CN115961086A - Tea tree flavonoid 3-O-methyltransferase gene and application thereof - Google Patents
Tea tree flavonoid 3-O-methyltransferase gene and application thereof Download PDFInfo
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Abstract
The invention discloses a tea tree flavonoid 3-O-methyltransferase gene and application thereof, belonging to the field of molecular biology. The invention discovers a gene CsCCoAOMT for controlling flavonoid methoxylation such as ECG, EGCG and the like by a whole genome correlation analysis method, wherein the nucleotide sequence of the gene CsCCoAOMT is shown as SEQ ID NO.3, and C/G mutation exists at the 314bp position of the sequence. The gene can encode a methoxytransferase, and the amino acid sequence of the methoxytransferase is shown in SEQ ID No. 4. By utilizing the gene, tea varieties with high ECG-3 ' -Me and EGCG-3 ' -Me contents can be rapidly screened for cultivation, the breeding period is shortened by 2-3 years, the breeding efficiency is improved, and meanwhile, the CsCCoAOMT1 protein can be used for in-vitro synthesis of ECG-3 ' -Me, and the synthesis efficiency is higher.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a tea tree flavonoid 3-O-methyltransferase gene and application thereof.
Background
Epigallocatechin gallate (EGCG) and epicatechin gallate (ECG) are high catechin monomers in tea, wherein EGCG accounts for about 10-20% of dry weight of tea plant tender leaves (one bud and two leaves), and ECG accounts for about 5-10% of dry weight of tea plant tender leaves. EGCG-3 ' -Me is methoxy derivative of EGCG, and a great deal of research shows that the EGCG-3 ' -Me has good antiallergic effect, so that the development of high-content EGCG-3 ' -Me tea has wide market.
EGCG-3' -Me is of great interest in the food and hygiene sector. In recent years, a lot of researchers try to perform in-vitro synthesis by using a protein coded by a caffeoyl-coenzyme AO-methyltransferase gene (GenBank accession No. DD361102), and the results of the existing in-vitro enzyme synthesis research show that the conversion rate of EGCG in an experiment of synthesizing EGCG-3' -Me by in-vitro expressed tea tree EGCG-O-methyltransferase is low. Therefore, the main method for obtaining EGCG-3' -Me at present is still extraction in tea. However, the ECG and EGCG are higher in content in most tea tree varieties and germplasm resources, but only a few tea tree germplasm resources have higher content of ECG-3 '-Me, EGCG-3' -Me.
The method for detecting metabolites such as ECG-3 '-Me and EGCG-3' -Me in tea trees is generally to measure the metabolites through LC-MS, however, as the metabolite detection result is influenced by various factors, a plurality of repeated experiments are needed to ensure the stability and reliability of the metabolites, time and labor are wasted, and larger system errors are easy to exist.
Therefore, a method for accurately and rapidly screening tea plant germplasm resources is needed to be developed so as to rapidly and accurately screen out tea plant germplasm resources with high ECG-3 '-Me, EGCG-3' -Me content.
Disclosure of Invention
The invention aims to provide a tea tree flavonoid 3-O-methyltransferase gene and application thereof, which are used for solving the problems in the prior art. The gene provided by the invention can be used for rapidly screening tea varieties with higher ECG-3 '-Me and EGCG-3' -Me contents for cultivation, the breeding period is shortened by 2-3 years, the breeding efficiency is improved, and meanwhile, the protein coded by the gene can be used for in vitro synthesis of ECG-3 '-Me and EGCG-3' -Me, and the synthesis efficiency is higher.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a molecular marker related to ECG-3 '-Me and EGCG-3' -Me contents in tea trees, wherein the molecular marker is a gene CsCCoAOMT, the nucleotide sequence of the gene CsCCoAOMT is shown as SEQ ID NO.3, and C/G mutation exists at the 314bp position of the sequence.
The invention also provides application of the molecular marker in tea tree breeding.
The invention also provides a method for screening the tea plant germplasm resources with high ECG-3 '-Me and EGCG-3' -Me contents, which comprises the following steps:
(1) Performing PCR amplification by using DNA of a tea tree to be detected as a template and adopting a primer pair shown in SEQ ID NO.1-2 to obtain a cloned gene CsCCoAOMT;
(2) And (3) sequencing analysis is carried out on the cloned gene CsCCoAOMT, and when a sequence shown as SEQ ID NO.3 appears in the cloned gene CsCCoAOMT, the tea tree is the tea tree germplasm with high ECG-3 '-Me and EGCG-3' -Me contents.
Further, in the step (3), the expression amount of the cloned gene is detected, and tea trees with FPKM values higher than 60 are selected as tea tree germplasm with high ECG-3 '-Me and EGCG-3' -Me contents.
The invention also provides tea tree flavonoid 3-O-methyltransferase, wherein the amino acid sequence of the tea tree flavonoid 3-O-methyltransferase is shown as SEQ ID NO.4 and is coded by the nucleotide sequence shown as SEQ ID NO. 3.
The invention also provides a method for synthesizing ECG-3 '-Me in vitro, which utilizes the 3-O-methyltransferase to perform in vitro catalytic reaction so as to convert ester catechin into ECG-3' -Me.
Further, in the in vitro catalytic reaction, ester catechin is an acceptor of a methoxy group, and flavonoid containing the methoxy group is used as a donor of the methoxy group.
Further, the ester type catechin includes ECG.
The invention also provides application of the molecular marker in screening of ECG-3 '-Me and EGCG-3' -Me high-content tea tree germplasm resources.
The invention also provides application of the gene CsCCoAOMT or the 3-O-methyltransferase in-vitro synthesis of ECG-3 '-Me and EGCG-3' -Me.
The invention discloses the following technical effects:
the invention discovers a gene CsCCoAOMT1 for controlling the methoxylation of flavonoids such as ECG, EGCG and the like by a whole genome correlation analysis method, can quickly screen tea varieties with higher ECG-3 '-Me and EGCG-3' -Me contents for cultivation by utilizing the gene, shortens the breeding period by 2-3 years, improves the breeding efficiency, simultaneously can code a methoxytransferase, and can be used for the in vitro synthesis of ECG-3 '-Me and EGCG-3' -Me, and the synthesis efficiency is higher.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required in the embodiments will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 shows the LC-MS detection results of metabolites ECG-3"-Me and EGCG-3" -Me;
FIG. 2 is a close correlation of the QTL where the 3-O-methyltransferase gene is located with metabolites of the tea plant with the methyloxy group;
FIG. 3 shows the content differences of metabolites ECG-3"-Me and EGCG-3" -Me corresponding to different alleles;
FIG. 4 shows the results of LC-MS detection of the supernatant product.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but rather as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the documents are cited. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Example 13 localization of the O-methyltransferase Gene CsCCoAOMT
The method comprises the steps of collecting 220 tea plant germplasm resources at home and abroad, including wild species and cultivars, sequencing transcriptome mRNA of the tea plant germplasm resources, comparing sequencing results to a tea plant reference genome G240, genotyping to obtain SNPs of expressed genes, performing qualitative and quantitative analysis on ECG-3 '-Me and EGCG-3' -Me in flowers and leaves of the 220 tea plant germplasm resources by using LC-MS, performing LC-MS detection results of metabolites ECG-3 '-Me and EGCG-3' -Me as shown in figure 1, performing correlation analysis on the metabolite content as phenotype data and genotype data to find a QTL located on a tea plant genome No. 7 chromosome, and locating a 3-O-methyltransferase gene CsCCoAOMT (a tea plant reference genome G240 annotation file is located from htps:// www.ncbi.nlm.nih.goCCoV/download, wherein the CsCCoC registration number is KAF 5945117.1), the gene comprises two non-mutated genes and has high corresponding SNP (SNP), and the two non-mutated genes are located on a high-synonym SNP (SNP/bp) of the tea plant genome, and the SNP of the genome No. 1932.
And (3) performing qualitative and quantitative analysis on ECG-3 '-Me and EGCG-3' -Me in leaves and flowers of 220 tea plant germplasm resources by annual repetition, and performing whole genome association analysis, wherein the results prove that QTL obtained by association analysis is stable.
Example 23 correlation of the O-methyltransferase Gene CsCCoAOMT with the ECG-3"-Me and EGCG-3" -Me content in tea
Designing a forward primer (SEQ ID NO. 1) 5 'ATGGCGGATAATATATTGTTTTA-3', a reverse primer (SEQ ID NO. 2) 5 'GAGTATGCGCCTGCACAATTGT 3', and carrying out PCR amplification reaction on the gene CsCCoAOMT by taking cDNA of tea plant germplasm resources as a template, wherein the amplification system is as follows: mu.L of template, 3. Mu.L of 10 × amplification buffer, 0.6. Mu.L of 4 kinds of dNTP mixtures (dATP, dGTP, dTTP, dCTP,), 0.6. Mu.L of forward and reverse primers, 2.5. Mu.L of Taq DNA polymerase, and 24. Mu.L of purified water. Amplification procedure step 1 temperature 94 ℃,2min; step 2, the temperature is 94 ℃ for 25s; step 3, the temperature is 56 ℃ and 20s; step 4, the temperature is 72 ℃ and 35s; step 5 the temperature is 72 ℃ for 5min, and the steps 2-4 are circulated for 35 times. Sequencing analysis and protein sequence translation are carried out on the amplification result, two alleles CsCCoAOMT1 and CsCCoAOMT2 of the gene are obtained, when the gene is CsCCoAOMT1, the SNP of the gene at 19390344bp of the No. 7 chromosome of the tea tree genome is C, and one alanine is coded; when the gene is CsCCoAOMT2, the SNP at 19390344bp of the No. 7 chromosome of the gene tea tree genome is G, and one glycine is coded. The amino acid sequences of the genes CsCCoAOMT1 and CsCCoAOMT2 and the 3-O-methyltransferase coded by the genes are shown in SEQ ID NO. 3-6.
Nucleotide sequence of gene CsCCoAOMT1 (SEQ ID NO. 3):
ATGGCGGATAATATTGTTTTAAAGACCATCCTGCAAAGCGAGGCTCTTCAAAAGTACATCTTTGATACCAATGTATACCCAAGAGAACATGAGCAGCTCAAGAGGATAAGAGATGCAACATTCAAGAAATATGGCGACAGAGCGGAGCTAAGTGTGCCCCCTGATGAAGGATTGTTCTTGTCTATGCTTTTGAAATTAATGAATGCCAAGAAGACATTGGAGATTGGTGTTTTCACTGGCTATTCTCTTCTTACCACTGCCCTTGCTTTACCTCATGATGGCCAGATAGTAGCAATAGATCCAAATCGAGAAGC<xnotran> ATTTGAAGTTGGACTGCCATTCATTCAGAAGGCTGGTGTGGAGCACAAGATCAATTTCATTGAATCAGATGCCATTTCTGTTCTCAATGAAATGTTGAGCGATGAGGGTAAGTTGCACTTGTGGATT. </xnotran> (SNP site in bold underline)
The amino acid sequence of 3-O-methyltransferase encoded by the gene CsCCoAOMT1 (SEQ ID NO. 4):
MQHSRNMATELSVPPDERLFLSMLLKLMNAKKILEIGVFTGYSLLTTALALPHD GQIVAIDPNREAFEVGLPFIQKVGVEHKIIFFIDSDAISVLNEMLSNVIVSNEEEVPEFLCI GRKPIIELNKYLASDPRIEIAQISDGVTLCRI. (the amino acid encoded by the nucleotide comprising the SNP site is underlined in bold)
Nucleotide sequence of gene CsCCoAOMT2 (SEQ ID NO. 5):
ATGGCGGATAATATTGTTTTAAAGACCATCCTGCAAAGCGAGGCTCTTCAAAAGTACATCTTTGATACCAATGTATACCCAAGAGAACATGAGCAGCTCAAGAGGATAAGAGATGCAACATTCAAGAAATATGGCGACAGAGCGGAGCTAAGTGTGCCCCCTGATGAAGGATTGTTCTTGTCTATGCTTTTGAAATTAATGAATGCCAAGAAGACATTGGAGATTGGTGTTTTCACTGGCTATTCTCTTCTTACCACTGCCCTTGCTTTACCTCATGATGGCCAGATAGTAGCAATAGATCCAAATCGAGAAGG<xnotran> ATTTGAAGTTGGACTGCCATTCATTCAGAAGGCTGGTGTGGAGCACAAGATCAATTTCATTGAATCAGATGCCATTTCTGTTCTCAATGAAATGTTGAGCGATGAGGGTAAGTTGCACTTGTGGATT. </xnotran> (SNP site in bold underline)
Amino acid sequence of 3-O-methyltransferase encoded by the gene CsCCoAOMT2 (SEQ ID NO. 6):
MQHSRNMATELSVPPDERLFLSMLLKLMNAKKILEIGVFTGYSLLTTALALPHD GQIVAIDPNREGFEVGLPFIQKVGVEHKIIFIDSDAISVLNEMLSNVIVSNEEEVPEFLCI GRKPIIELNKYLASDPRIEIAQISDGVTLCRI. (the amino acid encoded by the nucleotide comprising the SNP site is underlined in bold)
When a CsCCoAOMT1 gene (including only CsCCoAOMT1 or both CsCCoAOMT1 and CsCCoAOMT 2) appears in an amplification product, the content of ECG-3 '-Me and EGCG-3' -Me in the tea tree germplasm resource is relatively high; when only CsCCoAOMT2 gene appears in the amplification product or the gene is not cloned, the content of ECG-3 '-Me and EGCG-3' -Me in the tea tree germplasm resource is lower. (FIG. 3).
Example 33 correlation of expression level of the O-methyltransferase Gene CsCCoAOMT with the ECG-3"-Me and EGCG-3" -Me contents in tea Tree
The ECG-3 '-Me and EGCG-3' -Me contents in tea trees are influenced by the genotype and the gene expression quantity, and when the FPKM value of the gene relative expression quantity is higher than 60, the ECG-3 '-Me relative content of tea tree population metabolites is 1907.4, and the EGCG-3' -Me content is 1784.158617; when the relative gene expression FPKM value is lower than 60, the metabolites ECG-3 '-Me of the tea plant population are 368.3362396, and the EGCG-3' -Me content is 242.4492192. It can be seen that the ECG-3"-Me and EGCG-3" -Me contents are relatively high when the FPKM value is greater than 60. Therefore, specific germplasm resources with high ECG-3 '-Me and EGCG-3' -Me contents are screened for tea tree breeding by the combination of identification of genotype and gene expression quantity.
Example 43 functional analysis of O-methyltransferase
Enzymatic studies of csccooaomt were performed using purified recombinant proteins. 0.01mg of the purified protein was added to a 1.5mL reaction system comprising 0.05mmol/LEGCG, 0.16mmol/LSAM, 0.2mmol/LMgCl 2 And 100mmol/LTris-HCl (pH = 7.8). The reaction mixture was incubated at 30 ℃ for 30 minutes and then quenched by the addition of 200. Mu.L of methanol. The supernatant product was analyzed by LC-MS and the results are shown in FIG. 4.
As can be seen from FIG. 4, in vitro enzyme activity verification shows that the 3-O-methyltransferase synthesized under the control of CsCCoAOMT gene can catalyze the conversion of ECG into ECG-3' -Me by using ester catechins such as EGCG and ECG and flavonoids containing methoxy groups as substrates, ECG as an methoxy group acceptor and other flavonoids containing methoxy groups as a donor of the methoxy groups.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (10)
1. A molecular marker related to ECG-3 '-Me and EGCG-3' -Me contents in tea trees is characterized in that the molecular marker is a gene CsCCoAOMT, the nucleotide sequence of the gene CsCCoAOMT is shown as SEQ ID NO.3, and C/G mutation exists at the 314bp position of the sequence.
2. Use of a molecular marker as claimed in claim 1 in tea tree breeding.
3. A method for screening ECG-3 '-Me and EGCG-3' -Me high-content tea plant germplasm resources is characterized by comprising the following steps:
(1) Performing PCR amplification by using DNA of a tea tree to be detected as a template and adopting a primer pair shown in SEQ ID NO.1-2 to obtain a cloned gene CsCCoAOMT;
(2) And (3) sequencing analysis is carried out on the cloned gene CsCCoAOMT, and when a sequence shown as SEQ ID NO.3 appears in the cloned gene CsCCoAOMT, the tea tree is the tea tree germplasm with high ECG-3 '-Me and EGCG-3' -Me contents.
4. The method as claimed in claim 3, wherein in the step (3), the expression level of the cloned gene is detected, and tea plant with FPKM value higher than 60 is selected as tea plant germplasm with high ECG-3 '-Me and EGCG-3' -Me content.
5. Tea tree flavonoid 3-O-methyltransferase is characterized in that the amino acid sequence of the tea tree flavonoid 3-O-methyltransferase is shown as SEQ ID No.4 and is encoded by the nucleotide sequence shown as SEQ ID No. 3.
6. A method for in vitro synthesis of ECG-3 '-Me, characterized in that an ester type catechin is converted into ECG-3' -Me by an in vitro catalytic reaction using the 3-O-methyltransferase of claim 5.
7. The method of claim 6, wherein the ester catechin is an acceptor of a methine group and the flavonoid having the methine group is a donor of the methine group in the in vitro catalytic reaction.
8. The method according to claim 6 or 7, wherein said ester-type catechin comprises ECG.
9. Use of the molecular marker of claim 1 in screening of ECG-3"-Me and EGCG-3" -Me high content tea plant germplasm resources.
10. Use of the gene csccooaomt according to claim 1 or the 3-O-methyltransferase according to claim 2 for the in vitro synthesis of ECG-3"-Me and EGCG-3" -Me.
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