CN115961086B - A kind of tea tree flavonoid 3-O-methyltransferase gene and its application - Google Patents

Abstract

The invention discloses a tea tree flavonoid 3-O-methyltransferase gene and its application, belonging to the field of molecular biology. The present invention discovers a gene CsCCoAOMT that controls the methoxylation of flavonoids such as ECG and EGCG through the method of genome-wide association analysis. The nucleotide sequence of the gene CsCCoAOMT is shown in SEQ ID NO.3, in the sequence There is a C/G mutation at 314bp. The gene can encode a methoxytransferase, and the amino acid sequence of the methoxytransferase is shown in SEQ ID NO.4. The gene can be used to quickly screen tea tree varieties with high content of ECG-3”-Me and EGCG-3”-Me for breeding, shorten the breeding period by 2-3 years, and improve breeding efficiency. At the same time, CsCCoAOMT1 protein can be used for ECG-3” ‑Me is synthesized in vitro, and its synthesis efficiency is higher.

Description

A kind of tea tree flavonoid 3-O-methyltransferase gene and its application

technical field

The invention relates to the field of molecular biology, in particular to a tea tree flavonoid 3-O-methyltransferase gene and its application.

Background technique

Epigallocatechin gallate (Epigallocatechin gallate, EGCG) and epicatechin gallate (ECG) are catechin monomers with a relatively high content in tea, of which EGCG accounts for about the dry weight of young tea leaves (one bud and two leaves ), ECG accounts for about 5-10% of the dry weight of tea tree young leaves. EGCG-3”-Me is a methoxy derivative of EGCG. A large number of studies have shown that EGCG-3”-Me has good anti-allergic effects, and the development of tea with high content of EGCG-3”-Me has a broad market.

EGCG-3”-Me has received great attention in the fields of food and hygiene. In recent years, many researchers have attempted to use the protein encoded by the caffeoyl-coenzyme AO-methyltransferase gene (GenBankAccessionNo.DD361102) for in vitro synthesis. The existing in vitro enzymatic synthesis research results show that the EGCG conversion rate is low in the synthesis of EGCG-3”-Me by tea tree EGCG-O-methyltransferase expressed in vitro. Therefore, the main method to obtain EGCG-3”-Me is still to extract from tea leaves. However, ECG and EGCG are relatively high in most tea tree varieties and germplasm resources, but only a few tea tree germplasm resources have ECG-3 "-Me, EGCG-3"-Me content is higher.

The method of detecting metabolites such as ECG-3”-Me and EGCG-3”-Me in tea tree is usually to measure metabolites by LC-MS. However, because the results of metabolite detection are affected by many factors, many repeated experiments are required to ensure Its stability and reliability are time-consuming and labor-intensive and prone to large system errors.

Therefore, there is an urgent need to develop a precise and rapid screening method for tea tree germplasm resources to quickly and accurately screen out tea tree germplasm resources with high ECG-3”-Me and EGCG-3”-Me content.

Contents of the invention

The purpose of the present invention is to provide a tea tree flavonoid 3-O-methyltransferase gene and its application to solve the problems in the prior art. Utilize the gene provided by the invention to quickly screen the tea tree varieties with higher content of ECG-3"-Me and EGCG-3"-Me for cultivation, shorten the breeding period by 2-3 years, improve the breeding efficiency, and at the same time, the protein encoded by the gene can be It is used for the in vitro synthesis of ECG-3”-Me and EGCG-3”-Me, and its synthesis efficiency is higher.

To achieve the above object, the present invention provides the following scheme:

The present invention provides a molecular marker related to the content of ECG-3"-Me and EGCG-3"-Me in tea tree, said molecular marker is gene CsCCoAOMT, the nucleotide sequence of said gene CsCCoAOMT is as SEQ ID NO.3 As shown, there is a C/G mutation at the 314th bp of the sequence.

The invention also provides an application of the molecular marker in tea plant breeding.

The present invention also provides a method for screening ECG-3 "-Me and EGCG-3 "-Me high-content tea tree germplasm resources, comprising the following steps:

(1) using the DNA of the tea tree to be tested as a template, and using the primer pair shown in SEQ ID NO.1-2, to perform PCR amplification to obtain the cloned gene CsCCoAOMT;

(2) Sequencing and analyzing the cloned gene CsCCoAOMT, when the sequence shown in SEQ ID NO.3 appears in the cloned gene CsCCoAOMT, the tea tree is ECG-3"-Me and EGCG-3"-Me High content of tea tree germplasm.

Further, in step (3), it also includes detecting the expression level of the cloned gene, and selecting a tea tree with a FPKM value higher than 60 as a tea tree species with high content of ECG-3 "-Me and EGCG-3 "-Me quality.

The present invention also provides a tea tree flavonoid 3-O-methyltransferase, the amino acid sequence of the tea tree flavonoid 3-O-methyltransferase is shown in SEQ ID NO.4, represented by SEQ ID NO.3 The nucleotide sequence code is shown.

The invention also provides a method for synthesizing ECG-3"-Me in vitro, using the 3-O-methyltransferase to carry out catalytic reaction in vitro to convert ester catechin into ECG-3"-Me.

Further, in the in vitro catalytic reaction, the ester catechin is the acceptor of the methoxy group, and the flavonoid containing the methoxy group is the donor of the methoxy group.

Further, the ester catechins include ECG.

The present invention also provides an application of the molecular marker in screening tea tree germplasm resources with high content of ECG-3"-Me and EGCG-3"-Me.

The present invention also provides an application of the gene CsCCoAOMT or the 3-O-methyltransferase in synthesizing ECG-3"-Me and EGCG-3"-Me in vitro.

The invention discloses the following technical effects:

The present invention discovers a gene CsCCoAOMT1 that controls the methoxylation of flavonoids such as ECG and EGCG through the method of whole-genome association analysis, and uses this gene to quickly screen ECG-3”-Me and EGCG-3”-Me content higher Tea tree varieties are cultivated to shorten the breeding period by 2-3 years and improve the breeding efficiency. At the same time, CsCCoAOMT1 can encode a methoxytransferase, which can be used for ECG-3”-Me and EGCG-3”- The in vitro synthesis of Me has a higher synthesis efficiency.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required in the embodiments. Obviously, the accompanying drawings in the following description are only some of the present invention. Embodiments, for those of ordinary skill in the art, other drawings can also be obtained based on these drawings without any creative effort.

Fig. 1 is the LC-MS detection result of metabolite ECG-3 "-Me and EGCG-3 "-Me;

Figure 2 is the close correlation between the QTL where the 3-O-methyltransferase gene is located and the metabolites with methoxy groups in the tea plant;

Figure 3 is the content difference of metabolites ECG-3 "-Me and EGCG-3 "-Me corresponding to different alleles;

Figure 4 is the LC-MS detection result of the supernatant product.

Detailed ways

Various exemplary embodiments of the present invention will now be described in detail. The 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.

It should be understood that the terminology described in the present invention is only used to describe specific embodiments, and is not used to limit the present invention. In addition, regarding the numerical ranges in the present invention, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated value 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 from 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 the 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 to disclose and describe the methods and/or materials in connection with which the documents are described. In case of conflict with any incorporated document, the contents of this specification control.

It will be apparent to those skilled in the art that various modifications and changes can be made in the specific embodiments of the present invention described herein without departing from the scope or spirit of the present invention. Other embodiments will be apparent to the skilled person from the description of the present invention. The specification and examples in this application are exemplary only.

As used herein, "comprising", "comprising", "having", "comprising" and so on are all open terms, meaning including but not limited to.

Location of embodiment 13-O-methyltransferase gene CsCCoAOMT

The present invention collects 220 tea tree germplasm resources at home and abroad, including wild species and cultivated species, sequences its transcriptome mRNA, compares the sequencing results to the tea tree reference genome G240, performs genotyping, and obtains the SNPs of the expressed genes. Qualitative and quantitative analysis of ECG-3”-Me and EGCG-3”-Me in flowers and leaves of 220 tea tree germplasm resources by LC-MS, metabolites ECG-3”-Me and EGCG-3”- The LC-MS detection results of Me are shown in Figure 1. The content of metabolites was used as phenotype data and genotype data for correlation analysis, and a QTL located on chromosome 7 of the tea plant genome was found, and 3-O-methyl transfer was located in the QTL Enzyme gene CsCCoAOMT (tea tree reference genome G240 annotation file is downloaded from https://www.ncbi.nlm.nih.gov/, where the gene CsCCoAOMT registration number is KAF5945117.1), the gene contains two non-synonymous mutation SNP , and correspond to two genotypes with high and low metabolite content (Figure 2), the SNP is located at 19390344bp of chromosome 7 of the tea plant genome, and there is a C/G mutation.

The qualitative and quantitative analysis of ECG-3”-Me and EGCG-3”-Me in the leaves and flowers of 220 tea germplasm resources was repeated annually, and the genome-wide association analysis was carried out. The results proved that the QTL obtained by the association analysis was stable.

Correlation between the content of ECG-3 "-Me and EGCG-3 "-Me in embodiment 23-O-methyltransferase gene CsCCoAOMT and tea tree

Design the forward primer (SEQ ID NO.1) 5'-ATGGCGGATAATATTGTTTTA-3', the reverse primer (SEQ ID NO.2) 5'-GAGTATGCGCCTGCACAATGT-3', and use the cDNA of tea tree germplasm resources as a template to carry out the gene CsCCoAOMT PCR amplification reaction, the amplification system is: template 2 μL, 10× amplification buffer 3 μl, 4 kinds of dNTP mixture (dATP, dGTP, dTTP, dCTP, 0.6 μL in total, forward and reverse primers 0.6 μL each, TaqDNA polymerase 2.5 μl, pure water 24 μl. Amplification program step 1 temperature 94°C, 2min; step 2 temperature 94°C, 25s; step 3 temperature 56°C, 20s; step 4 temperature 72°C, 35s; step 5 temperature 72°C 5min, steps 2-4 cycled 35 times. Sequencing analysis and protein sequence translation of the amplification results obtained two alleles of the gene, CsCCoAOMT1 and CsCCoAOMT2. When the gene is CsCCoAOMT1, the SNP of the gene at 19390344bp on chromosome 7 of the tea tree genome is C, encoding a Alanine; when the gene is CsCCoAOMT2, the SNP at 19390344bp of chromosome 7 of the tea plant genome is G, encoding a glycine. The amino acid sequences of genes CsCCoAOMT1 and CsCCoAOMT2 and their encoded 3-O-methyltransferases are shown in SEQ ID NO.3-6.

Nucleotide sequence of gene CsCCoAOMT1 (SEQ ID NO.3):

ATGGCGGATAATATTGTTTTAAAGACCATCCTGCAAAGCGAGGCTCTTCAAAAGTACATCTTTGATACCAATGTATACCCAAAGAGAACATGAGCAGCTCAAGAGGATAAGAGATGCAACATTCAAGAAATATGGCGACAGAGCGGAGCTAAGTGTGCCCCCTGATGAAGGATTGTTCTTGTCTATGCTTTTGAAATTAATGAATGCCAAGAAGACATTGGAGATTG GTGTTTTCACTGGCTATTCTCTTCTTTACCACTGCCCTTGCTTTACCTCATGATGGCCAGATAGTAGCAATAGATCCAAATCGAGAAGC ATTTGAAGTTGGACTGCCATTCATTCAGAAGGCTGGTGTGGAGCACAAGATCAATTTCATTGAATCAGATGCCATTTCTGTTCTCAATGAAATGTTGAGCGATGAGGGTAAGTTGCACTTGTGGATT. (Bold and underlined are SNP sites)

The amino acid sequence (SEQ ID NO.4) of the 3-O-methyltransferase encoded by the gene CsCCoAOMT1:

MQHSRNMATELSVPPDERLFLSMLLKLMNAKKILEIGVFTGYSLLTTALALPHD GQIVAIDPNRE A FEVGLPFIQKVGVEHKIIFIDSDAISVLNEMLSNVIVSNEEEVPEFLCI GRKPIIELNKYLASDPRIEIAQISISDGVTLCRRI. (The amino acid encoded by the nucleotide containing the SNP site is underlined in bold)

Nucleotide sequence of gene CsCCoAOMT2 (SEQ ID NO.5):

ATGGCGGATAATATTGTTTTAAAGACCATCCTGCAAAGCGAGGCTCTTCAAAAGTACATCTTTGATACCAATGTATACCCAAAGAGAACATGAGCAGCTCAAGAGGATAAGAGATGCAACATTCAAGAAATATGGCGACAGAGCGGAGCTAAGTGTGCCCCCTGATGAAGGATTGTTCTTGTCTATGCTTTTGAAATTAATGAATGCCAAGAAGACATTGGAGATTG GTGTTTTCACTGGCTATTCTCTTTCTTACCACTGCCCTTGCTTTACCTCATGATGGCCAGATAGTAGCAATAGATCCAAATCGAGAAGG ATTTGAAGTTGGACTGCCATTCATTCAGAAGGCTGGTGTGGAGCACAAGATCAATTTCATTGAATCAGATGCCATTTCTGTTCTCAATGAAATGTTGAGCGATGAGGGTAAGTTGCACTTGTGGATT. (Bold and underlined are SNP sites)

The amino acid sequence (SEQ ID NO.6) of the 3-O-methyltransferase encoded by the gene CsCCoAOMT2:

MQHSRNMATELSVPPDERLFLSMLLKLMNAKKILEIGVFTGYSLLTTALALPHD GQIVAIDPNRE G FEVGLPFIQKVGVEHKIIFIDSDAISVLNEMLSNVIVSNEEEVPEFLCI GRKPIIELNKYLASDPRIEIAQISISDGVTLCRRI. (The amino acid encoded by the nucleotide containing the SNP site is underlined in bold)

When the CsCCoAOMT1 gene appears in the amplified product (including only CsCCoAOMT1 or both CsCCoAOMT1 and CsCCoAOMT2), the content of ECG-3"-Me and EGCG-3"-Me in the tea germplasm resources is relatively high; When only the CsCCoAOMT2 gene appeared in the amplified product or the gene was not cloned, the contents of ECG-3"-Me and EGCG-3"-Me in the tea germplasm resources were low. (image 3).

Correlation between the expression level of the embodiment 33-O-methyltransferase gene CsCCoAOMT and the content of ECG-3 "-Me and EGCG-3 "-Me in tea tree

The content of ECG-3”-Me and EGCG-3”-Me in tea tree is affected by genotype and gene expression. When the relative gene expression FPKM value is higher than 60, the metabolite ECG-3”- The relative content of Me is 1907.4, and the content of EGCG-3”-Me is 1784.158617; when the relative gene expression FPKM value is lower than 60, the relative content of metabolite ECG-3”-Me in the tea tree population is 368.3362396, and the content of EGCG-3”-Me is 242.4492192. It can be seen that when the FPKM value is greater than 60, the contents of ECG-3"-Me and EGCG-3"-Me are relatively high. Therefore, the specific germplasm resources with high ECG-3"-Me and EGCG-3"-Me content were screened for tea plant breeding by identifying the combination of genotype and gene expression.

Functional analysis of embodiment 43-O-methyltransferase

Enzymatic studies of CsCCoAOMT were performed using the purified recombinant protein. 0.01 mg of the purified protein was added to a 1.5 mL reaction system including 0.05 mmol/LEGCG, 0.16 mmol/LSAM, 0.2 mmol/LMgCl ₂ and 100 mmol/LTris-HCl (pH=7.8). The reaction mixture was incubated at 30 °C for 30 min, and then 200 μL of methanol was added to stop the reaction. The supernatant product was analyzed by LC-MS, and the results are shown in FIG. 4 .

It can be seen from Figure 4 that the in vitro enzyme activity verification shows that the 3-O-methyltransferase controlled by the CsCCoAOMT gene can use ester catechins such as EGCG and ECG and flavonoids containing methoxy groups as substrates, and ECG As a methoxy group acceptor, other flavonoids containing methoxy groups act as methoxy group donors to catalyze the conversion of ECG to ECG-3”-Me.

The above-mentioned embodiments are only to describe the preferred mode of the present invention, and are not intended to limit the scope of the present invention. Variations and improvements should fall within the scope of protection defined by the claims of the present invention.

Claims (4)

1. the application of a detection reagent of a molecular marker related to ECG-3 "-Me and EGCG-3 "-Me content in tea tree in ECG-3 "-Me and EGCG-3 "-Me content breeding in tea tree, It is characterized in that the molecular marker is the gene CsCCoAOMT , the nucleotide sequence of the gene CsCCoAOMT is shown in SEQ ID NO.3, there is a C/G mutation at the 314th bp position of the sequence, when the position is When the genotype at this position is CC or CG, the content of ECG-3”-Me and EGCG-3”-Me in the tea tree is high; when the genotype at this position is GG, the content of ECG-3”-Me and EGCG-Me in the tea tree is high The content of 3”-Me is low. 2. a method for screening ECG-3 "-Me and EGCG-3 "-Me high-content tea tree germplasm resources, is characterized in that, comprises the following steps: Taking the DNA of the tea tree to be tested as a template, and using the primer pair shown in SEQ ID NO.1-2, PCR amplification is carried out to obtain the cloned gene CsCCoAOMT ; Sequencing analysis is carried out on the cloned gene CsCCoAOMT , when the sequence shown in SEQ ID NO.3 appears in the cloned gene CsCCoAOMT , the tea tree is a tea tree with high content of ECG-3"-Me and EGCG-3"-Me Germplasm. 3. The method according to claim 2, characterized in that, in step (3), it also includes detecting the expression level of the cloned gene, and selecting the tea tree whose FPKM value is higher than 60 as ECG-3”-Me and EGCG-3"-Me high content tea germplasm. 4. A detection reagent for molecular markers related to the content of ECG-3”-Me and EGCG-3”-Me in tea tree in the screening of tea tree germplasm resources with high content of ECG-3”-Me and EGCG-3”-Me application, characterized in that the molecular marker is the gene CsCCoAOMT , the nucleotide sequence of the gene CsCCoAOMT is shown in SEQ ID NO.3, there is a C/G mutation at the 314th bp position of the sequence, when When the genotype at this position is CC or CG, the content of ECG-3"-Me and EGCG-3"-Me in tea plant is high.

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