Application of CsPRMT5 gene in resisting tea tree zonate spot

Abstract

The invention relates to the technical field of gene engineering, in particular to application of a CsPRMT5 gene to resistance of tea tree physalospora piricola, the base sequence of the CsPRMT5 gene is shown as SEQ ID NO.1, the CsPRMT5 gene is a negative regulation factor of the resistance of the tea tree physalospora piricola, and the resistance of the tea tree physalospora piricola is regulated by regulating expression of CsMAPK3; the disease resistance of the tea trees can be remarkably improved and enhanced by silencing CsPRMT5 in the tea trees, and the sensitivity of the tea trees to pathogenic bacteria can be improved by overexpression. The regulation and control mechanism of the CsPRMT5 gene to the resistance of the tea tree physalospora piricola disclosed by the invention provides a basis for subsequently applying the CsPRMT5 gene to the resistance of the tea tree physalospora piricola.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to application of CsPRMT5 gene in resistance to tea tree ring spot disease. Background Art

[0002] Tea tree (Camellia sinensis), belonging to the family Theaceae, is an evergreen woody plant with important economic value. Its young shoots are rich in various secondary metabolites, such as polyphenols, catechins, flavanones, caffeine, theanine, saponins, vitamins, minerals and volatile oils, and are widely used in the production of tea, a popular non-alcoholic beverage. As an evergreen plant, tea tree is susceptible to biotic stresses such as bacteria, fungi and viruses in terms of growth and tea quality, which in turn affects its normal growth and development, leading to a significant decline in global tea production and quality.

[0003] Tea ring spot disease, caused by Pestalotiopsis-like species, is one of the most serious leaf diseases, mainly affecting mature and old leaves, but also posing a threat to young buds. Although ring spot disease can be controlled chemically, the long-term use of chemical fungicides will make the pathogen increasingly resistant and also pollute the environment. In comparison, the selection of resistant varieties is considered to be a more economical, environmentally friendly and effective strategy for preventing and controlling ring spot disease, and the functional analysis of key genes for tea ring spot disease resistance is the basis for constructing a resistance genetic network and conducting molecular design breeding. Therefore, it is imperative to explore the genes that play an important role in the tea tree-ring spot disease interaction and reveal their potential molecular mechanisms.

[0004] At present, in order to cope with the attack of pathogens, plants have evolved various complex defense mechanisms. Epigenetic processes (such as DNA methylation and histone modification) play an important role in transcriptional regulation and significantly affect plant immune responses. Histone methylation is a widespread and critical epigenetic modification that occurs on lysine and arginine residues. Recent studies have shown that histone lysine methylation marks play a vital role in the response of plants to stress. For example, JMJ27 activates defense genes such as PR1 and PR3 by regulating H3K9 methylation, while inhibiting negative regulators of defense genes. Similarly, the H3K4 demethylase JMJ14 regulates the immune response by regulating the deposition of H3K4me3 marks, thereby affecting the transcription of key defense genes (such as PR1, FMO1 and SNI1). In addition, OsJMJ705 removes H3K27me3 from defense-related genes, thereby enhancing resistance. Application of Tea Tree Histone Arginine Methyltransferase in Molecular Breeding of Tea Trees

[0005] Recent studies have shown that protein arginine methyltransferase 5 (PRMT5) plays a role in immunity in a variety of plants. In Arabidopsis, PRMT5 (also known as AtSKB1) catalyzes the addition of two methyl groups to the third arginine residue of histone H4 to form H4R3sme2, a modification that serves as a repressive mark for transcriptional regulation. AtPRMT5 acts as a negative regulator to regulate immune responses to oomycete and AvrRpt pathogens. Specifically, bacterial infection leads to decreased PRMT5 expression and is accompanied by reduced arginine methylation levels of key proteins such as AGO2 and LSM4. By dually regulating AtAGO2, PRMT5 regulates plant immune responses to pathogens. In contrast, OsPRMT5 in rice plays a different role in resistance to rice blast. Studies have shown that OsPRMT5 acts as a positive regulator of rice immunity. After rice is infected with the rice blast fungus, OsPRMT5 regulates AGO2 activity through arginine methylation and interacts with miR1875 to affect rice immune responses. The results showed that PRMT5 may have different modes of action in different species, and its role in the disease resistance mechanism of tea plants still needs further study.

[0006] In view of the above-mentioned defects, the inventors of the present invention finally obtained the present invention after a long period of research and practice. Summary of the invention

[0007] The present invention aims to solve the problem of how to apply the CsPRMT5 gene to tea tree disease resistance, and provides the application of the CsPRMT5 gene in tea tree ring spot resistance.

[0008] In order to achieve the above object, the present invention discloses the use of CsPRMT5 gene in resistance to tea tree ring spot disease. The nucleotide sequence of the CsPRMT5 gene is shown in SEQ ID NO.1.

[0009] The amino acid sequence of the protein encoded by the CsPRMT5 gene is shown in SEQ ID NO.2.

[0010] The CsPRMT5 gene is a negative regulatory factor for tea tree ring spot disease resistance.

[0011] The CsPRMT5 gene regulates the ring spot disease resistance by regulating the expression of the CsMAPK3 gene. The regulation of the tea tree ring spot disease resistance is: overexpressing the CsMAPK3 gene to increase the resistance of the tea tree to the ring spot pathogen. The nucleotide sequence of the CsMAPK3 gene is shown in SEQ ID NO.3.

[0012] The nucleotide sequence of the antisense oligonucleotide for inhibiting the expression of the CsPRMT5 gene is shown in SEQ ID NO.4.

[0013] The antisense oligonucleotide provided by the present invention can inhibit the expression of CsPRMT5 gene, improve the activity of POD, a key enzyme in the ROS scavenging system, increase the expression of resistance genes such as CsMAPK3, and improve the resistance of tea leaves to wheel spot pathogens.

[0014] Compared with the prior art, the beneficial effect of the present invention is that: the present invention provides the application of CsPRMT5 gene in regulating the sensitivity of tea tree ring spot pathogen, and the nucleotide sequence of CsPRMT5 gene is shown in SEQ ID NO.1. The present invention studies the relationship between the expression of CsPRMT5 gene and disease resistance in different tea varieties, indicating that CsPRMT5 is a negative regulatory factor for tea tree defense, and overexpression of CsPRMT5 in tea trees shows a larger lesion area and weaker resistance to Ps, so CsPRMT5 can be used to negatively regulate the resistance of plants to ring spot disease. The present invention inhibits the expression of CsPRMT5 gene by antisense oligonucleotides and measures the physiological indicators after infection with ring spot pathogens. It is found that the DAB staining effect of tea leaves after inhibiting the expression of CsPRMT5 gene is weaker than that of the control group, the activity of the key enzyme POD of the ROS scavenging system is higher than that of the control group, and the expression of resistance-related genes such as CsMAPK3 is higher than that of the control group. The resistance of tea leaves to ring spot pathogens is increased after inhibiting the expression of CsPRMT5 gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a time course analysis of the methylation levels of histone H4 arginine residues in tea plants, where:

[0016] (a) Histones were isolated from tea leaves after 0, 24, 48, and 72 hours, and Western blot analysis was performed using antibodies against histone methylation marks. Different methylation modifications are represented by numbers relative to the plant levels at 0 hours of inoculation; (b) Expression patterns of PRMT family genes in infected leaves. FPKM values ​​are presented as heat maps; (c) Expression analysis of CsPRMT5 in tea leaves infected with Ps at different time points, relative to the double distilled water control group (CK); (d) Immunoblotting analysis of CsPRMT5 and H4R3sme2 levels after Ps infection, data are expressed as mean ± SD (n = 3), and statistical significance (p < 0.05) is indicated by different letters based on Duncan's multiple range test. *P < 0.05, **P < 0.01.

[0017] Figure 2 Correlation between disease resistance and CsPRMT5 expression in different tea varieties, including:

[0018] (a) Phenotypes of different tea varieties, scale = 1 cm; (b) Expression and protein levels of CsPRMT5 in different tea varieties; (c) Phenotypes of different tea varieties at 5 dpi; (d) Total lesion area of ​​different tea varieties after infection; (e) Correlation between disease resistance and CsPRMT5 expression level in different varieties, CsGAPDH as internal reference, data are expressed as mean ± standard deviation (n = 3), statistical significance (p < 0.05) is indicated by different letters, based on Duncan multiple range test. *P < 0.05, **P < 0.01;

[0019] Figure 3 The pathogenic phenotype and related gene expression in CsPRMT5-silenced tea plants, including:

[0020] (a) Schematic diagram of AsODN gene silencing; (b) Relative expression level of CsPRMT5 in AsODN and sODN plants; (c) Immunoblotting analysis of CsPRMT5 and H4R3sme2 levels in AsODN and sODN plants; (d) Disease symptoms of CsPRMT5-silenced tea plants and control (sODN) plants after fungal infection, scale bar = 2 mm; (e) Relative expression level of PR genes in sODN and AsODN tea plants; (f) Diaminobenzidine (DAB) staining of tea leaves 48 hours after Ps inoculation; (g) H2O2 content; (h) CAT activity; (i) POD activity; Data are expressed as mean ± standard deviation (n = 3), and statistical significance (p < 0.05) is indicated by different letters, based on Duncan's multiple range test. *P < 0.05, **P < 0.01;

[0021] Figure 4 The pathogenic phenotype and related gene expression in tea plants overexpressing CsPRMT5,

[0022] (a) Schematic diagram of transient overexpression of CsPRMT5 gene; (b) relative expression level of CsPRMT5 in 35S::CsPRMT5 and EV plants; (c) immunoblot analysis of CsPRMT5 and H4R3sme2 levels in 35S::CsPRMT5 and EV plants; (d) disease symptoms of CsPRMT5 overexpression and control plants after fungal infection, scale bar = 2 mm; (e) relative expression level of PR genes in 35S::CsPRMT5 and control (EV) tea plants, the significant difference between the treatment group and the control group was calculated by one-way ANOVA of SPSS21.0, *P<0.05, **P<0.01; (f) diaminobenzidine (DAB) staining of tea leaves 48 hours after Ps inoculation; (g) H2O2 content; CAT activity; POD activity.

[0023] Figure 5 is the interaction between transgenic Arabidopsis leaves and Ps, where:

[0024] (a) Western blot analysis of WT, skb1, and Com2 Arabidopsis plants; (b) Typical leaf disease symptoms of 5-week-old Arabidopsis WT, skb1, and Com2 plants after Ps infection. The control treatment (infection) was inoculated with water, and the photos were taken at 5 dpi; (c) Average lesion statistics at 7 dpi; (d) RT-qPCR analysis of AtPR1 transcription levels in WT, Com2, and skb1 Arabidopsis plants after Ps infection; (e) H2O2 content; (f) POD activity; (g) SOD activity. Data are expressed as mean ± standard deviation (n = 3), scale = 1 cm, and significant differences between the treatment groups and the control group were calculated by one-way analysis of variance using SPSS21.0, *P < 0.05, **P < 0.01, ***P < 0.001.

[0025] Figure 6 CsPRMT5 regulates immune genes related to gray mold response, including:

[0026] (a) is a bar graph showing differentially regulated genes in CsPRMT5-silenced plants. The transcriptome analysis of tea samples with effective CsPRMT5 silencing was performed by RNA sequencing; (b) is a GO enrichment analysis of differentially regulated genes in CsPRMT5-silenced plants. The screening criteria for differentially regulated genes were gene expression fold change > 2 and adjusted P value < 0.05; (c) is a heat map of pathogen-related genes generated based on the published RNAseq results of tea leaf ring spot disease. CK represents the control group, Ps represents the control group, and Ps represents the control group. 8, 24, and 72 hours correspond to different time points after infection, respectively. The color bar (from white to red) represents the TPM (log2) of the gene as the z-score; these genes are upregulated in CsPRMT5-silenced plants; (d) RT-qPCR analysis of the effect of CsPRMT5 silencing on potential target genes under control conditions (CK) and pathogen stress. Data are expressed as mean ± SD (n = 3). Statistical significance (p < 0.05) is marked with different letters. Based on Duncan's multiple range test, *P < 0.05, **P < 0.01.

[0027] Figure 7 The binding of CsPRMT5 to the chromatin of resistance genes and the decrease in H4R3sme2 levels in tea plants after wheel spot infection, among which:

[0028] (a) ChIP-seq analysis of the binding of CsPRMT5 to chromatin containing defense-related genes; (b) CsMAPK3 gene structure, black boxes represent exons, black lines represent introns, ChIP experiments of CsMAPK3 were performed using anti-CsPRMT5 and anti-H4R3sme2 antibodies, and chromatin extracted from tea leaves was treated with Ps for 24 hours and compared with the untreated control group; (c) CsMAPK3 expression levels after tea leaves were infected with Ps at different time points. For the double distilled water control group (CK); (d) for the fungal infection symptoms of CsMAPK3 overexpression and control plants, scale bar = 2 mm; (e) for the fungal infection symptoms of CsMAPK3 silenced tea plants (CsMAPK3-TRV) and control (TRV) plants, scale bar = 2 mm, data are expressed as mean ± standard deviation (n = 3), statistical significance (p < 0.05) is marked with different letters, based on Duncan multiple range test, *P < 0.05, **P < 0.01. DETAILED DESCRIPTION

[0029] The above and other technical features and advantages of the present invention are described in more detail below in conjunction with the accompanying drawings.

[0030] All the following experiments were performed with at least three independent biological replicates, each with three measurements. One-way analysis of variance (ANOVA) was used to compare the statistically significant differences between the control group plants and the experimental group plants, using the Duncan test (P<0.05), and the analysis was performed using SPSS software. The tea variety "Shucha Zao" used in the present invention comes from Niansheng Agricultural Ecology Co., Ltd., Chaohu City, Anhui Province, and is the main tea material for this study. The ring spot pathogen Pseudopestalotiopsis (Ps) was inoculated with it. The present invention also used the skb1-1 mutant (salk_065814) and its complement strain (35S:CsPRMT5 / skb1-1), which were obtained in the laboratory in the early stage. All plants were cultivated under suitable environmental conditions, with a growth temperature of 25±1°C and a photoperiod of 16 hours of light / 8 hours of darkness.

[0031] Example 1

[0032] Identification of the tea tree PRMT gene family

[0033] Based on the PRMT and PrmA domains (PF05185, PF06325), the incomplete structures and the domains not containing PRMT were eliminated, the candidate sequences were identified and verified, and finally the identified PRMT family members were used for subsequent analysis. (Table 1). A total of 9 PRMT genes were identified. Among them, PRMT1A-C, PRMT4A-B, and PRMT10A-C were 8 type I genes. The wheel spot transcriptome published on the tea tree genome website was used for heat map analysis of the above nine genes (Table 2).

[0034] Table 1 9 PRMT genes

[0035]

[0036] Table 2 Heat map analysis results of 9 PRMT genes

[0037]

[0038]

[0039] Example 2

[0040] Changes of histone arginine methylation after wheel spot infection

[0041] 1. The Ps strain was inoculated in a potato dextrose agar (PDA) medium at 28°C and cultured for 5 days, and then the spores were collected by centrifugation (6000 g, 10 minutes). The collected spores were resuspended in sterile water and their concentration was observed under a microscope and adjusted to 10 6 Spores / mL. Subsequently, 50 μL of spore suspension was inoculated on plant leaves by piercing the upper epidermis of the leaves with a sterile syringe. As a control group, plants were inoculated with an equal volume of sterile distilled water.

[0042] 2. Collect samples at 0, 24, 48 and 72 hours post infection (hpi) and verify them with RT-RT-qPCR, and perform Western Blot analysis using antibodies against H4R3sme2, H4R3ame2, H3R2ame2 and H3R17ame2. Histone H4 was used as a control. Immunoblot analysis used anti-histone H4 / anti-H4R3sme2 antibodies (Shanghai Abbot, predicted molecular mass of 10 / 15kDa). Proteins were separated in 12% SDS / PAGE gels and transferred to nitrocellulose membranes (Amersham Protran, 0.45μm). ACTIN was used as a loading control (Shanghai Bioengineering, predicted molecular mass 42kDa).

[0043] 3. Detailed steps for total protein extraction: First, take 0.5g of material and grind it into fine powder with liquid nitrogen. Add it to a centrifuge tube containing 600μl pre-cooled total protein extract, place it on ice for 20min, and centrifuge it at 4℃, 16000g for 20min. For tea tree samples, add an appropriate amount of PVPP powder during grinding to reduce the impact of polyphenols. Plant total protein extraction buffer: 20mM Tris-HCl pH 8.0, 1mM EDTA, 150mM NaCl, 0.2% Triton X-100, 10% Glycerol, 1mM PMSF, 1×Protease Inhibitor Cocktail.

[0044] 4. Detailed steps for histone extraction: Weigh 0.5g of sample, grind into fine powder with liquid nitrogen, add to 3mL pre-cooled nuclear protein extraction buffer, and shake on ice for 10min. Centrifuge at 4℃, 10000g for 10min, and keep the precipitate. Then, add 600μl 0.4M H2SO4, resuspend and continue to shake on ice for 1h. Centrifuge at 4℃, 10000g for 10min. Transfer the supernatant to 12 times the volume of pre-cooled acetone and place at -20℃ for more than 8h. Centrifuge at 4℃, 8000g for 15min. Finally, after drying in the fume hood, add 200μl 4M urea to dissolve. Tea tree nuclear protein extraction buffer: 0.25M Sucrose, 60mM KCl, 15mM NaCl, 5mM MgCl, 1mM CaCl, 15mM PIPES (pH 6.8), 0.8% Triton X-100, 0.1mM 1×ProteaseInhibitor Cocktail, 0.1mM PMSF, 0.035% β-Mercaptoethanol.

[0045] according to Figure 1 It can be seen that the methylation levels of each arginine site changed at different time points after Ps inoculation. The expression pattern of PRMT5 was consistent with the changing trend of H4R3sme2 levels after Ps infection. RT-qPCR results showed that the transcription level of CsPRMT5 was significantly reduced at 24 hours and 48 hours after infection, and recovered at 72 hours ( Figure 1 c).

[0046] Example 3

[0047] Correlation between disease resistance and CsPRMT5 transcription level in different tea varieties

[0048] Seven tea varieties were selected for this study, including: Longjing 43 (LJ43), Shucha Zao (SCZ), Wuniuzao (WNZ), Fuyun 6 (FY6), Longjing Changye (LJCY), Zhe Nong 113 (ZN113), and Pingyang Te Zao (PYTZ), all of which were from the National High-tech Agricultural Park of Anhui Agricultural University. The expression of CsPRMT5 in uninfected leaves of each variety was quantified, and the second leaf was selected for pathogen inoculation. The lesion area ( Figure 2 bd).

[0049] according to Figure 2 The results showed that the expression level of CsPRMT5 in varieties FY6 and PYTZ was low and showed high disease resistance, while the transcription level of CsPRMT5 in varieties SCZ and WNZ was high and had poor disease resistance. The lesion area and CsPRMT5 expression level were significantly positively correlated in each tea variety (r=0.83, **p<0.01)( Figure 2 e). These findings suggest that CsPRMT5 acts as a negative regulator of tea plant defense.

[0050] Example 4

[0051] AsODN validates the physiological function of CsPRMT5

[0052] 1. The candidate sequence of antisense oligonucleotides (AsODNs) targeting CsPRMT5 was designed using Soligo software (https: / / pubmed.ncbi.nlm.nih.gov / 31828806 / ) (SEQ ID NO.4), and standard oligonucleotides (sODNs) were used as controls. Young shoots were selected for the AsODN experiment and soaked in 100 μM AsODN or sODN solution. After 48 hours, the samples were collected to detect the silencing efficiency of CsPRMT5.

[0053] 2. CsPRMT5-silenced tea plants and control tea plants were inoculated with spore suspension of Ps. The lesion area was counted, and the genes and reactive oxygen species related to disease resistance were detected. The specific detection method of reactive oxygen species was as follows: the leaves inoculated with pathogens were placed in 3,3'-diaminobenzidine (DAB) solution (1 mg / mL, pH 3.8) and incubated at 37°C for 8 hours. Sample pretreatment: 0.02 g of sample was added to 180 μL phosphate buffer (pH 7.0) and ground on ice. The ground homogenate was centrifuged at 3500g for 10 min at 4°C. The supernatant was the total crude enzyme. The supernatant was collected for subsequent enzyme activity detection. The enzyme activities of superoxide dismutase (SOD) and peroxidase (POD) in the leaves were measured using a kit (Beijing Solebold Technology Co., Ltd., Beijing, China).

[0054] RT-qPCR and immunoblot analysis confirmed that CsPRMT5 transcript and protein levels were significantly decreased in AsODN-treated tea plants compared with the control group (sODN), accompanied by a significant decrease in H4R3sme2 levels ( Figure 3 ac). The lesion area of ​​tea leaves with CsPRMT5 silenced was significantly smaller than that of the control group 5 days after inoculation ( Figure 3 d). Pathology-related genes (PR genes) play an important role in the immune response of tea plants. In both CsPRMT5-silenced and control tea plants, these genes were induced at the early stage of Ps infection, but in CsPRMT5-silenced plants, the induced expression of PR genes was significantly enhanced ( Figure 3 e). H2O2 levels were lower and POD and SOD enzyme activities were higher in CsPRMT5-silenced plants. In contrast, ROS accumulation in leaves overexpressing CsPRMT5 was significantly higher than that in the control group (EV) ( Figure 4 ). These results indicate that CsPRMT5 acts as a negative regulator of tea plant ring spot disease.

[0055] Example 5

[0056] Overexpression of CsPRMT5 verifies its physiological function

[0057] 1. Take the young roots of the national-level fine Shucha Zaocha tree variety (planted in the Agricultural Industrial Park of Anhui Agricultural University, Hefei, Luyang District, Anhui Province) for RNA extraction. The total RNA was extracted using the RNAprep Pure Plant Kit (Tiangen, Beijing, China) and operated according to the instructions. After extraction, the RNA concentration was detected using a nucleic acid detector.

[0058] 2. Reverse transcription generates the first strand, and the first strand of cDNA is used as a template to amplify the CsPRMT5 gene by PCR. The PCR amplification system is as follows: 10 μL of KOD enzyme, 1 μL of upstream and downstream primers, 1 μL of template, 7 μL of ddH2O, a total of 20 μL;

[0059] The PCR amplification program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 2 min, 32 cycles; and extension at 72°C for 10 min.

[0060] 3. The nucleotide sequence of the upstream primer is as follows:

[0061] 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGCCTCTTGGAGAAGTAGTGA-3';

[0062] The nucleotide sequence of the downstream primer is shown below

[0063] 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAGAGGCCGACCCAATACGAA-3'. The PCR product obtained in the recovery step was ligated to the entry vector pDNOR207 vector using a gel recovery kit. After successful colony PCR verification, it was sent to Shanghai Bio-Sequencing Co., Ltd. for sequencing, as shown in SEQ ID NO.1. The amino acid sequence encoded by the CsPRMT5 gene is shown in SEQ ID NO.2.

[0064] 4. After sequencing verification, the genes were connected to pB2GW7 and pK7WGF vectors respectively, and then transferred into Agrobacterium GV3101 strain after successful construction. Among them, pB2GW7-CsPRMT5 was used to stably construct transgenic Arabidopsis, and pK7WGF-CsPRMT5 was used for transient overexpression experiments in tea plants.

[0065] 5. The floral dipping method was used for stable transformation of Arabidopsis thaliana. The specific steps are as follows:

[0066] (1) Agrobacterium GV3101 introduced with the pB2GW7-CsPRMT5 recombinant plasmid was taken out from -80°C and streaked. A single clone was then picked and shaken using liquid LB medium containing 50 mg / L rifampicin and spectinomycin hydrochloride, cultured overnight at 28°C, and the cells were collected.

[0067] (2) Suspend the cells in Arabidopsis infection buffer. Arabidopsis infection buffer (100 mL): 10% sucrose, 20 μl Silwet-L 77. Adjust OD600 to about 1.0, and take the mutant skb1-1 (salk_065814) that has just bloomed for infection by the floral dip method.

[0068] (3) After infection, the cells were cultured in the dark for 24 h and then cultured under normal light conditions.

[0069] (4) One week later, repeat the above steps and infect again.

[0070] (5) The seeds received from infected Arabidopsis were recorded as T0 generation. The received seeds were first dried in a 37°C oven for 3 days, and then stored in a 4°C cold storage with silica gel desiccant.

[0071] (6) Take an appropriate amount of seeds and sow them in the soil. After about 10 days, spray herbicides to screen positive plants.

[0072] (7) Three days later, spray once more. If no seedlings die, they are positive seedlings. Transplant and continue to cultivate individual plants to collect seeds of the T1 generation. Repeat the above operation until the T3 generation pure lines are screened and the seeds are collected and stored for subsequent inoculation experiments.

[0073] 6. Experiment on transient overexpression of CsPRMT5 in tea plants. The specific steps are as follows:

[0074] The constructed pK7WGF-CsPRMT5 and pK7WGF were transferred into GV3101 Agrobacterium, and after picking a single colony for PCR verification, it was placed in liquid LB with Spec+Rif and cultured until the OD600 value reached about 0.8. The cultured bacterial solution was centrifuged at 5000r / min for 10min, the supernatant was poured out, and the bacterial cell was resuspended with a resuspension (0.4066g MgCl2 6H2O (magnesium chloride hexahydrate), 0.4265g MES (2-ethanesulfonic acid), 200μl 1mol / L acetosyringone, and pure water was fixed to 200ml) to OD600 of 0.8. Use a syringe to inject into the back of the tea leaf, and perform an inoculation experiment 48 hours later. The area of ​​the lesions was counted later, and the genes and reactive oxygen species related to disease resistance were detected (as in Example 4).

[0075] RT-qPCR results showed that the expression of CsPRMT5 was significantly upregulated. Compared with the control group, tea plants with overexpression of CsPRMT5 showed larger lesion areas and weaker resistance to Ps. Arabidopsis skb1-1(salk_065814) mutants showed significantly enhanced resistance to Ps, while Com2 plants were more susceptible to infection ( Figure 5 b). Based on the percentage of green area loss on leaves, the disease severity was divided into four levels: level 1 (<10%), level 2 (10-25%), level 3 (25-50%), and level 4 (>50%). The results showed that most Com2 and WT plants were at level 4 severity, while most skb1-1 mutants were at level 1 severity ( Figure 5 c). Consistent with the phenotypic results, AtPR1 was significantly induced in Com2 plants ( Figure 5 d) By Figure 5 (e)- Figure 5 (g) It can be seen that H2O2 accumulation is stronger and SOD and POD activities are lower in Com2 leaves, while the H2O2 level in skb1-1 plants remains almost unchanged.

[0076] Example 6

[0077] CsPRMT5 participates in tea plant resistance to ring spot disease by regulating the expression of CsMAPK3

[0078] 1. Detailed steps of chromatin immunoprecipitation:

[0079] After the samples were infected with pathogens for 24 hours, they were rinsed with deionized water, dried with absorbent paper, placed in a 50 mL centrifuge tube, and 1% formaldehyde was added for cross-linking reaction, and incubated under vacuum for 30 minutes. Subsequently, 0.125 mol / L glycine was added to terminate the cross-linking reaction. The plant material was ground with liquid nitrogen, and the nuclei were separated after adding lysis buffer. The chromatin was then fragmented by ultrasonic disruption. The DNA fragments bound to the protein were immunoprecipitated with anti-H4R3sme2 antibody (Shanghai Abbot, 1:5000 dilution). The immunoprecipitated DNA fragments were extracted and purified for ChIP-qPCR analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene, and the primers used for ChIP-qPCR analysis are listed in Table 3. The ChIP-seq data were derived from previous laboratory research.

[0080] Table 3 Primers used in ChIP-qPCR analysis

[0081]

[0082] 2. Detailed steps for transient overexpression and silencing of CsMAPK3 in tea leaves:

[0083] The cDNA obtained in Example 5 was used as a template to perform PCR amplification using upstream primers and downstream primers.

[0084] Upstream primer:

[0085] 5'-aggacagcccagatcactagtATGGCTGACTCAAACACAGTCG-3';

[0086] Downstream primer:

[0087] 5'-gcccttgctcaccatggatccTGCAAACTCTGGATTGAGTGC-3';

[0088] The PCR amplification procedure was as follows: 94°C pre-denaturation for 3 min; 94°C denaturation for 30 sec, 58°C annealing for 30 sec, 68°C extension for 2 min, 32 cycles; 72°C extension for 10 min. The PCR product was recovered by agarose gel electrophoresis bands. The PCR product was ligated with the pCAMBIA1305 vector (recovered after digestion with SpeⅠ and BamHI restriction enzymes) using homologous recombinase and One Step Cloning kit (Nanjing Novogene Biotech Co., Ltd., Nanjing, China) at 37°C for 30 min. The recombinant product was used for Trans1-T1 competent cell transformation, and sequencing was performed to verify whether CsMAPK3 was ligated, resulting in pCAMBIA1305-CsMAPK3. The pCAMBIA1305-CsMAPK3 plasmid was transferred into GV3101 Agrobacterium separately from pCAMBIA1305. After selecting a single colony for PCR verification, it was cultured in liquid LB with Kan+Rif until the OD600 value reached about 0.8. Use a syringe to inject into the back of the tea leaf, and perform the inoculation experiment 48 hours later. The lesion area was counted later.

[0089] 3. VIGS method for silencing CsMAPK3 gene: Construction of pTRV2-CsMAPK3 vector:

[0090] The vector construction of pTRV2-CsMAPK3 was constructed by using a 274 bp CsMAPK3 fragment containing EcoRI and BamHI restriction enzyme sites. The PCR product was ligated with the pTRV2 vector (recovered after digestion with EcoRI and BamHI restriction enzymes) using homologous recombinase (Nanjing Novogene Biotech Co., Ltd., Nanjing, China) at 37 °C for 30 min and then transformed into Escherichia coli Trans-T1.

[0091] The results showed that CsPRMT5 was associated with the chromatin region 1000 bp upstream of the transcription start site of defense genes (CsMAPK3, CsMYB73, CsPP2C) Figure 7 a). During pathogen infection, the amount of chromatin DNA immunoprecipitated by anti-CsPRMT5 and anti-H4R3sme2 antibodies decreased significantly, corresponding to the third region of MAPK3 ( Figure 7 bc). In addition, the results of the Ps infection experiment showed that the lesion area of ​​leaves overexpressing CsMAPK3 was significantly smaller than that of the control group 5 days after inoculation, and the level of H4R3sme2 was significantly decreased ( Figure 7 d).

[0092] In summary, the present invention takes the CsPRMT5 gene as the research object, and through inhibiting the expression of the CsPRMT5 gene and conducting physiological indicators after infection with the wheel spot pathogen, it is found that the DAB staining effect of the tea leaves after the expression of the CsPRMT5 gene is inhibited is weaker than that of the control group, and the activity of the key enzyme POD of the ROS scavenging system is higher than that of the control group. The resistance of the tea leaves to the wheel spot pathogen is enhanced after the expression of the CsPRMT5 gene is inhibited.

[0093] The present invention discovered a target gene CsMAPK3 that is negatively regulated by CsPRMT5. By overexpressing the CsMAPK3 gene and infecting the tea leaves with the ring spot pathogen, it was found that the tea leaves overexpressing CsMAPK3 have enhanced resistance to the ring spot pathogen. In view of the effects of the antisense oligonucleotides provided by the present invention, the application of assisting the breeding of tea varieties resistant to the ring spot pathogen and enhancing the tea tree ring spot pathogen resistance model also falls within the scope of protection of the present invention.

[0094] The above description is only a preferred embodiment of the present invention, which is only illustrative and not restrictive of the present invention. Those skilled in the art understand that many changes, modifications, and even equivalences may be made to the present invention within the spirit and scope defined by the claims of the present invention, but all of them will fall within the scope of protection of the present invention.

Claims

1. Application of CsPRMT5 gene in regulating tea tree ring spot disease resistance, characterized in that: The nucleotide sequence of the CsPRMT5 gene is shown in SEQ ID NO.

1.

2. The use of the CsPRMT5 gene in regulating tea tree ring spot disease resistance according to claim 1, characterized in that: The amino acid sequence of the protein encoded by the CsPRMT5 gene is shown in SEQ ID NO.

2.

3. The use of the CsPRMT5 gene in resistance to tea tree ring spot disease as claimed in claim 1, characterized in that: The CsPRMT5 gene is a negative regulatory factor for tea tree ring spot disease resistance.

4. The use of the CsPRMT5 gene in resistance to tea tree ring spot disease as claimed in claim 1, characterized in that: The CsPRMT5 gene regulates the ring spot disease resistance by regulating the expression of the CsMAPK3 gene. The regulation of the tea tree ring spot disease resistance is: overexpressing the CsMAPK3 gene to increase the resistance of the tea tree to the ring spot pathogen. The nucleotide sequence of the CsMAPK3 gene is shown in SEQ ID NO.

3.

5. The use of the CsPRMT5 gene in resistance to tea tree ring spot disease as claimed in claim 1, characterized in that: The nucleotide sequence of the antisense oligonucleotide for inhibiting the expression of the CsPRMT5 gene is shown in SEQ ID NO.4.

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