Method for regulating synthesis of cis- catechin in camellia sinensis based on csahl20 gene and application thereof

Abstract

The application provides a method for regulating synthesis of catechin in tea tree based on CsAHL20 gene and application, and belongs to the field of plant molecular biological technology. The tea tree gene CsAHL20 screened in the application can combine with the CsANR gene promoter of a key enzyme for synthesis of catechin, and inhibit accumulation of catechin content in tea tree; through verification, silencing of the gene by using a virus-induced gene silencing technology can increase catechin content in tea tree, and overexpression of the gene by using an agrobacterium-mediated transient transformation technology can reduce catechin content in tea tree, so that the CsAHL20 gene can be used for regulating synthesis of catechin in tea tree.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology technology, and in particular relates to a method and application of regulating the synthesis of cis-catechins in tea plants based on the CsAHL20 gene. Background Technology

[0002] Catechins are one of the main components of tea, accounting for more than 70% of the total tea polyphenols. Catechins include catechin (C) gallocatechin (GC), epicatechin (EC), gallocatechin (EGC), gallocatechin gallate (ECG), gallocatechin gallate (GCG), gallocatechin gallate (EGCG), and catechin gallate (CG). C, CG, GC, and GCG are the corresponding cis-trans isomers of EC, ECG, EGC, and EGCG. Among them, cis-catechins (EC, EGC, ECG, and EGCG) are present in higher amounts and are the main catechins.

[0003] As research into catechins deepens, more mechanisms influencing catechin synthesis are being revealed. During tea plant growth, various biotic and abiotic factors frequently exert stress, with drought being one of the most critical abiotic factors limiting tea production. Plant responses to drought include morphological, biochemical, physiological, and molecular changes; importantly, the accumulation of secondary metabolites in plants is significantly affected by declining soil moisture. Studies have shown that under drought stress, the catechin content in tea decreases; simultaneously, the expression levels of some genes involved in catechin synthesis also change accordingly. Under drought treatment, catechin content is positively correlated with the expression of CsPAL, Cs4CL, and CsC4H; current research has found that the expression levels of CsCHS, CsLAR, and CsANR genes are positively correlated with total catechins, with ANR and LAR genes positively correlated with EGCG and ECG content. Under drought stress, numerous genes participate in the biosynthesis and metabolism of catechins; tea plants have formed a complex signaling network at the molecular level to cope with drought stress; transcription factors (TFs) are key regulators of gene expression.

[0004] Existing studies have identified more than 30 transcription factors (TFs) that may be involved in the regulation of catechins in tea plants. Many TFs have also been found to be closely related to the expression of catechin biosynthesis genes, including MADS box, R2R3-MYB, and bHLH. Some existing studies have also identified 35 TFs that may be involved in the regulation of catechin biosynthesis, such as ANL2, WRKY44, and AtMYB113. The ancient transcription factor AT-Hook Motif Nuclear Localized (AHL) gene family is present in all sequenced plant species; members of the AHL family contain two conserved structural units: the AT-hook motif and the PPC domain. The AT-hook motif can bind to AT-rich DNA and has been identified in various gene families in prokaryotes and eukaryotes. The PPC domain, also annotated as Unknown Functional Domain 296 (DUF296), is approximately 120 amino acids long and is located at the carboxyl terminus relative to the AT-hook motif. The PPC domain is responsible for the nuclear localization of AHL proteins and protein-protein interactions between AHL proteins and with other common interactors (such as transcription factors). AHLs play an important role in stress responses and have been shown to have drought resistance in various plants; for example, in rice, AHL expression is significantly upregulated under drought stress. However, most studies on the responses of AHLs to abiotic stresses have focused on model species, with few studies in woody species; as an important transcription factor in response to abiotic stresses, there are currently no reports on the CsAHL gene in tea.

[0005] Therefore, it is necessary to provide a method and application for regulating the synthesis of cis-catechins in tea plants based on the CsAHL20 gene, so as to solve or at least alleviate the technical problem of how to regulate the synthesis of cis-catechins in tea plants based on a new regulatory gene. Summary of the Invention

[0006] The main objective of this invention is to provide a method and application for regulating the synthesis of cis-catechins in tea plants based on the CsAHL20 gene, aiming to solve the technical problem of how to regulate the synthesis of cis-catechins in tea plants based on a new regulatory gene.

[0007] To achieve the above objectives, the present invention provides an application of the CsAHL20 gene as a target in regulating the synthesis of cis-catechins in tea plants, wherein the nucleotide sequence of the CsAHL20 gene is shown in SEQ ID NO.1.

[0008] Further, the CsAHL20 gene in tea plants can be silenced to promote the synthesis of cis-catechins in tea plants; or, the CsAHL20 gene in tea plants can be overexpressed to inhibit the synthesis of cis-catechins in tea plants.

[0009] The present invention also provides the application of a protein encoded by the CsAHL20 gene in inhibiting the synthesis of cis-catechins in tea plants, wherein the amino acid sequence of the protein encoded by the CsAHL20 gene is shown in SEQ ID NO.2.

[0010] This invention also provides an application of the CsAHL20 gene as a target in the preparation of a tea plant cis-catechin regulatory reagent, wherein the nucleotide sequence of the CsAHL20 gene is shown in SEQ ID NO.1.

[0011] The present invention also provides a tea plant cis-catechin regulatory reagent, which includes a silencing reagent for the CsAHL20 gene and / or an overexpression reagent for the CsAHL20 gene, the nucleotide sequence of the CsAHL20 gene being shown in SEQ ID NO.1.

[0012] This invention also provides a method for regulating the synthesis of cis-catechins in tea plants based on the CsAHL20 gene, comprising using any of the tea plant cis-catechin regulating reagents described above to regulate the synthesis of cis-catechins in tea plants.

[0013] The present invention also provides a method for regulating the synthesis of cis-catechins in tea plants based on the CsAHL20 gene, comprising: constructing a silencing vector of the CsAHL20 gene, and transferring the silencing vector into tea plants to silence the CsAHL20 gene; or, constructing an overexpression vector of the CsAHL20 gene, and transferring the overexpression vector into tea plants to overexpress the CsAHL20 gene.

[0014] Furthermore, the construction process of the gene silencing vector includes: using pTRV2 as the original vector, inserting a 252bp sequence of the CsAHL20 gene CDS into the multiple cloning site of pTRV2, with the insertion site located between the EcoRI and BamHI restriction sites on the original vector pTRV2; the nucleotide sequence of the 252bp sequence of the CsAHL20 gene CDS is shown in SEQ ID NO.24.

[0015] The construction process of the overexpression vector includes: using pCAMBIA2301-35S as the original vector, inserting the full-length CDS sequence of the CsAHL20 gene into the multiple cloning site of pCAMBIA2301-35S, with the insertion site located between the Kpn I and Xba I restriction sites on the original vector pCAMBIA2301-35S; the nucleotide sequence of the full-length CDS sequence of the CsAHL20 gene is shown in SEQ ID NO.1.

[0016] The present invention also provides a tea tree breeding method, comprising: transferring a silencing vector of the CsAHL20 gene into tea tree tissue to silence the CsAHL20 gene, and then culturing the tea tree tissue; or, transferring an overexpression vector of the CsAHL20 gene into tea tree tissue to overexpress the CsAHL20 gene, and then culturing the tea tree tissue; the nucleotide sequence of the CsAHL20 gene is shown in SEQ ID NO.1.

[0017] The present invention also provides an application of the CsAHL20 gene as a target in improving tea quality, wherein the nucleotide sequence of the CsAHL20 gene is shown in SEQ ID NO.1.

[0018] Compared with the prior art, the present invention has at least the following advantages:

[0019] This invention provides a tea tree gene, CsAHL20, involved in the regulation of cis-catechin synthesis in tea plants, and enables the regulation of cis-catechin synthesis based on this novel regulatory gene. This invention utilizes Agrobacterium-mediated transient transformation technology to overexpress CsAHL20, which reduces the content of cis-catechins in tea plants. Dual-luciferase (LUC) and electrophoretic mobility shift assay (EMSA) experiments demonstrate that CsAHL20 binds to the promoter of the key enzyme in cis-catechin synthesis, CsANR, inhibiting CsANR expression and thus suppressing cis-catechin synthesis and accumulation. Furthermore, this invention employs virus-induced gene silencing technology to silence CsAHL20, which increases the content of cis-catechins in tea plants. Compared to control plants, the silenced plants show a significant increase in cis-catechin content. In summary, this invention demonstrates that the CsAHL20 gene inhibits cis-catechin synthesis, and silencing the CsAHL20 gene significantly increases the cis-catechin content in tea plants, making it suitable for regulating cis-catechin synthesis in tea plants. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art 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 the structures shown in these drawings without creative effort.

[0021] Figure 1 shows the identification and analysis of CsAHL20 in Example 1 of this invention. Figure 1a A phylogenetic tree of AHL proteins, Figure 1b To analyze the expression pattern of the CsAHL gene in tea plants treated with 10% polyethylene glycol 6000 for different time periods using heatmaps, Figure 1cCorrelation analysis of CsAHL family genes and catechins in tea treated with 10% polyethylene glycol 6000. Figure 1d Sequence alignment of CsAHL20 protein with other similar proteins, as well as AT-hook motifs and PPC domains;

[0022] Figure 2 shows the catechin content in tea leaves after treatment with 10% polyethylene glycol 6000 for different times and the analysis by real-time quantitative PCR (RT-qPCR) in Example 1 of this invention. Figure 2a To address the condition of tea seedlings at different times. Figure 2b To determine the relative expression levels of the CsAHL20 gene in tea leaves at different time points, Figure 2c To address the total amount of catechins after different time periods, Figure 2d To process the cis-catechin content after different time periods, Figure 2e To determine the levels of cis-catechins EGC, EC, EGCG, and ECG after different time periods, Figure 2f The content of trans-catechins GC, C, GCG, and CG after different treatment times was determined.

[0023] Figure 3 This illustrates the subcellular localization of the CsAHL20 protein in tobacco leaf cells in Example 2 of this invention. Figure 3 In the diagram, Bright corresponds to the bright field image of tobacco leaf epidermal cells under a fluorescence microscope, RFP corresponds to the red fluorescence field image of tobacco leaf epidermal cells under a fluorescence microscope, GFP corresponds to the green fluorescence field image of tobacco leaf epidermal cells under a fluorescence microscope, and Merge corresponds to the superimposed image of bright field, red fluorescence field, and green fluorescence field of tobacco leaf epidermal cells under a fluorescence microscope.

[0024] Figure 4 shows the catechin content and RT-qPCR analysis of tea leaves after transient overexpression of the CsAHL20 gene in Example 3 of this invention. Figure 4a The relative expression level of CsAHL20. Figure 4b Total catechin content, Figure 4c The content of cis-catechins, Figure 4d The content of cis-catechins EGC, EC, EGCG, and ECG. Figure 4e For the catechin synthesis pathway, Figure 4f The correlation between the relative expression levels of genes related to catechin synthesis and the relative expression level of CsAHL20 was analyzed. Figure 4g This represents the relative expression level of CsC4H. Figure 4h This represents the relative expression level of Cs4CL. Figure 4i The relative expression level of CsCHS. Figure 4j The relative expression level of CsCHI. Figure 4k The relative expression level of CsF3'5'H. Figure 4l The relative expression level of CsF3'H. Figure 4m This represents the relative expression level of CsANS. Figure 4n (Relative expression level of CsANR); In Figure 4, Control corresponds to the empty control of the pCAMBIA2301-35S vector, and 35S:CsAHL20-1 to 35S:CsAHL20-3 correspond to the three experimental groups injected with pCAMBIA2301-35S-CsAHL2.

[0025] Figure 5 shows the determination of catechin content and relative gene expression level after virus-induced gene silencing of CsAHL20 in Example 4 of this invention. Figure 5a The relative expression level of CsAHL20 in tea leaf petals. Figure 5b The relative expression level of CsANR in tea leaf petals. Figure 5c Total catechins in tea leaves, Figure 5d The content of cis-catechins in tea leaves. Figure 5e (The content of cis-catechins EGC, EC, EGCG, and ECG in tea leaves); In Figure 5, WT corresponds to wild-type plants, pTRV2 corresponds to strains infected with pTRV1+pTRV2 bacterial solution, and pTRV2-CsAHL20-1 to pTRV2-CsAHL20-3 correspond to strains infected with pTRV1+pTRV2-CsAHL20 bacterial solution;

[0026] Figure 6 The figure shows the interaction detection of tea plant CsAHL20 with CsANR promoter in Example 5 of the present invention (EMSA); in the figure, (a) is the predicted binding sequence motif of AHL20, and (b) is the binding of recombinant protein His-CsAHL20 purified in the EMSA experiment with CsANR promoter. Figure 6 In the diagram, the positive probe corresponds to the CsANR active probe (SEQ ID NO. 26 and 27), the mutation probe corresponds to the CsANR mutant probe (SEQ ID NO. 28 and 29), the positive protein corresponds to the CsAHL20 protein, the swim lane corresponds to the electrophoresis lane, the bound probe corresponds to the binding probe, the free probe corresponds to the unbound free probe, "-" corresponds to the absence of the corresponding substance, "+" corresponds to the addition of the corresponding substance, the number of "+" corresponds to the amount of the added substance, and 1-5 correspond to 5 different electrophoresis lanes;

[0027] Figure 7The results of the dual-luciferase reporter assay for CsAHL20 and CsANR in tea plant in Example 5 of this invention show that CsAHL20 inhibits the CsANR promoter activity and the Luc / Ren ratio.

[0028] The realization of the objective, functional characteristics and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0031] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention, as well as the prior art known to those skilled in the art and the description of this invention, may be implemented using any prior art methods, devices, and materials similar to or equivalent to those described, used, or made of materials in the embodiments of this invention.

[0032] This invention provides an application of the CsAHL20 gene as a target in regulating the synthesis of cis-catechins in tea plants, wherein the nucleotide sequence of the CsAHL20 gene is shown in SEQ ID NO.1. In this invention, catechins include catechin (C), gallocatechin (GC), epicatechin (EC), gallocatechin (EGC), gallocatechin gallate (ECG), gallocatechin gallate (GCG), gallocatechin gallate (EGCG), and catechin gallate (CG); cis-catechins include or may include EGC, EC, EGCG, and ECG; trans-catechins include or may include the content of GC, C, GCG, and CG.

[0033] In this invention, the regulation can be directed to either promote or inhibit the synthesis of cis-catechins in tea plants. Studies have shown that catechins possess antioxidant, anticancer, lipid-lowering, antibacterial, antiviral, and weight-loss effects. This invention silences the CsAHL20 gene in tea plants to promote the synthesis of cis-catechins. However, in certain scenarios and studies where a lower requirement for cis-catechin content is needed, the CsAHL20 gene can be overexpressed to inhibit cis-catechin synthesis. In this invention, cis-catechins in tea plants can refer to the cis-catechin content in tea leaves.

[0034] This invention also provides the application of a protein encoded by the CsAHL20 gene in inhibiting the synthesis of cis-catechins in tea plants. The amino acid sequence of the protein encoded by the CsAHL20 gene is shown in SEQ ID NO.2. In this invention, the CsAHL20 protein inhibits the expression of the CsANR gene by binding to the CsANR gene promoter, thereby inhibiting the synthesis of cis-catechins in tea plants. In this invention, the CsANR gene promoter is present in the tea plant or tea plant tissue; the nucleotide sequence of the CsANR gene promoter is shown in SEQ ID NO.3.

[0035] The present invention also provides an application of the CsAHL20 gene as a target in the preparation of a cis-catechin regulatory reagent for tea plants, wherein the nucleotide sequence of the CsAHL20 gene is shown in SEQ ID NO.1; specifically, the present invention prepares a silencing reagent for the CsAHL20 gene and / or an overexpression reagent for the CsAHL20 gene by using the CsAHL20 gene as a target.

[0036] The present invention also provides a tea plant cis-catechin regulatory reagent, which includes a silencing agent for the CsAHL20 gene and / or an overexpression agent for the CsAHL20 gene, wherein the nucleotide sequence of the CsAHL20 gene is shown in SEQ ID NO.1; exemplaryly, the tea plant cis-catechin regulatory reagent is either a silencing agent for the CsAHL20 gene or an overexpression agent for the CsAHL20 gene.

[0037] This invention also provides a method for regulating the synthesis of cis-catechins in tea plants based on the CsAHL20 gene, comprising using any of the cis-catechin regulating reagents described above to regulate the synthesis of cis-catechins in tea plants. Specifically, this invention uses the CsAHL20 gene silencing reagent to silence the CsAHL20 gene in tea plants to promote the synthesis of cis-catechins; or uses the CsAHL20 gene overexpression reagent to overexpress the CsAHL20 gene in tea plants to inhibit the synthesis of cis-catechins.

[0038] This invention also provides a method for regulating cis-catechin synthesis in tea plants based on the CsAHL20 gene, comprising: constructing a silencing vector for the CsAHL20 gene and transferring the silencing vector into tea plants to silence the CsAHL20 gene; or, constructing an overexpression vector for the CsAHL20 gene and transferring the overexpression vector into tea plants to overexpress the CsAHL20 gene; the nucleotide sequence of the CsAHL20 gene is shown in SEQ ID NO.1. In this invention, the silencing vector or the overexpression vector for the CsAHL20 gene can be transferred into the leaves of tea plants; the overexpression in this invention can be transient overexpression.

[0039] In this invention, the construction process of the gene silencing vector includes: using pTRV2 as the original vector, inserting a 252bp sequence of the CDS of the CsAHL20 gene into the multiple cloning site of pTRV2, with the insertion site located between the EcoRI and BamHI restriction sites on the original vector pTRV2, and the resulting silencing vector is named pTRV2-CsAHL20; the nucleotide sequence of the 252bp sequence of the CDS of the CsAHL20 gene is shown in SEQ ID NO.24.

[0040] The construction process of the overexpression vector includes: using pCAMBIA2301-35S as the original vector, inserting the full-length CDS sequence of the CsAHL20 gene into the multiple cloning site of pCAMBIA2301-35S, with the insertion site located between the Kpn I and Xba I restriction sites on the original vector pCAMBIA2301-35S, and the resulting overexpression vector is named pCAMBIA2301-35S-CsAHL20; the nucleotide sequence of the full-length CDS sequence of the CsAHL20 gene is shown in SEQ ID NO.1.

[0041] This invention also provides a tea plant breeding method, comprising: transferring a silencing vector of the CsAHL20 gene into tea plant tissue to silence the CsAHL20 gene, and then culturing the tea plant tissue; or, transferring an overexpression vector of the CsAHL20 gene into tea plant tissue to overexpress the CsAHL20 gene, and then culturing the tea plant tissue; the nucleotide sequence of the CsAHL20 gene is shown in SEQ ID NO.1; the culture is for breeding purposes and may include: tissue culture for breeding purposes. In this invention, the tea plant tissue may be a tea plant leaf.

[0042] The present invention also provides an application of the CsAHL20 gene as a target in improving tea quality, wherein the nucleotide sequence of the CsAHL20 gene is shown in SEQ ID NO.1; the improvement of tea quality can be to increase the content of cis-catechins in tea; the application can include: silencing the CsAHL20 gene in tea plants to promote the synthesis of cis-catechins in tea plants, thereby increasing the content of cis-catechins in tea.

[0043] During the experiment, this invention screened CsAHL20 transcription factors containing AT-Hook Motif and PPC domains, which are significantly responsive to drought, from transcriptome data by treating tea seedlings with drought. CsAHL20 belongs to the AHL family.

[0044] In this invention, the nucleotide sequence of the CsAHL20 gene is shown in SEQ ID NO.1, and the nucleotide sequence of SEQ ID NO.1 is as follows:

[0045] .

[0046] In this invention, the amino acid sequence of the protein encoded by the CsAHL20 gene is shown in SEQ ID NO.2; the protein contains 265 amino acids and has an AT-Hook motif and a PPC domain; the amino acid sequence of SEQ ID NO.2 is as follows:

[0047] MLNKRDLEISMNETSARSTGQGDEDDDKDNGDEPKEGAVEVGTRRPRGRPPGSKNKPKPPIFVTRDSPNALRSHVMEVAGGTDVAESIAQFARRRQRGVCVLSGSGSVANVTLRQPAAPGAVMALHGRFEILS LTGAFLPGPAPPGSTGLTVYLAGGQGQVVGGSVVGSLVAAGPVMVIAATFSNATYERLPLEDDDEAGSAGQTQLPGTTGSSTPAIGSSGGQQQHGLPDPSSLQLYNLPPNLLPNGVQLSHDAYNWTHARPPY.

[0048] In this invention, the nucleotide sequence of the CsANR promoter used is shown in SEQ ID NO.3, and the nucleotide sequence of SEQ ID NO.3 is as follows:

[0049]

[0050] The relevant culture media were prepared as follows during the experiments of this invention:

[0051] (1) LB solid medium: 10 g / L tryptone + 10 g / L sodium chloride + 5 g / L yeast extract + 7.5 g / L agar powder;

[0052] (2) LB liquid medium: 10 g / L tryptone + 10 g / L sodium chloride + 5 g / L yeast extract;

[0053] (3) YEP solid medium: 10 g / L peptone + 10 g / L yeast extract + 5 g / L sodium chloride + 7.5 g / L agar powder;

[0054] (4) YEP liquid culture medium: 10 g / L yeast extract + 10 g / L peptone + 5 g / L sodium chloride;

[0055] (5) Resuspension: 4.74 g / L MS + 30 g / L sucrose.

[0056] All biological materials used in the experiments of this invention are commercially available.

[0057] The following are specific examples of the present invention:

[0058] Example 1: Identification and analysis of CsAHL20:

[0059] (I) Experimental Methods:

[0060] 1. Identification and analysis of CsAHL:

[0061] Thirty-one AHL proteins from Arabidopsis thaliana were used as seed sequences. Blast alignments were performed in Rice Gene Index (RGI; https: / / riceome.hzau.edu.cn / ) and Tea Plant Information Archive (TPIA; http: / / tpia.teaplants.cn / ) to obtain AHL protein sequences from rice and tea. A phylogenetic tree was constructed using MEGA-X software (version 7.0) with 1000 bootstrap replicates, and visualization was performed using the iTOL online platform (https: / / itol.embl.de / ). Based on catechin content and transcriptome data measured after previous drought treatment, catechin-related genes were identified. DNAMAN software was used for sequence alignment visualization.

[0062] 2. Drought treatment of tea seedlings, growth conditions, and material collection:

[0063] One-year-old Fuding Da Bai tea seedlings with consistent growth were collected from the same seedbed at Meitan Tea Factory in Zunyi City, Guizhou Province, China. After the soil around the seedlings' roots was cleaned, they were hydroponically cultured. The cultivation containers were 5L polyethylene plastic pots, covered with perforated foam boards. The seedlings were tightly wrapped with sponges and planted in each pot. At the Tea College of Guizhou University, the hydroponic seedlings were cultivated in an artificial climate chamber. After two weeks of cultivation in untreated nutrient solution, 10% PEG6000 was added to the normal nutrient solution. Second leaves were harvested after 0, 6, 12, 24, and 48 hours of treatment. To reduce individual differences among seedlings, three samples were taken as a pooled sample, and three groups were used as three biological replicates. Immediately after collection, the samples were flash-frozen in liquid nitrogen and stored at -80°C for further research.

[0064] 3. HPLC analysis of catechin content in tea leaves:

[0065] After freeze-drying the tea sample taken from -80℃, the test solution was prepared according to the method specified in GB / T 8313-2018 "Determination of Tea Polyphenols and Catechins in Tea". 0.2 g (accurate to 0.001 g) was weighed, ground, and extracted with 5 mL of 70% (v / v) methanol in a 70℃ water bath for 10 min, shaking every 5 min. After centrifugation at 3500×g and 4℃ for 10 min, the extracted solution was transferred to a clean 10 mL volumetric flask. The above steps were repeated until the final volume was adjusted to 10 mL, and the solution was filtered using a 0.45 μm Millipore filter. High-performance liquid chromatography (HPLC) analysis was performed.

[0066] Chromatographic conditions: Detection wavelength 278 nm; mobile phase A: 0.5% acetic acid aqueous solution, mobile phase B: methanol, mobile phase C: acetonitrile; elution gradient: 0–6 min, A 85%, B 10%, C 5%; 6–16 min, A 85–70%, B 10–20%, C 5–10%; 16–22 min, A 70–72%, B 20%, C 10–8%; 22–26 min, A 72–85%, B 20–10%, C 8–5%; flow rate 60 mL / h; column temperature 35 °C; injection volume 10 μL; analysis time 26 min.

[0067] 4. Total RNA extraction and real-time quantitative PCR (RT-qPCR) analysis:

[0068] After freezing, samples were treated with a modified cetyltrimethylammonium bromide (CTAB) technique to extract total RNA. Genomic DNA was removed from the extracted RNA, which was then reverse transcribed into cDNA, diluted 10-fold, and analyzed. The CsACTIN gene from tea leaves was used as an internal control in this study. RT-qPCR was performed using a Bio-Rad CFX Connect™ real-time quantitative PCR instrument. -ΔCt The relative expression levels of genes were detected. The primers used are listed in Table 1.

[0069] Table 1 Primer names and sequences

[0070]

[0071] (II) Experimental Results:

[0072] 1. Identification and analysis of CsAHL:

[0073] In rice, genes from the AHL family have been shown to enhance the plant's drought resistance. In this example, the identification and analysis of CsAHL20 are as follows: Figures 1a-1d As shown in the figure. In this embodiment, a phylogenetic tree was constructed using 31 AHL family genes from Arabidopsis thaliana and 27 from rice, and 37 CsAHL genes were found in tea plants. Combined with drought transcriptome data, it was found that this family basically responds to drought. After removing genes with FPKM values ​​less than 10, 12 CsAHL genes remained. Previous studies have found that drought leads to a decrease in catechins, so correlation analysis was performed on these 12 CsAHL genes. The results showed that CSS0021339, CSS0017050, CSS0016287, and CSS0024158 were strongly negatively correlated with cis-catechins; however, the FPKM values ​​of the latter three genes were relatively small, while the FPKM value of CSS0021339 was significantly higher than them. Therefore, it is reasonable to speculate that the CSS0021339 transcription factor plays a key role in cis-catechin synthesis.

[0074] CSS0021339 is named AT-hook motif nuclear localized protein 20 in the early-maturing tea genome, and will be referred to as CsAHL20 below. CsAHL20 has a CDS length of 798 bp, encoding a protein containing 265 amino acids; the protein has a molecular weight of 27.47 kDa and an isoelectric point of 6.02. Phylogenetic analysis shows that CsAHL20 has high sequence similarity to Os07g13100, Os08g44910, and Os02g57520 in rice, and CSS0007054, CSS0023327, and CSS0022210 in tea, and all of them contain AT-hook motifs and PPC domains. In rice, Os07g13100, Os08g44910, and Os02g57520 are significantly upregulated under drought stress. In conclusion, the CsAHL20 gene is highly likely to respond to drought stress and may be involved in the synthesis of cis-catechins under drought conditions.

[0075] 2.10% PEG6000 treatment upregulated the CsAHL20 gene in tea plants and reduced the content of cis-catechins.

[0076] To further verify the relationship between CsAHL20 and catechins under drought conditions, tea trees were subjected to drought treatment. In this example, the catechin content in tea leaves after treatment with 10% polyethylene glycol 6000 for different time periods and the results of real-time quantitative PCR (RT-qPCR) analysis are as follows: Figures 2a-2f As shown in the figure, in this embodiment, it was found that after 12 hours of treatment with 10% PEG6000, scorching began to appear on the edges of tea leaves, and by 48 hours, the leaves had severely wilted. RT-qPCR results showed that CsAHL20 was upregulated after 10% PEG6000 treatment, and its expression level continued to increase with the extension of treatment time. However, the total catechin content in tea decreased with the extension of treatment time. The trend of cis-catechins (EGC, EC, EGCG, ECG) was consistent with that of total catechins. Among trans-catechins, the CG content decreased after treatment, while the GC content increased. This result indicates that drought stress reduces the total catechin content in tea, and continuous drought leads to a continuous decrease in the cis-catechin content. This is consistent with the results obtained in the laboratory in the past, which confirms the above inference that the CsAHL20 gene responds to drought stress and may lead to a decrease in cis-catechins.

[0077] Example 2: Subcellular localization of the CsAHL20 gene in tea plants:

[0078] (I) Experimental Methods:

[0079] 1. The tobacco used is Benzoic tobacco.

[0080] 2. Construction of integrated expression vectors:

[0081] The CDS sequence of CsAHL20 was ligated to the SacI and SalI restriction sites of the PCAMBIA1300-35S-GFP vector using T4 DNA ligase. The resulting ligation product was transformed into DH5α competent cells. After PCR amplification, restriction enzyme digestion screening, and sequencing verification, positive clones were screened and plasmids were extracted to obtain the GFP-target gene fusion expression vector PCAMBIA1300-35S-CsAHL20-GFP. The subcellular localization vector was synthesized by Wuhan Transduction Biology Laboratory Co., Ltd.

[0082] 3. Instantaneous conversion steps of tobacco:

[0083] (1) The successfully detected Agrobacterium bacterial culture was amplified and shaken overnight at 28°C and 200 rpm;

[0084] (2) Take 1 mL of bacterial solution and add it to a sterile 1.5 mL centrifuge tube;

[0085] (3) 6000 rpm, 3 min, precipitate the bacterial cells (room temperature), remove the supernatant, add 1 mL of permeate, and suspend the bacterial cells;

[0086] (4) Repeat step 3 to further remove small amounts of antibiotics;

[0087] (5) Take a small amount of the bacterial suspension, dilute it 10 times, and measure the OD. 600 The value is multiplied by 10 to obtain the OD of the bacterial suspension. 600 value;

[0088] (6) Determine the titer of the suspended bacterial solution to the permeate, calculate the dilution factor, and make the final suspended bacterial solution (for infection) 5.0 mL, OD 600 The concentration is 0.4 (0.1-0.8 as needed, not exceeding 1), and usually 0.5-1.0 mL of final bacterial suspension is sufficient for infection;

[0089] (7) Prepare the final bacterial suspension in a 1.5 mL centrifuge tube, let it stand at room temperature for 2 hours, and prepare for infection;

[0090] (8) Before infection, place the tobacco under a white fluorescent lamp for 1 hour to open its stomata;

[0091] (9) Select the third and fourth leaves from the bottom for infection (infection between the two leaf veins). Select two leaves from one plant and infect with one bacterial solution.

[0092] (10) Gently rub the back of the blade to be rotated with a syringe without the needle, or pierce it with a small needle to remove its wax layer.

[0093] (11) Before infection, mark the area to be transferred with a marker pen;

[0094] (12) Aspirate the final suspension of bacteria from step (7) into a 1 mL syringe without a needle;

[0095] (13) Point the syringe at the area to be rotated on the back of the blade, press the blade with one hand and gently push the piston with the other hand until you see the liquid spread, then infect other parts. After infecting, circle the infected area with a marker.

[0096] (14) Spray the leaves with water, cover them with plastic bags, and put the infected tobacco back into the cultivation room and leave it in the dark overnight;

[0097] (15) Open the plastic bag the next day. The expression level was highest 2 days after the injection.

[0098] (16) Cut out the infected area, tear off the epidermis to make a slide, and observe it under a confocal laser microscope.

[0099] (II) Experimental Results:

[0100] In this embodiment, the subcellular localization of the CsAHL20 protein in tobacco leaf cells is as follows: Figure 3 As shown, the empty vector carrying GFP could be detected with fluorescence signals in all tobacco epidermal leaf cells, and the PCAMBIA1300-35S-CsAHL20-GFP fusion protein could be detected with fluorescence signals in the cell nucleus, proving that the CsAHL20 protein functions in the cell nucleus.

[0101] Example 3: Validation of CsAHL20 function using an Agrobacterium-mediated transient overexpression system in tea plants:

[0102] (I) Experimental Methods:

[0103] 1. Experimental materials: The materials were cuttings of the "Wuniuzao" tea tree from the Tea College of Guizhou University.

[0104] 2. Construction of plant overexpression vectors:

[0105] The plant overexpression vector was designed and constructed using the CDS sequence (SEQ ID NO.1) of CsAHL20, based on the initial vector pCAMBIA2301-35S. The recombinant plasmid was transformed into competent *E. coli* (DH5α), and positive clones were screened using 50 mg / L Kan. The recombinant plasmid was extracted and verified by double digestion with KpnI and XbaI. Recombinant plasmids with positive digestion results were transformed into competent *Agrobacterium* strain GV3101 cells. Positive *Agrobacterium* strains were screened using 50 mg / L Kan and 50 mg / L Rif. Colony PCR was performed on positive strains using primers. *Agrobacterium* strains with positive PCR results were expanded and preserved at -80℃. The plasmid containing the overexpression vector was transformed into *Agrobacterium* strain GV3101 using a freeze-thaw method to prepare engineered strains. Take GV3101 competent cells stored at -80℃ and thaw them on ice for 5 min; add 5 μL of plasmid DNA to each competent cell, gently shake to mix, and then incubate on ice for 5 min; after flash freezing in liquid nitrogen for 5 min, immediately incubate in water at 37℃ for 1 min, add 700 μL of YEP liquid medium, and culture at 28℃ and 200 rpm for 3 h with shaking; after centrifugation at 6000 rpm for 1 min at room temperature, discard the supernatant, leaving about 100 μL, mix it with a pipette tip, take an appropriate amount of bacterial culture and spread it on YEP agar plates containing 50 mg / L Kan and 50 mg / L Rif, invert and incubate in a constant temperature incubator at 28℃ for 2 days, pick single colonies and culture them on a shaker at 28℃ for 18 h, and then store the bacterial culture at -80℃.

[0106] 3. Tea tree infection:

[0107] The recombinant plasmid pCAMBIA2301-35S-CsAHL20 was introduced into Agrobacterium GV3101 competent cells using a freeze-thaw method. Each Agrobacterium strain was inoculated into solid YEP medium containing 50 mg / L Kan and 50 mg / L Lrif, and activated at 28°C for 48 h. Single colonies were cultured in the corresponding liquid YEP medium until OD... 600 The concentration was 1.0. Agrobacterium cells were centrifuged at 6000 rpm for 6 min. The bacterial suspension was collected and resuspended in 4.74 g / L MS, 30 mg / L sucrose, 150 μmol / L acetylsylgenone (AS), and 25 μmol / L LMES, pH 5.6. Agrobacterium with the pCAMBIA2301-35S vector was injected into the sixth leaf as a control, and the CsAHL20 overexpression vector was injected into the sixth leaf of the tea plant. Each experiment had at least 5 replicates. Samples were collected 3 days after injection, divided into two portions: one portion was flash-frozen in liquid nitrogen and stored at -80℃ for total RNA extraction, and the other portion was blanched, dried to constant weight, and ground into powder. The catechin content was determined by high-performance liquid chromatography.

[0108] 4. Total RNA extraction and real-time quantitative PCR (RT-qPCR) analysis (primers used are listed in Table 2).

[0109] Table 2 Primer names and sequences

[0110]

[0111]

[0112] (II) Experimental Results:

[0113] In this embodiment, the catechin content in tea leaves after transient overexpression of the CsAHL20 gene and RT-qPCR analysis are as follows: Figures 4a-4n As shown, the results indicated that, compared with the control, the expression level of the CsAHL20 gene in tea leaves transiently overexpressing the target gene was significantly upregulated. Compared with the control, the total catechin content in CsAHL20 transgenic leaves decreased by 16.26% (35S:CsAHL20-1), 30.23% (35S:CsAHL20-2), and 20.89% (35S:CsAHL20-3), respectively; among them, cis-catechins decreased by 12.75% (35S:CsAHL20-1), 29.27% ​​(35S:CsAHL20-2), and 22.40% (35S:CsAHL20-3), respectively. Among the genes involved in the catechin synthesis pathway, the expression level of CsANR showed a corresponding decrease; the results indicate that CsAHL20 negatively regulates the biosynthesis of cis-catechins and may inhibit the expression of CsANR.

[0114] Example 4: Verification of CsAHL20 function using virus-induced gene silencing (VIGS) technology:

[0115] (I) Experimental Methods:

[0116] 1. Experimental materials:

[0117] The material used was the "Fuding Dabai" tea tree cuttings from the Tea College of Guizhou University. The function of CsAHL20 was verified using virus-induced gene silencing (VIGS) technology.

[0118] 2. Construction of plant VIGS vectors:

[0119] A 252bp fragment of the pTRV2-CsAHL20 gene was constructed, containing EcoRI and BamHI restriction sites. The PCR product was ligated with the pTRV2 vector, which had been recovered after double digestion with EcoRI and BamHI, using T4 DNA ligase (Kewen Biotechnology Co., Ltd.). After ligation overnight at 16°C, the ligation was transformed into *E. coli* DH5α. The insertion of the product was verified by colony PCR using specific primers CsAHL20-F and CsAHL20-R. The CsAHL20 gene fragment was obtained using a whole-gene synthesis method and loaded into the pUC57-Simple vector to obtain the pUC57-Simple-CsAHL20 plasmid.

[0120] The sequence of the 252bp fragment of the CsAHL20 gene is shown in SEQ ID NO.24:

[0121] CGAAGCCATGTTATGGAGGTGGCCGGAGGCACTGATGTAGCGGAGAGCATAGCCCAATTCGCCCGGAGGCGTCAGAGAGGGGTTTGTGTACTAAGTGGAAGTGGTTCGGTTGCCAACGTGACACTG AGGCAGCCAGCTGCGCCAGGTGCTGTGATGGCGCTCCATGGTAGGTTTGAAATTTTATCACTGACTGGGGCTTTCCTGCCAGGACCCGCCCCGCCAGGTTCCACTGGCTTGACAGTGTACCTGGCA.

[0122] The sequence of the CsAHL20 gene containing flanking restriction enzyme sites is shown in SEQ ID NO.25:

[0123] CGGAATTCCG CGAAGCCATGTTATGGAGGTGGCCGGAGGCACTGATGTAGCGGAGAGCATAGCCCAATTCGCCCGGAGGCGTCAGAGAGGGGTTTGTGTACTAAGTGGAAGTGGTTCGGTTGCCAACGTGACACTG AGGCAGCCAGCTGCGCCAGGTGCTGTGATGGCGCTCCATGGTAGGTTTGAAATTTTATCACTGACTGGGGCTTTCCTGCCAGGACCCGCCCCGCCAGGTTCCACTGGCTTGACAGTGTACCTGGCA CGGGATCCCG .

[0124] Gene fragment structure: EcoRI-CsAHL20-BamHI.

[0125] Construction of the pTRV2-CsAHL20 plasmid: The pUC57-Simple-CsAHL20 plasmid was digested with EcoRI and BamHI to obtain the CsAHL20 gene fragment. The fragment was then processed using a gel electrophoresis and recovered. Simultaneously, the pTRV2 vector was digested with EcoRI and BamHI, and the vector fragment was recovered using a gel electrophoresis. The digested CsAHL20, gene fragment, and pTRV2 vector fragment were ligated. The ligation product was transformed into DH5α competent cells. Several clones were picked from the transformed plates, and plasmids were extracted and identified by EcoRI and BamHI digestion. The recombinant plasmids were further sequenced for identification (Note: All the above processes were outsourced to Changsha Kewen Biotechnology Co., Ltd.).

[0126] 3. Tea tree infection:

[0127] pTRV1, pTRV2, and pTRV2-CsAHL20 were introduced into Agrobacterium GV3101 competent cells using a freeze-thaw method. Each Agrobacterium strain was inoculated into solid YEP medium containing 50 mg / L kanamycin and 50 mg / L rifampin and incubated at 28°C for 2 days for activation. Single colonies were cultured in the corresponding liquid YEP medium until OD... 600 The concentration was 1.2. Agrobacterium cells were centrifuged at 6000 rpm for 6 min. The bacterial suspension was collected and resuspended in 4.74 g / L MS, 2 mol / L 6-BA, 2 mol / L acetylsuccine (AS), and 100 μmol / L naphthaleneacetic acid (NAA), pH 5.6. Vacuum permeation treatment was then applied to both tea plant materials. The OD... 600 Adjust to 1.2. At room temperature, mix pTRV1 with pTRV2 and pTRV2-CsAHL20 bacterial solutions at a 1:1 ratio (pTRV1+pTRV2, pTRV1+pTRV2-CsAHL20) and use this mixture to vacuum infiltrate tea cuttings. Cut the tea cuttings to a length of 20cm using pruning shears. Retain two mature leaves, then place the tea cuttings in Buchner flasks containing the mixed bacterial solutions for vacuum infiltration. Incubate them in the dark for three days, then grow them in a greenhouse at 25°C with a 16h / 8h light / dark cycle.

[0128] After lateral buds emerged from vacuum-infected pTRV2-CsAHL20 silent plants, pTRV2 plants, and wild-type plants, samples were collected and divided into two portions. One portion was flash-frozen in liquid nitrogen and stored at -80℃ for total RNA extraction, while the other portion was blanched, dried to constant weight, and ground into powder. The catechin content was determined by high-performance liquid chromatography.

[0129] 4. Detection of related gene expression levels: The detection method is the same as in Example 1.

[0130] (II) Experimental Results:

[0131] In this embodiment, the relative expression level of catechins and the gene after virus-induced gene silencing of CsAHL20 was measured as follows: Figures 5a-5e As shown, CsAHL20 was successfully silenced in tea plants compared to wild-type and empty vector controls; its expression levels decreased by 60.1%, 42.9%, and 29.3% compared to WT (wild-type), while the expression level of CsANR was significantly increased in CsAHL20-silenced plants. Analysis of catechin content in CsAHL20-silenced tea cuttings showed that, compared to WT (wild-type), the content of cis-catechins increased in CsAHL20-silenced plants; the cis-catechin content increased by 94.5% (pTRV-CsAHL20-1), 74.0% (pTRV-CsAHL20-2), and 45.5% (pTRV-CsAHL20-3), respectively. These data indicate that CsAHL20 silencing promotes the formation of cis-catechins in the leaves of Fuding Da Bai Cha tea, therefore CsAHL20 is a negative regulator of cis-catechin biosynthesis in tea plants.

[0132] Example 5: Detection of CsAHL20 and CsANR promoter binding using EMSA and DLRA methods:

[0133] (I) Experimental Methods:

[0134] 1. Electrophoretic mobility shift measurement (EMSA):

[0135] To determine the regulatory role of CsAHL20 TF in CsANR, multiple binding sites for the CsAHL20 motif were predicted using the JASPAR database. A probe was designed based on a site with high confidence. The purified AHL20 protein (Figure) was incubated with a DNA fragment from the CsANR promoter, followed by gel electrophoresis migration assays. The probe sequences are shown in Table 3.

[0136] Table 3 Probe Names and Sequences

[0137]

[0138]

[0139] 2. Dual-luciferase reporter assay (DLRA):

[0140] Transcriptional activity in tobacco was analyzed using a dual-luciferase assay system. CsAHL20 and its CDS were cloned as effectors into the pGreenII 62-SK vector, and the promoter fragment of CsANR was introduced into the pGreenII 0800-LUC vector as a reporter gene. These were used to transform Agrobacterium rhizogenes strain EHA105, followed by transient fusion expression of the recombinant plasmids in leaf cells infected with EHA105. Three days later, infected leaves were sprayed with 0.2 mg / mL D-luciferin potassium salt and incubated at 37°C for 10 min. Fluorescence images were obtained using a Fusion FX7 (VILBER, France) chemiluminescence instrument. Transcriptional activity based on LUC / REN was measured using a dual-luciferase reporter gene assay kit.

[0141] (II) Experimental Results:

[0142] For the detection results of CsAHL20 and CsANR promoter interaction in tea (EMSA) and the dual-luciferase reporter assay results of CsAHL20 and CsANR in tea, please refer to [link to relevant documentation]. Figure 6 and Figure 7 As shown. First, the purified CsAHL20 protein was incubated with the CsANR promoter DNA fragment, followed by electrophoretic gel migration assays; see [link to relevant documentation]. Figure 6 As shown, after the AHL20 protein attaches to the nucleic acid fragment, the protein + nucleic acid band lags significantly behind the nucleic acid-only band. Furthermore, the binding band becomes increasingly prominent with increasing protein concentration, while the free probe band becomes increasingly faint until it disappears. However, the mutant probe does not show a lag in its binding band with the AHL20 protein. This indicates that AHL20 and the CsANR promoter specifically bind, and the mutant probe does not show a binding band with the AHL20 protein. Gel electrophoresis results confirm that the AHL20 protein specifically binds to the CsANR promoter.

[0143] The CsANR promoter was integrated into the 0800-LUC plasmid, while the CsAHL20 CDS was integrated into the 62-SK plasmid. These plasmids were co-transfected into Agrobacterium and transformed into tobacco; see also Figure 7 As shown, compared with the luciferase signal produced by tobacco leaves co-transfected with CsAHL20 and CsANR promoters, the control group (③pro-CsANR-LUC+35S-62-SK) produced a stronger luciferase signal, with a fluorescence value significantly higher than that of the co-transfected CsAHL20 and CsANR promoters. This further indicates that CsAHL20 can inhibit CsANR at the transcriptional level. Therefore, CsAHL20 inhibits CsANR transcription by directly binding to the CsANR promoter, suggesting that CsAHL20 is a key regulator of CsANR-mediated catechin biosynthesis.

[0144] The above-described technical solutions provided by this invention are merely preferred embodiments and do not limit the scope of the patent. Any equivalent structural transformations made using the description and drawings of this invention under the technical concept of this invention, or direct / indirect applications in other related technical fields, are included within the scope of patent protection of this invention.

Claims

1. A method of modulating the synthesis of cis- catechins in tea plants by targeting the gene CsAHL20 characterized in that, Regarding the tea tree CsAHL20 Gene silencing promotes the synthesis of cis-catechins in tea plants; CsAHL20 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. A method according to claim 1, wherein the step of determining the presence of the target nucleic acid sequence comprises amplifying the target nucleic acid sequence. CsAHL20 Use of a gene as a target in regulating the synthesis of cis- catechins in tea plants, characterized in that, Regarding the tea tree CsAHL20 The gene was overexpressed to inhibit the synthesis of cis-catechins in tea plants; CsAHL20 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

3. A method according to claim 1 or 2, wherein the method comprises the step of: - determining the presence of a mutation in the gene encoding the protein in the tea plant. CsAHL20 Use of a protein encoded by a gene in inhibiting the synthesis of cis- catechins in a tea plant, characterized in that, The CsAHL20 The amino acid sequence of the protein encoded by the gene is shown as SEQ ID NO.

2.

4. A method of producing a tea plant with increased yield comprising CsAHL20 the use of a gene as a target in the preparation of a tea plant cis- catechin modulating agent, characterized in that, The tea tree cis- catechin modulating agent includes the CsAHL20 gene silencing agent or the CsAHL20 gene overexpression agent; The CsAHL20 silencing agent for a gene in a tea plant CsAHL20 silences a gene to promote the synthesis of cis- catechins in a tea plant; The CsAHL20 overexpression reagent of the gene is used to inhibit the synthesis of cis- catechins in the tea plant; the CsAHL20 nucleotide sequence of the gene is shown as SEQ ID NO.

1. CsAHL20 nucleotide sequence of the gene is shown as SEQ ID NO.

1.

5. A method according to any one of claims 1 to 4 wherein the method is based on the use of a compound of formula (I) or a salt thereof. CsAHL20 A method of genetically regulating the synthesis of cis- catechins in tea plants, characterized in that, By the method CsAHL20 Silencing agents for genes in tea plants CsAHL20 Silencing genes to promote the synthesis of cis- catechins in tea plants; or, by the method CsAHL20 overexpression reagent of the gene in tea tree CsAHL20 The gene is overexpressed to inhibit the synthesis of cis- catechin in tea tree; The CsAHL20 The nucleotide sequence of the gene is shown as SEQ ID NO.

1.

6. A method of producing a tea product based on CsAHL20 A method of genetically regulating the synthesis of cis- catechins in tea plants, characterized by, comprising: constructing the CsAHL20 silencing vector of the gene, transforming the silencing vector into the tea plant to silence the CsAHL20 gene, so as to promote the synthesis of the tea plant cis- catechin. or, constructing the overexpression vector of the gene, transforming the overexpression vector into the tea plant to make the gene overexpressed, so as to inhibit the synthesis of the catechin of the tea plant CsAHL20 or, constructing the overexpression vector of the gene, transforming the overexpression vector into the tea plant to make the gene overexpressed, so as to inhibit the synthesis of the catechin of the tea plant CsAHL20 or, constructing the overexpression vector of the gene, transforming the overexpression vector into the tea plant to make the gene overexpressed, The CsAHL20 The nucleotide sequence of the gene is shown as SEQ ID NO.

1.

7. The method of claim 6, wherein the compound is ###0002### CsAHL20 A method of genetically regulating the synthesis of cis- catechins in tea plants, characterized by, The construction process of the silencing vector comprises: taking pTRV2 as a raw vector, inserting the 252bp sequence of the CDS of the gene into the multiple cloning site of pTRV2, and the insertion position is between the EcoR I and BamH I enzyme cutting sites on the raw vector pTRV2. CsAHL20 The nucleotide sequence of the 252bp sequence of the CDS of the gene is shown as SEQ ID NO.

24. CsAHL20 The nucleotide sequence of the 252bp sequence of the CDS of the gene is shown as SEQ ID NO.

24. The construction process of the overexpression vector comprises the following steps: taking pCAMBIA2301-35S as a raw vector, inserting the CDS full sequence of the gene into the multiple cloning site of pCAMBIA2301-35S, and the insertion position is located between the Kpn I and Xba I enzyme cutting sites on the raw vector pCAMBIA2301-35S. CsAHL20 The nucleotide sequence of the CDS full sequence of the gene is shown as SEQ ID NO.

1. CsAHL20 ​ 8. A method of breeding tea plants, characterised by, comprising: Will CsAHL20 The gene silencing vector was transferred into the tea plant tissue to make the aforementioned CsAHL20 Gene silencing, followed by culturing tea plant tissue to promote the synthesis of cis-catechins in tea; or, the aforementioned CsAHL20 The gene overexpression vector was transferred into tea plant tissue to make the above CsAHL20 Gene overexpression was performed, followed by culturing tea plant tissues to inhibit the synthesis of cis-catechins in tea plants. The CsAHL20 The nucleotide sequence of the gene is shown as SEQ ID NO. 1.

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