Application of CsUGT84A22 gene in synthesis of ferulic acid-beta-glucoside and breeding of tea tree disease-resistant varieties

Abstract

The application discloses application of a CsUGT84A22 gene in synthesis of ferulic acid-beta-glucoside and breeding of tea tree disease-resistant varieties, and belongs to the technical field of tea tree gene utilization. The CDS nucleotide sequence of the tea tree glycosyltransferase gene CsUGT84A22 is as shown in SEQ ID No. 1. The protein coded by the CsUGT84A22 gene has an amino acid sequence as shown in SEQ ID NO: 2. The application has the beneficial effect that the tea tree glycosyltransferase CsUGT84A22 gene derived from tea trees is cloned, the gene is expressed in Escherichia coli in a prokaryotic manner, and a recombinant protein is obtained through affinity chromatography purification, the protein has high purity and activity, and can be applied to ferulic acid-beta-glucoside biosynthesis genetic engineering. Sequence differences in tea tree varieties with different resistance to brown spot are analyzed, and it is found that the promoter sequence of CsUGT84A22 in a variety with strong resistance is more than that in a variety with weak resistance, which helps to understand the reason for the difference in resistance of different tea trees and provides guidance for tea tree breeding.

Description

Technical Field

[0001] This invention belongs to the field of tea tree gene utilization technology, specifically involving the application of the CsUGT84A22 gene in the synthesis of ferulic acid-β-glucan and the selection of disease-resistant tea varieties. Background Technology

[0002] Tea is an important leaf crop in my country, but due to the continuous increase in the planting area of ​​tea gardens and temperature changes, the incidence and severity of tea leaf spot disease have expanded. As an important perennial woody crop, tea is rich in various secondary metabolites, among which phenolic acids and their derivatives are particularly complex and diverse. These secondary metabolites help the plant better resist biotic and abiotic stresses.

[0003] Tea leaf spot, as the earliest reported leaf disease in tea gardens, is a common and frequently occurring fungal leaf disease in tea gardens. It is widely distributed in tea-producing areas of my country, such as Anhui, Zhejiang, Jiangxi, Jiangsu, Hunan, Hubei, Yunnan, Guizhou, Sichuan, and Taiwan. It is also one of the most destructive leaf diseases of tea trees worldwide, including in India, Thailand, Sri Lanka, and Kenya.

[0004] Tea leaf spot disease is caused by a fungus of the genus *Pestalotiopsis-like*. The pathogen overwinters as mycelium or conidiophores in diseased leaf tissue. The following year, the pathogen produces spores, which are then spread and infect again by wind and rain. The incidence of tea leaf spot disease is closely related to ecological factors such as temperature and humidity. The pathogen can survive in the range of 5-35℃ and forms new lesions and produces more spores after 7-14 days. Under in vitro conditions of 25℃, the pathogen's spores begin to germinate after 4 hours. The pathogen of tea leaf spot disease prefers warm and humid conditions, growing most rapidly at around 28℃. In my country's tea-growing areas, the two peak periods for the incidence of tea leaf spot disease are the summer plum rain season (May-June) and autumn (August-October).

[0005] Plant pathogenic fungi must effectively detoxify antibacterial secondary metabolites in host tissues, such as phenols, steroids, terpenes, and alkaloids, in order to successfully infect and colonize plants. There are over 8,000 known phenolic compounds in the plant kingdom, mainly divided into three categories: flavonoids, phenolic acids, and tannins. Phenolic acids, as important secondary metabolites in plants, possess multiple functions including antibacterial, antitoxin, anti-inflammatory, antioxidant, and anticancer properties. Their presence is beneficial to plant growth, reproduction, and protection against pathogens. Ferulic acid is the most abundant phenolic acid in the raw materials of grasses. Phenolic acids often exhibit higher biological activity after structural modification. Their plasticity depends on various enzymes, among which the glycosylation modification by glycosyltransferases is more conducive to plant changes in a direction beneficial to their survival and development under adverse conditions.

[0006] Therefore, how to select tea varieties with stronger disease resistance and how to improve the resistance of tea trees to leaf spot disease are crucial issues that remain to be solved. Summary of the Invention

[0007] The technical problem to be solved by this invention is how to provide a method for improving and breeding resistance to leaf spot disease in tea trees.

[0008] The present invention solves the above-mentioned technical problems through the following technical means:

[0009] The first aspect of the present invention proposes the application of CsUGT84A22 protein in the preparation of ferulic acid-β-glucoside, wherein the CsUGT84A22 protein is a protein encoded by the CsUGT84A22 gene, and its amino acid sequence is shown in SEQ ID NO: 2; the CDS nucleotide sequence of the CsUGT84A22 gene is shown in SEQ ID No. 1.

[0010] Preferably, the method includes the following steps: using ferulic acid as a substrate and CsUGT84A22 protein as an enzyme, reacting at pH 4.5-7.5 and a reaction temperature of 20-45℃ to obtain ferulic acid-β-glucoside.

[0011] Preferably, the pH is 5.5 and the reaction temperature is 30°C.

[0012] Preferably, the CsUGT84A22 protein, when subjected to enzyme kinetic analysis, exhibits a Michaelis constant Km of 0.1 Mm, a maximum reaction rate Vmax of 1.87 nKat / mg, and a Kcat / Km of 18.7 ± 0.20 s. -1 ·mM -1 .

[0013] The second aspect of the present invention provides a method for preparing the above-mentioned CsUGT84A22 protein, comprising the following steps: adding isopropyl-β-D-thiogalactoside to a culture medium to induce expression in a recombinant expression strain, and collecting the protein; the recombinant expression strain is obtained by the following process: ligating the CsUGT84A22 gene into the pMAL-c5x vector to obtain the recombinant expression vector pMAL-c5x-CsUGT84A22, and then transforming the recombinant expression vector into Escherichia coli Rosetta.

[0014] A third aspect of the present invention proposes the application of ferulic acid-β-glucoside in improving resistance to tea leaf spot disease.

[0015] The fourth aspect of the present invention proposes the application of CsUGT84A22 as a drug target in the preparation of drugs for the prevention and control of tea leaf spot disease, wherein CsUGT84A22 includes the CsUGT84A22 gene and the CsUGT84A22 protein.

[0016] Preferably, the drug is a CsUGT84A22 overexpression reagent or a reagent that promotes CsUGT84A22 overexpression.

[0017] The fifth aspect of this invention provides a method for identifying tea varieties with strong resistance to leaf spot disease, comprising the following steps: extracting total DNA from tea plants, performing PCR amplification using upstream and downstream primers of the CsUGT84A22 promoter, performing sequencing after amplification, and comparing the sequencing results. If the CsUGT84A22 promoter lacks a CTACA segment at 777bp, the tea variety is Dan Gui, which has weak resistance to leaf spot disease; if it does not lack the segment, the tea variety is Jin Xuan, which has strong resistance to leaf spot disease.

[0018] Preferably, the sequences of the upstream and downstream primers of the CsUGT84A22 promoter are shown in SEQ ID No. 7-8.

[0019] The beneficial effects of this invention are as follows:

[0020] 1. This invention cloned the tea plant glycosyltransferase CsUGT84A22 gene, expressed it in prokaryotes in *E. coli*, and purified it by affinity chromatography to obtain a recombinant protein with high purity and activity, which can be applied to the genetic engineering of ferulic acid-β-glucan biosynthesis. Furthermore, the sequence differences among tea varieties with different resistance to leaf spot disease were analyzed. It was found that the promoter sequence of CsUGT84A22 in more resistant varieties had an additional insertion segment compared to less resistant varieties. This helps us understand the reasons for the differences in resistance among different tea varieties and provides guidance for tea breeding.

[0021] 2. In this invention, a prokaryotic expression recombinant vector pMAL-c5x-CsUGT84A22 was constructed and heterologously expressed in Escherichia coli Rosetta. The recombinant protein CsUGT84A22 was expressed under the induction of isopropyl-β-D-thiogalactoside and was found to be a soluble protein. The CsUGT84A22 protein was purified by affinity chromatography and its band was found to be single and without impurities by SDS-PAGE. Ferulic acid-β-glucoside can be synthesized using ferulic acid as a substrate and CsUGT84A22 as an enzyme. Optimal reaction conditions for the CsUGT84A22 enzymatic reaction were determined, showing that ferulic acid-β-glucoside could be generated at pH 4.5-7.5 and reaction temperatures 20-45℃, with the optimal pH being 5.5 and the optimal reaction temperature being 30℃. Liquid chromatography-mass spectrometry (LC-MS) analysis revealed that the characteristic ions of ferulic acid-β-glucoside were 355, 175, 271, and 193. Enzyme kinetic analysis of CsUGT84A22 under different substrate concentrations and optimal reaction conditions revealed a Michaelis constant Km of 0.1 Mm, a maximum reaction rate Vmax of 1.87 nKat / mg, and Kcat / Km of 18.7 ± 0.20 s. -1 ·mM -1 Inhibiting the expression of CsUGT84A22 in tea leaves reduces the production of ferulic acid-β-glucoside. When ferulic acid-β-glucoside standards were co-cultured with pathogenic mycelial blocks, the addition of ferulic acid-β-glucoside effectively inhibited mycelial block growth, indicating that it can improve tea plant resistance, thus completing this invention.

[0022] 3. Using *Dendrobium nobile*, a cultivar with weak resistance to *Dendrobium pseudotrichous*, and *Aeonium japonicum*, a cultivar with strong resistance, as materials, the promoter sequence of CsUGT84A22 was amplified. It was found that the promoter sequence of CsUGT84A22 in *Aeonium japonicum* had an additional sequence inserted before the MYB transcription factor binding site compared to *Dendrobium nobile*, which may cause the difference in resistance by affecting the binding ability of MYB transcription factors. Analysis of the expression pattern of CsUGT84A22 in both cultivars revealed that after infection with *Dendrobium pseudotrichous*, the expression pattern in *Aeonium japonicum* significantly increased compared to *Dendrobium nobile*. Attached Figure Description

[0023] Figure 1 Phylogenetic clustering analysis diagram of the CsUGT84A22 gene and the Arabidopsis AtUGTs family;

[0024] Figure 2SDS-PAGE gel image of CsUGT84A22 protein stained with Coomassie Brilliant Blue. In the image: 1, 120 kDa protein marker; 2, CsUGT84A22 before induction; 3, CsUGT84A22 after induction; 4, supernatant after disruption; 5, precipitate after disruption; 6, flow-through; 7, purified CsUGT84A22 protein. The target protein is indicated by a blue box.

[0025] Figure 3 Chromatograms of the products formed by recombinant CsUGT84A22 protein and empty vector for LC-MS identification;

[0026] Figure 4 A graph showing the optimal reaction temperature, time, pH, and concentration for the CsUGT84A22 enzymatic reaction;

[0027] Figure 5 The diagram shows the enzyme kinetics of CsUGT84A22.

[0028] Figure 6 The figure shows the effect of the CsUGT84A22 catalytic product on the growth of the ring spot pathogen;

[0029] Figure 7 The promoter sequence diagram of CsUGT84A22 in *Pseudomonas aeruginosa*, which is highly resistant to *Pseudomonas aeruginosa*, is shown in red. The differences between the promoter sequence and that of *Cinnamomum camphora*, which is less resistant, are marked in red. Analysis revealed that only CTACA may affect the binding ability of similar binding elements (blue box).

[0030] Figure 8 A comparative diagram showing the differences in expression patterns of CsUGT84A22 in different leaf spot resistant varieties, Osmanthus fragrans and Osmanthus fragrans var. chinensis. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. 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.

[0032] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available.

[0033] Unless otherwise specified in the embodiments, the techniques or conditions described in the literature in this field or in accordance with the product manual may be followed.

[0034] The main reagents and formulations used in the embodiments of this invention are as follows:

[0035] (1) Ferulic acid and ferulic acid-β-glucoside standards

[0036] (2) Amylose Resin

[0037] (3) Polysaccharide and polyphenol plant total RNA extraction kit

[0038] (4) RNA reverse transcription kit

[0039] (5) Gel recovery kit

[0040] (6) LB medium: Dissolve 5g yeast powder, 10g peptone, and 10g sodium chloride in 1L of pure water. Add 4-5g agar powder to every 250ml of solid LB medium. Autoclave for 20min.

[0041] (7) CTAB solution: 4g cetyltrimethylammonium bromide (CTAB), 2.42g tris(hydroxymethyl)aminomethane hydrochloride, 1.8986g disodium ethylenediaminetetraacetate, and 16.38g sodium chloride were diluted to 200ml with DEPC water, and the pH was adjusted to 8.0.

[0042] (8) Protein equilibration buffer: 200mM sodium chloride, 1mM disodium ethylenediaminetetraacetate, 20mM tris(hydroxymethyl)aminomethane hydrochloride, 1mM dithiothreitol, adjusted to pH 7.4.

[0043] (9) Protein elution buffer: 200mM sodium chloride, 1mM disodium ethylenediaminetetraacetate, 20mM tris(hydroxymethyl)aminomethane hydrochloride, 1mM dithiothreitol, 3g / L maltose, completely dissolved and adjusted to pH 7.4.

[0044] (10) 2xTBE buffer: Weigh 54.5g of tris(hydroxymethyl)aminomethane hydrochloride, 27.5g of boric acid, and 24.71g of disodium ethylenediaminetetraacetate and dissolve them in 2.5L of pure water. Generally, dilute with pure water to 0.5xTBE.

[0045] (11) 1.5% agarose gel formulation: Weigh 0.3g agarose into 20mL 0.5xTBE buffer, mix well, and heat until clear.

[0046] Example 1:

[0047] 1. Cloning of the CsUGT84A22 gene

[0048] (1) RNA extraction: Add 50 mg of sample to a 2 mL RNA extraction tube, add a steel ball, and grind thoroughly using a ball mill. Subsequent steps follow... The extraction was performed according to the instructions of the Universal Plant Total RNA Isolation Kit (Novizan).

[0049] After RNA extraction, the quality of RNA extraction was detected by 1.5% agarose gel electrophoresis at 130V. The quality of extracted nucleic acid was determined using a Nanodrop-2000 (Thermo Scientific) nucleic acid quantification instrument to check whether the OD260 / OD280 ratio was between 1.8 and 2.1.

[0050] (2) cDNA reverse transcription: according to PrimeScript TM The 1st Strand cDNA Synthesis Kit (Takara) instructions state that RNA is reverse transcribed into cDNA using conventional methods, which serves as a template for gene PCR amplification.

[0051] (3) PCR amplification: The CDS sequence of CsUGT84A22 was amplified using upstream and downstream primers for the CDS region. The required reagents were added sequentially according to the reaction system (1 μL cDNA template, 0.5 μL upstream primer, 0.5 μL downstream primer, 12.5 μL LA Taq premix, 10.5 μL ddH2O) for PCR amplification. The cloning program was 95℃ (3 min), 95℃ (30 s), 58℃ (30 s), 72℃ (1 min), 30 cycles, 72℃ (5 min), and the reaction product was stored at 4℃.

[0052] (4) Ligation and Sequencing: After electrophoresis of the PCR product on a 1.5% agarose gel, add the gel-recovered product of the target fragment, the gel-recovered product of the pMAL-c5x vector digested with EcoRI and SaLI, and the recombinase to the PCR tube, mix gently, and place in a PCR instrument at 37°C for 30 min for ligation.

[0053] ② Take Ecoli.BL21 competent cells out of the -80℃ ultra-low temperature freezer and place them on ice. Add the ligation product to the thawed competent cells, incubate on ice for 30 min, heat shock at 42℃ for 90 s, and then incubate on ice for 2 min.

[0054] ③ On a clean bench, add 400 μL of antibiotic-free LB medium to the transformed cells and culture them in a 37°C constant temperature shaker at 180 rpm for 1 h.

[0055] ④ On a clean bench, take 200 μL of bacterial culture from the cultured cells and spread it onto a plate containing 100 ng / mL ampicillin (Amp... + Incubate overnight in an inverted incubator at 37°C on solid LB medium.

[0056] ⑤ Select 5 single colonies for streaking culture and perform colony PCR verification using specific primers.

[0057] ⑥ Send the colonies that have been verified by PCR to GE for sequencing.

[0058] 2. Prokaryotic expression and purification of the CsUGT84A22 gene

[0059] Prokaryotic expression:

[0060] (1) Take the positive strain with correct sequencing and transform it into a culture containing Amp + In LB liquid medium, the culture was carried out at 37°C with shaking on a shaker (180 rpm) until the bacterial OD of the culture was reached. 600 =0.4~0.6. Take out the bacterial culture as the control group before induction, add isopropyl-β-D-thiogalactoside (IPTG) inducer to the remaining bacterial culture, and transfer it to a constant temperature shaker (180 rpm) at 28℃ for 12 h to induce culture to a final concentration of 0.1 mM.

[0061] (2) After induction, centrifuge the bacterial culture at 6000 rpm for 10 min, and take the supernatant as the soluble protein after induction. Collect the bacterial culture precipitate and resuspend the precipitate with 1× phosphate (PBS) buffer as the insoluble protein after induction. Add 5× protein loading buffer to the protein sample, mix thoroughly, and boil in a 100℃ water bath for denaturation.

[0062] (3) Prepare a 12% SDS-PAGE protein gel using an SDS-PAGE gel preparation kit. Load the denatured protein samples onto the gel. After electrophoresis, incubate the SDS-PAGE gel in Coomassie Brilliant Blue staining solution for destaining. Image the destaining SDS-PAGE gel.

[0063] Protein purification:

[0064] ① Based on the optimal conditions for prokaryotic expression described above, 100 mL of bacterial culture was induced for 16 h at 0.3 mM IPTG and 16 °C. The induced bacterial culture was then centrifuged at 4 °C and 5000 rpm for 10 min to collect the bacterial cells. The cells were resuspended in 15 mL of 1×PBS buffer and sonicated until clear. 1 mL of the lysed sample was centrifuged at 4 °C and 12000 rpm for 3 min to collect the supernatant and precipitate. The precipitate was resuspended in 100 μL of 1×PBS buffer. The remaining sample was centrifuged at 4 °C and 5000 rpm for 10 min, and the supernatant was collected.

[0065] ② Use a pipette tip to transfer an appropriate amount of amylose resin to an empty affinity column. Wait for the resin to settle. Excess buffer will flow out of the column by gravity. Equilibrate the column with 10 column volumes of column buffer.

[0066] ③ Add the clarified sample containing the target MBP-tagged protein to the column, seal it, and incubate it in a chromatography cabinet at 4°C for 2 hours. Open the bottom of the column and adjust the flow rate to 0.5 mL / min. Wash away any contaminating proteins using 5 column volumes of column buffer.

[0067] ④ Add 15 mL of maltose buffer to elute the target protein containing the MBP tag at a flow rate of 0.5 mL / min.

[0068] ⑤ Add pure water to the 50KD ultrafiltration tube and centrifuge at 3000 rpm for 5 min. Repeat the washing process 3 times. Centrifuge the eluted sample in the ultrafiltration tube at 3000 rpm until approximately 1 mL of sample remains. Calculate the protein concentration using the Coomassie Brilliant Blue assay kit, aliquot, and store at -80℃.

[0069] ⑥ Add the pre-induction, post-induction, post-lysis supernatant, precipitate, and purified protein samples to 5× protein loading buffer, mix well, and incubate in a 95–100℃ water bath for 10 min. Detect the recombinant protein purification results using SDS-PAGE protein gel chromatography.

[0070] 3. Enzymatic reaction of ferulic acid to ferulic acid-β-glucan catalyzed by CsUGT84A22 and its product detection

[0071] To determine whether CsUGT84A22 can catalyze the formation of ferulic acid-β-glucan, the product of CsUGT84A22 was identified using a QE-Foucs high-resolution liquid chromatography-mass spectrometry (HPLC-MS / MS). The reaction system consisted of 200 μL: 100 mM MES buffer (pH 5.5), 2.5 mM UDP-glucose, 0.5 mM primary screening substrate, and 10 μg purified protein. The control group consisted of empty purified protein. After reacting at 30 °C for 1 h, an equal volume of ethyl acetate was added for extraction twice. The organic solvent was then evaporated by rotary evaporation, and the mixture was dissolved in 300 μL of 80% methanol. The mixture was centrifuged at 12000 r / min for 10 min, and the supernatant was collected. After filtration through a 0.22 μm organic phase filter membrane, the supernatant was added to a sample vial for analysis. LC-MS detection conditions: Column: Phenomenex Synergi 4u Fusion-RP80 (100mm × 2.1mm); Flow rate: 0.3mL·min⁻¹; Injection volume: 2μL; Column temperature: 40℃. Mobile phase A: 0.075% acetic acid, B: 100% acetonitrile; Elution gradient: Phase B from 10% to 15% within 0–5 min, from 15% to 10% within 6–9 min, and from 30% to 10% within 10–15 min. The CsUGT84A22 enzymatic product was identified by comparing the peak times with the standard. Figure 3 We have successfully synthesized ferulic acid-β-glucoside.

[0072] 4. Enzymatic kinetics of CsUGT84A22 catalyzing the conversion of ferulic acid to ferulic acid-β-glucan.

[0073] After a preliminary scan of enzyme activity, the pH, temperature, concentration, and reaction time of the suitable substrate for the enzyme were determined. The temperature range was 20-45℃, the pH range was 4.5-7.5, and the reaction time range was 0-80 min. The enzyme kinetic parameters were determined according to the above system and method.

[0074] DNA was extracted from tea varieties with different resistance to leaf spot disease. The DNA from both samples was amplified by PCR using the same pair of PCR primers, and the amplified sequences were sequenced to compare the sequence differences.

[0075] 5. Antibacterial activity analysis of ferulic acid-β-glucoside

[0076] The resistance of CsUGT84A22 catalytic substrate and its products to *Tea leaf spot* causal agents was determined using the mycelial cake culture method, with standards as analytes. Mycelial cakes with a diameter of 6 mm were uniformly punched using a puncher. 20 μL of standard was added dropwise to the surface of PDA potato dextrose agar medium. The mycelial side of the pretreated mycelial cake was then gently placed on the area where the standard was added. 80% methanol and blank pure medium were used as controls. The entire procedure was performed under aseptic conditions. The culture was incubated at 28℃, and the mycelial growth area was observed to compare the inhibitory effect of the standard on the mycelial growth of *Tea leaf spot* causal agents.

[0077] 6. Amplification of the CsUGT84A22 promoter sequence

[0078] (1) Total DNA extraction: Add 100 mg of sample to an appropriate amount of PVPP in a 2 mL centrifuge tube, grind it into a fine powder using a high-speed ball mill, add 700 μL of CTAB (preheated at 65 °C) and 10 μL of β-mercaptoethanol, and vortex thoroughly to mix; heat the centrifuge tube in a 65 °C water bath for 15 min, and shake it every 5 min.

[0079] (3) Add 600 μL of chloroform:isoamyl alcohol (24:1) to the centrifuge tube and centrifuge at 12000 r / min for 10 min. Take 500 μL of supernatant and add an equal volume of isopropanol. Invert 6-8 times to mix well and centrifuge at 12000 r / min for 5 min. Discard the supernatant.

[0080] (4) Use 500 μL of 70% ethanol to blow the white precipitate at the bottom several times, centrifuge at 12000 r / min for 5 min, discard the supernatant and repeat once. After the ethanol has evaporated completely, add 100 μL of ddH2O.

[0081] (5) The quality of the extracted DNA samples was determined by 1% agarose gel electrophoresis.

[0082] (6) Using the DNA of Osmanthus fragrans and Achyranthes bidentata as templates, PCR amplification was performed using upstream and downstream primers of the CsUGT84A22 promoter. After amplification, sequencing was performed and the sequencing results were compared.

[0083] The comparison results are as follows Figure 7 As shown, several differences were found in the promoter sequences of the two groups ( Figure 7 (marked in red in the middle), but after analysis, it was found that only CTACA may affect the binding ability of similar binding elements (in the blue box). That is, Dan Gui lacks a CTACA insertion at 777bp compared to Jin Xuan. Through analysis, it was found that this insertion is located to the left of the MYB transcription factor binding element. Therefore, this insertion may affect the binding ability of MYB transcription factor, thus leading to different expression patterns of the CsUGT84A22 promoter in Dan Gui and Jin Xuan.

[0084] 7. Expression pattern analysis of CsUGT84A22

[0085] cDNA reverse transcription: RNA was quantitatively reverse transcribed into cDNA according to the instructions of the HiScript II One Step RT-PCR Kit (Vazyme) to serve as a template for qPCR amplification of the gene.

[0086] Quantitative real-time PCR: Amplification was performed using upstream and downstream primers of qPCR. The reaction system consisted of 2 μL cDNA template, 0.5 μL upstream primer, 0.5 μL downstream primer, 10 μL qPCR Master Mix, and 7 μL ddH2O. Detection was performed using a CFX quantitative real-time PCR instrument (Bio-Rad). Five biological replicates were set up for each sample, and each biological replicate had three technical replicates. CsGAPDH was used as an internal control. Results were analyzed using Bio-Rad CFX Manager software.

[0087] Without infection with *Pseudomonas pseudochaete*, the expression pattern of CsUGT84A22 showed no significant difference between *Osmanthus fragrans* and *Acer buergerianum*. However, after infection with both species, the expression level of CsUGT84A22 significantly increased in *Acer buergerianum*. This indicates that CsUGT84A22 can respond to pathogen infection and is one of the reasons for the difference in resistance between the two species.

[0088] CDS sequence of the glycosyltransferase CsUGT84A22 gene (SEQ ID NO.1)

[0089]

[0090] The amino acid sequence of glycosyltransferase CsUGT84A22 (SEQ ID NO.2)

[0091] MGSESLVHVFLVSFPGQGHVNPLLRLGKRLASRGLLVTFSAPESIGKDMRKASNCTDEPTPVGDGFIRFEFFEDGWDENEPRRQDLDQYLPQLELVGKDLLPKMLQKHADQDRPVSCLINNP FIPWVSDLAETLGLPSAMLWVQSCACFSAYYHYYHGIVPFPSEDNMEIDVQLPCMPLLKYDEVPSFLYPNTPYPFLRRAILGQYKNLSKPFCILMDTFQELEHEVIEYMSKISPIKPVGPLF KNPKAPNSNVRGDFVKADNCMEWLDSKPPGSVVYISFGSVVYLKQEHVDEIAHGILSSGVSFLWVMKPPHKDAGLELLVLPEGFLEKAGDKGRVVQWSPQEQVLVHPAVACFVTHCGWNSTM ESLASGMPVVAFPQWGDQVTDAKYLVDVFKVGIRMCRGEAEDRVIPREEVEMCLREATSGPKAAEMKANALKWKETAEAAVAEGGSSDRNMQAFVDEVRRRSLGITSKSTTPEIITTTTVVV

[0092] CDS region amplification upstream primer: ATGGGCTCTGAATCACTTGTC (SEQ ID NO.3)

[0093] CDS region amplification downstream primers: TTAAACAACAGTAGTAGTTGTGATA (SEQ ID NO.4)

[0094] qPCR upstream primer: TCTCCAATGGGCTCTGAATC (SEQ ID NO.5)

[0095] qPCR downstream primer: TGGGGCTGAAAAGGTGACGA (SEQ ID NO.6)

[0096] Promoter amplification upstream primer: CGTTCCTTCAAGTTTCTAAT (SEQ ID NO.7)

[0097] Promoter amplification downstream primer: GACATGGACAAGTGATTCAG (SEQ ID NO.8)

[0098] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of the CsUGT84A22 gene in the control of tea leaf spot disease, characterized in that, The CDS nucleotide sequence of the CsUGT84A22 gene is shown in SEQ ID No.

1. By promoting the expression of the CsUGT84A22 gene, the production of ferulic acid-β-glucoside is increased, thereby improving the resistance of tea tree to ring spot disease.

2. The application according to claim 1, characterized in that, The amino acid sequence of the protein encoded by the CsUGT84A22 gene is shown in SEQ ID NO:

2.

3. A method for identifying tea varieties with strong resistance to leaf spot disease, characterized in that, Includes the following steps: Total DNA was extracted from tea plants and amplified by PCR using upstream and downstream primers of the CsUGT84A22 gene promoter. After amplification, sequencing was performed, and the sequencing results were compared. If the CsUGT84A22 gene promoter lacked a CTACA segment at 777 bp, the tea plant variety was Dan Gui, which has weak resistance to leaf spot disease; if it did not lack the segment, the tea plant variety was Jin Xuan, which has strong resistance to leaf spot disease.

4. The identification method according to claim 3, characterized in that, The sequences of the upstream and downstream primers for the CsUGT84A22 gene promoter are shown in SEQ ID No. 7-8.

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