Method for regulating synthesis of tea tree cis-catechin based on CsAHL20 gene and application

Abstract

The invention provides a method for regulating synthesis of tea tree cis-catechin based on a CsAHL20 gene and application, and belongs to the technical field of plant molecular biology. The screened tea tree gene CsAHL20 can be combined with cis-catechin to synthesize a key enzyme CsANR gene promoter, and accumulation of the cis-catechin content of tea trees is inhibited; through verification, the CsAHL20 gene can increase the content of tea tree cis-catechin substances by silencing the gene through a virus-induced gene silencing technology, and can reduce the content of the tea tree cis-catechin substances by overexpressing the gene through an agrobacterium-mediated instantaneous transformation technology, so that the CsAHL20 gene can be used for regulating and controlling synthesis of the tea tree cis-catechin.

Description

Technical Field

[0001] The invention belongs to the field of plant molecular biotechnology, and in particular relates to a method and application of regulating the synthesis of tea tree cis-catechins based on CsAHL20 gene. Background Art

[0002] Catechins are one of the main components of tea, accounting for more than 70% of the total amount of tea polyphenols. Catechins include catechin (C), epicatechin gallate (GC), epicatechin gallate (EC), epigallocatechin gallate (EGC), epigallocatechin gallate (ECG), epigallocatechin gallate (GCG), epigallocatechin gallate (EGCG) and epigallocatechin 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 the main catechins with higher content.

[0003] With the deepening of the study of catechins, more mechanisms affecting catechin synthesis have been revealed. During the growth of tea plants, they are often stressed by various biological or abiotic factors. Drought is one of the most critical abiotic factors restricting tea production. The response of plants to drought includes morphological, biochemical, physiological and molecular changes. Importantly, the accumulation of secondary metabolites in plants is largely affected by the decline in soil moisture. Studies have shown that under drought stress, the catechin content in tea will decrease; at the same time, 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; existing studies have found that the expression levels of CsCHS, CsLAR and CsANR genes are positively correlated with total catechins, among which ANR and LAR genes are positively correlated with the content of EGCG and ECG. Under drought stress, many genes are involved in the biosynthesis and metabolism of catechins; tea plants form 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 TFs that may be involved in the regulation of tea catechins, and found that many TFs are closely related to the expression of catechin biosynthesis genes, including MADS box, R2R3-MYB and bHLH TFs; 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 exists in all sequenced plant species; members of the AHL family contain two conserved structural units: AT-hook motif and PPC domain. The AT-hook motif is able to bind to AT-rich DNA and has been identified in various gene families in prokaryotes and eukaryotes; the PPC domain has also been annotated as domain of unknown function 296 (DUF296), which 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 response and have been shown to have drought resistance in a variety of plants; for example, in rice, the expression of AHLs is significantly upregulated under drought stress. However, most studies on the response of AHLs to abiotic stresses have focused on model species, and few have been studied in woody species; as an important transcription factor that responds to abiotic stresses, there are currently no reports on CsAHL genes in tea plants.

[0005] In view of this, it is necessary to provide a method and application of regulating the synthesis of cis-catechins in tea trees 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 trees based on new regulatory genes. Summary of the invention

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

[0007] To achieve the above object, the present invention provides an application of 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] The CsAHL20 gene in the tea tree is further silenced to promote the synthesis of cis-catechins in the tea tree; or the CsAHL20 gene in the tea tree is overexpressed to inhibit the synthesis of cis-catechins in the tea tree.

[0009] The present invention also provides an application of a protein encoded by a 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.

[0010] The present invention also provides an application of the CsAHL20 gene as a target in the preparation of a tea tree cis-catechin regulating agent, wherein the nucleotide sequence of the CsAHL20 gene is shown in SEQ ID NO.1.

[0011] The present invention also provides a tea tree cis-catechin regulating reagent, which comprises a CsAHL20 gene silencing reagent and / or a CsAHL20 gene overexpression reagent, and the nucleotide sequence of the CsAHL20 gene is shown in SEQ ID NO.1.

[0012] The present invention also provides a method for regulating the synthesis of cis-catechins in tea plants based on the CsAHL20 gene, comprising regulating the synthesis of cis-catechins in tea plants using any of the above-mentioned tea tree cis-catechin regulating reagents.

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

[0014] Furthermore, the construction process of the gene silencing vector includes: using pTRV2 as the original vector, inserting the 252bp sequence of the CsAHL20 gene CDS into the multiple cloning site of pTRV2, and the insertion position is 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 SEQID NO.24.

[0015] The construction process of the overexpression vector includes: using pCAMBIA2301-35S as the original vector, inserting the CDS full sequence of the CsAHL20 gene into the multiple cloning site of pCAMBIA2301-35S, and the insertion position is located between the Kpn I and Xba I restriction sites on the original vector pCAMBI A2301-35S; the nucleotide sequence of the CDS full 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 CsAHL 20 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 the quality of tea leaves, 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] The present invention provides a tea tree gene CsAHL20 that participates in the regulation of tea tree cis-catechin synthesis, and can regulate tea tree cis-catechin synthesis based on a new regulatory gene. The present invention uses Agrobacterium-mediated transient transformation technology to overexpress CsAHL20 to reduce the content of tea tree cis-catechin substances, and through dual luciferase (LUC) and electrophoretic mobility shift assay (EMSA) experiments, it is proved that CsAHL20 binds to the promoter of the CsANR gene, a key enzyme for cis-catechin synthesis, to inhibit the expression of CsANR, thereby inhibiting the synthesis and accumulation of cis-catechins; the present invention uses virus-induced gene silencing technology to silence CsAHL20 to increase the content of tea tree cis-catechin substances, and compared with the control plant, the cis-catechin content in the silenced plant is significantly increased. In summary, the present invention finds that the CsAHL20 gene inhibits cis-catechin synthesis, and silencing the CsAHL20 gene can significantly increase the content of tea tree cis-catechins, and can be used to regulate the synthesis of tea tree cis-catechins. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0021] FIG. 1 is the identification and analysis of CsAHL20 in Example 1 of the present invention ( Figure 1a is the phylogenetic tree of AHL proteins, Figure 1b Heat map analysis of CsAHL gene expression patterns in tea plants treated with 10% polyethylene glycol 6000 for different time periods. Figure 1cThis is the correlation analysis between CsAHL family genes and catechins in tea leaves treated with 10% polyethylene glycol 6000. Figure 1d Sequence alignment of CsAHL20 protein with other similar proteins, as well as AT-hook motif and PPC domain);

[0022] Figure 2 shows the catechin content in tea leaves after treatment with 10% polyethylene glycol 6000 for different time periods and real-time fluorescence quantitative PCR (RT-qPCR) analysis in Example 1 of the present invention ( Figure 2a To deal with the status of tea seedlings after different time periods, Figure 2b is the relative expression level of CsAHL20 gene in tea leaves after treatment at different times. Figure 2c is the total amount of catechins after treatment at different times. Figure 2d To determine the cis-catechin content after different treatment times, Figure 2e To measure the contents of cis-catechins EGC, EC, EGCG, and ECG after different treatment times, Figure 2f is the content of trans-catechins GC, C, GCG, and CG after treatment for different time periods);

[0023] Figure 3 The subcellular localization of CsAHL20 protein in tobacco leaf cells in Example 2 of the present invention; Figure 3 In the figure, 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 superposition of the bright field, red fluorescence field and green fluorescence field of tobacco leaf epidermal cells under a fluorescence microscope;

[0024] FIG4 is a graph showing the catechin content in tea leaves and RT-qPCR analysis after transient overexpression of the CsAHL20 gene in Example 3 of the present invention ( Figure 4a is the relative expression level of CsAHL20, Figure 4b is the total catechin content, Figure 4c is the content of cis-catechins, Figure 4d is the content of cis-catechins EGC, EC, EGCG, and ECG, Figure 4e For the catechin synthesis pathway, Figure 4f is the correlation between the relative expression of catechin synthesis-related genes and the relative expression of CsAHL20, Figure 4g is the relative expression level of CsC4H, Figure 4h is the relative expression level of Cs4CL, Figure 4i is the relative expression level of CsCHS, Figure 4j is the relative expression level of CsCHI, Figure 4k is the relative expression level of CsF3'5'H, Figure 4l is the relative expression level of CsF3'H, Figure 4m is the relative expression level of CsANS, Figure 4n is the relative expression level of CsANR); in FIG4 , 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] FIG. 5 is a graph showing the relative expression level of catechin content and genes after virus-induced gene silencing CsAHL20 in Example 4 of the present invention ( Figure 5a is the relative expression level of CsAHL20 in tea leaves, Figure 5b is the relative expression level of CsANR in tea leaves, Figure 5c It is the total catechins in tea leaves. Figure 5d is the content of cis-catechins in tea leaves, Figure 5e is the content of cis-catechins EGC, EC, EGCG, and ECG in tea leaves); in FIG5 , WT corresponds to the wild-type plant, pTRV2 corresponds to the strain infected with pTRV1+pTRV2 bacterial solution, and pTRV2-CsAHL20-1 to pTRV2-CsAHL20-3 correspond to the strains infected with pTRV1+pTRV2-CsAHL20 bacterial solution;

[0026] Figure 6 The interaction detection (EMSA) between tea plant CsAHL20 and CsANR promoter in Example 5 of the present invention; in the figure, (a) is the predicted binding sequence motif of AHL20, and (b) is the binding of the purified recombinant protein His-CsAHL20 to the CsANR promoter in the EMSA experiment; Figure 6 In the figure, Positive probe corresponds to CsANR activity probe (SEQ ID NO.26 and 27), Mutation probe corresponds to CsANR mutation probe (SEQ ID NO.28 and 29), Positive protein corresponds to CsAHL20 protein, Swim lane corresponds to electrophoresis lane, Bound probe corresponds to binding probe, Free probe corresponds to unbound free probe, "-" corresponds to no addition of corresponding substance, "+" corresponds to addition of corresponding substance, the number of "+" corresponds to the amount of added substance, and 1-5 corresponds to 5 different electrophoresis lanes;

[0027] Figure 7This is the result of the dual luciferase reporter assay of CsAHL20 and CsANR in tea plant in Example 5 of the present invention. CsAHL20 inhibits the CsANR promoter activity and Luc / Ren ratio.

[0028] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with the implementation methods and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0029] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Furthermore, the technical solutions between the various embodiments of the present invention may be combined with each other, but this must be based on the fact that they can be implemented by ordinary technicians in the field. When the combination of technical solutions is mutually contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0031] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention are familiar to those skilled in the art and the description of the present invention, and any methods, equipment and materials of the prior art similar or equivalent to the methods, equipment and materials described in the embodiments of the present invention can also be used to implement the present invention.

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

[0033] In the present invention, the direction of the regulation can be to promote the synthesis of tea tree cis-catechins or inhibit the synthesis of tea tree cis-catechins; studies have shown that catechins have antioxidant, anti-cancer, lipid-lowering, antibacterial and antiviral, weight loss and other effects. The present invention silences the CsAHL20 gene in the tea tree to promote the synthesis of tea tree cis-catechins; but in some specific scenarios and specific studies, when there is a lower requirement for the content of tea tree cis-catechins, the CsAHL20 gene in the tea tree can also be overexpressed to inhibit the synthesis of tea tree cis-catechins. In the present invention, tea tree cis-catechins can be the cis-catechin content in tea leaves.

[0034] The present invention also provides an application of a protein encoded by a 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; in the present 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 the present invention, the tea plant or tea plant tissue has a CsANR gene promoter; 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 tea tree cis-catechin regulating reagent, 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 taking the CsAHL20 gene as a target.

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

[0037] The present invention also provides a method for regulating the synthesis of cis-catechins in tea trees based on the CsAHL20 gene, comprising using any of the above-described tea tree cis-catechin regulating reagents to regulate the synthesis of cis-catechins in tea trees. Specifically, the present invention silences the CsAHL20 gene in tea trees by using the CsAHL20 gene silencing reagent to promote the synthesis of cis-catechins in tea trees; or overexpresses the CsAHL20 gene in tea trees by using the CsAHL20 gene overexpression reagent to inhibit the synthesis of cis-catechins in tea trees.

[0038] 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, transferring the silencing vector into the tea plant to silence the CsAHL20 gene; or, constructing an overexpression vector of the CsAHL20 gene, transferring the overexpression vector into the tea plant to overexpress the CsAHL20 gene; the nucleotide sequence of the CsAHL20 gene is shown in SEQ ID NO. 1. In the present invention, the silencing vector of the CsAHL20 gene or the overexpression vector of the CsAHL20 gene can be transferred into the leaves of the tea plant; the overexpression in the present invention can be transient overexpression.

[0039] In the present invention, the construction process of the gene silencing vector includes: using pTRV2 as the original vector, inserting the 252bp sequence of the CsAHL20 gene CDS into the multiple cloning site of pTRV2, and the insertion position is located between the EcoRⅠ and BamHⅠ restriction sites on the original vector pTRV2. The obtained silencing vector is named pTRV2-CsAHL20; the nucleotide sequence of the 252bp sequence of the CsAHL20 gene CDS 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 CDS sequence of the CsAHL20 gene into the multiple cloning site of pCAMBIA2301-35S, and the insertion position is located between the Kpn I and Xba I restriction sites on the original vector pCAMBI A2301-35S. The obtained overexpression vector is named pCAMBIA2301-35S-CsAHL20; the nucleotide sequence of the full CDS sequence of the CsAHL20 gene is shown in SEQ ID NO.1.

[0041] The present invention also provides a tea tree breeding method, comprising: transferring a silencing vector of a 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 CsAHL 20 gene is shown in SEQ ID NO.1; the culturing is for the purpose of breeding, which may include: tissue culturing for the purpose of breeding. In the present invention, the tea tree tissue may be a leaf of a tea tree.

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

[0043] During the experiment, the present invention treated tea seedlings with drought and screened out CsAHL20 transcription factors containing AT-Hook Motif and PPC domain that significantly responded to drought from transcriptome data, which belonged to the AHL family members.

[0044] In the present 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:

[0045] .

[0046] In the present 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, has an AT-Hook motif and a PPC domain; the amino acid sequence of SEQ ID NO.2 is:

[0047] MLNKRDLEISMNETSARSTGQGDEDDDKDNGDEPKEGAVEVGTRRPRGRPPGSKNKPKPPIFVTRDSPNALRSHVMEVAGGTDVAESIAQFARRRQRGVCVLSGSGSVANVTLRQPAAPGAVMALHGRFEILS LTGAFLPGPAPPGSTGLTVYLAGGQGQVVGGSVVGSLVAAGPVMVIAATFSNATYERLPLEDDDEAGSAGQTQLPGTTGSSTPAIGSSGGQQQHGLPDPSSLQLYNLPPNLLPNGVQLSHDAYNWTHARPPY.

[0048] In the present 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:

[0049]

[0050] The relevant culture medium in the experimental process of the present invention is prepared as follows:

[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 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] The biological materials involved in the experimental process of the present invention are all commercially available.

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

[0058] Example 1 Identification and analysis of CsAHL20:

[0059] 1. Experimental methods

[0060] 1. Identification and analysis of CsAHL:

[0061] Using 31 AHL proteins from Arabidopsis thaliana as seed sequences, rice and tea plant AHL protein sequences were obtained by blast alignment in Rice Gene Index (RGI; https: / / riceome.hzau.edu.cn / ) and Tea Plant Information Archive (TPIA; http: / / tpia.teaplants.cn / ). Phylogenetic trees were constructed using MEGA-X software (version 7.0), with 1000 bootstrap replicates, and visualized using the iTOL online platform (https: / / itol.embl.de / ). Combined with the catechin content and transcriptome data measured after drought treatment in the early stage, genes related to catechins were identified. DNAMAN software was used to visualize the sequence alignment.

[0062] 2. Tea seedling drought treatment, growth conditions and material collection:

[0063] One-year-old Fuding Dabaicha tea seedlings with the same growth status were collected from Meitan Tea Factory in Zunyi, Guizhou Province, China. The soil at the roots of the tea seedlings was cleaned and then hydroponically cultivated. The cultivation container was a 5-L polyethylene plastic pot with a perforated foam board as the cover. The tea seedlings were tightly wrapped with sponges and planted in each cultivation pot. The hydroponic tea seedlings were cultivated in an artificial climate chamber at the School of Tea, Guizhou University. After two weeks of cultivation in untreated nutrient solution, 10% PEG6000 was added to the normal nutrient solution for cultivation. The second leaf was taken after 0, 6, 12, 24, and 48 h of treatment. To reduce individual differences among tea seedlings, 3 samples were taken as mixed samples, and 3 groups were taken as 3 biological replicates. After collection, they were immediately frozen in liquid nitrogen and stored in a refrigerator at -80°C for further study.

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

[0065] After freeze-drying the tea sample taken out from -80℃, prepare the test solution according to the method specified in GB / T 8313-2018 "Determination of Tea Polyphenols and Catechins in Tea". Weigh 0.2g (accurate to 0.001g) and grind it in a 70℃ water bath and extract it with 5mL 70% (v / v) methanol for 10min, shaking every 5min. After centrifugation at 3500×g and 4℃ for 10min, transfer the extracted solution to a clean 10mL volumetric flask. Repeat the above steps, finally set the volume to 10mL, and filter it using a 0.45μm Millipore filter. Analyze by high performance liquid chromatography.

[0066] Chromatographic conditions: detection wavelength 278nm; mobile phase A is 0.5% acetic acid aqueous solution, mobile phase B is methanol, and mobile phase C is acetonitrile; the elution gradient is 0-6min, A is 85%, B is 10%, and C is 5%; 6-16min, A is 85-70%, B is 10-20%, and C is 5-10%; 16-22min, A 70-72%, B 20%, C10-8% B; 22-26min, A72-85%, B20-10%, C 8-5%, flow rate is 60mL / h, column temperature is 35℃, injection volume is 10μL, and analysis is 26min.

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

[0068] After freezing, the samples were treated with a modified cetyltrimethylammonium bromide (CTAB) technique to extract total RNA. The extracted RNA was reverse transcribed into cDNA after removing genomic DNA and diluted 10 times before detection. The CsACTIN gene in tea was used as an internal reference in this study. RT-qPCR was performed using a Bio-Rad CFX ConnectTM real-time quantitative PCR instrument. -ΔCt The relative expression of genes was 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 of the AHL family have been shown to enhance drought resistance in plants. In this example, the identification and analysis of CsAHL20 Figure 1a-1d As shown. In this example, an evolutionary tree was constructed using 31 AHL family genes from Arabidopsis and 27 AHL family genes from rice, and 37 CsAHL genes were found in tea trees; combined with the drought transcriptome data, it was found that the family basically responded to drought, and after removing genes with FPKM values ​​less than 10, 12 CsAHL genes remained. In previous studies, it was found that drought caused a decrease in catechins, so a 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; but the FPKM values ​​of the latter three genes were small, while the FPKM value of CSS0021339 was significantly higher than them; so it is reasonable to speculate that the CSS0021339 transcription factor plays a key role in the synthesis of cis-catechins.

[0074] CSS0021339 was named AT-hook motif nuclear localizedprotein 20 in the Shuchazao genome, so it is referred to as CsAHL20 in the following text. The CDS of CsAHL20 is 798 bp long and encodes a protein containing 265 amino acids; the molecular weight of the protein is 27.47 kDa and the isoelectric point is 6.02. Phylogenetic analysis showed that CsAHL20 had high sequence similarity with Os07g13100, Os08g44910 and Os02g57520 in rice and CSS0007054, CSS0023327 and CSS0022210 in tea plants, and they all contained AT-hook motif and PPC domain. In rice, Os07g13100, Os08g44910 and Os02g57520 were significantly upregulated under drought stress. In summary, the CsAHL20 gene is very likely to respond to drought stress and may be involved in the synthesis of cis-catechins under drought.

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

[0076] In order to further verify the relationship between CsAHL20 and catechins under drought conditions, tea plants 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 fluorescence quantitative PCR (RT-qPCR) analysis are shown in Figure 2. Figure 2a-2f As shown; In this example, it was found that after 12 hours of treatment with 10% PEG6000, the edges of tea leaves began to burn, and the tea leaves were severely wilted at 48 hours; RT-qPCR results showed that after 10% PEG6000 treatment, CsAHL20 was upregulated, and the expression of CsAHL20 continued to increase with the extension of treatment time. However, the total catechin content in tea leaves decreased with the extension of treatment time, and the change trend of cis-catechins (EGC, EC, EGCG, ECG) was consistent with that of total catechins. The CG content in trans-catechins decreased after treatment, while the GC content increased. This result shows that drought stress can reduce the content of total catechins in tea leaves, and continuous drought will cause the content of cis-catechins to continue to decrease. This is consistent with the results obtained in the laboratory in the early stage, 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 tea plant CsAHL20 gene:

[0078] 1. Experimental methods

[0079] 1. The tobacco used is Nicotiana benthamiana.

[0080] 2. Construction of fusion expression vector:

[0081] The CDS sequence of CsAHL20 was connected to the SacI and SalI restriction sites of the PCAMBIA1300-35S-GFP vector by T4 DNA ligase, and the obtained ligation product was transformed into DH5a competent cells. After PCR amplification, restriction enzyme screening and sequencing verification, positive clones were screened and plasmids were extracted to obtain the GFP and target gene fusion expression vector PCAMBIA1300-35S-CsAHL20-GFP. The subcellular localization vector was synthesized by Wuhan Transduction Biological Laboratory Co., Ltd.

[0082] 3. Tobacco transient transformation steps:

[0083] (1) Shake the successfully detected Agrobacterium culture overnight at 28°C and 200 rpm;

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

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

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

[0087] (5) Take a small amount of suspended bacterial solution, dilute it 10 times, and measure the OD 600 The value was multiplied by 10 and used as the OD of the bacterial suspension. 600 value;

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

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

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

[0091] (9) Select the leaves with three or four leaves in the opposite direction for infection (infect between two leaf veins). Select two leaves from one plant and infect with one bacterial solution.

[0092] (10) Use a syringe without a needle to gently rub the back of the leaf to be rotated, 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;

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

[0095] (13) Point the syringe at the area to be transferred on the back of the blade, press the top of the blade with one hand, and gently push the piston with the other hand until the liquid spreads, then infect other parts. After infection, circle the infected area with a marker;

[0096] (14) Spray the leaves with water, put them in a fresh-keeping bag, and return the infected tobacco to the culture room and leave them in the dark overnight;

[0097] (15) The next day, the fresh-keeping bag was opened and the expression level was highest 2 days after injection;

[0098] (16) Excise the infected area, tear off the epidermis for preparation, and observe under a confocal laser microscope.

[0099] (II) Experimental results:

[0100] In this example, the subcellular localization of CsAHL20 protein in tobacco leaf cells is as follows Figure 3 As shown, the fluorescent signal of the empty vector carrying GFP was detected in the entire tobacco epidermal leaf cells, and the fluorescent signal of the PCAMBIA1300-35S-CsAHL20-GFP fusion protein was detected in the cell nucleus, proving that the CsAHL20 protein functions in the cell nucleus.

[0101] Example 3 Agrobacterium-mediated tea plant transient overexpression system verifies the function of CsAHL20:

[0102] 1. Experimental methods

[0103] 1. Experimental materials: The materials are cuttings of “Wuniuzao” tea trees from the Tea College of Guizhou University.

[0104] 2. Construction of plant overexpression vector:

[0105] The plant overexpression vector was based on the initial vector pCAMBIA2301-35S, and the CDS sequence of CsAHL20 (SEQ ID NO.1) was used to design and construct the pCAMBIA2301-35S-CsAHL20 vector. The recombinant plasmid was transformed into competent Escherichia coli (DH5α), and positive clones were screened using Kan 50 mg / L. The recombinant plasmid was extracted and double-digested with KpnI and XbaI for verification. The recombinant plasmid with positive results of enzyme digestion was transformed into competent cells of Agrobacterium strain GV3101, and positive Agrobacterium strains were screened with Kan 50 mg / L and Rif50 mg / L. The positive strains were tested by colony PCR using primers, and the Agrobacterium strains with positive PCR results were expanded and cultured at -80°C for seed preservation. The plasmid containing the overexpression vector was transformed into Agrobacterium strain GV3101 by freeze-thaw method to prepare the engineering strain. Take the GV3101 competent cells stored at -80℃ and place them in ice to thaw for 5 minutes; add 5μL of plasmid DNA to each competent cell, gently flick to mix, and then place in an ice bath for 5 minutes; after quick freezing in liquid nitrogen for 5 minutes, immediately place in a 37℃ water bath for 1 minute, add 700μL YEP liquid culture medium, and culture at 28℃, 200rpm shaking for 3 hours; after centrifugation at room temperature, 6000rpm for 1 minute, discard the supernatant, keep about 100μL, blow and mix with a pipette tip, take an appropriate amount of bacterial solution and spread it on the YEP plate culture medium containing 50mg / LKan and 50mg / L Rif, invert it in a 28℃ constant temperature incubator and culture for 2 days, pick a single colony and culture it on a 28℃ shaking incubator for 18 hours, and then store the bacterial solution at -80℃.

[0106] 3. Tea Tree Infection:

[0107] The pCAMBIA2301-35S-CsAHL20 recombinant plasmid was introduced into the competent Agrobacterium GV3101 by freeze-thaw method. Each Agrobacterium strain was inoculated into solid YEP medium containing 50 mg / L Kan and 50 mg / L R Fif and activated and cultured at 28°C for 48 h. A single colony was cultured in the corresponding liquid YEP medium until OD 600 is 1.0. The Agrobacterium cells were centrifuged at 6000rpm for 6min. The bacterial solution was collected and suspended in 4.74g / L MS, 30mg / L sucrose, 150μmol / L acetosyringone (AS), 25μmol / LMES, pH 5.6. The Agrobacterium carrying the pCAMBIA2301-35S vector was injected into the sixth leaf as a control (Control), and the CsAHL20 overexpression vector was injected to infect the sixth leaf of the tea tree. Each experiment was repeated at least 5 times. The samples were collected 3 days after injection and divided into two parts. One part was quickly frozen in liquid nitrogen and stored at -80℃ for total RNA extraction, and the other part was dried to constant weight after being sterilized and ground into powder. The catechin content was determined by high performance liquid chromatography.

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

[0109] Table 2 Primer names and sequences

[0110]

[0111]

[0112] (II) Experimental results:

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

[0114] Example 4 Virus-induced gene silencing (VIGS) technology verifies the function of CsAHL20:

[0115] 1. Experimental methods

[0116] 1. Experimental materials:

[0117] The materials were "Fuding Dabai" tea tree cuttings from the School of Tea of ​​Guizhou University, and the virus-induced gene silencing (VIGS) technology was used to verify the function of CsAHL20.

[0118] 2. Construction of plant VIGS vector:

[0119] The 252 bp fragment of pTRV2-CsAHL20 gene was constructed with EcoRI and BamHI restriction sites. The PCR product and the pTRV2 vector recovered by double digestion with EcoRI and BamHI were ligated with T4 DNA ligase (Kewen Biotechnology Co., Ltd.). After overnight ligation at 16°C, Escherichia coli DH5α was transformed. The insertion of the product was verified by bacterial liquid PCR using specific primers for CsAHL20-F and CsAHL20-R. The CsAHL20 gene fragment was obtained by total gene synthesis and loaded into the pUC57-Simple vector to obtain the pUC57-Simple-CsAHL20 plasmid.

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

[0121] CGAAGCCATGTTATGGAGGTGGCCGGAGGCACTGATGTAGCGGAGAGCATAGCCCAATTCGCCCGGAGGCGTCAGAGAGGGGTTTGTGTACTAAGTGGAAGTGGTTCGGTTGCCAACGTGACACTG AGGCAGCCAGCTGCGCCAGGTGCTGTGATGGCGCTCCATGGTAGGTTTGAAATTTTATCACTGACTGGGGCTTTCCTGCCAGGACCCGCCCCGCCAGGTTCCACTGGCTTGACAGTGTACCTGGCA.

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

[0123] CGGAATTCCG CGAAGCCATGTTATGGAGGTGGCCGGAGGCACTGATGTAGCGGAGAGCATAGCCCAATTCGCCCGGAGGCGTCAGAGAGGGGTTTGTGTACTAAGTGGAAGTGGTTCGGTTGCCAACGTGACACTG AGGCAGCCAGCTGCGCCAGGTGCTGTGATGGCGCTCCATGGTAGGTTTGAAATTTTATCACTGACTGGGGCTTTCCTGCCAGGACCCGCCCCGCCAGGTTCCACTGGCTTGACAGTGTACCTGGCA CGGGATCCCG .

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

[0125] Construction of pTRV2-CsAHL20 plasmid: Double-digest the pUC57-Simple-CsAHL20 plasmid with EcoRI and BamHI to obtain the CsAHL20 gene fragment, run the gel and recover the fragment. At the same time, double-digest the pTRV2 vector with EcoRI and BamHI, run the gel and recover the vector fragment. Connect the CsAHL20, gene fragment and pTRV2 vector fragment after enzyme digestion. Transform the ligation product into DH5α competent cells. Pick several clones on the transformed plate, extract the plasmid, and identify it by enzyme digestion with EcoRI and BamHI. The recombinant plasmid is further sequenced and identified (Note: The above process was commissioned to Changsha Kewen Biotechnology Co., Ltd.).

[0126] 3. Tea Tree Infection:

[0127] pTRV1, pTRV2, and pTRV2-CsAHL20 were introduced into Agrobacterium GV3101 competent cells by freeze-thaw method. Each Agrobacterium strain was inoculated into solid YEP medium containing 50 mg / L kanamycin and 50 mg / L rifampicin and activated and cultured at 28°C for 2 days. Single colonies were cultured in corresponding liquid YEP medium until OD 600 The Agrobacterium cells were centrifuged at 6000 rpm for 6 min. The bacterial suspension was collected and suspended in 4.74 g / L MS, 2 mol / L 6-BA, 2 mol / L acetosyringone (AS), 100 umol / L naphthaleneacetic acid (NAA), pH 5.6. The two tea tree materials were vacuum infiltrated. The OD 600 Adjust to 1.2. Mix pTRV1 with pTRV2 and pTRV2-CsAHL20 bacterial solutions in a ratio of 1:1 at room temperature (pTRV1+pTRV2, pTRV1+pTRV2-CsAHL20), and vacuum infiltrate the tea tree cuttings. Cut the tea tree cuttings to a length of 20 cm with pruning shears. Keep two mature leaves, and then place the tea tree cuttings in a Buchner bottle filled with the mixed bacterial solution for vacuum infiltration. Cultivate them in the dark for three days, and then grow them in a greenhouse at 25°C with a light / dark cycle of 16h / 8h.

[0128] After the pTRV2-CsAHL20 silenced plants, pTRV2 plants, and wild-type plants were cultured for 38 days and lateral buds grew, samples were collected and divided into two parts, one was quickly frozen in liquid nitrogen and stored at -80°C for total RNA extraction, and the other was dried to constant weight after being sterilized 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 similar to that in Example 1.

[0130] (II) Experimental results:

[0131] In this example, the relative expression level of catechin content and gene expression after virus-induced gene silencing CsAHL20 was determined as follows: Figure 5a-5e As shown, CsAHL20 was successfully silenced in tea plants compared with wild-type and empty vector controls; its expression levels were reduced by 60.1%, 42.9% and 29.3% respectively compared with WT (wild type), while the expression level of CsANR in CsAHL20 silenced plants was significantly increased. Detection of catechin content in CsAHL20 silenced tea cuttings showed that compared with WT (wild type), the cis-catechin content in CsAHL20 silenced plants increased; the cis-catechin content increased by 94.5% (pTRV-CsAHL20-1), 74.0% (pTRV-CsAHL20-2) and 45.5% (pTRV-CsAHL20-3), respectively; the above data indicate that silencing of CsAHL20 promotes the formation of cis-catechins in Fuding Dabai tea leaves, so CsAHL20 is a negative regulator of cis-catechin biosynthesis in tea plants.

[0132] Example 5 EMSA and DLRA methods to detect the binding of CsAHL20 to the promoter of CsANR:

[0133] 1. Experimental methods

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

[0135] In order to determine the regulatory effect of CsAHL20 TF on CsANR, multiple sites that can bind to the CsAHL20 motif were predicted using the JASPAR database, and a probe was designed for a site with a higher confidence level. The purified AHL20 protein (Figure) was incubated with the DNA fragment of the CsANR promoter, and then a gel electrophoresis migration experiment was performed. The probe sequence is shown in Table 3.

[0136] Table 3 Probe names and sequences

[0137]

[0138]

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

[0140] The transcriptional activity in tobacco was analyzed by the dual luciferase assay system. The CDS of CsAHL20 and was cloned into the pGreenII 62-SK vector as an effector, and the promoter fragment of CsANR was introduced into the pGreenII 0800-LUC vector as a reporter gene, which were used to transform the EHA105 strain of Agrobacterium tumefaciens, and then the transient fusion expression of the recombinant plasmid was performed in the leaf cells infected with EHA105. After 3 days, the 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 chemiluminescence instrument Fusion FX7 (VILBER, France). The transcriptional activity based on LUC / REN was measured using a dual luciferase reporter gene assay kit.

[0141] (II) Experimental results:

[0142] For the interaction detection of tea plant CsAHL20 and CsANR promoter (EMSA) and the dual luciferase reporter assay of tea plant CsAHL20 and CsANR, see Figure 6 and Figure 7 As shown. First, the purified CsAHL20 protein was incubated with the CsANR promoter DNA fragment, and then the electrophoresis gel shift assay was performed; see Figure 6 As shown in the figure, after the AHL20 protein attached to the nucleic acid fragment, the lane band of protein + nucleic acid obviously lagged behind the lane band of nucleic acid only, and as the protein concentration increased, the binding band became more and more obvious, the free probe band became fainter and fainter, until it disappeared, and the mutant probe had no lagging binding band with the AHL20 protein. This shows that AHL20 and CsANR promoter have specific binding, and the mutant probe has no binding band with AHL20 protein. The gel electrophoresis results show that the AHL20 protein has specific binding with the CsANR promoter.

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

[0144] The present invention provides the above technical solutions of the present invention, which are only preferred embodiments of the present invention and do not limit the patent scope of the present invention. All equivalent structural changes made by using the contents of the present invention specification and drawings under the technical concept of the present invention, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. An application of CsAHL20 gene as a target in regulating the synthesis of cis-catechins in tea plants, characterized in that: The nucleotide sequence of the CsAHL20 gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The CsAHL20 gene in the tea plant is silenced to promote the synthesis of cis-catechins in the tea plant; or, the CsAHL20 gene in the tea plant is overexpressed to inhibit the synthesis of cis-catechins in the tea plant.

3. Use of a protein encoded by the CsAHL20 gene in inhibiting the synthesis of cis-catechins in tea plants, characterized in that: The amino acid sequence of the protein encoded by the CsAHL20 gene is shown in SEQ ID NO.

2.

4. An application of CsAHL20 gene as a target in the preparation of tea tree cis-catechin regulating reagents, characterized in that: The nucleotide sequence of the CsAHL20 gene is shown in SEQ ID NO.

1.

5. A tea tree cis-catechin regulating agent, characterized in that: The tea tree cis-catechin regulating reagent comprises a CsAHL20 gene silencing reagent and / or a CsAHL20 gene overexpression reagent, and the nucleotide sequence of the CsAHL20 gene is shown in SEQ ID NO.

1.

6. A method for regulating the synthesis of cis-catechins in tea plants based on the CsAHL20 gene, characterized in that: The method comprises using the tea tree cis-catechin regulating agent as claimed in claim 5 to regulate the synthesis of cis-catechin in tea tree.

7. A method for regulating the synthesis of cis-catechins in tea plants based on CsAHL20 gene, characterized in that: include: Constructing a silencing vector of the CsAHL20 gene, and transferring the silencing vector into tea plants to silence the CsAHL20 gene; Alternatively, an overexpression vector of the CsAHL20 gene is constructed, and the overexpression vector is transferred into tea plants to overexpress the CsAHL20 gene.

8. The method for regulating the synthesis of cis-catechins in tea plants based on CsAHL20 gene according to claim 7, characterized in that: The construction process of the silencing vector includes: using pTRV2 as the original vector, inserting the 252 bp sequence of the CsAHL20 gene CDS into the multiple cloning site of pTRV2, and the insertion position is located between the EcoRⅠ and BamHI restriction sites on the original vector pTRV2; the nucleotide sequence of the 252 bp sequence of the CsAHL20 gene CDS is shown in SEQ ID NO.24; The construction process of the overexpression vector includes: using pCAMBIA2301-35S as the original vector, inserting the CDS full sequence of the CsAHL20 gene into the multiple cloning site of pCAMBIA2301-35S, and the insertion position is located between the Kpn I and Xba I restriction sites on the original vector pCAMBIA2301-35S; the nucleotide sequence of the CDS full sequence of the CsAHL20 gene is shown in SEQ ID NO.

1.

9. A tea tree breeding method, characterized in that: include: The silencing vector of the CsAHL20 gene is transferred into tea plant tissue to silence the CsAHL20 gene, and then the tea plant tissue is cultured; Alternatively, the overexpression vector of the CsAHL20 gene is transferred into tea plant tissue to overexpress the CsAHL20 gene, and then the tea plant tissue is cultured; the nucleotide sequence of the CsAHL 20 gene is shown in SEQ ID NO.

1.

10. An application of CsAHL20 gene as a target in improving tea quality, characterized in that: The nucleotide sequence of the CsAHL20 gene is shown in SEQ ID NO.1.

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