Application of CsmiR390a, CsARF2s and CsOPR2-1 as drug targets in prevention and treatment of tea tree anthracnose

Abstract

The invention discloses application of CsmiR390a, CsARF2s and CsOPR2-1 as drug targets in prevention and treatment of tea tree anthracnose, and belongs to the technical field of prevention and treatment of tea tree anthracnose. According to the application of CsOPR2-1 as a drug target in preparation of drugs for preventing and / or treating tea tree anthracnose, the resistance of tea trees to colletotrichum gloeosporioides is enhanced by promoting expression of CsOPR2-1. The CsmiR390 gene has the beneficial effects that the CsmiR390 gene is obviously up-regulated, and the downstream CsARFs (CsARF2.1, CsARF2.2, CsARF3 and CsARF4.1) of the CsmiR390 gene are all obviously down-regulated. Wherein the inhibition effect on CsOPR2 is weakened by down-regulation of CsARF2.1 and CsARF2.2, so that the anti-venereal disease spot area of the tea tree on colletotrichum gloeosporioides is obviously reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of tea tree anthracnose prevention and treatment, and particularly to the application of CsmiR390a, CsARF2s and CsOPR2-1 as drug targets in the prevention and treatment of tea tree anthracnose. Background Art

[0002] Anthracnose is a serious semi-lethal fungal disease of tea leaves that causes great harm to tea production.

[0003] Increasing evidence supports the key role of noncoding RNA genes in enhancing plant immunity. The mechanisms of action of lncRNAs in resistance regulation are gradually being elucidated. For example, the long noncoding RNA (lncRNA) ALEX1 in rice directly binds to the intrinsically disordered middle region of ARF3, promoting ARF3 homodimerization. This enhances its ability to transcriptionally repress the expression of downstream target genes (such as JAZ13), thereby regulating the jasmonic acid signaling pathway and enhancing pathogen resistance in rice. As an important ncRNA, miRNAs also play an indispensable role in plant defense mechanisms. For example, the rice miRNA Os-miR169y, induced by Sclerotinia sclerotiorum, can target the 60S ribosomal protein L19 (SsRPL19) of Sclerotinia sclerotiorum, affecting ribosome assembly and thereby inhibiting the growth and pathogenicity of the pathogen. NB-LRR (nucleotide binding and leucine rich repeat) genes are the main type of plant innate immune receptors. A large number of miRNAs have been found to regulate the expression of NB-LRR genes in different plants. These miRNAs trigger phasi-RNA synthesis by targeting NB-LRR genes to enhance their silencing effect. The participation of mRNA and ncRNA interactions is indispensable in conferring resistance traits on tea plants, but research on their mechanism of action is still very limited. We previously identified an evolutionarily conserved lncRNA OPRL in tea plants, which negatively regulates tea plants' resistance to anthracnose by forming a triplex with the target gene OPR to inhibit JA synthesis.

[0004] Defects and shortcomings of existing technology:

[0005] ① The existing tea tree anthracnose prevention technology relies on chemical control. The long-term and large-scale use of chemical agents such as pyraclostrobin emulsifiable concentrate, difenoconazole water-dispersible granules, and thiophanate-methyl wettable powder can easily lead to pesticide residues in tea leaves and soil, which not only affects the quality of tea, but also pollutes the ecological environment and threatens the balance of the tea garden ecosystem.

[0006] ② The research on the mechanism of LncRNA regulating anthrax resistance at the molecular level is not in-depth enough, and it is impossible to effectively prevent anthrax at the biomolecular level.

[0007] Chinese patent application publication number CN109221277A discloses a drug for preventing and treating tea anthracnose. The drug is made from the following ingredients, by weight: 100 parts Patchouli, 110-180 parts Lavender, 120-200 parts Taraxacum, 130-210 parts Xanthium sibiricum, 120-190 parts Plantain, 200-280 parts Ficus carica leaves, 100-140 parts Sophora japonica, 20-25 parts Sophora japonica seeds, and 150-220 parts Costus root. This drug can prevent and treat tea anthracnose and also acts as a foliar fertilizer. However, this patent utilizes a traditional Chinese medicine composition, rather than a genetically engineered approach, which has limitations and warrants further improvement. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to propose a drug target and its application in preventing and treating tea tree anthracnose.

[0009] The present invention solves the above technical problems through the following technical means:

[0010] The first aspect of the present invention proposes the use of CsOPR2-1 as a drug target in the preparation of a drug for preventing and / or treating tea tree anthracnose. The sequence of CsOPR2-1 is shown in SEQ ID No. 1.

[0011] Preferably, the resistance of tea plants to anthracnose is enhanced by promoting the expression of CsOPR2-1.

[0012] The second aspect of the present invention provides a use of a CsmiR390a upregulator in preventing and treating tea anthracnose. The precursor cDNA sequence of the CsmiR390a is shown in SEQ ID No. 2, and the mature RNA sequence is shown in SEQ ID No. 3.

[0013] Preferably, the CsmiR390a upregulator refers to a substance that increases the level of CsmiR390a.

[0014] The CsmiR390a upregulator may be CsmiR390a and its analogs, that is, it may be CsmiR390a or a substance having a functional activity similar to that of CsmiR390a.

[0015] Preferably, the CsmiR390a upregulator is at least one of CsmiR390a, a CsmiR390a mimetic, a CsmiR390a analog, and a CsmiR390a modifier.

[0016] CsmiR390a mimetics refer to nucleotide sequences synthesized by chemical synthesis methods that can enhance the function of endogenous CsmiR390a.

[0017] A CsmiR390a analog refers to a nucleotide sequence that is similar to or homologous to CsmiR390a in nucleotide sequence, and can be, for example, a CsmiR390a agonist.

[0018] CsmiR390a modifiers refer to the nucleotide sequence design nucleic acid sequence of CsmiR390a and its modifications, wherein the modifications include one or more combinations of ribose modification, base modification and phosphate backbone modification of any nucleotide or the addition, deletion or replacement of any nucleotide, as long as the modified nucleotide sequence still has functional activity similar to that of CsmiR390a.

[0019] The third aspect of the present invention provides a use of a CsARF2s down-regulator in preventing and treating tea anthracnose. The CsARF2s include CsARF2.1 and CsARF2.2, and the sequences are shown in SEQ ID No. 4-5.

[0020] Preferably, the CsARF2s downregulator is an interfering molecule that specifically interferes with the expression of the CsARF2s gene.

[0021] Preferably, the interfering molecule that specifically interferes with the expression of the CsARF2s gene is a dsRNA, antisense nucleic acid, small interfering RNA, microRNA that inhibits or silences the CsARF2s gene or its transcript, or a construct that can express or form the dsRNA, antisense nucleic acid, small interfering RNA, microRNA.

[0022] A fourth aspect of the present invention provides a pharmaceutical composition for preventing and treating tea tree anthracnose, the active ingredient of which includes at least one of the above-mentioned CsmiR390a upregulator and CsARF2s downregulator.

[0023] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0024] "Pharmaceutically acceptable" means a non-toxic material that does not reduce the active ingredient. Such pharmaceutically acceptable carriers are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, edited by AR Gennaro, Mack Publishing Company (1990) and handbook of Pharmaceutical Excipients, 3rd edition, edited by A. Kibbe, Pharmaceutical Press (2000)).

[0025] The beneficial effects of the present invention are:

[0026] 1. We used sRNA transcriptome sequencing to perform enrichment analysis of differentially expressed miRNAs and their target genes in tea leaves 6 days after infection with the anthrax strain C. camelliae, clarifying that the plant-pathogen interaction pathway and the plant hormone signal transduction pathway are regulated by the miRNA-target network.

[0027] 2. CsmiR390a, one of the miRNAs significantly induced by anthrax, inhibits the expression of ARFs by cleaving lncRNA TAS3 to produce conserved tasiRNAs. Based on transient overexpression and gene silencing techniques in tea plants, the CsmiR390a-TAS3-ARF2s module was characterized in response to anthrax stress, confirming its function in resistance regulation. It was found that among the CsARFs regulated by the CsmiR390a-TAS3 module in tea plants, CsARF2.1 and CsARF2.2 have the function of enhancing tea plant disease resistance. CsOPR2-1 is the disease resistance target of the CsmiR390a-TAS3-ARF2s module. Specifically, ARF2s negatively regulates tea plant disease resistance by inhibiting the promoter activity of CsOPR2-1.

[0028] 3. Transcriptome-based correlation analysis was conducted between the expression patterns of seven CsARFs regulated by the CsmiR390a-TAS3 module and genes related to the jasmonic acid pathway in tea plants. The results showed that CsARF2s were significantly negatively correlated with CsOPR2 family members. In our previous study, overexpression of CsOPR2-1 did increase JA content and enhance tea plant resistance. To further confirm the correlation between CsARF2s and CsOPR2, qRT-PCR confirmed that when CsARF2.1 and CsARF2.2 were overexpressed in tea plants, CsOPR2-1 was significantly downregulated, while when CsARF2s were silenced, CsOPR2-1 was significantly upregulated.

[0029] 4. Three potential ARF binding targets, namely auxin response elements, were identified on the CsOPR2-1 promoter. These two sites, TGTCNN and TGTCTC, are located 173 bp, 180 bp, and 417 bp upstream of the TSS, respectively. Therefore, we speculated that CsARF2s may attenuate tea plant resistance by inhibiting CsOPR2-1 transcription. To verify this, we performed a dual-LUC reporter assay. The results showed that when CsARF2s was co-expressed with the CsOPR2-1 promoter, the fluorescence intensity on the leaves was significantly lower than that of the combination of empty vector and CsOPR2-1 promoter co-expression, indicating that CsARF2s can indeed inhibit the CsOPR2-1 promoter. These results suggest that the MiR390-TAS3 module reduces the expression level of CsARF2s by inhibiting it, thereby weakening the inhibitory effect on CsOPR2 and enhancing tea plant resistance to anthracnose. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Figures 1 and 2 show the GO and KEGG enrichment analysis of the differentially expressed anthrax-induced miRNA target genes in Example 1. A shows the GO enrichment analysis of the differentially expressed miRNA target genes, and B shows the KEGG enrichment analysis of the differentially expressed miRNA target genes. The figure shows the top 20 pathways, sorted in ascending order by q-value.

[0031] Figure 2 Figure 1 shows the role of CsmiR390a and CsTAS3.1 in tea plant anthracnose resistance in Example 1 of the present invention. A is a workflow diagram for studying tea plant anthracnose resistance based on Agrobacterium-mediated transient gene expression and antisense oligonucleotide-mediated gene silencing. B is a functional diagram of CsmiR390a analyzed in tea plant anthracnose resistance by Agrobacterium-mediated transient expression. C and D are functional diagrams of CsTAS3.1 analyzed in tea plant anthracnose resistance by Agrobacterium-mediated transient expression and antisense oligonucleotide-mediated gene silencing. Data are presented as mean ± standard deviation (n ≥ 5), and two-way statistical significance was analyzed using the Student's t-test (*P < 0.05). Dots represent biological replicates, and the red line indicates normalization to 1.

[0032] Figure 3Figure 1 shows the role of CsARFs in tea plant anthracnose resistance in Example 1 of the present invention. Figures A and B show the functional analysis of CsARF2.1, CsARF2.2, CsARF3, and CsARF4 in tea plant anthracnose resistance based on Agrobacterium-mediated transient expression, and Figure E shows the role of CsARF2s in tea plant anthracnose resistance through AsODN-mediated gene silencing. Data are presented as mean ± SD (n ≥ 5), and statistical significance was assessed using a two-tailed Student's t-test (*P < 0.05). Individual data points in the figures represent biological replicates.

[0033] Figure 4 This is a diagram for the identification and functional analysis of downstream genes regulated by CsARF2s in tea plants in Example 1 of the present invention, wherein A is a correlation analysis diagram between CsARF and key genes in the JA pathway. The color depth indicates the size of the correlation coefficient, and the significance was determined by Student's t test (*P<0.05, **P<0.01). B is a qRT-PCR detection of the expression of CsOPR2-1 in tea plants after Agrobacterium-mediated transient overexpression and AsODN gene silencing of CsARF2s (CsARF2.1 / CsARF2.2). C is a schematic diagram of the putative auxin response elements (AuxREs) in the CsOPR2-1 promoter, and the AuxREs in specific regions are marked with boxes. D is a design diagram of the dual-luciferase reporter experiment, in which the effector genes CsARF2.1 and CsARF2.2 were co-expressed with a firefly luciferase (LUC) reporter vector containing the CsOPR2-1 promoter, with Renilla luciferase (REN) as the internal control, and CaMV The 35S promoter drives effector gene expression. EF is a luciferase assay verifying that CsARF2.1 and CsARF2.2 activate the CsOPR2-1 promoter. Data are mean ± SD (n = 3). Significance was assessed by two-tailed Student's t-test (*P < 0.05, **P < 0.01). Individual data points represent biological replicates. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Unless otherwise defined, the professional terms used below have the same meaning as those understood by professional and technical personnel in this field.

[0035] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources or prepared by known methods.

[0036] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the quantitative tests in the following examples were repeated three times and the results were averaged.

[0037] Example 1:

[0038] This example provides a group of applications of the CsmiR390 / TAS3 / ARFs / OPR gene axis in tea plants for improving anthracnose resistance.

[0039] The following steps are involved:

[0040] Based on the transcriptome, a correlation analysis was conducted between the expression patterns of seven CsARFs regulated by the CsmiR390-TAS3 module and jasmonic acid pathway-related genes in tea plants.

[0041] 1. Target gene correlation analysis based on transcriptome sequencing of tea leaves with different stages of anthracnose disease

[0042] (1) Zhongcha 108 was inoculated with Colletotrichum spp. Tea leaves were sampled at 24, 48, 72, 96, and 6 days post-infection. Blank cultured tea leaves were inoculated at the corresponding time points as controls. Each sample was subjected to three biological replicates, and a total of 30 samples were sequenced.

[0043] (2) Transcriptome data were analyzed to extract the expression data of seven CsARFs (CsARF2.1, CsARF2.2, CsARF2.3, CsARF2.4, CsARF3, CsARF4.1 and CsARF4.2) regulated by the CsmiR390-TAS3 module in tea plants and jasmonic acid pathway-related genes, including CsLOXs, CsAOSs, CsAOCs, CsOPR2s, CsOPR1s, CsJMT, and CsJAZs, and the average of three replicates was taken.

[0044] (3) ORIGIN software was used to perform correlation analysis on the expression data obtained above. The color depth indicated the size of the correlation coefficient. The significance was determined by Student's t test (*P<0.05, **P<0.01)

[0045] 2. Gene cloning and vector construction of CsmiR390a, CsTAS3.1, CsARF2.1, CsARF2.2, CsARF3, and CsARF4

[0046] (1) Extract RNA from leaves of Zhongcha 108. The RNA extraction was performed according to the instructions of Fruit-mate™ for RNA Purification Kit of Novozymes Biotech Co., Ltd. 1000 ng of extracted RNA was extracted according to PrimeScript TM Perform reverse transcription according to the RT Master Mix reagent instructions. Add 4 μL of reverse transcriptase to an RNAse-free PCR tube and add RNAse-free water to a total reaction volume of 20 μL. Mix thoroughly and centrifuge. The reverse transcription PCR program is: 37°C for 15 minutes, 85°C for 5 seconds, and 4°C forever. Obtain the first-strand cDNA.

[0047] (2) Design primers for PCR amplification. The primer sequences are as follows:

[0048] CsmiR390a-1305-F (SEQ ID NO:6):

[0049] 5'-GTCCGGAGCTAGCTCTAGAATAGAAGTGACTCGTGAATGG-3'

[0050] CsmiR390a-1305-R (SEQ ID NO:7):

[0051] 3'-CTTGCTCACCATGGATCCGACCAGAGATAGTAAGAAGA-5'

[0052] CsTAS3.1-1305-F (SEQ ID NO: 8):

[0053] 5'-GTCCGGAGCTAGCTCTAGAAGTCTTGTCTATCCCTCCTGAGCTGTT-3';

[0054] CsTAS3.1-1305-R (SEQ ID NO:9):

[0055] 3'-CTTGCTCACCATGGATCCACAGCTCAGGAGGGATAGACAAGAC-5';

[0056] CsARF2.1-1305-F (SEQ ID NO:10):

[0057] 5'-GTCCGGAGCTAGCTCTAGAAATGGCTTCTTCAGAGCTGT-3';

[0058] CsARF2.1-1305-R(SEQ ID NO:11):

[0059] 3’-CTTGCTCACCATGGATCCGTAAAACCACCATATCTG-5’;

[0060] CsARF2.2-1305-F(SEQ ID NO:12):

[0061] 5’-GTCCGGAGCTAGCTCTAGAAATGGCTTCTTCAGAGGTTTC-3’;

[0062] CsARF2.2-1305-R(SEQ ID NO:13):

[0063] 3’-CTTGCTCACCATGGATCCTATATCCGAAATCAAGTAGGA-5’;

[0064] CsARF3-1305-F(SEQ ID NO:14):

[0065] 5’-GTCCGGAGCTAGCTCTAGAAATGTGTAGTTTGATTGATCTC-3’;

[0066] CsARF3-1305-R(SEQ ID NO:15):

[0067] 3’-CTTGCTCACCATGGATCCACTCTGCATGTTGAATGG-5’;

[0068] CsARF4-1305-F(SEQ ID NO:16):

[0069] 5’-GTCCGGAGCTAGCTCTAGAAATGGAAATTGATCTGAACCATG-3’;

[0070] CsARF4-1305-R(SEQ ID NO:17):

[0071] 3’-CTTGCTCACCATGGATCCATTGATGTTTGGGGGAGA-5’;

[0072] The reaction system of PCR is based on PrimeSTAR TMHSDNA Polymerase (Taraka) was used according to the instructions. The PCR amplification program was as follows: 98°C for 10 seconds, 62°C for 10 seconds, 72°C for 1 minute, 30 cycles, 72°C for 6 minutes, and 16°C for termination. The resulting PCR products were verified by agarose gel electrophoresis, and the bands at the target positions were recovered from the gel.

[0073] (3) Gel recovery: refer to the instructions of the recovery kit TaKaRa Mini BEST Agarose Gel DNA Extraction Kit.

[0074] (4) Vector ligation: Take the 1305 vector and perform double digestion with restriction endonucleases BamhI and XbaI to recover the vector backbone. The amplified product recovered in step 2) was ligated with the recovered vector backbone to obtain recombinant plasmids 35S::CsmiR390a, 35S::CsTAS3.1, 35S::CsARF2.1, 35S::CsARF2.2, 35S::CsARF3 and 35S::CsARF4. The ligation process was based on the ClonExpress II One Step Cloning Kit from Nanjing Novizan Biotechnology Co., Ltd. 2 μL of the recovered product was ligated to 0.5 μL of the cloning vector. The reaction was carried out at 25°C for 15 min, followed by an ice bath for 5 min. 20 μL of DH5α competent medium was added, heat-shocked at 42°C for 1 min, and then ice-bathed for 2 min. The resulting product was added to 200 μL of Incubate the culture medium in a 37°C shaker at LB medium for 1 hour. Finally, spread the bacterial solution on a plate containing Kan antibiotics and culture it in a 37°C incubator. Observe the colony growth the next day, pick a single colony for PCR verification, and sequence the bacteria with the correct band position.

[0075] 3. Transformation of Agrobacterium Competent Cells

[0076] Mix 20 μL of competent cells Agrobacterium GV3101 with 2 uL of plasmid, place on ice for 5 minutes; freeze in liquid nitrogen for 5 minutes; place in a 37°C water bath for 5 minutes; place on ice for 5 minutes, add 500 μL of LB liquid medium (without antibiotics), and culture at 28°C, 220g for 3 hours; draw 200 μL and apply it to a plate containing LB solid medium containing Kan and Rif antibiotics, and culture at 28°C for 3 days; screen positive clones by colony PCR.

[0077] 4. Transient Overexpression in Tea Leaves

[0078] (1) Activation culture of recombinant Agrobacterium of 35S::CsmiR390a, 35S::CsTAS3.1, 35S::CsARF2.1, 35S::CsARF2.2, 35S::CsARF3 and 35S::CsARF4 to OD 600 is 0.8;

[0079] (2) The cells were collected by centrifugation at room temperature and resuspended twice in a solution containing 10 mM MgCl2, 10 mM MES, and 100 μM acetosyringone (pH = 5.6);

[0080] (3) Adjust the OD of Agrobacterium suspension 600 To 0.8 injection of 108 tea leaves.

[0081] (4) Anthrax inoculation and gene quantitative detection and analysis after 24 hours

[0082] 5. Antisense oligonucleotide silencing experiments of CsTAS3.1 and CsARF2s

[0083] (1) The following candidate antisense oligonucleotides (AsODNs) targeting CsTAS3.1 and CsARF2s (CsARF2.1 and CsARF2.2) were selected using SOLIGO software and synthesized by General Biosystems:

[0084] CsTAS3.1-sODN (SEQ ID NO:18): 5'-CTCCTTCCTTGTCTATCCCTCCTG-3';

[0085] CsTAS3.1-AsODN1 (SEQ ID NO:19): 3'-CCGGTGGCACTCCGATCACTGGAG-5';

[0086] CsTAS3.1-AsODN2 (SEQ ID NO:20): 3'-CACAACGGAGTCCCCAACGCC-5';

[0087] CsTAS3.1-AsODN3 (SEQ ID NO:21): 3'-CAGGAGGGATAGACAAGGAAGGAG-5';

[0088] CsARF2s-sODN (SEQ ID NO:22): 5'-GAACCTATGCCACCTCCACC-3';

[0089] CsARF2s-AsODN1 (SEQ ID NO:23): 3'-AACAGTGGAATAACCCTTCG-5';

[0090] CsARF2s-AsODN2 (SEQ ID NO:24): 3'-CCATTTGTCCGATGCATATT-5';

[0091] CsARF2s-AsODN3 (SEQ ID NO:25): 3'-GGTGGAGGTGGCATAGGTTC-5'.

[0092] (2) 1 mL of a 100 μM AsODN mixed solution was injected into the leaves of 2-year-old cutting seedlings, while those injected with sense oligonucleotides (sODNs) served as controls;

[0093] (3) Real-time fluorescence quantitative PCR (qRT-PCR) detection and analysis were performed 48 hours later.

[0094] 6. Fluorescence quantitative (qRT-PCR) experiment

[0095] (1) Based on the gene sequence and mature body sequence of the local transcriptome CsmiR390a, the following reverse transcription primers and fluorescence quantitative primers were set using the stem-loop method, with the CsU6 gene as the internal reference.

[0096] CsmiR390a-cDNA (SEQ ID NO:26):

[0097] 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGGCGCT-3'

[0098] CsmiR390a-qF (SEQ ID NO:27): 5'-GCGGCAAGCTCAGGAGGGAT-3';

[0099] CsmiR390a-qR (SEQ ID NO:28): 3'-CCAGTGCAGGGTCCGAGGTA-5';

[0100] CsU6-qF (SEQ ID NO:29): 5'-CGGGGACATCCGATAAAATTG-3';

[0101] CsU6-qR (SEQ ID NO:30): 3'-GGACCATTTCTCGATTTGTGC-5';

[0102] (2) The following qRT-PCR primers were designed according to the MIQE guidelines. The reaction mixture included 1 μL cDNA template, 10 μL MonoAmp TM ChemoHS qPCR Mix and 1 μL of gene-specific primers were added, and RNA-free water was added to a final volume of 20 μL. The reaction procedure was as follows: pre-denaturation at 95°C for 10 min, denaturation at 95°C for 10 sec, annealing at 60°C for 10 sec, and extension at 72°C for 30 sec, for 40 cycles. The instrument default procedure was used for melting curve acquisition. Three biological replicates were set for each material, and three technical replicates were set for each PCR reaction. The relative expression of genes was calculated using the Pfaffl method, using the tea tree housekeeping gene CsGAPDH as an internal reference.

[0103] CsTAS3.1-qF (SEQ ID NO:31): 5'-GAGATAGGAGCCGATAAAGTTGG-3';

[0104] CsTAS3.1-qR (SEQ ID NO:32): 3'-GCAATGAAAGTGCCCTTGAAAGC-5';

[0105] CsOPR2-1-qF (SEQ ID NO:33): 5'-GAGATAGGAGCCGATAAAGTTGG-3';

[0106] CsOPR2-1-qR (SEQ ID NO:34): 3'-GCAATGAAAGTGCCCTTGAAAGC-5';

[0107] CsARF2.1-qF (SEQ ID NO:35): 5'-GAGATAGGAGCCGATAAAGTTGG-3';

[0108] CsARF2.1-qR (SEQ ID NO:36): 3'-GCAATGAAAGTGCCCTTGAAAGC-5';

[0109] CsARF2.2-qF (SEQ ID NO:37): 5'-CAATGCCTTCTGGTCTCTCCC-3';

[0110] CsARF2.2-qR (SEQ ID NO:38): 3'-TTTCGTTGCTGTGCTCTGCT-5';

[0111] CsARF3-qF (SEQ ID NO:39): 5'-ATCTGCTGACTACTGGTTGGA-3';

[0112] CsARF3-qR (SEQ ID NO:40): 3'-AAGGCTCTTTGAGAATTTACG-5';

[0113] CsARF4.1-qF (SEQ ID NO:41): 5'-AAGAAGAATGCATGTGGCAAT-3';

[0114] CsARF4.1-qR (SEQ ID NO:42): 3'-GGTGGAAAAGGTGAGGAAGAG-5';

[0115] CsGAPDH-qF (SEQ ID NO:43): 5'-TTGGCATCGTTGAGGGTCT-3';

[0116] CsGAPDH-qR (SEQ ID NO:44): 3'-CAGTGGGAACACGGAAAGC-5'.

[0117] 6. Anthracnose infection of Zhongcha 108 tea leaves

[0118] (1) C. camelliae was cultured on PDA medium at 28°C for 5 days.

[0119] (2) Collect spores by centrifugation at 6000 g for 10 minutes. After collection, resuspend the spores in sterile water and adjust the concentration to 10^6 spores / mL under microscopic monitoring for subsequent inoculation.

[0120] (3) A total of 50 μL of conidia suspension was inoculated into the upper epidermis of tea leaves using a sterile syringe. Control plants were inoculated with an equal amount of sterile distilled water. The inoculated leaves were covered with plastic film to maintain high humidity and promote fungal growth.

[0121] (4) After a 24-hour incubation period, the films were removed and the symptoms of infected leaves were recorded within 6 days after inoculation.

[0122] The results indicate that the CsmiR390a-TAS3-ARF2s module plays a crucial role in tea plant resistance to anthracnose. Transient overexpression and antisense oligonucleotide experiments in tea plants demonstrated that CsmiR390a and CsTAS3.1 positively regulate tea plant resistance to anthracnose, significantly reducing lesion area compared to controls. Analysis of the anthracnose resistance-related phenotypes of the ARFs regulated by the miR390-TAS3 module (CsARF2.1, CsARF2.2, CsARF3, and CsARF4) revealed that only ARF2s (CsARF2.1 and CsARF2.2) negatively regulate tea plant resistance.

[0123] Further correlation analysis revealed that CsARF2s were significantly negatively correlated with CsOPR2 family members. In our previous research, overexpression of CsOPR2-1 indeed increased JA content and enhanced resistance in tea plants. To further confirm the correlation between CsARF2s and CsOPR2, qRT-PCR confirmed that overexpression of CsARF2.1 and CsARF2.2 in tea plants significantly downregulated CsOPR2-1, while silencing CsARF2s resulted in a significant upregulation of CsOPR2-1.

[0124] Three potential ARF binding targets, namely auxin response elements, were identified in the CsOPR2-1 promoter. These two, TGTCNN and TGTCTC, are located 173, 180, and 417 bp upstream of the TSS, respectively. Therefore, we hypothesized that CsARF2s might enhance tea plant resistance by inhibiting CsOPR2-1 transcription. To verify this, we performed a dual-LUC reporter assay.

[0125] 1. Construction of overexpression vector of CsOPR2-1 promoter

[0126] (1) Using the cDNA obtained from Zhongcha 108 as the material, PCR amplification was performed using the following primers, and the amplified product was recovered:

[0127] CsOPR2-1pro-0800-F(SEQ ID NO:45):

[0128] 5'-CGAATTCCTGCAGCCCGGGGTCTCAAATAAGTGGTTCACCAAC-3';

[0129] CsOPR2-1pro-0800-R(SEQ ID NO:46):

[0130] 3'-GCTCTAGAACTAGTGGATCCGGGGAGGAAATATTTGTCGC-5'

[0131] (2) Take the pGreenII 0800 vector and perform single enzyme digestion with the restriction endonuclease BamhI to recover the vector backbone. Then, ligate the amplified product recovered in step (1) with the vector backbone to obtain the recombinant plasmid CsOPR2-1pro::LUC.

[0132] (3) The recombinant plasmid CsOPR2-1pro::LUC was introduced into Agrobacterium GV3101.

[0133] 2. Dual luciferase reporter assay in tobacco

[0134] (1) The recombinant Agrobacterium containing effectors (35S::CsARF2.1 and 35S::CsARF2.1) and reporter gene (CsOPR2-1pro::LUC) and the control recombinant Agrobacterium (pGreenII 0800 vector introduced into GV3101) were activated and cultured to OD600 = 0.8. 600 is 0.8.

[0135] (2) The cells were collected by centrifugation at room temperature and resuspended twice in a solution containing 10 mM MgCl2, 10 mM MES, and 100 μM acetosyringone (pH 5.6);

[0136] (3) Adjust the OD of Agrobacterium suspension 600 The pH was adjusted to 0.8 and used to infiltrate 5-6 week old Nicotiana benthamiana leaves. The plants were then incubated for another 3 days.

[0137] (4) Luciferase substrate was sprayed onto Nicotiana benthamiana leaves, and the luminescence signal was detected using a CCD imaging device (Lumazone Pylon 2048B). Simultaneously, the activities of firefly LUC and Renilla (REN) were measured using a dual-luciferase assay reagent (Promega, Madison, WI, USA). The LUC / REN ratio represents the promoter activity.

[0138] The results showed that when CsARF2s was co-expressed with the CsOPR2-1 promoter, the fluorescence intensity on the leaves was significantly lower than when the empty vector was co-expressed with the CsOPR2-1 promoter, indicating that CsARF2s can indeed inhibit the CsOPR2-1 promoter. These results suggest that CsARF2s regulated by the miR390-TAS3 module can mediate disease resistance in tea plants by inhibiting the CsOPR2-1 promoter. Overall, this study identified a set of applications of the miR390 / TAS3 / ARFs / OPR gene axis in tea plants for improving anthracnose resistance.

[0139] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. Use of CsOPR2-1 as a drug target in the preparation of a drug for preventing and / or treating tea tree anthracnose, characterized in that: The sequence of CsOPR2-1 is shown in SEQ ID No.

1.

2. The use according to claim 1, characterized in that By promoting the expression of CsOPR2-1, the resistance of tea plants to anthracnose is enhanced.

3. Use of a CsmiR390a upregulator in preventing and treating tea anthracnose, characterized in that: The precursor cDNA sequence of CsmiR390a is shown in SEQ ID No. 2, and the mature RNA sequence is shown in SEQ ID No.

3.

4. The use according to claim 3, characterized in that The CsmiR390a upregulator refers to a substance that increases the level of CsmiR390a.

5. The use according to claim 3, characterized in that The CsmiR390a upregulator is at least one of CsmiR390a, a CsmiR390a mimetic, a CsmiR390a analog, and a CsmiR390a modifier.

6. A use of a CsARF2s down-regulator in preventing and treating tea anthracnose, characterized in that: The CsARF2s include CsARF2.1 and CsARF2.2, and the sequences are shown in SEQ ID No. 4-5.

7. The use according to claim 6, characterized in that The CsARF2s downregulator is an interfering molecule that specifically interferes with the expression of the CsARF2s gene.

8. The use according to claim 7, characterized in that The interfering molecule that specifically interferes with the expression of the CsARF2s gene is a dsRNA, antisense nucleic acid, small interfering RNA, microRNA that inhibits or silences the CsARF2s gene or its transcript, or a construct that can express or form the dsRNA, antisense nucleic acid, small interfering RNA, microRNA.

9. A pharmaceutical composition for preventing and treating tea tree anthracnose, characterized in that: The active ingredient thereof includes at least one of the CsmiR390a upregulator according to claim 3 and the CsARF2s downregulator according to claim 6.

10. The pharmaceutical composition according to claim 9, characterized in that The pharmaceutical composition further includes a pharmaceutically acceptable carrier.

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