Tea tree CsPR10-5 gene and application thereof in enhancing anthracnose resistance

Abstract

The invention discloses a tea tree CsPR10-5 gene and application thereof in enhancing anthracnose resistance of a tea tree, and belongs to the technical field of gene engineering. The nucleotide sequence of the CDS region of the gene is as shown in SEQ ID NO. 1. The invention further provides an application in regulating the anthracnose sensitivity of the tea tree, an anthracnose-sensitive tea tree model and an anthracnose-resistant tobacco model. The tea tree CsPR10-5 gene has the beneficial effects that the effect of the tea tree CsPR10-5 gene in regulation and control of tea tree anthracnose infection is provided, the tea tree CsPR10-5 gene has different expression quantities at different time points of anthracnose infected tea trees and can respond to germ infection at different time points, so that the biological function of mediating the anthracnose resistance of the tea trees is verified, and a key gene resource is provided for disease-resistant breeding of the tea trees.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, in particular to a tea tree CsPR10-5 gene and its application in enhancing the resistance of tea trees to anthracnose. BACKGROUND

[0002] Tea tree is one of the most important economic crops in China. Tea contains many substances beneficial to the human body, such as flavonoids, tea polyphenols, alkaloids, and soluble sugars. The biological characteristics of tea trees, which prefer warm and humid environments, determine that their growth cannot be separated from warm and humid environments, which provides favorable conditions for the occurrence of anthracnose. Tea tree anthracnose is a fungal disease that is prone to spread in late spring, early summer, and autumn, mainly damaging tea tree leaves and tender shoots, leading to leaf loss, weakened tree vigor, reduced tea yield, and decreased tea quality. Currently, research on tea tree anthracnose has mainly focused on differential expression analysis based on transcriptomics and proteomics, but the mining of defense-related genes and their biological functions in mediating tea tree resistance to disease are not clear, and their application in disease resistance is insufficient.

[0003] Chinese patent application document with publication number CN118440954A discloses a tea tree CsLAC23 gene and its application in resisting infection by anthracnose fungus. The patent also proposes the application of tea tree CsLAC23 gene in regulating tea tree anthracnose infection and in breeding new tea tree varieties resistant to anthracnose. It also proposes an antisense oligonucleotide that inhibits the expression of CsLAC23 gene. After inhibiting the expression of CsLAC23 gene and performing anthracnose fungus infection, it is found through tissue staining and determination of active oxygen content that the DAB and NBT staining effects of tea tree leaves after inhibiting the expression of CsLAC23 gene are more obvious than those of the control group, and the activities of key enzymes POD and SOD in the ROS scavenging system are lower than those of the control group. Tea trees are more sensitive to anthracnose infection after inhibiting the expression of CsLAC23 gene, indicating that CsLAC23 enhances the resistance of tea trees to anthracnose stress. However, there is no report on whether other tea tree genes also have the effect of resisting anthracnose fungus infection. SUMMARY

[0004] The technical problem to be solved by the present application is how to propose a new tea tree gene that can resist anthracnose fungus and apply it to resist anthracnose fungus infection.

[0005] The present application solves the above technical problems through the following technical means:

[0006] The first aspect of the present application proposes a tea tree CsPR10-5 gene, and the nucleotide sequence of the CDS region is shown in SEQ ID NO. 1.

[0007] The second aspect of the present application proposes a protein encoded by the above-mentioned tea tree CsPR10-5 gene, and the amino acid sequence is shown in SEQ ID NO. 2.

[0008] The third aspect of the present application provides the use of the tea tree CsPR10-5 gene in regulating the susceptibility of tea tree to anthracnose.

[0009] The fourth aspect of the present application provides the use of the tea tree CsPR10-5 gene in improving the anthracnose resistance of tea tree in breeding.

[0010] The fifth aspect of the present application provides a model of anthracnose-susceptible tea tree, which contains a product inhibiting the expression of CsPR10-5 gene.

[0011] Preferably, the product includes an antisense oligonucleotide with the sequence shown in SEQ ID NO. 3.

[0012] The sixth aspect of the present application provides a model of anthracnose-resistant tobacco, which contains a product over-expressing the tea tree CsPR10-5 gene in tobacco.

[0013] The seventh aspect of the present application provides a tea tree expression vector pCAMBIA1305-CsPR10-5, which is obtained by enzyme cutting the fragment shown in SEQ ID NO: 1 to the pCAMBIA1305 vector.

[0014] The eighth aspect of the present application provides a method for cultivating an anthracnose-resistant plant variety, which introduces the tea tree CsPR10-5 gene into a target plant to obtain a transgenic plant with improved anthracnose resistance.

[0015] Preferably, the method specifically includes the following steps:

[0016] (1) cloning the tea tree CsPR10-5 gene;

[0017] (2) constructing a tea tree CsPR10-5 gene over-expression vector;

[0018] (3) transforming the target plant with the tea tree CsPR10-5 gene over-expression vector, and obtaining a transgenic plant with improved anthracnose resistance through identification.

[0019] Preferably, the plant includes one or more of tea tree and tobacco.

[0020] The present application has the following beneficial effects:

[0021] 1. The present application proposes the use of tea tree CsPR10-5 gene in regulating the infection of tea tree by anthracnose, which has different expression levels at different time points of anthracnose infection, can respond to the infection of pathogenic bacteria at different time points, and further verifies its biological function in mediating the resistance of tea tree to anthracnose, thereby providing key gene resources for the breeding of tea tree for disease resistance.

[0022] 2. The application is found that the lesion area is significantly larger than that of the control group after the inhibition of CsPR10-5 gene expression in tea plants and the infection of anthracnose; the staining effect of tea leaves after the inhibition of CsPR10-5 gene expression is more obvious after NBT and DAB staining; the key enzymes POD and SOD for ROS scavenging activity are significantly lower than those of the control group, and the content of hydrogen peroxide is significantly increased.

[0023] 3. The application is found that the leaf necrosis area is significantly smaller than that of the control after transient overexpression of CsPR10-5 gene in N. benthamiana and infection of anthracnose; the content of hydrogen peroxide, the main component of ROS, is significantly decreased, and the activities of key enzymes POD and SOD for ROS scavenging are significantly higher than those of the control group. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Figure is the expression pattern diagram of tea CsPR10-5 gene at different time points of anthracnose infection in Example 1 of the application;

[0025] Figure 2 Figure is the expression pattern diagram of tea CsPR10-5 gene in different tissues in Example 1 of the application;

[0026] Figure 3 Figure is the subcellular localization diagram of tea CsPR10-5 protein in tobacco in Example 1 of the application;

[0027] Figure 4 Figure is the verification diagram of CsPR10-5 protein having DNase activity in Example 1 of the application;

[0028] Figure 5 Figure is the phenotype, lesion area and antioxidant enzyme activity diagram of tobacco leaves after transient overexpression of CsPR10-5 gene in tobacco and infection of anthracnose in Example 1 of the application; wherein A is the position diagram of tobacco leaf injected with vector bacterial liquid of treatment group and control group, B is the leaf phenotype diagram under natural light, C is the leaf phenotype diagram under chlorophyll fluorescence irradiation, D is the tobacco leaf lesion area statistical diagram of fusion expression vector (treatment group, Cg) and empty load (control, CK) injection, E is the POD enzyme activity diagram of tobacco leaves of treatment group and control group, F is the SOD enzyme activity diagram of tobacco leaves of treatment group and control group, G is the H2O2 content diagram of tobacco leaves of treatment group and control group;

[0029] Figure 6To reduce the resistance of tea plant to anthracnose by antisense inhibition of CsPR10-5 gene in Example 1 of the present application; wherein A is the qRT-PCR analysis of the expression level of CsPR10-5 in antisense inhibition treated tea plant leaves; B is the leaf phenotype chart of the treated group (AsODN) and the control group (H2O and sODN) respectively infected and not infected with anthracnose, including chlorophyll fluorescence imaging, NBT and DAB staining chart; C is the average lesion size statistics of tea plant leaves inoculated with anthracnose after H2O, sODN and AsODN treatment (n = 28); D-F are the contents of H2O2, POD and SOD in anthracnose infected leaves after antisense inhibition of CsPR10-5 gene; different letters represent significant differences between groups (P < 0.05, n = 5). DETAILED DESCRIPTION

[0030] To make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application. Unless otherwise defined, the professional terms used below are consistent with the meanings understood by those skilled in the art.

[0031] The test materials and reagents used in the following examples, unless otherwise specified, can be obtained commercially or prepared by known methods.

[0032] Unless otherwise specified, the quantitative tests in the following examples all set up more than three repeated experiments, and the results are the average values.

[0033] Example 1:

[0034] 1. Cloning of CsPR10-5 gene of tea plant

[0035] (1) Fresh Shuchaya tea plant samples were quickly transferred to centrifuge tubes with pre-added steel balls, and a ball mill was used to grind them into powder. The subsequent steps were carried out according to the instructions of Universal Plant Total RNA Isolation Kit (Norgen Biotek Corporation). Universal Plant Total RNA Isolation Kit extraction kit (Norgen Biotek Corporation) instructions, extract its RNA.

[0036] (2) Reverse transcription to generate first strand: according to the PrimeScript II 1st Strand cDNA Synthesis Kit (Takara Biotech, China) kit instructions. Take 1 μg of RNA as a template, add 1 μL Random 6mers, 1 μL dNTP Mixture, RNase-Free H2O to 10 μL, 65 °C denaturation for 5 min, immediately placed on ice for 2 min; then add 4 μL 5x PrimerScript buffer, 0.5 μL RNase Inhibitor, 1 μL PrimerScript RTase to the above reaction solution, ddH2O to 20 μL, 30 °C for 10 min, 42 °C for 1 h, 95 °C for 5 min, 70 °C for 15 min. Take an appropriate amount of reverse transcription product (i.e. cDNA) for subsequent PCR amplification.

[0037] (3) PCR amplification of CsPR10-5 gene using the cDNA first strand as a template.

[0038] wherein the upstream primer is 5'-CTCATTTCCTACACCTCTGT-3' (SEQ ID NO. 4),

[0039] the downstream primer is 5'-AAGGTAGCTCTTCGAACAACT-3' (SEQ ID NO. 5).

[0040] The 25 μL reaction system is: LA Taq premix 12.5 μL, 1 μL of each of the upstream and downstream primers, 1 μL of template, and 9.5 μL of ddH2O. The PCR amplification program is: 94 °C for 3 min, 94 °C for 30 sec, 60 °C for 30 sec, 72 °C for 40 sec, 72 °C for 10 min, 30 cycles.

[0041] (4) The PCR product obtained in (3) is recovered using a gel recovery kit, ligated to a pEASY-T1 vector (Promega, Shanghai, China), and a recombinant plasmid (denoted as pEASY-T1 :: CsPR10-5) is obtained. The recombinant plasmid is transformed into E. coli competent cells Trans1-T1, and the universal biological company is contacted for sequencing to obtain the nucleotide sequence of the CsPR10-5 gene, which is specifically shown in SEQ ID NO. 1, and the amino acid sequence encoded by the CsPR10-5 gene is specifically shown in SEQ ID NO. 2.

[0042] 2. Expression pattern of CsPR10-5 gene under infection of C. gloeosprioides

[0043] Select 2-year-old 'Longjing 43' cuttings with consistent growth and no pests and diseases, and cultivate them in an artificial climate chamber at Anhui Agricultural University (temperature 25°C, humidity 70%, 16h light, 8h darkness). Use an inoculation needle to carefully inoculate the mycelium on a PDA plate, and cultivate it at 28°C in the dark for 7-10 days. Perform aseptic operations throughout. Add ddH2O to the plate, and use a sterile spreader to scrape the pathogenic fungal spores from the plate, filter them with sterile gauze, and then transfer them to a sterile conical flask. Use a vortex to evenly distribute the fungal spores in the liquid. Use a hemocytometer to count the spores under an optical microscope, dilute them with sterile water, and prepare a spore suspension of 10 7 CFU / mL. Wipe the two leaves of the tea seedlings with 75% ethanol, and then wipe them again with ddH2O. Let the water on the leaves evaporate. Use a large needle to make a rosette-shaped hole in the leaves, and add 50uL of the spore suspension to each side. The control group is treated with sterile water. Then wrap the leaves with plastic wrap, and cover the entire tea seedling with a large plastic bag. Place it in the artificial climate chamber and maintain a humidity of 70% or higher. Take samples at 1, 4, 7, 10, and 13 days, and immediately freeze them in liquid nitrogen. Store them at -80°C for later use.

[0044] Extract the RNA and reverse transcribe it into cDNA. Dilute the reverse transcription product 9-fold as a template, use 2x AceQ Universal qPCR Master Mix (Vazyme, Nanjing, China), and prepare a 10uL reaction system: 5uL 2x AceQ Universal qPCR Master Mix, 1.2uL diluted reverse transcription product, 0.3uL of each upstream and downstream primer, and 3.2uL ddH2O. Perform three biological replicates and three technical replicates for each sample. Then perform quantitative analysis on a Bio-rad CFX instrument, with the following program: 95°C for 3min, 95°C for 10sec, 56°C for 30sec, 65°C for 5sec, 95°C for 5sec, 39 cycles. The qRT-PCR technique detects the expression level of CsPR10-5 gene at different time points of the infection of the anthracnose disease, as shown in Figure 1 , and it can be seen from Figure 1 that, compared with the water treatment control group (CK), the expression level of CsPR10-5 gene in the anthracnose fungus infection treatment group (Cg) is significantly increased at 1, 4, 7, 10, and 13d, with the most obvious increase at 7d.

[0045] 3. Expression pattern and subcellular localization of CsPR10-5 gene in tea plants

[0046] (1) Expression pattern of CsPR10-5 gene in different tissues of tea plants

[0047] Different tissues including bud, one leaf, two leaves, three leaves, mature leaf, stem, flower and root were collected from the national tea cultivar 'Shuchazao'. Eight samples were used for total RNA extraction and cDNA first strand synthesis. Quantitative analysis was performed as described above, and the results are shown in Fig. 1. The CsPR10-5 gene was expressed in all eight organs, with the highest expression in flowers, followed by fruits, and the expression in mature leaves was significantly higher than that in young leaves and buds. Figure 2

[0048] The upstream primer was 5'-ACCCAGCAGGATGTTCAAG-3'(SEQ ID NO. 6), and the downstream primer was 5'-CTTGATGCTTCCAACACCAC-3'(SEQ ID NO. 7).

[0049] The downstream primer was 5'-CTTGATGCTTCCAACACCAC-3'(SEQ ID NO. 7).

[0050] (2) Subcellular localization of tea CsPR10-5 protein

[0051] ① Construction of the expression vector of the target gene

[0052] The pCAMBIA1305 vector was double-digested with Spe I and BamH I restriction enzymes, and then subjected to agarose gel electrophoresis and gel recovery after 37°C reaction for 30 min to obtain a linearized vector with a restriction enzyme site. The plasmid was amplified with primers with a restriction enzyme site using the target gene plasmid as a template to obtain a target gene fragment with a restriction enzyme site. The upstream primer was 5'-GACAGCCCAGATCACTAGTATGGGTGTAACTACTTTTACC-3'(SEQ ID NO. 8), and the downstream primer was 5'-CTTGCTCACCATGGATCCAGCATAAACATCAGGGTTTGCT-3'(SEQ ID NO. 9). The product with a restriction enzyme site linker sequence and the linearized vector were recombined using recombinant enzyme, and the reaction system was 10 μL: linearized pCAMBIA1305 vector 4 μL, product with a restriction enzyme site linker sequence 2 μL, recombinant enzyme E×nase II 1 μL, 5×CE II Buffer 2 μL, added in order in a 200 μL PCR tube. The reaction was performed at 37°C for 60 min. The recombined product was transformed into E. coli competent cells Trans1-T1, and the pCAMBIA1305-CsPR10-5 plasmid was obtained by sequencing by General Biosystems.

[0053] ② Agrobacterium transformation and tobacco infection

[0054] ​The above recombinant plasmid 1 uL was added to EHA105 Agrobacterium competent, placed on ice for 5 min, liquid nitrogen for 5 min, 37°C for 5 min, then placed on ice for 5 min, added 400 uL liquid LB medium, then shaken in a 28°C shaker for 2 h, finally 200 uL bacterial liquid was absorbed and coated on solid LB medium containing Kan + resistance, 28°C dark culture for 48 h. Single colonies were picked for PCR verification, and colonies with identical band positions to the target band positions were added to 400 uL liquid LB medium containing Kan + resistance, 28°C shaker culture for 8 h. 100 uL bacterial liquid was absorbed into 50 mL liquid LB medium containing Kan + resistance, 28°C shaker culture for 12 h until the OD value was between 0.8 and 1. The bacterial liquid was centrifuged (5000 r / min, 10 min), the supernatant was discarded, the bacterial body was resuspended with Agrobacterium resuspension liquid, and the resuspended bacterial liquid was made to have an OD value of 0.4-0.6 at 600 nm by ultraviolet spectrophotometry. Room temperature standing for 1 h. The bacterial liquid was injected into tobacco with a syringe to fill the entire leaf, and was placed in a dark artificial climate chamber for 48 h.

[0055] The infected part of the tobacco leaf was carefully cut with a blade, and the back was made into a slide with the back facing up, and the GFP signal was observed under laser confocal. The subcellular localization results are shown in Figure 3 GFP: green fluorescent protein, excitation wavelength 488 nm, emission wavelength 509 nm; DAPI: nuclear localization marker; Bright: bright field, used to show cell structure; mCherry: membrane localization marker, excitation wavelength 587 nm, emission wavelength 610 nm; Merge: GFP, DAPI, Bright and mCherry fusion photo. It can be seen that the CsPR10-5 protein is located in the cytoplasm and cell membrane.

[0056] 4. DNAse activity verification of CsPR10-5 protein

[0057] The tea tree 'Shuchaya' genome DAN as a reference template, with 10 mM Tris-HCl (PH7.5), 2.5 mM Mgcl2, 0.5 mM CaCl2 mixture 10 ul as reaction buffer, each hole is added 2 uL tea tree DNA and 10 ul reaction buffer, 5 ul of 6 x DNA loading buffer, positive control for 2 uL DNase enzyme (brand white shark), experimental group for 10 ng CsPR10-5 protein, negative control for GST empty load 10 ng, 100 ℃ boiled denaturation CsPR10-5 protein 10 ng. Reaction conditions for 37 ℃ water bath reaction 10 min, after the reaction, each PCR tube is added 4 uL stop solution (50 mM EDTA), mix well and place on ice for 5 min, then all sample on 1% agarose gel, in 1 x TBE solution at 80 V voltage run gel 30 min, under UV light to observe the band. Results as shown in Figure 4 The active CsPR10-5 protein is added, the DNA is completely degraded, and the DNA is not degraded in the inactive protein control group (GST and boiled CsPR10-5), indicating that the CsPR10-5 protein has DNA enzyme degradation activity.

[0058] 5. Tobacco transient expression verifies the function of CsPR10-5 against anthrax

[0059] (1) The pCAMBIA1305-CsPR10-5 plasmid is transformed into Agrobacterium, and the growth is verified. Select the nicotiana benthamiana which grows for about 3 weeks and has consistent growth, and divide the tobacco leaves into 4 parts, as shown in Figure 5 . Use a syringe to inject pCAMBIA1305 and pCAMBIA1305-CsPR10-5 bacterial solution into the tobacco body, and ensure that the injection amount of the two bacterial solutions is equal.

[0060] (2) After 48 h, observe whether it is expressed under the laser confocal microscope. After confirming the expression, use a large needle to prick the injection site into a flower shape, and then use the anthrax mycelium block to infect, and wrap it with preservative film. The blank culture medium is used as a control. After 2 d, observe the phenotype of tobacco and take pictures. As shown in Figure 5 B, the tobacco leaves without anthrax mycelium block infection do not show obvious phenotype, and the corresponding chlorophyll fluorescence also does not show obvious change; after the anthrax mycelium block infection, the tobacco leaves are obviously yellow, and the leaves of the control part are extremely dark and have begun to necrosis. As shown in Figure 5 C, the chlorophyll fluorescence shows that the diseased area of the tobacco leaves after overexpression of CsPR10-5 protein is smaller and the disease degree is lighter than that of pCAMBIA-1305 empty load, and the yellow part is also less; it is found that the diseased area of the tobacco leaves after overexpression of CsPR10-5 protein is significantly lower than that of the control group (as shown in Figure 5Figure 6 shows the expression of CsPR10-5 in tea plant leaves after inoculation with P. theae.

[0061] (3) Peroxidase activity and hydrogen peroxide content determination

[0062] The tobacco leaves were sampled and immediately frozen in liquid nitrogen. The contents of active oxygen and hydrogen peroxide were then determined according to the method described in 5 above. Compared with the pCAMBIA-1305 empty vector, the overexpression of CsPR10-5 resulted in less damage to the tobacco leaves, and the activities of peroxidase POD and superoxide dismutase SOD were significantly increased (as shown in Figs. 6E-F), while the content of H2O2 was significantly reduced (as shown in Fig. 6G). This indicates that CsPR10-5 mediates the resistance of tea plants to P. theae infection. Figure 5 Figure 5 6. Anti-sense inhibition verifies the anti-P. theae function of CsPR10-5

[0063] 6. Anti-sense inhibition verifies the anti-P. theae function of CsPR10-5

[0064] (1) Design of anti-sense inhibition probe

[0065] According to the gene sequence of CsPR10-5, an anti-sense oligonucleotide and a control positive-sense oligonucleotide were designed, and the specific names and sequences are as follows:

[0066] AsODN: 5'-TGGTGGTGAACTCTTTGGTA-3'; (SEQ ID NO. 3)

[0067] sODN: 5'-TTGGCATCGTTGAGGGTCT-3'. (SEQ ID NO. 8)

[0068] The designed probes were sent to Shanghai Shengong Biological Company for synthesis, and the concentration was diluted to 100 μM with ddH2O.

[0069] (2) Anti-sense inhibition and P. theae infection

[0070] ① One-year-old 'Longjing 43' cuttings with consistent growth and no pests and diseases were selected, and 1 mL of the diluted probe was injected into the second leaf with a syringe. Water and random chains were used as controls, and 12 h later, the second leaf was frozen in liquid nitrogen, RNA was extracted, and cDNA obtained by reverse transcription was used as a template for qPCR verification to screen the best probe (SEQ ID NO. 3). The best probe selected in the previous step was injected into the tea seedlings, and water and random primers were also injected. RNA was extracted at 8, 12, and 24 h, and after reverse transcription, fluorescent quantitative PCR verification was performed to screen the best time point for inhibition effect. As shown in Fig. 6A, the expression level of CsPR10-5 was significantly lower than that of the control group (H2O and sODN) 12 h after anti-sense inhibition. Figure 6

[0071] ​​② The best antisense probe was used to treat tea seedlings, and water and random chain were used as controls. 12h later, the tea leaves were infected with the spore suspension of Colletotrichum globerum. The leaves were taken 3d after infection and the control leaves were used for phenotype observation and IMAGE-PAM modulated fluorescence instrument (Germany, WALZ) observation.

[0072] ③ Tissue chemical staining of tea leaves after antisense inhibition

[0073] The tea leaves in which the CsPR10-5 gene was inhibited for 12h were infected with the spore suspension of Colletotrichum globerum, and the infected leaves were carefully taken out 3d later for analysis of the damage degree. The specific steps are as follows: prepare 50mM sodium phosphate buffer (16mL 1M sodium dihydrogen phosphate, 84mL 1M disodium hydrogen phosphate, and pure water to 2L, pH adjusted to 7.5), nitro blue tetrazolium (NBT) staining solution (0.1g NBT dissolved in 50mL sodium phosphate buffer), 3,3'-diaminobenzidine (DAB) staining solution (0.05g DAB dissolved in 45mL sodium phosphate buffer, pH adjusted to 3.8, and finally made up to 50mL with sodium phosphate buffer); take the inoculated leaves and put them into the NBT and DAB staining solutions, respectively, incubate the NBT staining at 37℃ for 3h and the DAB staining at 37℃ for 8h; the stained leaves are placed in 95% alcohol to remove chlorophyll, and the leaf phenotype is observed (as shown in Figure 6 Fig. B).

[0074] (3) Physiological index determination of tea leaves infected with Colletotrichum globerum after CsPR10-5 gene silencing

[0075] ① The area of the lesion after antisense inhibition was counted, and the ImageJ software was used for area calculation, and the number of samples for each treatment was 28 tea leaves. As shown in Figure 6 Fig. C, the lesion area of the antisense inhibition group (AsODN) was significantly larger than that of the control group (H2O and sODN).

[0076] ② Determination of hydrogen peroxide content and peroxidase activity

[0077] The H2O2 content and POD and SOD enzyme activity of the tea leaves after 3d of antisense inhibition and pathogen infection were determined. The specific steps are as follows: total protein extraction, weigh 0.06g of sample and grind into powder with liquid nitrogen, then transfer to a pre-cooled mortar on ice and add 540uL of 10×PBS; centrifuge at 12,000g for 10min, and take the supernatant for determination; according to the instruction manual of the H2O2, POD and SOD test kit (Nanjing Jiancheng), detect the H2O2 content, POD and SOD enzyme activity. The results are as shown in Figure 6As shown in D-F, after the anthracnose fungus infects the tea plant leaves, compared with ddH2O and sODN treatment, the H2O2 content significantly increases, and the POD and SOD enzyme activities significantly decrease after the expression of CsPR10-5 gene is inhibited. These results show that the CsPR10-5 gene enhances the resistance of tea plants to anthracnose by positively regulating the antioxidant enzyme system and maintaining the balance of active oxygen metabolism. Its silence not only reduces the activity of antioxidant enzymes, but also exacerbates the accumulation of active oxygen, leading to enhanced oxidative stress, and ultimately weakens the disease resistance of tea plants.

[0078] The nucleotide sequence represented by SEQ ID NO. 1 in the application is specifically:

[0079] 5'-ATGGGTGTAACTACTTTTACCAAAGAGTTCACCACCACAGTAGCACCCAGC AGGATGTTCAAGGCTTTGATCCTTGACTCCCACAATCTCATCCCTAAGCTTCTCCCTCAATCCATCAAGAGCATCGAGTACGTTCAAGGTGATGGTGGTGTTGGAAGCATCAAGCAAACTAACTTCCCTGAAGGGGGTCACATGAAGTGCTTGAAGCACAAGATTGATTCACTTGATGTCGAGAACTACGAGTGCAAGTACACTTTGATTGAAGGCGATGCATTGGGCGGCAAGCTTGAATCTCTCTCTTATGAGGTTAAGTTTGAGGCTATTGGTGATGGAAGTAAGGTGACAAGCACAAGCTACTATCACGCCAAGGGTGAGATTGAGCTCAATGAAGAGGAAATGAATGCTGGAAAGGACAAGGCCATGGGGATGTTCAAGGTTGTCGAAGAATACCTCCTAGCAAACCCTGATGTTTATGCT-3'

[0080] The amino acid sequence represented by SEQ ID NO. 2 in the application is specifically: MGVTTFTKEFTTTVAPSRMFKALILDSHNLIPKLLPQSIKSIEYVQGDGGVGSIKQTNFPEGGHMKCLKHKIDSLDVENYECKYTLIEGDALGGKLESLSYEVKFEAIGDGSKVTSTSYYHAKGEIELNEEEMNAGKDKAMGMFKVVEEYLLANPDVYA

[0081] The nucleotide sequence represented by SEQ ID NO. 3 is specifically 5'-TGGTGGTGAACTCTTTGGTA-3'

[0082] The above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions recorded in the foregoing examples can be modified, or some technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A tea tree CsPR10-5 gene, characterized in that, The nucleotide sequence of the CDS region thereof is shown as SEQ ID NO.

1.

2. The protein encoded by the CsPR10-5 gene of Camellia sinensis as claimed in claim 1, characterized in that, The amino acid sequence thereof is shown as SEQ ID NO.

2.

3. The tea tree CsPR10-5 gene of claim 1 is applied to regulate the susceptibility of tea tree to anthracnose.

4. The tea tree CsPR10-5 gene of claim 1 is applied to improve the breeding of tea tree anthracnose resistance.

5. A model of anthracnose-susceptible tea plant, characterized by, The model contains the product inhibiting the expression of the tea tree CsPR10-5 gene of claim 1.

6. The anthracnose susceptible tea plant model of claim 5, wherein, The product includes an antisense oligonucleotide with the sequence shown as SEQ ID NO.

3.

7. A tobacco model resistant to anthracnose, characterized in that, The model contains the product overexpressing the tea tree CsPR10-5 gene of claim 1 in tobacco.

8. A tea plant expression vector pCAMBIA1305-CsPR10-5, characterized in that, It is obtained by enzyme digestion of the fragment shown as SEQ ID NO: 1 into a pCAMBIA1305 vector.

9. A method of breeding a plant variety resistant to anthracnose, characterized by, The tea tree CsPR10-5 gene of claim 1 is introduced into a target plant to obtain a transgenic plant with improved anthracnose resistance.

10. The breeding method according to claim 9, characterized by, Specifically comprising the following steps: (1) cloning the tea tree CsPR10-5 gene; (2) constructing a tea tree CsPR10-5 gene overexpression vector; (3) transforming a target plant with the tea tree CsPR10-5 gene overexpression vector to obtain a transgenic plant with improved anthracnose resistance; the plant includes one or more of tea tree and tobacco.

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