CN115961086B - Tea plant flavonoid 3-O-methyltransferase gene and application thereof - Google Patents
Tea plant flavonoid 3-O-methyltransferase gene and application thereof Download PDFInfo
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Abstract
The invention discloses a tea plant flavonoid 3-O-methyltransferase gene and application thereof, belonging to the field of molecular biology. The invention discovers a gene CccoAOMT for controlling flavonoid methoxylation such as ECG, EGCG and the like by a whole genome association analysis method, the nucleotide sequence of the gene CccoAOMT is shown as SEQ ID NO.3, and a C/G mutation exists at the 314bp position of the sequence. The gene can code a methoxytransferase, and the amino acid sequence of the methoxytransferase is shown as SEQ ID NO. 4. The gene can be used for rapidly screening tea tree 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 simultaneously, 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 plant flavonoid 3-O-methyltransferase gene and application thereof.
Background
Epigallocatechin gallate (EGCG) and epicatechin gallate (ECG) are catechin monomers with high content in tea, wherein EGCG accounts for about 10-20% of tea tender leaf dry weight (one bud and two leaves), and ECG accounts for about 5-10% of tea tender leaf dry weight. EGCG-3 ' -Me is methoxy derivative of EGCG, and a great deal of research shows that EGCG-3 ' -Me has good antiallergic effect, and the development of tea with high content of EGCG-3 ' -Me has wide market.
EGCG-3"-Me is of great interest in the food and hygiene fields. In recent years, several researchers have tried to utilize the protein coded by caffeoyl-coenzyme AO-methyltransferase gene (GenBank accession No. DD361102) to synthesize in vitro, and the results of the existing in vitro enzymatic synthesis research show that the EGCG conversion rate of in vitro expressed tea tree EGCG-O-methyltransferase in the EGCG-3' -Me experiment is lower. Thus, the main method for obtaining EGCG-3' -Me is still extracted from tea leaves. However, ECG and EGCG are higher in most tea tree varieties and germplasm resources, but only a few tea tree germplasm resources have a higher ECG-3"-Me, EGCG-3" -Me content.
The method for detecting the metabolites such as ECG-3 '-Me and EGCG-3' -Me in tea trees is usually to detect the metabolites through LC-MS, however, since the detection result of the metabolites is influenced by various factors, a plurality of repeated experiments are needed to ensure the stability and the reliability, and the method is time-consuming, labor-consuming and easy to have large systematic errors.
Therefore, there is a need to develop a precise and rapid screening method for tea plant germplasm resources, so as to rapidly and accurately screen out tea plant germplasm resources with higher ECG-3 '-Me, EGCG-3' -Me content.
Disclosure of Invention
The invention aims to provide a tea plant flavonoid 3-O-methyltransferase gene and application thereof, so as to solve the problems in the prior art. The gene provided by the invention can be used for rapidly screening tea tree 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 encoded 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 above object, the present invention provides the following solutions:
the invention provides a molecular marker related to the content of ECG-3 '-Me and EGCG-3' -Me in tea trees, wherein the molecular marker is a gene CccoAOMT, the nucleotide sequence of the gene CccoAOMT is shown as SEQ ID NO.3, and a 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 high-content tea tree germplasm resources of the ECG-3 '-Me and the EGCG-3' -Me, which comprises the following steps:
(1) Taking DNA of tea tree to be detected as a template, and adopting a primer pair shown in SEQ ID NO.1-2 to carry out PCR amplification to obtain a clone gene CccoAOMT;
(2) Sequencing analysis is carried out on the cloned gene CccoAOMT, and when the sequence shown as SEQ ID NO.3 appears in the cloned gene CccoAOMT, the tea tree is the tea tree germplasm with high content of ECG-3 '-Me and EGCG-3' -Me.
Further, in the step (3), the method further comprises detecting the expression level of the cloned gene, and selecting tea trees with FPKM value higher than 60 as high-content tea tree germplasm of ECG-3 '-Me and EGCG-3' -Me.
The invention also provides tea plant flavonoid 3-O-methyltransferase, the amino acid sequence of which is shown as SEQ ID NO.4 and is encoded 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 to convert ester catechin into ECG-3' -Me.
Further, in the in vitro catalytic reaction, the ester catechin is an acceptor of methoxy group, and flavonoid containing methoxy group is used as a donor of methoxy group.
Further, the ester-type catechin includes ECG.
The invention also provides application of the molecular marker in screening ECG-3 '-Me and EGCG-3' -Me high-content tea tree germplasm resources.
The invention also provides an application of the gene CccoAOMT or the 3-O-methyltransferase in synthesizing ECG-3 '-Me and EGCG-3' -Me in vitro.
The invention discloses the following technical effects:
the invention discovers a gene CccoAOMT 1 for controlling flavonoid methoxylation such as ECG and EGCG by a whole genome association analysis method, and can be used for rapidly screening tea tree varieties with higher ECG-3 '-Me and EGCG-3' -Me content for cultivation, shortening the breeding period by 2-3 years, improving the breeding efficiency, and simultaneously, the CccoAOMT 1 can code a methoxytransferase which can be used for in vitro synthesis of ECG-3 '-Me and EGCG-3' -Me and has higher synthesis efficiency.
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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 that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the LC-MS detection results of metabolites ECG-3"-Me and EGCG-3" -Me;
FIG. 2 shows the close correlation of the QTL where the 3-O-methyltransferase gene is located with the metabolites with methoxy groups in tea tree;
FIG. 3 shows the content differences of the corresponding metabolites ECG-3 '-Me and EGCG-3' -Me of different alleles;
FIG. 4 shows the LC-MS detection results of the supernatant products.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions 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. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, 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 by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. 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 invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
Example 13 localization of the O-methyltransferase Gene CccoAOMT
The invention collects 220 tea plant germplasm resources at home and abroad, comprising wild species and cultivated species, sequences transcriptome mRNA thereof, compares the sequencing result to a tea plant reference genome G240, carries out genotyping to obtain SNPs of expressed genes, simultaneously uses LC-MS to carry out qualitative and quantitative analysis on ECG-3 '-Me and EGCG-3' -Me in flowers and leaves in 220 tea plant germplasm resources, the LC-MS detection results of metabolites ECG-3 '-Me and EGCG-3' -Me are shown as figure 1, carries out correlation analysis on the content of metabolites as phenotype data and genotype data, discovers QTL of chromosome 7 in the tea plant genome, locates 3-O-methyltransferase gene CsCCoAOMT (CsCCoAOMT annotated file of tea plant reference genome G240 is downloaded from https:// www.ncbi.nlm.nih.gov/wherein the CsCCoMT registration number is KAF 5945117.1), and corresponds to two genotypes with high and low content of metabolites, and locates at 35 bp C84 of chromosome C in the genome.
Quantitative and qualitative analysis is carried out on ECG-3 '-Me and EGCG-3' -Me in leaves and flowers of 220 tea plant germplasm resources through annual repetition, and whole genome association analysis is carried out, so that the results prove that the QTL obtained by the association analysis is stable.
Example 23 correlation of the O-methyltransferase Gene CccoAOMT with the content of ECG-3"-Me and EGCG-3" -Me in tea plant
Designing a forward primer (SEQ ID NO. 1) 5'-ATGGCGGATAATATTGTTTTA-3' and a reverse primer (SEQ ID NO. 2) 5'-GAGTATGCGCCTGCACAATGT-3', and carrying out PCR (polymerase chain reaction) amplification reaction on a gene CsCCoAOMT by taking cDNA of tea plant germplasm resources as a template, wherein an amplification system is as follows: 2. Mu.L of template, 3. Mu.L of 10 Xamplification buffer, 0.6. Mu.L of 4 dNTP mixture (dATP, dGTP, dTTP, dCTP), 0.6. Mu.L of forward and reverse primers, 2.5. Mu.L of TaqDNA polymerase, and 24. Mu.L of pure water. Amplification procedure step 1, 94℃for 2min; step 2, the temperature is 94 ℃ and 25s; step 3, the temperature is 56 ℃ and 20s; step 4, the temperature is 72 ℃ for 35s; step 5, namely, cycling for 35 times in the steps 2-4 at the temperature of 72 ℃ for 5 min. Sequencing analysis and protein sequence translation are carried out on the amplification result, two alleles CsCCoAOMT1 and CsCCoAOMT2 of the gene are obtained, and when the gene is CsCCoAOMT1, the SNP of the gene at 19390344bp of chromosome 7 of the tea genome is C, and an alanine is encoded; when the gene is CccoAOMT 2, the SNP at 19390344bp of chromosome 7 of the gene tea tree genome is G, and codes for a glycine. The amino acid sequences of the genes CsCCoAOMT1 and CsCCoAOMT2 and the coded 3-O-methyltransferase are shown in SEQ ID NO. 3-6.
Nucleotide sequence of gene csccooaomt 1 (SEQ ID No. 3):
ATGGCGGATAATATTGTTTTAAAGACCATCCTGCAAAGCGAGGCTCTTCAAAAGTACATCTTTGATACCAATGTATACCCAAGAGAACATGAGCAGCTCAAGAGGATAAGAGATGCAACATTCAAGAAATATGGCGACAGAGCGGAGCTAAGTGTGCCCCCTGATGAAGGATTGTTCTTGTCTATGCTTTTGAAATTAATGAATGCCAAGAAGACATTGGAGATTGGTGTTTTCACTGGCTATTCTCTTCTTACCACTGCCCTTGCTTTACCTCATGATGGCCAGATAGTAGCAATAGATCCAAATCGAGAAGCATTTGAAGTTGGACTGCCATTCATTCAGAAGGCTGGTGTGGAGCACAAGATCAATTTCATTGAATCAGATGCCATTTCTGTTCTCAATGAAATGTTGAGCGATGAGGGTAAGTTGCACTTGTGGATT. (SNP locus is underlined in bold)
The amino acid sequence of the 3-O-methyltransferase encoded by the gene CccoAOMT 1 (SEQ ID NO. 4):
MQHSRNMATELSVPPDERLFLSMLLKLMNAKKILEIGVFTGYSLLTTALALPHD GQIVAIDPNREAFEVGLPFIQKVGVEHKIIFIDSDAISVLNEMLSNVIVSNEEEVPEFLCI GRKPIIELNKYLASDPRIEIAQISISDGVTLCRRI. (amino acids encoded by nucleotides comprising SNP sites are underlined in bold)
Nucleotide sequence of gene csccooaomt 2 (SEQ ID No. 5):
ATGGCGGATAATATTGTTTTAAAGACCATCCTGCAAAGCGAGGCTCTTCAAAAGTACATCTTTGATACCAATGTATACCCAAGAGAACATGAGCAGCTCAAGAGGATAAGAGATGCAACATTCAAGAAATATGGCGACAGAGCGGAGCTAAGTGTGCCCCCTGATGAAGGATTGTTCTTGTCTATGCTTTTGAAATTAATGAATGCCAAGAAGACATTGGAGATTGGTGTTTTCACTGGCTATTCTCTTCTTACCACTGCCCTTGCTTTACCTCATGATGGCCAGATAGTAGCAATAGATCCAAATCGAGAAGGATTTGAAGTTGGACTGCCATTCATTCAGAAGGCTGGTGTGGAGCACAAGATCAATTTCATTGAATCAGATGCCATTTCTGTTCTCAATGAAATGTTGAGCGATGAGGGTAAGTTGCACTTGTGGATT. (SNP locus is underlined in bold)
The amino acid sequence of the 3-O-methyltransferase encoded by the gene CccoAOMT 2 (SEQ ID NO. 6):
MQHSRNMATELSVPPDERLFLSMLLKLMNAKKILEIGVFTGYSLLTTALALPHD GQIVAIDPNREGFEVGLPFIQKVGVEHKIIFIDSDAISVLNEMLSNVIVSNEEEVPEFLCI GRKPIIELNKYLASDPRIEIAQISISDGVTLCRRI. (amino acids encoded by nucleotides comprising SNP sites are underlined in bold)
When the CsCCoAOMT1 gene appears in the amplified product (comprising the cases of only CsCCoAOMT1 or both CsCCoAOMT1 and CsCCoAOMT 2), the content of ECG-3 '-Me and EGCG-3' -Me in the tea plant germplasm resource is relatively high; when only the csccooaomt 2 gene is present in the amplified product or is not cloned into this gene, the tea germplasm resources have lower levels of ECG-3"-Me and EGCG-3" -Me. (FIG. 3).
Example 33 correlation of expression level of CsCCoAOMT gene with ECG-3"-Me and EGCG-3" -Me content in tea plant
The content of ECG-3 '-Me and EGCG-3' -Me in tea trees is jointly influenced by genotype and gene expression quantity, and when the relative gene expression quantity FPKM value is higher than 60, the relative content of the metabolite ECG-3 '-Me in tea tree population is 1907.4, and the content of EGCG-3' -Me is 1784.158617; when the relative expression quantity FPKM value of the gene is lower than 60, the metabolite ECG-3 '-Me relative content is 368.3362396, and EGCG-3' -Me content is 242.4492192. It can be seen that ECG-3"-Me and EGCG-3" -Me content is relatively high when the FPKM value is greater than 60. Therefore, specific germplasm resources with high ECG-3 '-Me and EGCG-3' -Me content are screened for tea tree breeding by combining the 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 purified protein was added to 1.5mL of a 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 the reaction was terminated by adding 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 by CsCCoAOMT gene control can catalyze the conversion of ECG into ECG-3' -Me by taking ester catechin such as EGCG and ECG and flavonoid containing methoxy group as substrates, taking ECG as methoxy group acceptor and other flavonoid containing methoxy group as donor of methoxy group.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (4)
1. The application of a detection reagent of molecular markers related to ECG-3 '-Me and EGCG-3' -Me content in tea trees in breeding of ECG-3 '-Me and EGCG-3' -Me content in tea trees is characterized in that the molecular markers are genesCsCCoAOMTThe geneCsCCoAOMTThe nucleotide sequence of (2) is shown as SEQ ID NO.3, C/G mutation exists at the position 314 and bp of the sequence, when the genotype at the position is CC or CG, the content of ECG-3 '-Me and EGCG-3' -Me in tea trees is high, and when the genotype at the position is GG, the content of ECG-3 '-Me and EGCG-3' -Me in tea trees is low.
2. A method for screening high-content tea tree germplasm resources of ECG-3 '-Me and EGCG-3' -Me, which is characterized by comprising the following steps:
using DNA of tea tree to be detected as template, adopting primer pair shown as SEQ ID NO.1-2 to make PCR amplification so as to obtain the cloned geneCsCCoAOMT；
For the cloned geneCsCCoAOMTSequencing analysis is performed when the cloned geneCsCCoAOMTWhen the sequence shown as SEQ ID NO.3 appears, the tea tree is the tea tree germplasm with high content of ECG-3 '-Me and EGCG-3' -Me.
3. The method according to claim 2, further comprising detecting the expression level of the cloned gene in step (3), wherein the tea plants having an FPKM value higher than 60 are selected as the germplasm of tea plants having a high content of ECG-3"-Me and EGCG-3" -Me.
4. The application of a detection reagent of molecular markers related to ECG-3 '-Me and EGCG-3' -Me content in tea trees in screening ECG-3 '-Me and EGCG-3' -Me high-content tea tree germplasm resources is characterized in that the molecular markers are genesCsCCoAOMTThe geneCsCCoAOMTNucleotide sequences of (A) such asAs shown in SEQ ID NO.3, there is a C/G mutation at position 314 and bp of the sequence, and when the genotype at this position is CC or CG, the content of ECG-3 '-Me and EGCG-3' -Me in tea trees is high.
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