0039] Construction of E. coli prokaryotic expression vector: Based on the principles of In-Fusion gene cloning technology, the target gene was constructed into the pET32a prokaryotic expression vector (purchased from the BioVector plasmid vector strain cell protein antibody gene preservation center). BamHI and SalHI were selected as restriction enzyme sites, and homologous arm primers were designed using Snapgene software. The obtained homologous arm primers are shown in Table 2. PCR amplification was performed using the high-fidelity enzyme 2×KeyPo master Mix, and the target fragment was purified using the FastPure Gel DNAExtraction Mini Kit (Nanjing Novizan Biotechnology Co., Ltd., product number DC301). The pET32a plasmid was digested using BamHI and SalI restriction enzymes (purchased from Shanghai Youyizhonglian Biotechnology Co., Ltd., NEB brand product numbers R3136S and R3138S, respectively) to obtain the pET32a linear vector. The purified product was ligated into the pET32a linear vector using the ClonExpress Ultra One Step Cloning Kit ligase (purchased from Nanjing Novizan Biotechnology Co., Ltd., product number C115). The total volume of the ligation system was 5 μL, containing 2.5 μL of 2×ClonExpress Mix, 1.5 μL of linear vector, and 1 μL of purified product. The mixture was incubated at 37°C for 30 min. The constructed recombinant plasmid was transformed into *E. coli* DH5α competent cells and cultured overnight at 37°C. Single colonies were picked, and the upstream primer pET32a-T7-F (TAATACGACTCACTATAGGG (SEQ ID NO. 19)) and the downstream primer pET32a-T7ter-R (TGCTAGTTATTGCTCAGCGG (SEQ ID NO. 20)) of the pET32a expression vector were selected for PCR identification. The total volume of the PCR system was 25 μL, containing 13 μL of 2×Taq PCR Mix, 1 μL of template, 1 μL of forward primer, 1 μL of reverse primer, and 9 μL of water. The PCR reaction program was: 94°C, 3 min; 94°C, 30 s, 55°C, 30 s, 72°C, 90 s, 32 cycles; 72°C, 5 min. The PCR amplification products were detected by 1% agarose gel electrophoresis (results are shown in the figure). Figure 3 As shown in B), the product size is around 1500 bp, indicating that the target gene was successfully constructed into the pET32a expression vector. Plasmids were extracted from the positive strain using the FastPure Plasmid Mini Kit (purchased from Nanjing Novizan Biotechnology Co., Ltd., product number DC201) and stored at -20℃.

[0040] Table 2 Homologous arm primers

[0041] Note: Lowercase letters in the sequence are homologous arms.

[0042] Example 2: Gene sequence and its encoded protein sequence The positive gene cloned in Example 1 was sequenced by Beijing Nuosai Genome Research Center Co., Ltd. The sequencing results were compared with the sequence obtained from the tartary buckwheat genome, and the results showed that: FtOMT19 The sequencing sequence is completely identical to the genome acquisition sequence (C_AA168263.1). FtOMT19 A protein containing 756 nucleotides and encoding 251 amino acids, the nucleotide and amino acid sequences of which are shown in SEQ ID NO. 4 and SEQ ID NO. 1, respectively; FtOMT20 The sequencing sequence is completely identical to the genome acquisition sequence (C_AA168264.1). FtOMT20 A protein containing 1098 nucleotides and encoding 365 amino acids, the nucleotide and amino acid sequences of which are shown in SEQ ID NO. 5 and SEQ ID NO. 2, respectively; FtOMT24 The sequencing sequence is completely identical to the genome acquisition sequence (C_AA168265.1). FtOMT24 The protein contains 753 nucleotides and encodes 250 amino acids, and its nucleotide and amino acid sequences are shown in SEQ ID NO. 6 and SEQ ID NO. 3, respectively.

[0043] Example 3: Verification of gene function Transformation of BL21 *E. coli*: *E. coli* was transformed using a chemical transformation method. BL21 competent *E. coli* cells (purchased from Shanghai Weidi Biotechnology Co., Ltd., product number EC1002) were removed from a -80℃ freezer, thawed on ice, and 1 μL of plasmid DNA extracted in Example 1 was added to 30 μL of competent *E. coli* cells and mixed. The mixture was incubated on ice for 20 min, followed by heat shock at 42℃ for 45 s, and then incubated on ice again for 3 min. 500 μL of antibiotic-free LB agar (containing 10 g / L trypsin, 5 g / L yeast extract, and 10 g / L NaCl) was added, and the mixture was incubated at 37℃ with shaking for 30 min. After centrifugation at 4000 rpm for 2 min, the supernatant was discarded, and 100 μL of the bacterial culture was resuspended and plated on monoclonal antibody LB agar (containing 100 mg / L Amp antibiotic). The plates were then incubated at 37℃ inverted for 24 h. Single colonies were picked from LB agar plates using sterile toothpicks and added to 500 μL of liquid monoclonal antibody LB medium (containing 10 g / L trypsin, 5 g / L yeast extract, 10 g / L NaCl, and 100 mg / L Amp antibiotic). The plates were incubated at 37°C with shaking for 12 h. The upstream primer pET32a-T7-F (TAATACGACTCACTATAGGG (SEQ ID NO. 19) and the downstream primer pET32a-T7ter-R (TGCTAGTTATTGCTCAGCGG (SEQ ID NO. 20)) of the pET32a expression vector were used for bacterial PCR identification. The total volume of the system was 25 μL: containing 13 μL of 2×Taq PCR Mix, 1 μL of bacterial culture, 1 μL of forward primer, 1 μL of reverse primer, and 9 μL of water. The PCR reaction program was: 94°C, 3 min; 94°C, 30 s; 55°C, 30 s; 72°C, 90°C. s, 32 cycles; 72℃, 5 min. The PCR amplification products were detected by 1% agarose gel electrophoresis (results are shown below). Figure 3 As shown in C), the product size is around 1500bp, indicating that the recombinant plasmid has been successfully transformed into BL21 Escherichia coli. The positive strain will proceed to the next step of in vitro expression experiment.

[0044] Induction of in vivo protein expression in *E. coli*: BL21 strain containing recombinant plasmid was inoculated into 1 mL of monoclonal antibody LB liquid medium (containing 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and 100 mg / L Amp antibiotic) and cultured at 37°C with shaking for 12 h. Then, 1 mL of the bacterial culture was transferred to 500 mL of monoclonal antibody LB liquid medium (containing 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and 100 mg / L Amp antibiotic) and cultured at 37°C with shaking for 3–6 h. When the bacteria reached the logarithmic growth phase with an OD60 of approximately 0.6–0.8, IPTG (isopropyl-3-4-H2O) was added to a final concentration of 0.3 mmol / L. β - D (-thiogalactopyranoside), induced culture at 16℃ and 110 rpm for 24 h, centrifuged at 4℃ and 8000 rpm for 10 min, discarded the supernatant, and collected the bacterial cells. The bacterial cells were resuspended in 5 mL of 1×PBS (0.01 mol / L phosphate buffer, pH 7.5) and lysed on ice using a cell disruptor (27W, 5 sec on, 5 sec off) for 20 min. After disruption, centrifuged at 4℃ and 12000 rpm for 20 min, and the supernatant was transferred to a new centrifuge tube as the crude enzyme solution. The protein purification kit BeyoGold was used. TM His-tag Purification Resin (purchased from Beyotime Biotechnology Co., Ltd., product number P2210) was used to purify the target protein according to the manufacturer's instructions. Baicalein, 7,8-dihydroxyflavone, myricetin, quercetin, quercetin, and rutin were used for gene function verification. The total enzymatic reaction volume was 200 μL, including 50 mmol / L Tris-HCl buffer (pH 7.5), 1.25 mmol / L SAM, 50 μmol / L substrate, and 20 μL of purified protein. The reaction mixture was incubated at 37°C for 2 h, and then an equal volume of methanol was added to terminate the reaction. The reaction solution was then dried and stored in a vacuum centrifuge.

[0045] Catalytic product extraction and identification: The dried sample obtained above was added to 200 μL of methanol, shaken and mixed, and then extracted by ultrasonication for 30 min. After centrifugation at 12000 rpm for 5 min, the supernatant was filtered through a 0.22 μm organic phase filter membrane and transferred to an Agilent sample vial for HPLC-QTOF-MS / MS detection.

[0046] Preparation of Standards: (1) Accurately weigh 2.7024 mg, 2.5424 mg, 3.1824 mg, 3.0224 mg, 4.4838 mg, and 6.1052 mg of baicalein, 7,8-dihydroxyflavone, myricetin, quercetin, quercetin glycoside, and rutin standard powder respectively, and dissolve each in 1 mL of methanol to prepare a stock solution with a concentration of 10 mmol / L. When using, dilute the above stock solution to 50 μmol / L as a substrate for enzyme catalytic reaction. (2) Accurately weigh 0.001 g of isorhamnetin, narcissin, 6-oxomethylbaicalein, and 8-hydroxy-7-oxomethylflavone standard powder respectively, and dissolve each in 1 mL of methanol. Take 10 μL of the above solution, dilute it 100 times with methanol, mix them, and transfer 200 μL of each solution to an Agilent sample vial for HPLC-QTOF-MS / MS detection. Detection was performed using an Agilent 6546 triple quadrupole-time-of-flight tandem liquid chromatograph-mass spectrometer (HPLC-QTOF-MS / MS) and an Agilent 120-EC HPLC C18 column (2.1 mm × 50 mm, 1.8 μm). Mobile phase A was 0.1% formic acid in water (v / v), and mobile phase B was acetonitrile. The gradient settings were: 0–2 min, 5% B; 2–2.5 min, 5%→18.5% B; 2.5–10.5 min, 18.5%→41% B; 10.5–11 min, 41%→59% B; 11–12 min, 59%→100% B; 12–16 min, 100% B. The mobile phase flow rate was 0.3 mL / min, and the column temperature was 40 °C. Mass spectrometry conditions: Electrospray ionization (ESI) source was used for detection in negative ion mode. MS parameters were set as follows: spray voltage 2.50 kV; capillary temperature 250 °C; MS scan range 100–1200 m / z, scan rate 1000 Da / s; heater temperature 500 °C; sheath gas flow rate 50.0 L / min, auxiliary gas flow rate 13.0 L / min, and purge gas flow rate 3.0 L / min. The acquired ion data were saved in Centroid mode.

[0047] LC-QTOF-MS / MS analysis results are as follows Figure 4 As shown. FtOMT19 and FtOMT24 It can catalyze the formation of isorhamnetin and 3',4'-dioxymethylquercetin from quercetin. FtOMT20 It can catalyze the formation of isorhamnetin from quercetin ( Figure 4 -A); FtOMT19 and FtOMT24 It can catalyze the formation of quercetin from 3'-oxomethylquercetin and 3',4'-dioxomethylquercetin. FtOMT20It can catalyze the formation of quercetin from 3'-oxymethylquercetin ( ). Figure 4 -B); FtOMT19 and FtOMT24 It can catalyze the formation of narcissin from rutin ( Figure 4 -C); FtOMT19 and FtOMT24 It can catalyze the formation of myricetin into syringin, 3',4'-dioxymethylmyricetin, and 3',4',5'-trioxymethylmyricetin. FtOMT20 It can catalyze the formation of myricetin into syringin ( Figure 4 -D); FtOMT19 and FtOMT24 It can catalyze the formation of 6-oxomethylbaicalein from baicalein. FtOMT20 It can catalyze the formation of 6-oxomethylbaicalein and 7-oxomethylbaicalein from baicalein ( Figure 4 -E); FtOMT19 , FtOMT20 and FtOMT24 7,8-Dihydroxyflavone is converted into 8-hydroxy-7-oxomethylflavone and 7-hydroxy-8-oxomethylflavone ( Figure 4 -F).

[0048] As can be seen from the above embodiments, this invention provides a class of OMT genes, primer sets, and applications for catalyzing the methylation of flavonoids in tartary buckwheat. Based on the analysis of tartary buckwheat genome, whole-genome resequencing, transcriptomics, spatial transcriptomics, metabolomics, and spatial metabolomics data, this invention has identified OMT genes that catalyze the methylation of baicalin, 7,8-dihydroxyflavone, myricetin, quercetin, quercetin, or rutin, and verified these genes using an E. coli prokaryotic expression system. FtOMT19 , FtOMT20 , FtOMT24 All of these can catalyze the formation of oxymethylflavonoids from flavonoids. This not only provides an important gene element for the biosynthesis of oxymethylflavonoids, but also provides a key gene locus for molecular breeding of tartary buckwheat, and provides a theoretical basis for the methylation modification of other flavonoids.

[0049] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A class of oxymethyltransferases catalyzing the synthesis of oxymethylflavonoids from tartary buckwheat, characterized in that, include FtOMT19, FtOMT20 or FtOMT24 The FtOMT19 The amino acid sequence is shown in SEQ ID NO. 1; FtOMT20 The amino acid sequence is shown in SEQ ID NO. 2; FtOMT24 The amino acid sequence is shown in SEQ ID NO.

3.

2. The gene encoding the oxygen methyltransferase of claim 1, characterized in that, The FtOMT19 The nucleotide sequence is shown in SEQ ID NO.

4. FtOMT20 The nucleotide sequence is shown in SEQ ID NO.

5. FtOMT24 The nucleotide sequence is shown in SEQ ID NO.

6.

3. A primer set for amplifying the gene of claim 2, characterized in that, Including upstream primers with nucleotide sequences as shown in SEQ ID NO.7 FtOMT19 -F and downstream primers with nucleotide sequences as shown in SEQ ID NO. 8 FtOMT19 -R; or Including upstream primers with nucleotide sequences as shown in SEQ ID NO. 9 FtOMT20 -F and downstream primers with nucleotide sequences as shown in SEQ ID NO. 10 FtOMT20 -R; or Including the upstream primer with a nucleotide sequence as shown in SEQ ID NO.

11. FtOMT24 -F and downstream primers with nucleotide sequences as shown in SEQ ID NO. 12 FtOMT24 -R.

4. The application of the oxymethyltransferase of claim 1 in the catalytic methylation of tartary buckwheat flavonoids.

5. The use of the oxymethyltransferase of claim 1 in catalyzing the methylation of baicalin, 7,8-dihydroxyflavone, myricetin, quercetin, quercetin or rutin.

6. The application according to claim 5, characterized in that, The baicalin is catalyzed to produce 6-oxomethylbaicalin and 7-oxomethylbaicalin; The 7,8-dihydroxyflavone was catalyzed to generate 8-hydroxy-7-oxomethylflavone and 7-hydroxy-8-oxomethylflavone; The myricetin is catalyzed to generate syringin, 3',4'-dioxymethylmyricetin, and 3',4',5'-trioxymethylmyricetin; The quercetin catalyzes the formation of isorhamnetin and 3',4'-dioxymethylquercetin; The quercetin is catalyzed to produce 3'-oxymethylquercetin and 3',4'-dioxymethylquercetin; The rutin catalyzes the formation of narcissin.

7. The use of the oxymethyltransferase of claim 1 in the synthesis of oxymethylflavonoids.

8. The application of the oxymethyltransferase described in claim 1 in marker-assisted breeding of tartary buckwheat.

9. The application of the oxymethyltransferase described in claim 1 in the breeding of new varieties of tartary buckwheat.

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