CN115838703B - Tea tree methylated catechin synthase, coding gene and application thereof - Google Patents

Abstract

The present invention belongs to the field of bioengineering technology, and relates to methylated catechin synthase, encoding gene and application thereof. The present invention discovers related genes through a series of methods and techniques, constructs recombinant engineering bacteria, obtains recombinant proteins, and verifies the genes of O-methyltransferase CsFAOMT1 and CsFAOMT2 related to catechin synthesis in tea trees by means of enzyme reaction methods. The present invention describes for the first time two O-methyltransferase genes that can catalyze the synthesis of methylated catechins, and their application value in the selection and breeding of excellent tea varieties and synthetic biology.

Description

Tea tree methylated catechin synthase, encoding gene and application thereof

Technical Field

The invention belongs to the field of biotechnology, and in particular relates to tea tree methylated catechin synthase, encoding genes and applications thereof.

Background Art

Tea is one of the most consumed beverages in the world. Tea’s popularity lies in its unique flavor and health benefits, which are conferred by a variety of bioactive compounds in tea leaves, such as caffeine and catechins. Catechins, which account for more than 12% of the dry weight of tea, are a class of bioactive polyphenols, of which (-)-epigallocatechin-3-gallate (EGCG) is the most abundant component. Extensive studies have confirmed the health-promoting effects of EGCG, including anti-cancer, anti-inflammatory, and cardioprotective effects. However, the therapeutic effects of EGCG are greatly reduced due to its instability in the neutral to alkaline conditions of the intestine. In addition, the highly polar nature of the structure also makes it difficult to penetrate the intestinal wall, further reducing its bioavailability in the human body.

O-methylated EGCG is a series of methylated derivatives formed by modifying the phenolic hydroxyl group on the benzene ring of EGCG with a methyl group. In tea plants, natural methylation mainly occurs at the 3″ and 4″ positions of EGCG to produce (-)-epigallocatechin-3-O-(3-O-methyl)-gallate (EGCG3″Me) and (-)-epigallocatechin-3-O-(4-O-methyl)-gallate (EGCG4″Me). In previous studies, the inventors investigated the levels of EGCG3″Me and EGCG4″Me in 27 tea germplasm resources with different genetic backgrounds, and detected EGCG3″Me in about half of the germplasm resources, and EGCG4″Me was only detected in 3 resources. Clinical trials have shown that tea containing EGCG3″Me (Benifuki) has stronger anti-allergic and anti-hypertensive effects than tea without EGCG3″Me (Yabukita).

Although tea plants are rich in EGCG, tea plants that are naturally rich in methylated catechins are rare. Tea plants have a long juvenile period, which makes it challenging and time-consuming to develop new varieties with high methylated catechins through traditional breeding methods. Previous studies have shown that O-methyltransferases (OMTs) can catalyze the formation of methylated catechins. In fact, caffeoyl-CoA-O-methyltransferase (CCoAOMT) has been cloned from tea plants. However, in vitro enzymatic reactions using recombinant CCoAOMT produced a variety of methylated catechins, including EGCG3″Me, EGCG4″Me, (-)-epigallocatechin-3-O-(3,5-O-dimethyl)-gallate, and (-)-3-O-methyl-epigallocatechin-3-O-(3,5-O-dimethyl)-gallate, which is inconsistent with the fact that the main O-methylated EGCG detected in tea plants is EGCG3″Me and/or EGCG4″Me. This suggests that the key OMTs involved in the specific O-methyl groups at the 3″ and 4″ positions remain unidentified.

Summary of the invention

In view of the problems existing in the prior art, the purpose of the present invention is to provide a technical solution for tea tree methylcatechin synthase, encoding gene and application thereof.

The present invention specifically adopts the following technical solutions:

The first aspect of the present invention provides a methylated catechin synthase CsFAOMT1, whose amino acid sequence is shown in SEQ ID No. 2;

Or an amino acid sequence having the same protein function as the amino acid sequence shown in SEQ ID No. 2 after substitution and/or deletion and/or addition of several amino acid residues;

Or an amino acid sequence derived from the amino acid sequence shown in SEQ ID No. 2, having more than 98% homology and having the same protein function.

The second aspect of the present invention provides a gene encoding the above-mentioned methylated catechin synthase CsFAOMT1, the nucleotide sequence of which is shown in SEQ ID No.1;

or a DNA sequence that hybridizes with the DNA sequence shown in SEQ ID No.1 and encodes a protein with the same function;

Or a DNA molecule having more than 95% homology with the DNA sequence shown in SEQ ID No.1 and encoding a protein with the same function.

The third aspect of the present invention provides a methylated catechin synthase CsFAOMT2, whose amino acid sequence is shown in SEQ ID No.4;

Or an amino acid sequence having the same protein function as the amino acid sequence shown in SEQ ID NO.4 after substitution and/or deletion and/or addition of several amino acid residues;

Or an amino acid sequence derived from the amino acid sequence shown in SEQ ID No. 4, having more than 98% homology and having the same protein function.

The fourth aspect of the present invention provides a gene encoding the above-mentioned methylated catechin synthase CsFAOMT2, the nucleotide sequence of which is shown in SEQ ID No.3;

or a DNA sequence that hybridizes with the DNA sequence shown in SEQ ID No.3 and encodes a protein with the same function;

Or a DNA molecule having more than 95% homology with the DNA sequence shown in SEQ ID No.3 and encoding a protein with the same function.

The fifth aspect of the present invention provides the use of the above-mentioned methylated catechin synthase CsFAOMT1 and the above-mentioned methylated catechin synthase CsFAOMT2 as O-methyltransferase.

The sixth aspect of the present invention provides the use of the above-mentioned methylated catechin synthase CsFAOMT1 and the above-mentioned methylated catechin synthase CsFAOMT2 in the breeding of superior tea varieties.

The seventh aspect of the present invention provides the use of the above-mentioned methylated catechin synthase CsFAOMT1 and the above-mentioned methylated catechin synthase CsFAOMT2 in catalyzing the conversion of EGCG into EGCG3"Me and/or EGCG4"Me.

The present invention has the following beneficial effects:

The present invention describes for the first time two O-methyltransferase genes that can catalyze the synthesis of methylated catechins, and their application value in the breeding of superior tea tree varieties and tea processing.

The invention obtains recombinant protein by constructing recombinant engineering bacteria, and verifies the genes encoding O-methyltransferase CsFAOMT1 and CsFAOMT2 in tea trees by using the recombinant protein of genetic engineering bacteria as catalytic enzyme by means of enzyme reaction method.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 Identification of key candidate genes involved in the synthesis of O-methylated catechins in tea plants. (a) Biosynthetic pathway of major O-methylated catechins (EGCG3″Me and EGCG4″Me). (b) Chromosome distribution of Euclidean distance (ED) values based on SNPs and InDels. (c) Phylogenetic tree analysis of O-methyltransferases.

Fig. 2 Amino acid sequence differences between CsFAOMT1 and CsFAOMT2.

Fig. 3 SDS-PAGE analysis of empty vector (EV), CsFAOMT1 and CsFAOTT2 and other recombinant proteins.

Fig. 4 Total ion chromatogram and MS ² ion fragments of reaction products of in vitro O-methyltransferase activity assay. In vitro reaction products of recombinant CsFAOMT1 and CsFAOMT2: (-)-epigallocatechin gallate (1) as substrate and empty vector (EV) as control. 2, (-)-epigallocatechin-3-O-(4-O-methyl)-gallate; 3, (-)-epigallocatechin-3-O-(3-O-methyl)-gallate. ESI ⁺ mode.

DETAILED DESCRIPTION

The present invention is further described in detail below in conjunction with specific examples, which are intended to explain the present invention rather than to limit it.

In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with the examples. The equipment and reagents used in each embodiment and test example can be obtained from commercial sources unless otherwise specified. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

The present invention relates to two methylated catechin synthase genes and their encoded proteins, which are named CsFAOMT1 and CsFAOMT2 respectively. The nucleotide sequence of CsFAOMT1 gene is shown in SEQ ID No.1, and the amino acid sequence is shown in SEQ ID No.2; the nucleotide sequence of CsFAOMT2 gene is shown in SEQ ID No.3, and the amino acid sequence is shown in SEQ ID No.4.

The specific sequence of SEQ ID No.1 is:

The specific sequence of SEQ ID No.2 is:

The specific sequence of SEQ ID No.3 is:

The specific sequence of SEQ ID No.4 is:

Example 1: Discovery and cloning of two methylated catechin synthase genes

We performed mixed segregant transcriptome sequencing (BSR-Seq) using an F1 population constructed by crossing Jinxuan (JX) and Zijuan (ZJ). In the F1 population, 24 individuals with high EGCG3″Me levels (>5 mg/g) and 24 individuals with low EGCG3″Me levels (<0.1 mg/g) were selected to form two extreme groups. BSR-Seq analysis was performed on the Illumina NovaSeq platform, and approximately 250,000 single nucleotide polymorphisms (SNPs) or insertions/deletions (InDels) were identified. We found that variants associated with EGCG3″Me content were enriched in a region around 46 Mb on chromosome 6 (GWHAZTZ00000004) of the ‘Huangdan’ reference genome, suggesting that the target gene might be localized in this region (Fig. 1b). In addition, a total of 217 differentially expressed genes (DEGs) were identified, including 94 up-regulated DEGs and 123 down-regulated DEGs. Among the 94 up-regulated DEGs, five DEGs (HD.03G0010060, HD.03G0009980, HD.03G0009970, HD.03C0009860, HD.06002937) were putative OMTs located in the region identified on chromosome 6. RNA-seq analysis was performed on 27 tea accessions with significantly different genetic backgrounds to analyze the correlation between the transcript levels of 35,628 tea genes, including the five OMTs, and the levels of EGCG3″Me. Finally, we found that tea plants expressing higher levels of OMT (HD.03G0010060) usually contained more EGCG3″Me, indicating a positive correlation between them (R=0.89, P=2.75×10 ^-28 ).

To identify the sequence of the candidate OMT, we designed primers FAOMTF: AGTCTYATCTGRGAAAACAT (SEQ ID No. 5) and FAOMTR: GATAACAACATTAYAAACGG (SEQ ID No. 6) and cloned the coding region from the cDNA of Jinxuan, which encodes a protein containing 236 amino acids (Figure 2). We used the same primers to further isolate a novel O-methyltransferase gene from the cDNA of Jinxuan, which was not found in any of the reported reference genomes of tea plants. Considering that Jinxuan is also rich in EGCG4″Me, we believe that this gene may be involved in the biosynthesis of EGCG4″Me. Phylogenetic analysis of these two O-methyltransferases identified in tea plants and other different species showed that they are closely related to the flavonol and anthocyanin O-methyltransferase (FAOMT) gene in grapes (Figure 1c), but relatively distant from the CsCCoAOMT cloned from tea plants. Therefore, the two O-methyltransferases were named CsFAOMT1 and CsFAOMT2, respectively.

Example 2: Functional verification of CsFAOMT1 and CsFAOMT2

In order to verify the functions of CsFAOMT1 and CsFAOMT2, the coding regions of CsFAOMT1 and CsFAOMT2 were fully connected to pMAL-c5x, and the recombinant expression vectors were introduced into BL21 (DE3) Escherichia coli, and the two proteins were purified using maltose tag (MBP) (Figure 3). Under the premise of exogenous addition of Mg ²⁺ , EGCG was used as a substrate and SAM was used as a methyl donor for incubation at 40°C for 10 minutes, and the reaction products were detected by LC-MS. As shown in Figure 4, the retention time of the substrate EGCG was 13.2 minutes; CsFAOMT1 specifically generated product 3 with a retention time of 15.1 minutes; CsFAOMT2 generated two products 2 and 3, and product 2 was significantly more than product 3, with retention times of 14.8 minutes and 15.1 minutes, respectively. The mass-to-charge ratios (m/z) of the characteristic daughter ions of the two products 2 and 3 were 139, 151, 167 and 289 (Figure 4). By comparing the retention time and ion fragments of the reaction product with those of EGCG4″Me and EGCG3″Me standards, it can be determined that the substance at 14.8min is EGCG4″Me, and the substance at 15.1min is EGCG3″Me. The above results show that CsFAOMT1 can specifically catalyze EGCG to form EGCG3″Me, and CsFAOMT2 can specifically catalyze EGCG to form EGCG3″Me and EGCG4″Me, with EGCG4″Me being the main component.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims (4)

1. A methylated catechin synthase CsFAOMT1, characterized in that the methylated catechin synthase CsFAOMT1 can specifically catalyze the conversion of EGCG into EGCG3″Me, and its amino acid sequence is shown in SEQ ID No.2. 2. A gene encoding a methylated catechin synthase CsFAOMT1 as claimed in claim 1, characterized in that its nucleotide sequence is shown in SEQ ID No. 1. 3. Use of the methylated catechin synthase CsFAOMT1 as claimed in claim 1 in specifically catalyzing the conversion of EGCG into EGCG3"Me as an O-methyltransferase. 4. Use of the methylated catechin synthase CsFAOMT1 as claimed in claim 1 in the breeding of superior tea varieties.