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

Abstract

The invention discloses application of a CsUGT84A22 gene in synthesis of ferulic acid-beta-glucoside and breeding of disease-resistant varieties of tea trees, and belongs to the technical field of tea tree gene utilization. The CDS nucleotide sequence of the tea tree glycosyl transferase gene CsUGT84A22 is as shown in SEQ ID No. 1 (sequence identifier number 1). The amino acid sequence of the protein coded by the CsUGT84A22 gene is as shown in SEQ ID NO: 2. The tea tree glycosyl transferase CsUGT84A22 gene has the beneficial effects that the tea tree glycosyl transferase CsUGT84A22 gene from a tea tree is cloned and is subjected to prokaryotic expression in escherichia coli and affinity chromatography purification to obtain the recombinant protein, and the recombinant protein is high in 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 physalospora piricola are analyzed, and it is found that more segments are inserted in the promoter sequence of CsUGT84A22 in varieties with high resistance compared with varieties with weak resistance, which is helpful for people to understand reasons causing resistance differences of different tea trees and provides guidance for tea tree breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of tea tree gene utilization, and specifically relates to application of a CsUGT84A22 gene in synthesizing ferulic acid-β-glucoside and breeding tea tree disease-resistant varieties. Background Art

[0002] Tea trees are important leaf crops in my country, but due to the continuous increase in the domestic tea plantation area and changes in temperature, the occurrence range and severity of tea ring spot disease have expanded. As an important perennial woody crop, tea trees are rich in various secondary metabolites, among which phenolic acids and their derivatives are even more complex and diverse. These secondary metabolites help plants better resist biotic and abiotic stresses.

[0003] Tea ring spot is the earliest reported leaf disease in tea gardens. It is a common and frequent leaf fungal disease in tea gardens. It is widely distributed in my country's tea-growing areas, such as Anhui, Zhejiang, Jiangxi, Jiangsu, Hunan, Hubei, Yunnan, Guizhou, Sichuan and Taiwan. It has been reported there. It is also one of the most destructive leaf diseases to tea trees in the world, including India, Thailand, Sri Lanka and Kenya.

[0004] Wheel spot is caused by Pestalotiopsis-like fungi. The pathogen overwinters in the diseased leaf tissue as mycelium or conidiophores. The pathogen produces spores the following year, which are spread and infected again with wind and rain. The incidence index of tea wheel spot is closely related to ecological factors such as temperature and humidity. The pathogen can survive in the range of 5-35℃, and form new lesions and produce more spores after 7-14 days; under in vitro conditions of 25℃, the pathogen spores begin to germinate after 4 hours. The pathogen of tea wheel spot prefers warmth and humidity, and grows most rapidly at around 28℃. In my country's tea-growing areas, the two peak periods of tea wheel spot are the summer rainy season (May-June) and autumn (August-October).

[0005] Plant pathogenic fungi must effectively detoxify secondary metabolites with antibacterial activity in host tissues, such as phenols, steroids, terpenes and alkaloids, in order to successfully infect and colonize plants. There are more than 8,000 known phenolic compounds in the plant kingdom, which are mainly divided into three categories: flavonoids, phenolic acids and tannins. As important secondary metabolites in plants, phenolic acids have multiple functions such as antibacterial, anti-toxin, anti-inflammatory, antioxidant, and anti-cancer. Their existence is beneficial to plant growth, reproduction and protection from pathogens. Ferulic acid is the most important phenolic acid in the raw materials of Gramineae plants. Phenolic acids often have higher biological activity after structural modification. Their plasticity depends on a variety of enzymes. Among them, the glycosylation modification of glycosyltransferase is more conducive to the change of plants in adverse conditions in a direction that is beneficial to their own survival and development.

[0006] Therefore, how to breed tea tree varieties with stronger disease resistance and how to improve the tea tree's resistance to ring spot disease are crucial and have become issues that need to be resolved. Summary of the invention

[0007] The technical problem to be solved by the present invention is how to provide a method for improving and breeding the resistance of tea trees to ring spot disease.

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

[0009] The first aspect of the present invention provides the use 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 comprises the following steps: using ferulic acid as a substrate and CsUGT84A22 protein as an enzyme, reacting at a pH of 4.5-7.5 and a reaction temperature of 20-45° C. 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 was subjected to enzyme kinetic analysis, and its Michaelis constant Km was 0.1 Mm, the maximum reaction rate Vmax was 1.87 nKat / mg, and Kcat / Km was 18.7±0.20S -1 mM -1 .

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

[0014] The third aspect of the present invention provides the use of ferulic acid-β-glucoside in improving the resistance of tea tree to ring spot disease.

[0015] The fourth aspect of the present invention proposes the use of CsUGT84A22 as a drug target in the preparation of drugs for preventing and treating tea tree ring spot disease, wherein the CsUGT84A22 includes a CsUGT84A22 gene and a CsUGT84A22 protein.

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

[0017] The fifth aspect of the present invention proposes a method for identifying tea tree varieties with strong resistance to ring spot disease, comprising the following steps: extracting total DNA of tea trees, performing PCR amplification using upstream and downstream primers of the CsUGT84A22 promoter, sequencing after the amplification is completed, and comparing the sequencing results. If the CsUGT84A22 promoter lacks a section of CTACA at 777bp, the tea tree variety is Dangui with weak resistance to ring spot disease; if it does not lack, the tea tree variety is Jinxuan with strong resistance to ring 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 the present invention are:

[0020] 1. The present invention cloned the tea glycosyltransferase CsUGT84A22 gene from tea trees, expressed it in Escherichia coli prokaryotically, and purified it by affinity chromatography to obtain a recombinant protein with high purity and activity, which can be applied to ferulic acid-β-glucoside biosynthesis genetic engineering. In addition, the sequence differences in tea varieties with different resistance to ring spot disease were analyzed, and it was found that the promoter sequence of CsUGT84A22 in varieties with stronger resistance had more insertions than that in varieties with weaker resistance, which helps us understand the reasons for the differences in resistance of different tea trees and provides guidance for tea breeding.

[0021] 2. The present invention constructs a prokaryotic expression recombinant vector pMAL-c5x-CsUGT84A22, heterologously expresses it in Escherichia coli Rosetta, and expresses the CsUGT84A22 recombinant protein under the induction of isopropyl-β-D-thiogalactoside, and finds that it is a soluble protein; the CsUGT84A22 protein is obtained by affinity chromatography purification, and SDS-PAGE detects that the band is single and free of impurity bands. When ferulic acid is used as substrate and CsUGT84A22 is used as enzyme, ferulic acid-β-glucoside can be synthesized; the optimal reaction conditions of CsUGT84A22 enzymatic reaction were explored and it was found that ferulic acid-β-glucoside can be generated at pH 4.5-7.5 and reaction temperature 20-45°C, with the optimal pH being 5.5 and the optimal reaction temperature being 30°C; ferulic acid-β-glucoside was detected by liquid chromatography-mass spectrometry, and its characteristic ions were 355, 175, 271, and 193. The reaction was carried out under the optimal reaction conditions with different substrate concentrations, and the enzyme kinetic analysis of CsUGT84A22 found that the Michaelis constant Km was 0.1Mm, the maximum reaction rate Vmax was 1.87nKat / mg, and Kcat / Km was 18.7±0.20S -1 mM -1 Inhibiting the expression of CsUGT84A22 in tea leaves can reduce the production of ferulic acid-β-glucoside. When ferulic acid-β-glucoside standard products are co-cultured with pathogenic fungus hyphae, it is found that the addition of ferulic acid-β-glucoside can effectively inhibit the growth of hyphae, indicating that it can improve the resistance of tea trees, thereby completing the present invention.

[0022] 3. Using Dangui, a variety with weaker resistance to Pseudodisco hirsutum, and Jinxuan, a variety with stronger resistance, as materials, the promoter sequence of CsUGT84A22 was amplified. It was found that the promoter sequence of CsUGT84A22 in Jinxuan had an additional sequence inserted before the binding site of MYB transcription factor compared with Dangui, which may cause the difference in resistance by affecting the binding ability of MYB transcription factor. Analysis of the expression pattern of CsUGT84A22 in the two varieties found that when infected by Pseudodisco hirsutum, the expression pattern in Jinxuan increased significantly compared with Dangui. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the phylogenetic cluster analysis diagram of CsUGT84A22 gene and Arabidopsis AtUGTs family;

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

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

[0026] Figure 4 The optimal reaction temperature, time, pH and concentration of CsUGT84A22 enzymatic reaction;

[0027] Figure 5 This is a diagram of the enzyme kinetic analysis of CsUGT84A22;

[0028] Figure 6 This is a graph showing the effect of CsUGT84A22 catalytic products on the growth of wheel spot pathogens;

[0029] Figure 7 This is the promoter sequence diagram of CsUGT84A22 in Jinxuan, which has strong resistance to Pseudodiscopolysporus. The red mark indicates the difference between the promoter sequence and that of Dangui, which has weaker resistance. After analysis, it was found that only CTACA may have an effect on the binding ability of similar binding elements (in the blue box);

[0030] Figure 8 This is a comparison chart of the expression patterns of CsUGT84A22 in the different wheel spot disease resistant varieties Dangui and Jinxuan. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are 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.

[0032] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0033] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0034] The main reagents and formulas used in the embodiments of the present invention are:

[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: 5 g of yeast powder, 10 g of peptone, and 10 g of sodium chloride were dissolved in 1 L of pure water. 4-5 g of agar powder was added to every 250 ml of solid LB medium. Sterilize with high temperature and high pressure for 20 min.

[0041] (7) CTAB solution: 4 g hexadecyltrimethylammonium bromide (CTAB), 2.42 g tris(hydroxymethyl)aminomethane hydrochloride, 1.8986 g disodium ethylenediaminetetraacetate, 16.38 g sodium chloride. Make up to 200 ml with DEPC water and adjust the pH to 8.0.

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

[0043] (9) Protein elution buffer: 200 mM sodium chloride, 1 mM disodium ethylenediaminetetraacetic acid, 20 mM tris(hydroxymethyl)aminomethane hydrochloride, 1 mM dithiothreitol, 3 g / L maltose. Adjust pH to 7.4 after complete dissolution.

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

[0045] (11) 1.5% agarose gel formula: Weigh 0.3 g agarose in 20 mL 0.5x TBE buffer, mix well, and heat to dissolve until clear.

[0046] Embodiment 1:

[0047] 1. CsUGT84A22 gene cloning

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

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

[0050] (2) cDNA reverse transcription: according to PrimeScript TM 1st Strand cDNA Synthesis Kit (Takara) instructions: RNA is reverse transcribed into cDNA as a template for PCR amplification of the gene.

[0051] (3) PCR amplification: The CDS sequence of CsUGT84A22 was amplified using the upstream and downstream primers in the CDS region. The required reagents were added in sequence 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) and PCR amplification was performed. The cloning program was 95°C (3 min), 95°C (30 s), 58°C (30 s), 72°C (1 min), 30 cycles, 72°C (5 min), and the reaction product was stored at 4°C.

[0052] (4) Ligation, transformation and sequencing: After electrophoresis of the PCR product on 1.5% agarose gel, add the target fragment gel recovery product, the pMAL-c5x vector gel recovery product after EcoRI and SaLI digestion, and the recombinase into a PCR tube, gently beat to mix, and place in a PCR instrument at 37°C for 30 minutes for ligation.

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

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

[0055] ④ On the clean bench, take 200 μL of bacterial solution from the cultured cells and apply it to 100 ng / mL ampicillin (Amp + ) on solid LB medium and cultured inverted in a 37°C incubator overnight.

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

[0057] ⑥ Send the colonies verified correct by PCR to Universal Company for sequencing.

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

[0059] Prokaryotic expression:

[0060] (1) Take the positive strain with correct sequencing and transfer it into the Amp + LB liquid medium was added and cultured in a 37°C constant temperature shaker (speed 180 rpm) until the bacterial solution OD 600 =0.4-0.6. The bacterial solution was taken from it as the pre-induction control group, and the remaining bacterial solution was added with isopropyl-β-D-thiogalactoside (IPTG) inducer, and transferred to a 28°C constant temperature shaker (speed 180 rpm) for induction culture for 12 hours to a final concentration of 0.1 mM.

[0061] (2) After the induction, the bacterial solution was centrifuged at 6000 rpm for 10 min, and the supernatant was collected as the soluble protein after induction. The bacterial solution precipitate was collected and resuspended with 1× phosphate-buffered saline (PBS) buffer as the insoluble protein after induction. 5× protein loading buffer was added to the protein sample, mixed thoroughly, and boiled in a 100°C water bath for denaturation.

[0062] (3) Prepare 12% SDS-PAGE protein gel using SDS-PAGE electrophoresis gel preparation kit. Load the denatured protein sample. After electrophoresis, incubate the SDS-PAGE gel in Coomassie Brilliant Blue staining solution and decolorize it. Take an image of the decolorized SDS-PAGE gel.

[0063] Protein purification:

[0064] ① According to the optimal conditions for prokaryotic expression mentioned above, induce 100mL of bacterial solution at 0.3mM IPTG and 16℃ for 16h. Centrifuge the induced bacterial solution at 4℃, 5000r / min for 10min to collect the bacteria. Resuspend the bacteria with 15ml 1×PBS buffer and use an ultrasonic disruptor to clarify. Take 1mL of the broken sample and centrifuge at 4℃, 12000r / min for 3min to collect the sample supernatant and precipitate, and resuspend the precipitate with 100μL 1×PBS buffer. Centrifuge the remaining sample at 4℃, 5000r / min for 10min and take the supernatant.

[0065] ② Use a pipette to transfer an appropriate amount of amylose resin to an empty affinity column, wait for the resin to settle, and allow excess buffer to flow out of the column by gravity. Use 10 times the column volume of column buffer to balance the column.

[0066] ③ Add the clarified sample containing the target MBP-tagged protein to the column, seal it and incubate it in a 4°C chromatography cabinet for 2 hours. Open the lower end of the chromatography column and adjust the flow rate to 0.5 mL / min. Use 5 times the column volume of column buffer to wash the impurities.

[0067] ④Add 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 3000r / min for 5min, repeat the washing 3 times, and centrifuge the eluted sample in the ultrafiltration tube at 3000r / min until the sample remains about 1mL. Use the Coomassie Brilliant Blue Kit to determine and calculate the protein concentration, and store it at -80℃ after aliquoting.

[0069] ⑥ Add 5× protein loading buffer to the supernatant, precipitate, and purified protein samples before and after induction, and mix well. Incubate in a 95-100℃ water bath for 10 min. Detect the purification results of the recombinant protein by SDS-PAGE protein gel.

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

[0071] In order to determine whether CsUGT84A22 can catalyze the production of ferulic acid-β-glucoside, the product of CsUGT84A22 was identified by high-resolution liquid chromatography-mass spectrometry (QE-Foucs). The reaction system was 200 μL: 100 mM MES buffer (pH 5.5), 2.5 mM UDP-glucose, 0.5 mM primary screening substrate, 10 μg purified protein, and the control group was empty purified protein. After reacting at 30 ° C for 1 hour, an equal volume of ethyl acetate was added for extraction twice, and the organic solvent was rotary evaporated and 300 μL of 80% methanol was added for dissolution. Centrifugation was performed at 12000 r / min for 10 minutes, and the supernatant was taken, filtered through an organic phase filter membrane with a pore size of 0.22 μm, and added to the injection bottle for analysis. LC-MS detection conditions: chromatographic 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: B phase from 10% to 15% within 0-5min, B phase from 15% to 10% within 6-9min, B phase from 30% to 10% within 10-15min. The enzymatic product of CsUGT84A22 was identified by comparing the peak time of the standard. Figure 3 It can be seen that we successfully prepared ferulic acid-β-glucoside.

[0072] 4. Enzymatic kinetics of ferulic acid to ferulic acid-β-glucoside catalyzed by CsUGT84A22

[0073] After the initial scan of enzyme activity, the pH, temperature, concentration and reaction time of the enzyme's suitable substrate are measured. The temperature measurement range is 20-45°C, the pH measurement range is 4.5-7.5, and the reaction time measurement range is 0-80min. The enzyme kinetic parameters are determined according to the above system and method.

[0074] DNA was extracted from tea varieties with different resistance to ring spot disease respectively; the DNA of the two samples was amplified by PCR using the same pair of PCR primers, and the amplified sequences were sequenced to compare the differences between the two sequences.

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

[0076] The bacterial cake culture method was used to detect whether the substrate catalyzed by CsUGT84A22 and its product were analytes with their standard products as analytes to detect whether they were resistant to the genus of tea ring spot pathogens. A 6mm diameter bacterial cake was evenly punched with a hole puncher, and 20μL of the standard product was suspended and dropped on the surface of PDA potato dextrose agar medium. The mycelium surface of the pretreated bacterial cake was gently placed on the place where the standard product was dropped. 80% methanol and blank pure culture medium were used as controls. The entire operation process was carried out under sterile conditions. The culture was carried out in an incubator at 28℃, and the growth area of ​​the mycelium was observed to compare the inhibitory effect of the standard product on the mycelium growth of tea ring spot pathogens.

[0077] 6. CsUGT84A22 promoter sequence amplification

[0078] (1) Total DNA extraction: Add an appropriate amount of PVPP to 100 mg of sample in a 2 mL centrifuge tube and grind into 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 every 5 min.

[0079] (3) Add 600 μL of chloroform:isoamyl alcohol (24:1) to the centrifuge tube and centrifuge at 12,000 r / min for 10 min. Pipette 500 μL of supernatant and add an equal volume of isopropanol. Invert 6-8 times to mix well and centrifuge at 12,000 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 evaporates completely, add 100 μL of ddH2O.

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

[0082] (6) Using the DNA of Dangui and Jinxuan as templates, PCR amplification was performed using the 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 in Figure 2, it was found that although there were many differences in the promoter sequences of the two genes ( Figure 7 However, after analysis, it was found that only CTACA may affect the binding ability of similar binding elements (in the blue box), that is, Dangui lacks a CTACA insertion at 777bp compared with Jinxuan. Through analysis, it was found that this insertion is located on the left side of the MYB transcription factor binding element. Therefore, this insertion may affect the binding ability of the MYB transcription factor, thereby causing the different expression patterns of the promoter of CsUGT84A22 in Dangui and Jinxuan.

[0084] 7. Analysis of CsUGT84A22 expression pattern

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

[0086] Fluorescence quantitative PCR: Amplification was performed using the upstream and downstream primers of qPCR. The reaction system (2 μL cDNA template, 0.5 μL upstream primer, 0.5 μL downstream primer, 10 μL qPCR Master Mix, 7 μL ddH2O) was used, and the CFX fluorescence quantitative PCR instrument (Bio-Rad) was used for detection. Five biological replicates were set for each sample, and each biological replicate had three technical replicates. CsGAPDH was used as the internal reference, and the results were analyzed using Bio-Rad CFX Manager software.

[0087] When not infected with Pseudodiscus polytrichosporium, there was no significant difference in the expression pattern of CsUGT84A22 in Dangui and Jinxuan, but after infection of both, the expression level of CsUGT84A22 in Jinxuan increased significantly. This indicates that CsUGT84A22 can respond to the infection of pathogens and is one of the reasons affecting the difference in resistance between the two.

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

[0089]

[0090] 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 primer: 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, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of CsUGT84A22 protein in the preparation of ferulic acid-β-glucoside, characterized in that: 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.

2. The use according to claim 1, characterized in that: The method comprises the following steps: taking 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 DEG C to obtain ferulic acid-β-glucoside.

3. The use according to claim 2, characterized in that: The pH was 5.5 and the reaction temperature was 30°C.

4. The use according to claim 2, characterized in that: The enzyme kinetics analysis of the CsUGT84A22 protein showed that the Michaelis constant Km was 0.1 Mm, the maximum reaction rate Vmax was 1.87 nKat / mg, and Kcat / Km was 18.7±0.20 S -1 mM -1 .

5. The method for preparing the CsUGT84A22 protein according to claim 1, characterized in that: The following steps are involved: Isopropyl-β-D-thiogalactoside is added to the culture medium to induce the expression of the recombinant expression strain, and the protein is collected; the recombinant expression strain is obtained by the following process: the CsUGT84A22 gene is connected to the pMAL-c5x vector to obtain the recombinant expression vector pMAL-c5x-CsUGT84A22, and then the recombinant expression vector is transferred into Escherichia coli Rosetta to obtain it.

6. Use of the ferulic acid-β-glucoside according to claim 1 in improving the resistance of tea tree to ring spot disease.

7. Application of CsUGT84A22 as a drug target in the preparation of a drug for preventing and treating tea tree ring spot disease, characterized in that: The CsUGT84A22 comprises the CsUGT84A22 gene and CsUGT84A22 protein according to claim 1.

8. The use according to claim 7, characterized in that: The drug is a CsUGT84A22 overexpression agent or an agent that promotes CsUGT84A22 overexpression.

9. A method for identifying tea tree varieties with strong resistance to ring spot disease, characterized in that: The following steps are involved: The total DNA of the tea tree was extracted, and PCR amplification was performed using the upstream and downstream primers of the CsUGT84A22 promoter. After the amplification, sequencing was performed and the sequencing results were compared. If the CsUGT84A22 promoter lacked a CTACA segment at 777bp, the tea tree variety was Dangui with weak resistance to ring spot disease; if it did not lack a CTACA segment, the tea tree variety was Jinxuan with strong resistance to ring spot disease.

10. The identification method according to claim 9, characterized in that: The sequences of the upstream and downstream primers of the CsUGT84A22 promoter are shown in SEQ ID No.7-8.

Images