Application of CsPRMT5 gene in resistance to tea annular spot disease

Abstract

The application relates to the technical field of genetic engineering, and particularly relates to application of a CsPRMT5 gene in resisting camellia sinensis ring spot disease, a base sequence of the CsPRMT5 gene is shown as SEQ ID NO. 1, the CsPRMT5 gene is a negative regulation factor of the camellia sinensis ring spot disease resistance, and the ring spot disease resistance is regulated by regulating CsMAPK3 expression; silencing CsPRMT5 in the camellia sinensis can significantly improve the disease resistance, and overexpression can increase the sensitivity of the camellia sinensis to pathogenic bacteria. The regulation mechanism of the CsPRMT5 gene to the camellia sinensis ring spot disease resistance disclosed in the application provides a basis for applying the CsPRMT5 gene to resisting the camellia sinensis ring spot disease.

Description

TECHNICAL FIELD

[0001] The application relates to the field of genetic engineering, in particular to application of CsPRMT5 gene in resisting tea tree ring spot disease. BACKGROUND

[0002] Camellia sinensis, belonging to Theaceae, is an evergreen woody plant with important economic value. Its tender buds are rich in various secondary metabolites, such as polyphenols, catechins, flavanones, caffeine, theanine, saponins, vitamins, minerals and volatile oils, and are widely used to produce the popular non-alcoholic beverage-tea. As an evergreen plant, tea tree is susceptible to biological stress such as bacteria, fungi and viruses in terms of growth and tea quality, thereby affecting its normal growth and development, and leading to a significant decrease in global tea yield and quality.

[0003] Tea tree ring spot disease caused by Pestalotiopsis-like species is one of the most serious leaf diseases, mainly affecting mature and old leaves, and also threatening tender buds. Although ring spot disease can be chemically controlled, long-term use of chemical fungicides will make the pathogenic bacteria increasingly resistant, and also pollute the environment. In contrast, breeding of resistant varieties is considered to be a more economical, more environmentally friendly and more effective strategy for preventing ring spot disease, and functional analysis of key genes of tea tree ring spot disease resistance is the basis for constructing a genetic network and carrying out molecular design breeding. Therefore, it is urgent to mine genes that play an important role in the interaction between tea tree and ring spot disease and reveal the potential molecular mechanism.

[0004] At present, in order to cope with the attack of pathogenic bacteria, 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 response. Histone methylation is a widely existing and key epigenetic modification, which occurs on lysine and arginine residues. Recent studies have shown that histone lysine methylation markers play a crucial role in plant response 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, H3K4 demethylase JMJ14 regulates immune response by regulating the deposition of H3K4me3 markers, thereby affecting the transcription of key defense genes (such as PR1, FMO1 and SNI1). In addition, OsJMJ705 removes H3K27me3 of defense-related genes, thereby enhancing resistance.

[0005] Recent studies have shown that protein arginine methyltransferase 5 (PRMT5) plays a role in the immunity of various 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, which is a modified inhibitory marker for transcriptional regulation. AtPRMT5 acts as a negative regulator to regulate the immune response to oomycetes and AvrRpt pathogenic bacteria. Specifically, bacterial infection leads to a decrease in PRMT5 expression, accompanied by a decrease in the level of arginine methylation of key proteins such as AGO2 and LSM4. By dual regulation of AtAGO2, PRMT5 regulates the immune response of plants to pathogens. In contrast, the role of OsPRMT5 in rice against Magnaporthe oryzae is different. Studies have shown that OsPRMT5 acts as a positive regulator of rice immunity. After rice is infected with Magnaporthe oryzae, OsPRMT5 regulates AGO2 activity through arginine methylation and interacts with miR1875, affecting the immune response of rice. The results show that PRMT5 may have different roles in different species, and its role in the disease resistance mechanism of tea plants still needs further study.

[0006] In view of the above defects, the inventor has finally obtained the present application after a long period of research and practice. SUMMARY

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

[0008] In order to achieve the above-mentioned purpose, the application discloses the application of CsPRMT5 gene in tea tree Cercospora resistance, and the nucleotide sequence of the CsPRMT5 gene is shown as SEQ ID NO. 1.

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

[0010] The CsPRMT5 gene is a negative regulator of tea tree Cercospora resistance.

[0011] The CsPRMT5 gene regulates Cercospora resistance by regulating the expression of CsMAPK3 gene, and the regulation of tea tree Cercospora resistance is that overexpression of CsMAPK3 gene increases the resistance of tea tree to Cercospora fungus, and the nucleotide sequence of the CsMAPK3 gene is shown as SEQ ID NO. 3.

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

[0013] The antisense oligonucleotide provided by the application can inhibit CsPRMT5 gene expression, improve the activity of key enzymes POD of ROS clearance system, increase the expression of resistance genes such as CsMAPK3, and improve the resistance of tea tree leaves to Cercospora.

[0014] Compared with the prior art, the beneficial effects of the application are that the application provides the application of CsPRMT5 gene in regulating the sensitivity of tea tree to Cercospora, and the nucleotide sequence of CsPRMT5 gene is shown as SEQ ID NO. 1. The application studies the relationship between the expression of CsPRMT5 gene in different tea tree varieties and disease resistance, and shows that CsPRMT5 is a negative regulatory factor for tea tree defense, and overexpression of CsPRMT5 in tea tree shows a larger lesion area and weaker Ps resistance, so CsPRMT5 can be used to negatively regulate the resistance of plants to Cercospora. The application inhibits the expression of CsPRMT5 gene by antisense oligonucleotide and the physiological indexes after Cercospora infection, and finds that the DAB staining effect of tea tree leaves after inhibiting the expression of CsPRMT5 gene is weaker than that of the control group, the activity of key enzymes POD of ROS clearance system is higher than that of the control group, the expression amount of resistance related genes such as CsMAPK3 is higher than that of the control group, and the resistance of tea tree leaves after inhibiting the expression of CsPRMT5 gene to Cercospora is increased. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The time course analysis of histone H4 arginine residue methylation level in tea tree is as follows:

[0016] (a) is the separation of histone from tea tree leaves after 0, 24, 48 and 72 hours, Western blot analysis is carried out, and an antibody against histone methylation marker is used. Different methylation modifications are represented by numbers relative to the plant level at the 0 hour point of inoculation; (b) is the expression pattern of PRMT family genes in infected leaves. FPKM values are represented in the form of a heat map; (c) is the expression analysis of CsPRMT5 in tea tree leaves infected with Ps at different time points, relative to the double distilled water control group (CK); (d) is the immunoblot analysis of CsPRMT5 and H4R3sme2 levels after Ps infection, the data is represented as mean ± standard deviation (n = 3), and statistical significance (p < 0.05) is indicated by different letters, based on Duncan multiple range test. *P < 0.05, **P < 0.01.

[0017] Figure 2 The correlation between disease resistance and CsPRMT5 expression in different tea tree varieties is as follows:

[0018] (a) is the phenotype of different tea varieties, scale = 1 cm; (b) is the expression and protein level of CsPRMT5 in different tea varieties; (c) is the phenotype of different tea varieties at 5 dpi; (d) is the total lesion area of different tea varieties after infection; (e) is the correlation between disease resistance and CsPRMT5 expression level in different varieties, CsGAPDH as internal control, data 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 Pathogenic phenotype and related gene expression in CsPRMT5-silenced tea plants, wherein

[0020] (a) is the schematic diagram of AsODN gene silencing; (b) is the relative expression level of CsPRMT5 in AsODN and sODN plants; (c) is the immunoblot analysis of CsPRMT5 and H4R3sme2 levels in AsODN and sODN plants; (d) is the disease symptoms of CsPRMT5-silenced tea plants and control (sODN) plants after fungal infection, scale = 2 mm; (e) is the relative expression level of PR genes in sODN and AsODN tea plants; (f) is the diaminobenzidine (DAB) staining of tea leaves 48 hours after Ps inoculation; (g) is the H2O2 content; (h) is the CAT activity; (i) is the POD activity; data 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;

[0021] Figure 4 Pathogenic phenotype and related gene expression in CsPRMT5-overexpressed tea plants, wherein

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

[0023] Figure 5 For the interaction of transgenic Arabidopsis leaves with Ps, wherein:

[0024] (a) is the analysis by immunoblotting of WT, skb1, Com2 Arabidopsis plants; (b) is the 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, the photos were taken at 5 dpi; (c) is the average statistics of lesions at 7 dpi; (d) is the RT-qPCR analysis of AtPR1 transcription levels in WT, Com2 and skb1 Arabidopsis plants after Ps infection; (e) is the H2O2 content; (f) is the POD activity; (g) is the SOD activity, the data are expressed as mean ± standard deviation (n = 3), the scale = 1 cm, the significant differences between the treatment and control groups were calculated by one-way ANOVA of SPSS 21.0, *P < 0.05, **P < 0.01, ***P < 0.001.

[0025] Figure 6 For the regulation of CsPRMT5 on immune genes related to gray mold disease response, wherein:

[0026] (a) is a bar graph showing the differentially regulated genes in CsPRMT5 silenced plants, transcriptome analysis was performed on tea samples effectively silenced CsPRMT5 by RNA sequencing; (b) is the GO enrichment analysis of differentially regulated genes in CsPRMT5 silenced plants, the screening criteria for differentially regulated genes are fold change of gene expression > 2, and adjusted P value < 0.05; (c) is a pathogen-related gene heat map generated based on the published RNAseq results of tea leaf brown spot disease, CK represents the control group, Ps 8, 24, 72 hours correspond to different time points after infection, the color bar (from white to red) represents the TPM (log2) of the gene as z-score; these genes are up-regulated in CsPRMT5 silenced plants; (d) is the effect of CsPRMT5 silencing on potential target genes under control (CK) and pathogen stress by RT-qPCR analysis, the 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.

[0027] Figure 7 For the binding of CsPRMT5 to the chromatin of resistance genes and the decrease of H4R3sme2 levels in tea after brown spot disease infection, wherein:

[0028] (a) CsPRMT5 binding to chromatin containing defense-related genes for ChIP-seq analysis; (b) CsMAPK3 gene structure, black boxes represent exons, and black lines represent introns, ChIP experiment of CsMAPK3 using anti-CsPRMT5 and anti-H4R3sme2 antibodies, chromatin extracted from tea leaves treated with Ps for 24 hours, compared with the untreated control group; (c) CsMAPK3 expression levels in tea leaves infected with Ps at different time points, relative to the double distilled water control group (CK); (d) fungal infection disease symptoms of CsMAPK3 overexpression and control plants, scale = 2 mm; (e) fungal infection disease symptoms of CsMAPK3 silenced tea plants (CsMAPK3-TRV) and control (TRV) plants, scale = 2 mm, data are expressed as mean ± standard deviation (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. DETAILED DESCRIPTION

[0029] The above and other technical features and advantages of the present application will be more apparent from the following detailed description, taken in conjunction with the accompanying drawings.

[0030] All the following experiments were performed with at least three independent biological replicates, each measured three times. Statistical significant differences between control plants and experimental plants were compared using one-way analysis of variance (ANOVA), with Duncan's test (P < 0.05), and the analysis was performed by SPSS software. The tea variety "Shuchazao" used in the present application is from Niancheng Agricultural Ecology Co., Ltd. of Chaohu City, Anhui Province, and is the main tea material for this study. The tea was inoculated with the pathogen Pseudopestalotiopsis (Ps) for the experiment. The skb1-1 mutant (salk_065814) and its complemented line (35S:CsPRMT5 / skb1-1) were also used in the present application, which were obtained by the laboratory in the early stage. All plants were cultivated under suitable environmental conditions, with a growth temperature of 25 ± 1°C and a light cycle of 16 hours light / 8 hours dark.

[0031] Example 1

[0032] Identification of tea PRMT gene family

[0033] Based on the PRMT and PrmA domains (PF05185, PF06325), the incomplete structure, which does not contain the PRMT-related domain, is eliminated, and the candidate sequence is identified and verified, and finally the identified PRMT family members are used for subsequent analysis. (Table 1). Nine PRMT genes were identified. Among them, PRMT1A-C, PRMT4A-B, PRMT10A-C are 8 type I genes. The heat map analysis of the above nine genes was performed using the published transcriptome of the leaf spot disease of the tea tree genome website (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 in histone arginine methylation after leaf spot disease infection

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

[0042] 2. Samples were collected at 0, 24, 48 and 72 hours (hpi) after infection, and verified by RT-RT-qPCR, and Western Blot analysis was performed 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 Aibio Antibody, predicted molecular weight 10 / 15 kDa). Proteins were separated in 12% SDS / PAGE gels and transferred to nitrocellulose membranes (Amersham Protran, 0.45 μm). ACTIN was used as a loading control (Shanghai Bioengineering, predicted molecular weight 42 kDa).

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

[0044] 4. Detailed steps of histone extraction: Weigh 0.5g sample, grind into fine powder with liquid nitrogen, then add to 3mL pre-cooled nuclear protein extraction buffer, shake on ice for 10min. 4°C, 10000g, centrifuge for 10min, leave the precipitate. Then, add 600ul 0.4M H2SO4, resuspend and continue to shake on ice for 1h. Again, 4°C, 10000g, centrifuge for 10min. Transfer the supernatant to a container containing 12 times the volume of pre-cooled acetone, place at -20°C for more than 8h. Again, 4°C, 8000g, centrifuge for 15min. Finally, after blowing dry in a fume hood, add 200ul 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 1x Protease Inhibitor Cocktail, 0.1mM PMSF, 0.035% β-Mercaptoethanol.

[0045] According to Figure 1 It can be seen that the methylation level of each arginine site changes at different time points after Ps inoculation. The expression pattern of PRMT5 is consistent with the trend of change of H4R3sme2 level after Ps infection, and the RT-qPCR result shows that the transcription level of CsPRMT5 is significantly reduced at 24h and 48h after infection, and recovered at 72h Figure 1 c).

[0046] Example 3

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

[0048] Seven tea cultivars were selected, including “Longjing 43” (LJ43), “Shuchazao” (SCZ), “Wuniuzao” (WNZ), “Fuyun 6” (FY6), “Longjingchangye” (LJCY), “Zhenong 113” (ZN113), and “Pingyangtezao” (PYTZ), which were all from the National High-tech Agricultural Park of Anhui Agricultural University. The expression of CsPRMT5 in uninfected leaves of each cultivar was quantified, and the second leaf was selected for pathogen inoculation. The lesion area was measured 5 days post inoculation (dpi) Figure 2 b-d).

[0049] According to Figure 2 The results showed that the CsPRMT5 expression levels of FY6 and PYTZ were lower, and they showed higher resistance; while the CsPRMT5 transcription levels of SCZ and WNZ were higher, and they showed lower resistance. The lesion area and CsPRMT5 expression level were significantly positively correlated in each tea cultivar (r = 0.83, **p < 0.01) Figure 2 e). These findings indicated that CsPRMT5 acts as a negative regulator of defense in tea.

[0050] Example 4

[0051] AsODN verified the physiological function of CsPRMT5

[0052] 1. The antisense oligonucleotide (AsODNs) candidate sequence for CsPRMT5 was designed using Soligo software (https: / / pubmed.ncbi.nlm.nih.gov / 31828806 / ) (SEQ ID NO. 4), and a standard oligonucleotide (sODNs) was used as a control. The AsODN experiment was performed on the tender buds, which were soaked in 100 mM AsODN or sODN solution. After 48 hours, the samples were collected to detect the silencing efficiency of CsPRMT5.

[0053] 2. The tea plants with CsPRMT5 silenced and the control tea plants were inoculated with the spore suspension of Ps. The lesion area was counted, and the disease resistance-related genes and reactive oxygen species were detected. The reactive oxygen species were detected by placing the pathogen-inoculated leaves in 3,3'-diaminobenzidine (DAB) solution (1 mg / mL, pH 3.8) and incubating at 37°C for 8 hours. The sample was pretreated by adding 0.02 g of sample to 180 pL of phosphate buffer (pH 7.0) and grinding on ice, and the ground homogenate was centrifuged at 3500 g for 10 min at 4°C. 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 Solapbio Science and Technology Co., Ltd., Beijing, China).

[0054] RT-qPCR and immunoblotting analysis confirmed that CsPRMT5 transcript and protein levels were significantly reduced in AsODN-treated tea plants compared to the control (sODN), accompanied by a clear decrease in H4R3sme2 levels Figure 3 a-c). Tea leaves with CsPRMT5 silenced had significantly smaller lesion areas than the control at 5 days post inoculation Figure 3 d). Pathogenesis-related genes (PR genes) play an important role in the immune response of tea. These genes were induced at the early stage of Ps infection in both CsPRMT5-silenced and control tea plants, but the induction of PR genes was significantly enhanced in CsPRMT5-silenced plants Figure 3 e). H2O2 levels were lower and POD and SOD enzyme activities were higher in CsPRMT5-silenced plants. In contrast, ROS accumulation was significantly higher in CsPRMT5-overexpressing leaves than in the control (EV) Figure 4 These results suggest that CsPRMT5 acts as a negative regulator of Ps in tea.

[0055] Example 5

[0056] Verification of the physiological function of CsPRMT5 overexpression

[0057] 1. Young roots of the national good variety Shuchaozao tea plant (grown in the agricultural industrial park of Anhui Agricultural University in Hefei, Anhui Province, China) were used for RNA extraction. Total RNA was extracted using the RNAprep Pure Plant Kit (Tiangen, Beijing, China) according to the manufacturer's instructions, and the RNA concentration was determined using a nucleic acid detector after extraction.

[0058] 2. First-strand cDNA was generated by reverse transcription, and the first-strand cDNA was used as a template for PCR amplification of the CsPRMT5 gene. The PCR amplification system was as follows: 10 μL of KOD enzyme, 1 μL of each upstream and downstream primer, 1 μL of template, 7 μL of ddH2O, and a total volume of 20 μL.

[0059] The PCR amplification program was as follows: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 sec, 55°C annealing for 30 sec, 72°C extension for 2 min, 32 cycles; and 72°C extension 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 step is recovered by using a gel recovery kit, ligated to the entry vector pDNOR207 vector, and after colony PCR verification, sent to Shanghai Shengong Sequencing Company for sequencing. The specific sequence is shown in SEQ ID NO. 1, and the amino acid sequence encoded by the CsPRMT5 gene is shown in SEQ ID NO. 2.

[0064] 4, After sequencing verification, it is respectively connected to pB2GW7 and pK7WGF vectors, and after successful construction, it is transformed into Agrobacterium GV3101 strain. Among them, pB2GW7-CsPRMT5 is used for stable construction of transgenic Arabidopsis, and pK7WGF-CsPRMT5 is used for transient overexpression experiment of tea tree.

[0065] 5, The dipping flower method is used for stable transformation of Arabidopsis, and the specific operation steps are as follows:

[0066] (1) The Agrobacterium GV3101 introduced with the pB2GW7-CsPRMT5 recombinant plasmid is taken out from -80℃, and streaked. Then single colonies are picked and cultured in liquid LB medium containing 50 mg / L rifampicin and hydrochloric acid spectinomycin, and incubated at 28℃ overnight. The bacterial cells are collected.

[0067] (2) The bacterial cells are suspended in Arabidopsis infection Buffer, and the Arabidopsis infection Buffer (100 mL) is 10% sucrose and 20 μl Silwet-L 77. The OD600 is adjusted to about 1.0, and the just flowering mutant skb1-1 (salk_065814) is used for infection by the dipping flower method.

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

[0069] (4) After one week, the above operation is performed again for one time of infection.

[0070] (5) The seeds of the infected Arabidopsis are recorded as T0 generation. The received seeds are first dehydrated in a 37℃ oven for 3 d, and then stored in a 4℃ refrigerator with silica gel desiccant.

[0071] (6) An appropriate amount of seeds is sown in the soil, and after about 10 d, herbicide is sprayed for positive plant screening.

[0072] (7) Three days later, continue to spray once, and there is no death as positive seedlings, and the seedlings are transplanted for single plant culture and seed collection of T1 generation. The same operation is performed until the T3 generation is screened for seed collection, and the seeds are preserved for subsequent inoculation experiments.

[0073] 6. The specific operation steps of tea tree transient overexpression of CsPRMT5 are as follows:

[0074] The constructed pK7WGF-CsPRMT5 and pK7WGF are respectively transferred into GV3101 Agrobacterium, and single colonies are picked for PCR verification, and then they are placed in liquid LB with Spec+Rif for culture until the OD600 value reaches about 0.8. The cultured bacteria liquid is centrifuged at 5000 r / min for 10 min, the supernatant is discarded, and the bacteria are resuspended to OD600 of 0.8 with resuspension solution (0.4066 g MgCl2 6H2O (magnesium chloride hexahydrate), 0.4265 g MES (2-ethanesulfonic acid), 200 μl 1 mol / L acetyl-syringone, and pure water to 200 ml). Using a syringe, inject into the back of tea tree leaves, and perform inoculation experiments after 48 hours. In the later stage, the lesion area is counted, and the disease resistance related genes and active oxygen are detected (such as Example 4).

[0075] RT-qPCR results show that the expression of CsPRMT5 is significantly up-regulated. Compared with the control group, CsPRMT5 overexpressed tea trees show larger lesion areas and weaker resistance to Ps. The resistance of Arabidopsis skb1-1 (salk_065814) mutant to Ps is significantly enhanced, while Com2 plants are more susceptible to Ps infection Figure 5 b) According to the percentage of loss of green area of leaves, the disease severity is divided into four levels: level 1 (<10%), level 2 (10-25%), level 3 (25-50%) and level 4 (>50%). The results show that most of Com2 and WT plants belong to level 4 severity, while most of skb1-1 mutants belong to level 1 severity Figure 5 c) Consistent with the phenotype results, AtPR1 in Com2 plants is significantly induced Figure 5 d) It can be seen from Figure 5 (e)- Figure 5 (g) that H2O2 accumulates more strongly in Com2 leaves, and SOD and POD activities are lower, while H2O2 levels in skb1-1 plants are almost unchanged.

[0076] Example 6

[0077] CsPRMT5 participates in tea tree resistance to Ps through regulating the expression of CsMAPK3

[0078] 1. Detailed steps of chromatin immunoprecipitation:

[0079] The samples 24 hours after pathogenic bacteria infection were washed with deionized water, dried with absorbent paper, placed in 50 mL centrifuge tubes, and cross-linked with 1% formaldehyde for 30 minutes under vacuum conditions. Then 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. Then the chromatin was fragmented by ultrasonic disruption. The DNA fragments bound to proteins were immunoprecipitated with anti-H4R3sme2 antibody (Shanghai Abmart, 1:5000 dilution). The immunoprecipitated DNA fragments were extracted and purified for ChIP-qPCR analysis. Glycerolaldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene, and the primers used for ChIP-qPCR analysis are listed in Table 3. ChIP-seq data was derived from previous laboratory studies.

[0080] Table 3 Primers used for 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 for PCR amplification using the upstream primer and the downstream primer.

[0084] Upstream primer:

[0085] 5'-aggacagcccagatcactagtATGGCTGACTCAAACACAGTCG-3';

[0086] Downstream primer:

[0087] 5'-gcccttgctcaccatggatccTGCAAACTCTGGATTGAGTGC-3';

[0088] PCR amplification procedure was as follows: 94°C pre-denaturation 3 min; 94°C denaturation 30 sec, 58°C annealing 30 sec, 68°C extension 2 min, 32 cycles; 72°C extension 10 min. The PCR product was recovered by agarose gel electrophoresis. The PCR product and pCAMBIA1305 vector (recovered after digestion with Spe I and BamHI restriction enzymes) were ligated using homologous recombination enzyme (One Step Cloning kit, Nanjing NorgenBiotek Co., Ltd., Nanjing, China) at 37°C for 30 min, and the recombination product was used for Trans1-T1 competent cell transformation. Sequencing verification was performed to confirm whether CsMAPK3 was ligated to obtain pCAMBIA1305-CsMAPK3. The pCAMBIA1305-CsMAPK3 plasmid was transformed into GV3101 Agrobacterium with pCAMBIA1305, and single colonies were picked for PCR verification. Then, they were cultured in liquid LB with Kan+Rif until the OD600 value reached about 0.8. A syringe was used to inject the tea tree leaves on the back, and inoculation experiments were performed 48 hours later. The lesion area was counted in the later stage.

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

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

[0091] It was found that CsPRMT5 was associated with the chromatin region 1000 bp upstream of the transcription start site of the 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 b-c). In addition, the results of 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 decreased significantly Figure 7 d).

[0092] In summary, the application takes CsPRMT5 gene as the research object, and finds that the tea tree leaf after inhibiting CsPRMT5 gene expression has weaker DAB staining effect compared with the control group, the key enzyme POD activity of the ROS scavenging system is higher than that of the control group, and the tea tree leaf after inhibiting CsPRMT5 gene expression has enhanced resistance to the algal leaf spot fungus.

[0093] The application finds that the target gene CsMAPK3 is negatively regulated by CsPRMT5, and finds that the tea tree leaf after overexpressing CsMAPK3 gene has enhanced resistance to the algal leaf spot fungus. In view of the effect of the antisense oligonucleotide provided by the application, the application of the antisense oligonucleotide in assisting the breeding of tea tree varieties resistant to the algal leaf spot fungus and the enhancement of the model of the tea tree resistance to the algal leaf spot fungus also belongs to the protection scope of the application.

[0094] The above description is only the preferred embodiment of the application, which is only illustrative but not limiting. It is understood by those skilled in the art that many changes, modifications and even equivalents can be made to the application within the spirit and scope defined by the claims of the application, and all of them will fall within the protection scope of the application.

Claims

1. The application of the CsPRMT5 gene in regulating resistance to tea leaf spot disease, characterized in that, The regulation is as follows: by silencing the CsPRMT5 gene in tea plants, the resistance of tea plants to leaf spot disease is improved; the nucleotide sequence of the CsPRMT5 gene is shown in SEQ ID NO.

1.

2. The application of the CsPRMT5 gene as described in claim 1 in regulating resistance to tea leaf spot disease, characterized in that, The amino acid sequence of the protein encoded by the CsPRMT5 gene is shown in SEQ ID NO.

2.

3. The application of the CsPRMT5 gene as described in claim 1 in resistance to tea leaf spot disease, characterized in that, The nucleotide sequence of the antisense oligonucleotide that inhibits the expression of the CsPRMT5 gene is shown in SEQ ID NO.4.

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