Application of Cslnc924 gene in diagnosis or prevention and treatment of tea tree anthracnose

Abstract

The application discloses application of a Cslnc924 gene in diagnosis or prevention and treatment of anthracnose of tea trees, and relates to the technical field of anthracnose prevention and treatment of tea trees. Expression of the Cslnc924 gene is promoted to enhance the resistance of the tea tree to anthracnose bacteria, and the full-length sequence of the cDNA of the Cslnc924 gene is shown as SEQ ID No. 1. Advantageous effects: Based on an agrobacterium-mediated transient overexpression technology and an antisense oligonucleotide-mediated gene silencing technology, the Cslnc924 gene is overexpressed and silenced in tea tree leaves respectively, and anthracnose bacteria inoculation experiments are carried out. The results show that when the Cslnc924 is overexpressed, CsmiR390 is significantly up-regulated, and downstream CsARFs (CsARF2.1, CsARF2.2, CsARF3 and CsARF4.1) are significantly down-regulated compared with a control, at this time, the resistance of the tea tree to anthracnose bacteria is enhanced, and a lesion area is significantly reduced.

Description

Technical Field

[0001] This invention relates to the field of anthracnose control technology for tea trees, specifically to the application of the Cslnc924 gene in the diagnosis or control of anthracnose in tea trees. Background Technology

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

[0003] Mounting evidence supports the crucial role of non-coding RNA genes in enhancing plant immune function. The mechanisms by which lncRNAs regulate resistance are gradually being elucidated. For example, in rice, the long non-coding RNA (lncRNA) ALEX1 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 (e.g., JAZ13), regulating the jasmonic acid signaling pathway and strengthening pathogen resistance in rice. As an important ncRNA, miRNAs also play an indispensable role in plant defense mechanisms. One example is the miRNA Os-miR169y induced by *Sclerotinia sclerotiorum* in rice, which targets the 60S ribosomal protein L19 (SsRPL19) of *Sclerotinia sclerotiorum*, affecting the pathogen's ribosome assembly and thus inhibiting its growth and pathogenicity. NB-LRR (nucleotide binding and leucine-rich repeat) genes are a major type of innate immune receptor in plants. Numerous miRNAs have been found in different plants to regulate NB-LRR gene expression. These miRNAs enhance their silencing effect by targeting the NB-LRR gene and triggering phasiRNA synthesis. The interaction between mRNA and ncRNA is indispensable in conferring resistance traits in tea plants, but research on their mechanisms is still limited. We previously identified an evolutionarily conserved lncRNA, OPRL, in tea plants that negatively regulates tea resistance to anthracnose by forming a triplet with the target gene OPR to inhibit JA synthesis.

[0004] For example, lncRNA 81246 can act as a ceRNA to negatively regulate tea plant resistance to tea leaf spot by influencing the miR164d-CsNAC1 regulatory module. We performed sRNA transcriptome sequencing on tea leaves infected with C. camelliae, and enriched differentially expressed miRNAs and their target genes using GO and KEGG pathways, clarifying that the plant-pathogen interaction pathway and plant hormone signal transduction pathway are regulated by the miRNA-target network.

[0005] Defects and shortcomings of existing technology:

[0006] ① Existing tea anthracnose prevention techniques rely on chemical control. Long-term and large-scale use of chemical agents such as pyraclostrobin EC, difenoconazole WATER granules, and chlorothalonil WP can easily lead to pesticide residues in tea leaves and soil, which not only affects the quality of tea leaves but also pollutes the ecological environment and threatens the balance of the tea garden ecosystem.

[0007] ② Research on the molecular mechanism of LncRNA regulation of anthrax resistance is not in-depth enough, making it impossible to effectively prevent anthrax at the biomolecular level.

[0008] Chinese patent application document CN108718838A discloses a method for preventing and controlling anthracnose in tea trees, including the following steps: (1) Strengthen tea garden management and do a good job of ditching and draining waterlogged tea gardens, and remove fallen leaves in autumn and winter; (2) Appropriately increase the application of phosphorus and potassium fertilizers, and apply 30-35 kg of farmyard manure, 0.9-1.1 kg of urea, 0.5-0.7 kg of phosphate fertilizer, 0.6-0.9 kg of nitrogen fertilizer, and 2-3 kg of potassium fertilizer per tree per year to improve disease resistance; (3) Enhance disease resistance by selecting disease-resistant varieties; (4) Remove diseased leaves: remove diseased branches and leaves in time; (5) Drug control: in the early stage of the disease, you can use 1000-1200 times diluted ash frost anthracnose + Bacillus spores for even spraying, and the tea leaves can be picked after 14-16 days. This patent can effectively prevent and control anthracnose in tea trees, so that tea trees can grow and develop normally, and ensure the quality and yield of tea. However, this patent still focuses on preventing and controlling anthracnose in tea trees through physical or chemical methods, rather than at the genetic level. This approach is time-consuming, labor-intensive, and has poor control efficiency, so further improvements are needed. Summary of the Invention

[0009] The technical problem to be solved by this invention is to propose a new use of a gene in the prevention and control of anthracnose in tea trees.

[0010] The present invention solves the above-mentioned technical problems through the following technical means:

[0011] The first aspect of the present invention provides a use of the Cslnc924 gene to promote the expression of the Cslnc924 gene in order to enhance the resistance of tea plants to anthracnose, wherein the full-length cDNA sequence of the Cslnc924 gene is shown in SEQ ID No. 1.

[0012] Preferably, the Cslnc924 gene is a long non-coding RNA, and its RNA sequence is shown in SEQ ID No. 2.

[0013] A second aspect of the present invention proposes the application of a reagent for detecting the expression level of the Cslnc924 gene in the diagnosis of resistance to anthracnose in tea trees.

[0014] A third aspect of this invention proposes the application of the Cslnc924 gene in the preparation of drugs for the prevention and treatment of anthracnose in tea trees.

[0015] The fourth aspect of this invention proposes the application of the Cslnc924 gene in the breeding of anthracnose-resistant tea varieties.

[0016] The fifth aspect of the present invention provides a drug for preventing and treating anthracnose in tea trees, comprising a reagent that promotes the expression of the Cslnc924 gene.

[0017] The sixth aspect of the present invention provides an anthrax-sensitive tea tree model, wherein the anthrax-sensitive tea tree model contains a product that inhibits the expression of the Cslnc924 gene.

[0018] A seventh aspect of the present invention provides an expression vector containing the aforementioned Cslnc924 gene.

[0019] The eighth aspect of the present invention provides a method for breeding anthracnose-resistant tea plants, comprising the following steps: obtaining tea plants with higher anthracnose resistance than the target plant by promoting the expression of the Cslnc924 gene in the target plant.

[0020] Preferably, the method for promoting the expression of the Cslnc924 gene in the target plant includes: constructing a Cslnc924 gene overexpression vector and introducing it into the target plant.

[0021] The beneficial effects of this invention are as follows:

[0022] 1. This invention proposes the application of the Cslnc924 gene in the diagnosis or control of anthracnose in tea trees. The 257bp-511bp portion of the Cslnc924 gene binds to the (-825bp)-(-1053bp) promoter of CsmiR390a and activates the transcription of CsmiR390a. Figure 1The Cslnc924 gene positively regulates the miR390-TAS3-ARF2s module, reducing the expression levels of ARF2s (ARF2.1 and ARF2.2) and thereby enhancing the resistance of tea plants to anthracnose. This study proposes a new use for the Cslnc924 gene and expands our understanding of the regulation of the miR390-TAS-ARF2s module.

[0023] 2. This invention aims to verify whether Cslnc924 affects the resistance of tea plants to anthracnose by regulating the MiR390-TAS3-ARF2s module. Based on Agrobacterium-mediated transient overexpression and antisense oligonucleotide-mediated gene silencing technologies, Cslnc924 was overexpressed and silenced on tea leaves, respectively, followed by anthracnose inoculation experiments. The results showed that when Cslnc924 was overexpressed, compared with the control, both Cslnc924 and CsmiR390 genes were significantly upregulated, while their downstream CsARFs (CsARF2.1, CsARF2.2, CsARF3, and CsARF4.1) were significantly downregulated. At this time, the resistance of tea plants to anthracnose was enhanced, and the lesion area was significantly reduced. Figure 2 ).

[0024] 3. Conversely, when Cslnc924 is silenced, both Cslnc924 and CsmiR390 genes are significantly downregulated, while the transcriptional levels of their downstream CsARFs (CsARF2.1, CsARF2.2, CsARF3, and CsARF4.1) are significantly upregulated. At this time, the tea plant's resistance to anthracnose is weakened, and the lesion area is significantly larger. It is noteworthy that when Cslnc924tu, which lacks the CsmiR390 promoter binding site, is overexpressed in tea plants, the expression levels of CsmiR390a and its downstream CsARFs (CsARF2.1, CsARF2.2, CsARF3, and CsARF4.1) show no significant changes, and the lesion size is not significantly different from the control. Figure 2 ). Attached Figure Description

[0025] Figure 1This is an experimental verification diagram of Cslnc924 activating CsmiR390a in Example 1 of the present invention. A shows a schematic diagram of the gene structure of Cslnc924, the CsmiR390a precursor, and the CsmiR390a promoter. The reverse complementary sequence is marked in red. B is a molecular interaction model diagram of Cslnc924 and the CsmiR390a promoter predicted by AlphaFold3 software. C is a secondary structure diagram of Cslnc924 and its truncated variant (Cslnc924tu), with the stem-loop structure of the complementary region marked in red. D is a schematic diagram of the truncated CsmiR390a promoter construct in a dual-luciferase reporter assay. E shows the activation effect of Cslnc924 and Cslnc924tu on the truncated CsmiR390a promoter fragment in a dual-luciferase reporter assay.

[0026] Figure 2 This is a functional verification diagram of Cslnc924 and Cslnc924tu in regulating tea plant disease resistance in Example 1 of this invention. A and B are functional diagrams analyzing the role of Cslnc924 in tea plant resistance to anthracnose through transient overexpression by Agrobacterium and AsODN-mediated gene silencing. C is a diagram verifying the regulation of tea plant resistance by Cslnc924tu based on transient overexpression of tea leaves by Agrobacterium. The gene expression levels of CsmiR390a and CsARFs were quantitatively detected by qRT-PCR. Data are presented as mean ± standard deviation (n≥6). Significance was assessed using a two-tailed Student's t-test (*P<0.05, **P<0.01). Each data point represents a biological replicate. Detailed Implementation

[0027] To make the objectives, 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 conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical terms used below have the same meaning as understood by those skilled in the art.

[0028] Unless otherwise specified, the test materials and reagents used in the following examples are commercially available or prepared by known methods.

[0029] Unless otherwise specified, all techniques or conditions described in the embodiments can be performed in accordance with the techniques or conditions described in the literature in this field or in the product manual. Unless otherwise specified, the quantitative experiments in the following embodiments are all repeated three times or more, and the results are averaged.

[0030] Example 1:

[0031] This embodiment provides an application of Cslnc924 regulating the miR390 / TAS3 / ARFs module in tea plants to improve anthracnose resistance. The steps include:

[0032] To verify whether Cslnc924 affects the resistance of tea plants to anthracnose by regulating the MiR390-TAS3-ARF2s module, we first confirmed through a dual-luciferase reporter assay that Cslnc924 can bind to the promoter of CsmiR390a via base complementarity and activate its promoter activity.

[0033] The dual-luciferase reporter assay includes the following steps:

[0034] 1. Construction of overexpression vectors for Cslnc924 and its mutant forms

[0035] (1) Using RNA obtained from Zhongcha 108 as material, 5′ and 3′ RACE-Ready cDNA templates required for RACE cloning were synthesized using the SMARTer RACE 5′ / 3′ Kit (Takara). PCR amplification was performed using the following primers, referring to the kit instructions:

[0036] Cslnc924-5'RACE-R (SEQ ID NO:3): 3'-GGACATCTGCTCAAGATCACTGG-5';

[0037] Cslnc924-3'RACE-F (SEQ ID NO:4): 5'-GCTATAAGCTGCATTGTTAAGC-3';

[0038] Cslnc924-F (SEQ ID NO:5): 5'-AAGCAGTGGTATCAACGCAGAGTACA-3';

[0039] Cslnc924-R (SEQ ID NO:6): 3'-GCAGTGGTATCAACGCAGAGTAC-5';

[0040] The PCR reaction system is based on PrimeSTAR. TM The PCR amplification was performed according to the instructions for HSDNA Polymerase (Taraka). The PCR amplification program was as follows: 98℃ for 10 sec, 62℃ for 10 sec, 72℃ for 1 min, 30 cycles, 72℃ for 6 min, and 16℃ to finish. The obtained PCR products were verified by agarose gel electrophoresis, and the bands at the target positions were recovered from the gel.

[0041] (2) Using the amplification product recovered in step (1) as a template, Cslnc924 was mutated using the following primers to obtain the cloned product Cslnc924tu:

[0042] Cslnc924tu-F1 (SEQ ID NO:7): 5'-ACATGGGCTACAACGGTCCGAC-3'

[0043] Cslnc924tu-R1 (SEQ ID NO:8): 3'-GGTGTTTTATTACCCACAGC-5'

[0044] Cslnc924tu-F2 (SEQ ID NO:9): 5'-GCTGTGGGTAATAAAACACC-3'

[0045] Cslnc924tu-R2 (SEQ ID NO:10): 3'-GCAGTGGTATCAACGCAGAGTAC-5'

[0046] (3) Take the 1305 vector and perform double digestion with restriction endonucleases BamhI and XbaI to recover the vector backbone;

[0047] (4) The amplification products recovered in steps (1) and (2) and the vector backbone recovered in step (3) were ligated to obtain recombinant plasmids 35S::Cslnc924 and 35S::Cslnc924tu. The ligation process was performed according to the ClonExpress II One Step Cloning Kit of Nanjing Novizan Biotechnology Co., Ltd. 2 μL of the recovered product was ligated into 0.5 μL of the cloning vector, reacted at 25℃ for 15 min, incubated on ice for 5 min, 20 μL of DH5α competent cells were added, heat-shocked at 42℃ for 1 min, incubated on ice for 2 min, and the resulting product was added to 200 μL of LB medium and incubated on a shaker at 37℃ for 1 h. Finally, the bacterial culture was spread on a plate containing Kan antibiotic and incubated at 37℃. The colony growth was observed the next day, and single colonies were picked for PCR verification. The bacteria with the correct band position were sequenced.

[0048] 2. Construction of CsmiR390a promoter and its truncated overexpression vector

[0049] (1) Using the DNA obtained from Zhongcha 108 as material, PCR amplification was performed according to the description in step 1 using the following primers, and the amplification products were recovered:

[0050] ProCsmiR390a-F (SEQ ID NO:11):

[0051] 5'-GAGTGTGAGGTAACGTAGTCGGCAG-3'

[0052] ProCsmiR390a-R (SEQ ID NO:12):

[0053] 3'-TGAGTTTGTATATGTGATGCATGTGT-5'

[0054] (2) Using the amplification product from step (1) as a template, the CsmiR390a promoter sequence was truncated using the following primers to obtain cloning products of different truncated forms of the CsmiR390a promoter, namely P1, P2, P3 and P4:

[0055] ProCsmiR390a(P1)-0800-F(SEQ ID NO:13):

[0056] 5'-CGAATTCCTGCAGCCCGGGGGAGTGTGAGGTAACGTAGTCGG-3'

[0057] ProCsmiR390a-0800-R (SEQ ID NO:14):

[0058] 3'-GCTCTAGAACTAGTGGATCGTTTGTATATGTGATGCA-5'

[0059] P2-0800-F (SEQ ID NO:15):

[0060] 5'-CGAATTCCTGCAGCCCGGGGTCCACAGCTTTACCAAACAAG-3'

[0061] P3-0800-F (SEQ ID NO:16):

[0062] 5'-CGAATTCCTGCAGCCCGGGGTGCATCACATATACAAACTCA-3'

[0063] P4-0800-F (SEQ ID NO:17):

[0064] 5'-CGAATTCCTGCAGCCCGGGGTCCACAGCTTTACCAAACAAG-3'

[0065] P4-F (SEQ ID NO:18): 5'-ATTATATATTATTCCACAGCTTTAC-3'

[0066] P4-R (SEQ ID NO:19): 3'-GTAAAGCTGTGGAATAATATATAAT-5'

[0067] (3) Take the pGreenII 0800 vector, digest it with the restriction endonuclease BamhI, and recover the vector backbone.

[0068] (4) The amplification products recovered in steps (1) and (2) are ligated with the vector backbone recovered in step (3) to obtain recombinant plasmids ProCsmiR390a::LUC, P1-LUC, P2-LUC, P3-LUC and P4-LUC.

[0069] 3. Transformation of Agrobacterium competent cells

[0070] Mix 20 μL of competent Agrobacterium GV3101 cells with 2 μL of plasmid and incubate on ice for 5 min; freeze in liquid nitrogen for 5 min; incubate in a 37°C water bath for 5 min; incubate on ice for 5 min; add 500 μL of LB liquid medium (antibiotic-free) and incubate at 28°C and 220g for 3 h; plate 200 μL onto LB solid medium containing Kan and Rif antibiotics and incubate at 28°C for 3 days; screen positive clones by colony PCR.

[0071] 4. Dual-luciferase reporter assay

[0072] (1) Recombinant Agrobacterium containing effectors (35S::Cslnc924 and 35S::Cslnc924tu) and reporter genes (ProCsmiR390a::LUC, P1-LUC, P2-LUC, P3-LUC, and P4-LUC), as well as control recombinant Agrobacterium (pGreenII 0800 vector introduced into GV3101), were activated and cultured to OD600 = 0.8. 600 It is 0.8.

[0073] (2) Collect the bacterial cells by centrifugation at room temperature and resuspend them twice in a solution containing 10-mM MgCl2, 10-mM MES and 100-μM acetylsyringone (pH 5.6);

[0074] (3) Adjust the OD of Agrobacterium suspension 600 A concentration of 0.8 was applied to 5-6 week old leaves of Nicotiana benthamiana. The plants were then cultured for another 3 days.

[0075] (4) The luciferase substrate was sprayed onto the leaves of *Tobacco Bungei*, and the luminescence signal was detected using a CCD imaging device (Lumazone Pylon2048B). At the same time, the activities of LUC and Renilla (REN) in fireflies were measured using dual luciferase assay reagents (Promega, Madison, WI, USA). The LUC / REN ratio represents the activity of the promoter.

[0076] Secondly, based on Agrobacterium-mediated transient overexpression and antisense oligonucleotide-mediated gene silencing technologies, the Cslnc924 gene was overexpressed and silenced on tea leaves, respectively, and anthrax inoculation experiments were conducted.

[0077] Specifically, the following steps are included:

[0078] 1. Transient overexpression of tea leaves

[0079] (1) Recombinant Agrobacterium tumefaciens 35S::Cslnc924 and 35S::Cslnc924tu were activated and cultured to OD. 600 It is 0.8;

[0080] (2) Collect the bacterial cells by centrifugation at room temperature and resuspend them twice in a solution containing 10-mM MgCl2, 10-mM MES and 100-μM acetylsyringone (pH=5.6);

[0081] (3) Adjust the OD of Agrobacterium suspension 600 0.8g of tea leaves were injected into the tea plant.

[0082] (4) Anthrax inoculation and gene quantitative detection and analysis were performed 24 hours later.

[0083] 2. Cslnc924 antisense oligonucleotide silencing experiment

[0084] (1) Using SOLIGO software, the following candidate antisense oligonucleotides (AsODNs) targeting Cslnc924 were selected and synthesized by General Biosystems.

[0085] Cslnc924-sODN (SEQ ID NO:20): 5'-GGGCTGCCCAAGGGGGTGACCAAGC-3';

[0086] Cslnc924-AsODN1 (SEQ ID NO:21): 3'-GTCGGACCGTGGTAGCAGGACAG-5';

[0087] Cslnc924-AsODN2 (SEQ ID NO:22): 3'-CCATTTGTCCGATGCATATT-5';

[0088] Cslnc924-AsODN3 (SEQ ID NO:23): 3'-GCTTGGTCACCCCCTTGGGCAGCCC-5'.

[0089] (2) 1 mL of 100 μM AsODN mixed solution with a final concentration was injected into the leaves of 2-year-old cuttings, while those injected with positive oligonucleotides (sODNs) served as controls.

[0090] (3) Real-time quantitative PCR (qRT-PCR) detection and analysis were performed 48 hours later.

[0091] 3. Anthracnose infection of tea leaves from Zhongcha 108 tea plantations

[0092] (1) The anthrax strain (Camelliae camelliae) was cultured on PDA medium for 5 days at a culture temperature of 28℃;

[0093] (2) Collect spores by centrifugation at 6000g for 10 minutes. After collection, resuspend the spores in sterile water and adjust their concentration to 10^6 spores / mL under microscopic monitoring for subsequent inoculation;

[0094] (3) A total of 50 μL of conidial suspension was inoculated into the upper epidermis of tea leaves using a sterile syringe. Control plants were inoculated with an equal volume of sterile distilled water. The inoculated leaves were covered with plastic film to maintain high humidity and promote fungal growth.

[0095] (4) After a 24-hour incubation period, remove the film and record symptoms of infected leaves within 6 days after inoculation.

[0096] 3. qRT-PCR assay was used to detect the gene expression levels of CsmiR390a, Cslnc924, and CsARFs.

[0097] (1) Based on the gene sequence and mature sequence of the local transcriptome CsmiR390a, the following reverse transcription primers and quantitative fluorescence primers were set using the stem-loop method, with the CsU6 gene as an internal reference.

[0098] CsmiR390a-cDNA (SEQ ID NO:24):

[0099] 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGGCGCT-3'

[0100] CsmiR390a-qF (SEQ ID NO:25): 5'-GCGGCAAGCTCAGGAGGGAT-3';

[0101] CsmiR390a-qR (SEQ ID NO:26): 3'-CCAGTGCAGGGTCCGAGGTA-3';

[0102] CsU6-qF (SEQ ID NO:27): 5'-CGGGGACATCCGATAAAATTG-3';

[0103] CsU6-qR (SEQ ID NO:28): 3'-GGACCATTTCTCGATTTGTGC-3';

[0104] (2) The qRT-PCR primers for Cslnc924 and CsARFs were designed according to the MIQE guidelines as follows:

[0105] Cslnc924-qF (SEQ ID NO:29): 5'-ATCTTCTTGTGGTAGGTTGCT-3';

[0106] Cslnc924-qR (SEQ ID NO:30): 3'-TTGGGTTGTTTGAATGTTAAA-3';

[0107] CsARF2.1-qF (SEQ ID NO:31): 5'-GAGATAGGAGCCGATAAAGTTGG-3';

[0108] CsARF2.1-qR (SEQ ID NO:32): 3'-GCAATGAAAGTGCCCTTGAAAGC-3';

[0109] CsARF2.2-qF (SEQ ID NO:33): 5'-CAATGCCTTCTGGTCTCTCCC-3';

[0110] CsARF2.2-qR (SEQ ID NO:34): 3'-TTTCGTTGCTGTGCTCTGCT-3';

[0111] CsARF3-qF (SEQ ID NO:35): 5'-ATCTGCTGACTACTGGTTGGA-3';

[0112] CsARF3-qR (SEQ ID NO:36): 3'-AAGGCTCTTTGAGAATTTACG-3';

[0113] CsARF4.1-qF (SEQ ID NO:37): 5'-AAGAAGAATGCATGTGGCAAT-3';

[0114] CsARF4.1-qR (SEQ ID NO:38): 3'-GGTGGAAAAGGTGAGGAAGAG-3';

[0115] CsGAPDH-qF (SEQ ID NO:39): 5'-TTGGCATCGTTGAGGGTCT-3';

[0116] CsGAPDH-qR (SEQ ID NO:40): 3'-CAGTGGGAACACGGAAAGC-5'.

[0117] (3) The qRT-PCR reaction mixture includes 1 μL of cDNA template and 10 μL of MonAmp. 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 program was: 95°C pre-denaturation for 10 min, 95°C denaturation for 10 sec, 60°C annealing for 10 sec, and 72°C extension for 30 sec, for a total of 40 cycles. Melting curves were acquired using the instrument's default program. Three biological replicates were set up for each material, and three technical replicates were set up for each PCR reaction. The relative expression levels of genes were calculated using the Pfaffl method.

[0118] The results showed that when Cslnc924 was overexpressed, both Cslnc924 and CsmiR390a genes were significantly upregulated compared to the control, while their downstream CsARFs (CsARF2.1, CsARF2.2, CsARF3, and CsARF4.1) were significantly downregulated. Under these conditions, tea plants exhibited enhanced resistance to anthracnose, and the lesion area was significantly reduced (e.g., ...). Figure 2 (As shown). Conversely, when Cslnc924 is silenced, both Cslnc924 and CsmiR390a genes are significantly downregulated, while the transcriptional levels of their downstream CsARFs (CsARF2.1, CsARF2.2, CsARF3, and CsARF4.1) are significantly upregulated. At this time, the tea plant's resistance to anthracnose is weakened, and the lesion area significantly increases (e.g., ...). Figure 2(As shown) It is noteworthy that when Cslnc924tu, which lacks the CsmiR390Pro binding site, was overexpressed in tea plants, the expression levels of CsmiR390a and its downstream CsARFs (CsARF2.1, CsARF2.2, CsARF3, and CsARF4.1) did not change significantly, and the lesion size was not significantly different from the control. These results further confirm that the 257bp-511bp region of Cslnc924 plays a crucial role in its resistance-conferring function.

[0119] In summary, this invention identifies a long non-coding RNA (lncRNA) Cslnc924 involved in regulating resistance to anthracnose in tea plants. Specifically, the anthracnose-induced significantly upregulated lncRNA Cslnc924 enhances tea plant resistance to anthracnose by activating the transcription of CsmiR390a and positively regulating the miR390-TAS3-ARF2s module, thereby reducing the expression level of CsARF2s.

[0120] SEQ ID NO.1:

[0121]

[0122] SEQ ID NO.2:

[0123]

[0124] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An application of the Cslnc924 gene, characterized in that, The expression of the Cslnc924 gene is promoted to enhance the resistance of tea plants to anthracnose. The full-length cDNA sequence of the Cslnc924 gene is shown in SEQ ID No.

1.

2. The use of the Cslnc924 gene according to claim 1, characterized in that, The Cslnc924 gene is a long non-coding RNA, and its RNA sequence is shown in SEQ ID No.

2.

3. Application of the Cslnc924 gene in the preparation of drugs for the prevention and control of anthracnose in tea trees, wherein the full-length cDNA sequence of the Cslnc924 gene is shown in SEQ ID No.

1.

4. Application of the Cslnc924 gene in the breeding of anthracnose-resistant tea varieties, wherein the full-length cDNA sequence of the Cslnc924 gene is shown in SEQ ID No.

1.

5. A tea tree model susceptible to anthrax, characterized in that, The anthrax-sensitive tea tree model contains a product that inhibits the expression of the Cslnc924 gene; the full-length cDNA sequence of the Cslnc924 gene is shown in SEQ ID No.

1.

6. An expression carrier, characterized in that, It contains the Cslnc924 gene; the full-length cDNA sequence of the Cslnc924 gene is shown in SEQ ID No.

1.

7. A breeding method for anthracnose-resistant tea trees, characterized in that, Includes the following steps: By promoting the expression of the Cslnc924 gene in the target plant, plants with higher resistance to anthracnose in tea trees than the target plant were obtained; the full-length cDNA sequence of the Cslnc924 gene is shown in SEQ ID No.

1.

8. The breeding method for anthracnose-resistant tea trees according to claim 7, characterized in that, The method for promoting the expression of the Cslnc924 gene in the target plant includes: constructing a Cslnc924 gene overexpression vector and introducing it into the target plant; the full-length cDNA sequence of the Cslnc924 gene is shown in SEQ ID No. 1.

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