Application of CsTSR gene in cucumber corynespora leaf spot resistance

Abstract

The invention belongs to the technical field of plant biology, and particularly provides application of a CsTSR gene in cucumber corynespora leaf spot resistance. A transient expression strain and a cstsr mutant are respectively obtained through transient transformation of the CsTSR gene and a Tnt1 reverse transcription transposon mutant library, and the CsTSR gene is found to positively regulate the resistance of the cucumber to the corynespora leaf spot disease. The miR164d can regulate and control the CsTSR transcription factor in a targeted manner. A dual luciferase report and a GUS (glucuronidase) staining experiment result show that the miR164d negatively regulates and controls the CsTSR, and the CsTSR-mediated cucumber disease resistance can be enhanced by silencing the miR164d. The invention discloses a functional mechanism of the miR164d-CsTSR molecular module in cucumber pathogenic bacterium infection resistance, and provides a new reference gene resource for cultivation of cucumber disease-resistant varieties.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology, and relates to genetic engineering technology, and specifically provides a... CsTSR Application of genes in resistance to Cucumber Corynebacterium leaf spot disease. Background Technology

[0002] Cucumber is one of the important greenhouse vegetable crops in my country. In recent years, due to increased planting density and continuous cropping, it has become increasingly susceptible to various fungal diseases. Among them, cucumber leaf spot disease manifests as yellow-brown spots on the leaves. As the disease progresses, the spots merge, causing the leaves to wither and fall off, severely affecting photosynthesis, yield, and quality. This disease is highly prone to outbreaks in hot and humid environments and is rapidly and widely spread through the air. Currently, chemical control is the main approach, but this poses risks such as pesticide residues and environmental hazards from pesticide resistance enhancers.

[0003] Plant resistance to pathogens is regulated by multiple signaling pathways, with transcription factors playing a central role. NAC family transcription factors are widely involved in plant growth, development, and responses to abiotic stresses. Previous studies have shown that microRNAs (miRNAs) can target and regulate NAC transcription factors in response to plant stress defenses; however, whether miR164d targets and regulates these factors in cucumber remains to be seen. CsTSR The mechanisms by which it affects resistance to Cladosporium leaf spot have not yet been reported. Therefore, it is necessary to explore the mechanisms by which it has a clear disease resistance function. CsTSR The discovery of the gene and the elucidation of its molecular mechanism of regulation by miR164d are of great significance for breeding disease-resistant cucumber varieties. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides an NAC-type transcription factor. CsTSR Gene( CsaV4_ 4G001697 Application of ) in resistance to Cucumber Corynebacterium leaf spot disease.

[0005] This invention is achieved by providing a gene that regulates resistance to *Cercospora leaf spot* disease in cucumbers. CsTSR Its nucleotide sequence is shown in SEQ ID NO.1.

[0006] Provided is a protein encoded by the gene regulating cucumber resistance to Corynebacterium leaf spot, the amino acid sequence of which is shown in SEQ ID NO.2.

[0007] Provided is a mutant of the gene regulating cucumber resistance to Corynebacterium leaf spot, which is a mutant. cstsr Its nucleotide sequence has a Tnt retrotransposon insertion relative to SEQ ID NO.1, resulting in the loss of function of the CsTSR gene.

[0008] A silenced fragment of the gene regulating resistance to Cucumber leaf spot disease is provided, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0009] The application of the gene regulating cucumber resistance to Corynebacterium leaf spot is provided, for regulating cucumber resistance to Corynebacterium leaf spot fungus.

[0010] Preferably, overexpression in cucumber plants CsTSR Genes were used to enhance cucumber resistance to Corynebacterium leaf spot disease.

[0011] The fungus *Cladosporium*, which causes leaf spot, is a plant fungal pathogen, with the Latin name... Corynespora cassiicola .

[0012] A microRNA molecule miR164d gene that regulates NAC transcription factors is provided, the nucleotide sequence of which is shown in SEQ ID NO.4.

[0013] A target mimic sequence STTM-miR164d for silencing the miR164d gene is provided, the nucleotide sequence of which is shown in SEQ ID NO.5.

[0014] The application of the miR164d gene, a microRNA molecule that regulates the NAC transcription factor, is provided for targeting and negatively regulating the expression of the gene that regulates cucumber resistance to Corynebacterium leaf spot.

[0015] Preferably, the expression of the miR164d gene is suppressed in cucumber plants to enhance the resistance of cucumbers to Corynebacterium leaf spot.

[0016] Provided CsTSR The gene, the miR164d gene or the target mimic sequence STTM-miR164d recombinant expression vector and recombinant bacteria.

[0017] Compared with the prior art, the advantages of the present invention are as follows: This invention clones transcription factor genes from cucumbers. CsTSR The study identified the gene and its encoded protein CsTSR, and constructed transient overexpression and transient silencing vectors. Using an Agrobacterium-mediated transient transformation system of cucumber cotyledons, it was found that… CsTSR Transient overexpression of the gene enhanced cucumber resistance to Corynebacterium leaf spot. Based on a library of cucumber Tnt1 retrotransposon mutants, the 9930 cucumber strain was obtained. cstsr The mutant, functional analysis showed that, cstsr The mutants exhibited high susceptibility to Cucumber leaf spot disease caused by Corynebacterium hupensis. Furthermore, the microRNA molecule miR164d, which targets and regulates NAC transcription factors, was discovered. Dual-luciferase reporter assays and GUS staining experiments showed that miR164d targets and negatively regulates...CsTSR Gene expression. Further utilizing transient conversion technology, silencing miR164d promoted... CsTSR mediated resistance of cucumber to Corynebacterium leaf spot.

[0018] This invention, through analysis of disease-resistance genes in cucumbers, discovered and identified novel genes related to cucumber scab leaf spot disease, and further determined... CsTSR The disease resistance function of the miR164d-CsTSR module and its regulation of cucumber disease resistance provide new reference gene resources for the breeding of disease-resistant cucumber varieties, and also provide new ideas for the study of the molecular mechanism of plant immunity mediated by transcription factors. Attached Figure Description

[0019] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments: Figure 1 for CsTSR qRTR-PCR quantification results after transient gene overexpression (A) and transient silencing (B); Figure 2 Wild-type strains WT and GFP: CsTSR Phenotypic characteristics of leaves inoculated with Corynebacterium leaf spot 5 dpi in transiently overexpressing and transiently silent lines; Figure 3 for CsTSR qRT-PCR quantification results of genes mediating disease course; Figure 4 Insertion of Tnt retrotransposon CsTSR A schematic diagram; Figure 5 Wild-type strain WT and homozygous mutant strain csTSR Phenotypic characteristics of cucumber leaves inoculated with Corynebacterium leaf spot at 5 dpi; Figure 6 To analyze the transcriptional regulation of CsTSR by miR164d in a dual-luciferase reporter assay, (A) represents the 35S reporter gene: CsTSR -Schematic diagram of the vector construction of LUC and effector 35S:miR164d, (B) is in vivo imaging of LUC, (C) is LUC enzyme activity assay; Figure 7 For GUS staining experiments and GUS enzyme activity analysis, miR164d regulates CsTSR, where (A) is a schematic diagram of the miR164d binding site and site mutations, and (B) is the reporter gene 35S. CsTSR -GUC, site-mutated reporter gene 35S: CsTSRtb - Vector construction map of GUS and effector 35S:miR164d, (C) is GUS staining, (D) is GUS enzyme activity assay; Figure 8(A) Schematic diagram of the structure of STTM-miR164d; qRTR-PCR quantification results after transient overexpression (B) and transient silencing (C) of miR164d; Figure 9 Transient overexpression of *Cladosporium leaf spot* under stress CsTSR Phenotypes of miR164d and co-transfected cells; Figure 10 Transient silencing under stress from *Cladosporium cladosporum* leaf spot pathogen CsTSR The phenotypes of STTM-miR164d and co-transfected cells. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0021] In recent years, multi-master spirochete ( Corynespora cassiicola Corynebacterium leaf spot, caused by *Corynebacterium*, has become a major biological stressor for greenhouse cucumbers. Currently, there is limited research both domestically and internationally on the resistance mechanisms of cucumbers to this disease. Therefore, identifying more genes related to resistance to Corynebacterium leaf spot and elucidating its molecular mechanisms is of great significance for controlling the occurrence of Corynebacterium leaf spot and increasing cucumber yield.

[0022] This application uses a combination of miRNA sequencing and transcriptome sequencing to predict the target gene of miRNA164d, which is involved in regulating resistance to cucumber corynebacterium leaf spot disease. CsTSR For further research CsTSR The molecular mechanism of cucumber resistance to Corynebacterium leaf spot mediated by this application. CsTSR The effect of this drug on cucumber resistance to Corynebacterium leaf spot and its targeted regulation by upstream miR164d were analyzed. This was achieved by constructing... CsTSR Transient transformation lines and cstsr Mutant evidence, CsTSR It positively regulates cucumber resistance to Corynebacterium leaf spot. Dual-luciferase reporter assays and GUS staining experiments showed that miR164d targets and negatively regulates the transcriptional level of CsTSR. Furthermore, using Agrobacterium-mediated transient transformation of cucumber cotyledons, it was found that inhibition of miR164d expression promoted... CsTSR The ability of cucumbers to resist Corynebacterium leaf spot. Therefore, this invention provides a new perspective on the molecular mechanisms of plant immunity mediated by transcription factors.

[0023] Example 1: Key Materials and Their Descriptions The pRI101-AN-eGFP expression vector, pTRV expression vector, pCAMBIA3301-LUC expression vector, and pRI101-AN-GUS expression vector were stored in the laboratory for use in constructing... CsTSR, Recombinant expression vectors of miR164d and STTM-miR164d.

[0024] *E. coli* DH5α competent cells and *Agrobacterium* EHA105 competent cells were purchased from Beijing Coollab Technology Co., Ltd.; the "Eastep® Super Total RNA Extraction Kit" (plant total RNA extraction kit), "Evo M-MLV RT for PCR Kit" (reverse transcription kit), and "SYBR Green Premix SupTaq HS qPCR Kit (Low Rox Plus)" (fluorescent quantitative PCR premix) were purchased from Hunan Aikerui Biotechnology Co., Ltd.; the "ClonExpress Ultra One Step Cloning Kit V3" (rapid cloning kit) was purchased from Nanjing Novizan Biotechnology Co., Ltd.; and the "2×Taq Master Mix" (DNA polymerase premix) was purchased from Liaoning Zhongke Saitu Biotechnology Co., Ltd. All amplification primers used were synthesized by Suzhou Genewiz Biotechnology Co., Ltd. Table 1. List of primers used in this invention Example 2 Construction CsTSR The recombinant expression vector and recombinant bacteria required for miR164d.

[0025] CsTSRThe nucleotide sequence of the gene coding region is as follows: ATGGAAAATAATAATAATAACAATAATATAAGCATGGTTGAGGCAAAACTCCCACCTGGATTTAGGTTTCATCCAAGAGATGAAGAATTGGTTTGTGATTATTTGATGAAGAAAATTGGGTCTAATTCTTCTTCTTCTTCTTCTCTATTGATTGAAGTTGACCTCAACAAGTGTGAGCCTTGGGATATTCCAAGAGAGGCATGCGTCGGTGGAAAAGAGTGGTACTTCTTCAGCCAGAGGGACCGTAAGTACGCGACTGGGCTTAGAACAAACCGCGCCACAGCCTCTGGGTATTGGAAGGCCACTGGCAAGGACAGGCCTGTTTTTCATAAGGCTAATCAACTCGTTGGGATGAGGAAGACCCTTGTTTTCTACCAAGGTAGGGCTCCTAAAGGCCGAAAAACTGAGTGGGTTATGCATGAATTTCGTCTTGAGGGTCCATTTTCTCCTATTACAGACCCATCTCCAAAGGAGGACTGGGTTCTGTGCAGATTGTTCTGTAAACAAAAGGAAGTTACCCCTCAGCCGAGCACAGGAAGCAGCAGCTGCTACAACGACACCATTGGGTCGTCGTCGTCTCTCCCAGCTTTAATGGATTCATACATCAGTTTTGACCAAAATCCAAATAGTCATTTAAACGAGTATGAGCAAGTGCCCTGCTTCTCCATTTTCTCTCATAATCAAACCATCCCAACTCTCACAAACCTCATACAAATGGAGGCAAACACAGGCAATAACATCAAGAACCTTAGCACCATGTTTGGAGGAGGAATGCCAAATTCAACCACTTGTTCTTCAAATATTGACCCTTTTACGTGTGACTCTAAAGTACTTAAAGTTGTTCTAAACAATATTACTAAGATGGAAACAAATGGAAGTTCCTTCATCGGGCAAACTAGCATGGGAGAAGGCAGCTCTGATAGCTACTTATCCGAGGTCGGAGTCGGAGACGACATTGCCAGCTTATGGAACAGATAA (SEQ IDNO.1); CsTSR The amino acid sequence of the protein encoded by the gene is as follows: MENNNNNNNISMVEAKLPPGFRFHPRDEELVCDYLMKKIGSNSSSSSSLLIEVDLNKCEPWDIPREACVGGKEWYFFSQRDRKYATGLRTNRATASGYWKATGKDRPVFHKANQLVGMRKTLVFYQGRAPKGRKTEWVMHEFRLEGPFSPITDPSP KEDWVLCRLFCKQKEVTPQPSTGSSSCYNDTIGSSSSLLPALMDSYISFDQNPNSHLNEYEQVPCFSIFSHNQTIPTLTNLIQMEANTGNNIKNLSTMFGGGMPNSTTCSSNIDPFTCDSKVLKVVLNNITKMETNGSSFIGQTSMGEGSSDSYLSEVGVGDDIASLWNR (SEQ IDNO.2); CsTSR The silencing sequence of the gene is as follows: GAGTATGAGCAAGTGCCCTGCTTCTCCATTTTCTCTCATAATCAAACCATCCCAACTCTCACAAACCTCATACAAATGGAGGCAAACACAGGCAATAACATCAAGAACCTTAGCACCATGTTTGGAGGAGGAATGCCAAATTCAACCACTTGTTCTTCAAATATTG ACCCTTTTACGTGTGACTCTAAAGTACTTAAAGTTGTTCTAAACAATATTACTAAGATGGAAACAAATGGAAGTTCCTTCATCGGGCAAACTAGCATGGGAGAAGGCAGCTTGATAGCTACTTATCCGAGGTCGGAGTCGGAGACGACATTGCCAGCTTATGGAACAGATAA (SEQ IDNO.3); The miR164d nucleotide sequence is as follows: TGGAGAAGCAGGGCACGTGCA (SEQ ID NO.4) (U is not allowed in the ST.26 standard, so to maintain consistency with the sequence listing readable vector, U is represented by T here). The STTM-miR164d sequence is as follows: AAGGTTACCGAATTCTGGAGAAGCAGCTAGGCACGTGCAGTTGTTGTTGTTATGGTCTAATTTAAATATGGTCTAAAGAAGAAGAATTGGAGAAGCAGCTAGGCACGTGCAGAGCTCACGCGTCTC (SEQ ID NO.5). The STTM-miR164d sequence used in this invention was synthesized by Suzhou Genewiz.

[0026] RNA was extracted from cucumber cotyledons and reverse transcribed to obtain cucumber cDNA. This cDNA was then used as a template for PCR amplification with Taq polymerase using primers designed in Example 1. CsTSR The gene fragment sequence size was 627 bp, with a gene silencing fragment length of 339 bp and an STTM-miRNA silencing fragment length of 126 bp. Corresponding bands were detected by agarose gel electrophoresis, indicating successful amplification of the target fragment. Large fragments of pRI101-AN-eGFP, pTRV2, pCAMBIA3301-LUC, and pRI101-AN-GUS stored in the laboratory were ligated with the corresponding target fragment gel recovery products and transformed into DH5α competent cells. Single colonies were selected for PCR identification. Positive single colonies were cultured overnight in LB medium at 37°C using a shaker. Plasmids were extracted and sent to Suzhou Genewiz Biotechnology Co., Ltd. for sequencing. Correct sequencing results confirmed successful vector construction.

[0027] Example 3 CsTSR Its role in the response of cucumber to infection by *Cyclospora leaf spot* This invention employs virus-induced gene silencing (VIGS) and transient overexpression technology to induce gene silencing in cotyledons of 10-day-old cucumber seedlings grown under a photoperiod of 16 h light / 8 h dark and a temperature of 25°C. CsTSR Genes are used for functional verification.

[0028] Cucumber cotyledons treated with recombinant vector injection were selected as experimental materials. Specifically, the treatment included injection of empty GFP:00 vector and GFP:00 vector. CsTSR The recombinant vector was treated for 3 days, and TRV:00 empty vector and TRV:00 were injected. CsTSR The recombinant vector was injected 7 days later. Real-time quantitative q-PCR was used to analyze the results. CsTSR Gene expression levels were analyzed. The results showed that, through the two techniques described above, CsTSR The gene achieved significant transient overexpression and effective transient silencing in cucumber cotyledons. Figure 1 ).

[0029] Seven days after injection treatment, cucumber cotyledons were inoculated with *Cladosporium* leaf spot pathogens, and a perforator was used to ensure that the initial lesion area at each inoculation site was uniform. Five days after inoculation, the disease incidence on the cotyledons was observed, the lesion area was measured, and the disease index was calculated. Figure 2 The results showed that compared to the TRV:00 empty vector control group, TRV: CsTSR The treated leaves showed a significant increase in lesion area, indicating transient silencing. CsTSR The gene reduced cucumber resistance to Cladosporium leaf spot. Conversely, GFP: CsTSR The lesion area in the treatment group was significantly smaller than that in the GFP:00 empty vector control group, indicating transient overexpression. CsTSR The gene can enhance the cucumber's resistance to this pathogen. These results confirm that... CsTSR Positively modulates cucumber resistance to Cladosporium leaf spot disease.

[0030] To explore CsTSR The role of this gene in cucumber resistance to Corynebacterium leaf spot was analyzed using qRT-PCR to examine the effects of transient overexpression and silencing of this gene on disease-related protein genes, including... PR1-1a (AB698861) PR2 (XM_011661051) and PR3 (HM015248). The result is as follows: Figure 3 As shown, compared to TRV:00, the cucumber cotyledons in the transient silence treatment group had lower concentrations of [missing information]. PR1-1a , PR2 and PR3 Expression levels were significantly reduced; while compared to the GFP:00 empty vector control, GFP: CsTSR The expression levels of the aforementioned PR genes were significantly upregulated in the transient overexpression treatment group. The results indicate that... CsTSR It can positively regulate the expression of multiple PR genes, thereby participating in the defense response of cucumber against Corynebacterium leaf spot disease.

[0031] 9930 were obtained from the Tnt1 retrotransposon mutant library. cstsr The mutant was obtained directly through multiple generations of self-crossing. cstsr Homozygous mutant. cstsr This is due to the insertion of Tnt1 retrotransposon into... CsTSR Formed on the second exon, homozygosity was determined using primers Chr4G6F, TN5R, and Chr4G6F. Figure 4 ).

[0032] For three-week-old WT wild-type plants, cstsr Homozygous mutant plants were inoculated with *Cladosporium* leaf spot pathogen, and resistance was assessed 5 days later. The results showed that... cstsr Leaf spot lesions on the leaf surface of *Cladosporium* were significantly more severe than those on WT plants. Figure 5 ).

[0033] Example 4 miR164d target gene CsTSR Regulation analysis This invention analyzes the effect of miR164d on LUC and GUS experiments. CsTSR Targeted regulatory effects. With 35S: CsTSR -LUC is the reporter gene, and 35S:miR164d is the effector. The two genes, along with the empty vector, were mixed in a 1:1 ratio to prepare the infection solution, yielding 35S: CsTSR -LUC / 35S: with 35S: CsTSR -LUC / 35S:miR164d. The above mixed infection solution was co-transformed into tobacco leaves, and fluorescence intensity and LUC enzyme activity were measured after transient expression for 24-72 h. Results showed that co-transformation with 35S: CsTSR Tobacco leaves with -LUC / 35S: showed a clear fluorescence signal, while those with co-transformed 35S: CsTSR The leaves of -LUC / 35S:miR164d showed almost no fluorescence; the LUC enzyme activity test results were consistent, with the former's enzyme activity being significantly higher than the latter's. Figure 6 ).

[0034] Based on the predicted binding sites, reporter vectors 35S containing complete binding sites were constructed. CsTSR -GUS and the corresponding mutated binding site vector 35S: CsTSRtb -GUS, see Figure 7 (A). Tobacco leaves were co-transformed with the two reporter vectors mentioned above using 35S:miR164d as the effector. The results showed that co-transformation of 35S:miR164d... CsTSR -GUS staining in tobacco leaves with 35S:miR164d was the lightest, significantly lighter than that with co-transformed 35S: CsTSR -GUS and no-load 35S: blades; while co-rotating 35S: CsTSRtb -GUS and 35S:miR164d staining depth of tobacco leaves compared with co-transformed 35S: CsTSRtb -GUS was similar to that of leaves without 35S. GUS enzyme activity assays were consistent with staining findings; under mutant binding site conditions, there was no significant change in enzyme activity after co-transfection with miR1 for 4 days ( Figure 7 The above results indicate that miR164d can specifically target... CsTSR It binds to the binding site and inhibits its expression.

[0035] The LUC and GUS experiments described above confirm that miR164d can interact with... CsTSR The specific binding site of miR164d significantly inhibits its expression activity, indicating that miR164d is a key binding site in cucumber. CsTSR The negative regulatory factor.

[0036] Example 5: miR164d-CsTSR module regulates resistance to Corynebacterium leaf spot disease in cucumber cotyledons. To efficiently inhibit miRNAs and explore their functions, this invention constructs the STTM-miR164d recombinant vector based on TRV. The structure of STTM-miR164d is as follows: Figure 8 (A) shows the simulated miR164d binding sites at both ends of the structure. Each target site has three base protrusions to prevent cleavage, and the restriction enzyme sites at both ends are EcoRI and SacI, respectively. The connecting region in the middle is a specific 48 nt sequence. Cucumber cotyledons treated with recombinant vectors were selected as experimental materials, specifically including: injection of pRI-101AN empty vector and 35:miR164d recombinant vector for 2 days, and injection of TRV:00 empty vector and TRV:STTM-miR164d recombinant vector for 7 days. The expression level of the miR164d gene was analyzed using real-time quantitative PCR. The results showed that, through the above two techniques, miR164d achieved significant transient overexpression and effective transient silencing in cucumber cotyledons, respectively. Figure 8 ).

[0037] To investigate the function of the miR164d-CsTSR regulatory module in cucumber response to infection by *Cladosporium cladosporium*, this invention employs a co-injection technique. Overexpression of miR164d was combined with GFP: CsTSR Co-injected into cucumber cotyledons, simultaneously containing empty GFP:00 vector and miR164d and GFP: CsTSR A single injection treatment served as a control. Three days after injection, the cotyledons of each treatment were inoculated with *Cladosporium* leaf spot pathogen, using a perforator to ensure uniform inoculation site area. Five days after inoculation, the lesion area was measured and the disease index was calculated. Results are as follows: Figure 9 As shown, GFP: CsTSR The lesion area in the single-treatment group was significantly smaller than that in the GFP:00 control group; the lesion area in the miR164d-only treatment group was significantly larger than that in the GFP:00 control group; while the lesion area in the miR164d-only treatment group was significantly larger than that in the GFP:00 control group; CsTSR The lesion area in the co-injection group was between that of the two groups mentioned above, that is, smaller than that in the miR164d-only treatment group but larger than that in the GFP group. CsTSR Individual treatment group. This result indicates that miR164d can partially inhibit... CsTSR The disease resistance mediated by this reduces the resistance of cucumber to Corynebacterium leaf spot.

[0038] This invention further utilizes STTM to inhibit miR164d function and performs gene silencing and functional complementation experiments. STTM-miR164d is then compared with TRV: CsTSRThe same vector was injected into cucumber cotyledons, with TRV:00 empty vector and single treatments used as controls. Seven days after injection, the cotyledons of each treatment were inoculated with *Cladosporium leaf spot*, and the lesion area and disease index were recorded five days later. Results are as follows: Figure 10 As shown, TRV: CsTSR The area of ​​leaf spot lesions in the treatment group was significantly larger than that in the TRV:00 control group, indicating that the silencing effect was significant. CsTSR Significantly reduced cucumber disease resistance; the lesion area in the TRV:STTM-miR164d treatment group was significantly smaller than that in the TRV:00 control group, indicating that inhibiting miR164d can enhance resistance. However, the treatment group treated with TRV:STTM-miR164d showed significantly smaller lesion area compared to the TRV:00 control group. CsTSR The lesion area in the co-injection group was between that of the two groups mentioned above. This result indicates that inhibition of miR164d can restore lesions to some extent. CsTSR The decreased disease resistance caused by silencing further confirms that miR164d negatively regulates... CsTSR Mediates resistance of cucumber to Corynebacterium leaf spot disease.

[0039] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A gene regulating resistance to cyclophosphamide leaf spot disease in cucumber, characterized in that, for CsTSR Gene number is CsaV4_ 4G001697 Its nucleotide sequence is shown in SEQ ID NO.

1.

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

2.

3. The mutant of the gene regulating resistance to Cucumber leaf spot disease as described in claim 1, characterized in that, mutant cstsr Its nucleotide sequence has a Tnt retrotransposon insertion relative to SEQ ID NO.1, resulting in the loss of function of the CsTSR gene.

4. A silencing fragment for silencing the gene regulating resistance to Cucumber leaf spot disease as described in claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

3.

5. The application of the gene regulating cucumber resistance to Corynebacterium leaf spot as described in claim 1, characterized in that, Used to regulate cucumber resistance to Cladosporium leaf spot pathogen.

6. The application of the gene regulating cucumber resistance to Corynebacterium leaf spot according to claim 5, characterized in that, Overexpression in cucumber plants CsTSR Genes were used to enhance cucumber resistance to Corynebacterium leaf spot disease.

7. The miR164d gene, a microRNA molecule that regulates NAC transcription factors, is characterized by, Its nucleotide sequence is shown in SEQ ID NO.

4.

8. A target mimic sequence STTM-miR164d for silencing the miR164d gene of claim 7, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

5.

9. The application of the miR164d gene, a microRNA molecule regulating NAC transcription factors, as described in claim 7, is characterized in that... This is used to target and negatively regulate the expression of the gene for regulating cucumber resistance to Corynebacterium leaf spot as described in claim 1.

10. The claim 1 CsTSR The gene, the miR164d gene of claim 7, or the target mimic sequence STTM-miR164d of claim 8, and the recombinant bacteria.

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