Cucumber csmyb82 gene and application thereof

Abstract

The application belongs to the field of plant biotechnology, and particularly relates to a cucumber CsMYB82 gene and application thereof, and is an MYB transcription factor capable of being combined with a CsCSE1 promoter through yeast one-hybrid screening. It is proved through EMSA and tobacco transient expression analysis that the CsMYB82 gene directly and specifically combines with a "CAACCA" sequence in the CsCSE1 promoter, and inhibits the expression of the CsCSE1 gene. The CsMYB82 gene can be induced to express by powdery mildew, and is differentially expressed in cucumber infected by the powdery mildew. It is found through a cucumber cotyledon transient transformation system mediated by agrobacterium that the CsMYB82 gene negatively regulates the biosynthesis of lignin and the resistance of cucumber to the powdery mildew. The application provides a new reference gene resource for the cultivation of a cucumber disease-resistant variety, and provides a new thought for the research on the molecular mechanism of plant immunity mediated by the lignin pathway.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology, specifically relating to the cucumber CsMYB82 gene and its application, used to regulate the biosynthesis of cucumber lignin and resistance to powdery mildew. Background Technology

[0002] Cucumber (Cucumis sativus L.) is one of the world's most widely cultivated economic crops. Efficient and stable cucumber production is crucial for ensuring global vegetable security. Powdery mildew, caused by the biotrophic fungal pathogen Podosphaera xanthii, is one of the most destructive diseases in cucumber cultivation, causing significant losses in both yield and quality. Although powdery mildew can be controlled chemically, long-term use of chemical fungicides leads to increasing resistance in the pathogen and also pollutes the environment. In contrast, genetic selection of resistant cucumber varieties is considered a more economical, environmentally friendly, and effective strategy for controlling powdery mildew, and functional analysis of key resistance genes in cucumbers is fundamental to constructing resistance genetic networks and conducting molecular design breeding. Therefore, identifying genes that play important roles in the cucumber-powdery mildew interaction and elucidating their underlying molecular mechanisms is of paramount importance.

[0003] Currently, plants have evolved various complex defense mechanisms to cope with pathogen attacks. As a crucial component of plant cell walls, lignin provides an important physical and chemical barrier against the spread of pathogens. When plants are infected by pathogens, large amounts of lignin accumulate in the cell walls. This accumulation effectively hinders pathogen spread and limits the entry of fungal enzymes and toxins into the plant cell walls. Furthermore, lignin-related compounds can render pathogens incapable of infecting the host, thereby preventing pathogen proliferation and invasion. Given the critical role of lignin in plant disease resistance, its biosynthesis mechanism has been a focus of extensive research. Lignin biosynthesis is regulated by a highly complex regulatory network, currently generally considered to be based on the NAC-MYB gene regulatory network model. In this model, NAC transcription factors are the first-layer regulators, acting as molecular switches for xylem cell differentiation. They can participate in lignin biosynthesis by regulating second-layer regulators or bypassing the second-layer regulators to directly regulate third-layer genes. The second layer of regulators consists of MYB transcription factors, including AtMYB46 and AtMYB83. These are direct targets of the first layer of regulators and also directly participate in the transcriptional regulation of key enzyme genes in the lignin biosynthesis pathway and other lignin biosynthesis-related MYB transcription factor genes (such as AtMYB58, AtMYB63, and MYB103). Besides NAC and MYB transcription factors, other transcription factors such as bHLH and WRKY have also been reported to participate in the regulation of lignin biosynthesis. In summary, the mechanism of lignin biosynthesis is extremely complex.

[0004] Despite significant progress in elucidating the role of lignin in plant immunity and the mechanisms of lignin biosynthesis, systematic research is still lacking on how key factors at various levels of the lignin biosynthesis regulatory network mediate plant defense responses against pathogens by regulating lignin biosynthesis. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a cucumber CsMYB82 gene and its application, used to regulate the biosynthesis of cucumber lignin and resistance to powdery mildew.

[0006] This invention is achieved by providing a cucumber CsMYB82 gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] A protein encoded by the cucumber CsMYB82 gene is provided, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] A recombinant expression vector for the cucumber CsMYB82 gene is provided.

[0009] A recombinant strain of cucumber with the CsMYB82 gene is provided.

[0010] This invention provides an application of the cucumber CsMYB82 gene for regulating the expression of the CsCSE1 gene.

[0011] Preferably, in the above applications, the cucumber CsMYB82 gene or its specific sequence specifically binds to the MYB element "CAACCA" in the CsCSE1 promoter, thereby inhibiting the expression of the CsCSE1 gene.

[0012] This invention provides an application of the cucumber CsMYB82 gene for regulating cucumber resistance to powdery mildew.

[0013] Preferably, in the above applications, the powdery mildew fungus is a vivotrophic fungal pathogen with the Latin name Podosphaera xanthii.

[0014] This invention provides an application of the cucumber CsMYB82 gene for regulating the biosynthesis of cucumber lignin.

[0015] A method for regulating the biosynthesis of cucumber lignin and resistance to powdery mildew is provided, wherein the cucumber CsMYB82 gene or its specific sequence is introduced into cucumber, and the specific sequence of the CsMYB82 gene is shown in SEQ ID NO.3.

[0016] Compared with the prior art, the advantages of the present invention are as follows:

[0017] The CsMYB82 gene provided in this invention was screened using yeast one-hybrid technology to identify a MYB transcription factor that binds to the CsCSE1 promoter. Further EMSA and transient expression analysis in tobacco demonstrated that the CsMYB82 gene directly and specifically binds to the "CAACCA" sequence in the CsCSE1 promoter, inhibiting CsCSE1 gene expression. The CsMYB82 gene can be induced by powdery mildew and exhibits differential expression in B21-a-2-1-2 and B21-a-2-2-2 cucumbers infected with powdery mildew. Using an Agrobacterium-mediated transient transformation system in cucumber cotyledons, it was found that the CsMYB82 gene negatively regulates lignin biosynthesis and cucumber resistance to powdery mildew. This invention provides a new reference gene resource for the breeding of disease-resistant cucumber varieties and offers new insights into the molecular mechanisms of plant immunity mediated through the lignin pathway. Attached Figure Description

[0018] Figure 1Phylogenetic analysis and homologous sequence alignment of cucumber CsMYBs and Arabidopsis AtMYBs, where (a) is the phylogenetic analysis of CsMYBs and AtMYBs; and (b) is the sequence alignment of CsMYB46, CsMYB58, CsMYB82, CsODO1, AtMYB46, AtMYB63, AtMYB83 and AtMYB85.

[0019] Figure 2 The screening process for CsMYB binding to the CsCSE1 promoter is shown in (a) as a schematic diagram of the CsCSE1 promoter segmentation; (b) as the detection of the binding of CsMYB46, CsMYB58, CsMYB82 and CsODO1 to the CsCSE1 promoter by yeast one-hybrid assay; and (c) as the EMSA analysis of the binding of CsMYB82 and CsODO1 to the CsCSE1 promoter.

[0020] Figure 3 The transcriptional regulation of CsCSE1 by CsMYB82 and CsODO1 is shown in the figure. (a) is a schematic diagram of the effector and reporter recombinant vectors used in the transient expression analysis experiment in tobacco; (b) is the histochemical staining and activity analysis of GUS; and (c) is the luminescence imaging and quantitative comparison of LUC signal.

[0021] Figure 4 Expression analysis of CsMYB82 and CsODO1 genes in B21-a-2-1-2 and B21-a-2-2-2 cucumbers infected with powdery mildew;

[0022] Figure 5 To identify powdery mildew resistance in cucumber cotyledons with silenced CsMYB82 and CsODO1 genes, (a) is a schematic diagram of the silenced recombinant expression vector; (b) is the symptom of cucumber cotyledons infected with Tobacco Crisp Virus (TRV); (c) is the expression analysis of CsMYB82 and CsODO1 silenced plants; and (d) is the identification of resistance of cucumber cotyledons with silenced CsMYB82 and CsODO1 genes 7 days after inoculation with powdery mildew by phenotypic observation, Coomassie brilliant blue staining, and DI survey.

[0023] Figure 6 To identify the powdery mildew resistance of cucumber cotyledons overexpressing CsMYB82 and CsODO1, (a) shows a schematic diagram of the overexpression recombinant vector and PCR analysis of the overexpressing plants; (b) shows the expression analysis of plants overexpressing CsMYB82 and CsODO1 genes; and (c) shows the resistance of cucumber cotyledons overexpressing CsMYB82 and CsODO1 to powdery mildew 7 days after inoculation, identified by phenotypic observation, Coomassie brilliant blue staining, and DI.

[0024] Figure 7The regulation of lignin biosynthesis by CsMYB82 and CsODO1 is shown; (a) shows the lignin content and lignin staining analysis of cucumber cotyledons with CsMYB82 gene silence / overexpression; (b) shows the lignin content and lignin staining analysis of cucumber cotyledons with CsODO1 gene silence / overexpression. Detailed Implementation

[0025] The present invention will be further illustrated below by describing the embodiments in detail, but this is not intended to limit the invention and is only for illustrative purposes.

[0026] Currently, systematic research on how key factors at various levels of the lignin biosynthesis regulatory network mediate plant defense responses against pathogens through the regulation of lignin biosynthesis remains lacking. Previous studies disclosed a potential regulatory gene for cucumber powdery mildew resistance, CsCSE, which is named CsCSE1 in this invention. This gene encodes caffeoyl shikimate esterase (CSE), a key member of the lignin biosynthesis pathway. To further investigate the molecular mechanism of CsCSE1-mediated powdery mildew resistance, this application screened two MYB transcription factors, CsMYB82 and CsODO1, in cucumber. In vitro and in vivo experiments demonstrated that CsMYB82 and CsODO1 can directly bind to the "CAACCA" sequence in the CsCSE1 promoter and inhibit CsCSE1 gene expression. Furthermore, silencing CsMYB82 and CsODO1 in cucumber promotes lignin accumulation and resistance to powdery mildew, while overexpression of CsMYB82 and CsODO1 in cucumber weakens lignin accumulation and resistance to powdery mildew. Therefore, this invention provides an important reference for research on the molecular mechanisms of plant immunity regulation through the lignin pathway.

[0027] Example 1: Obtaining Experimental Samples

[0028] I. Plant materials and growing conditions

[0029] Three cucumber germplasms (B21-a-2-1-2, B21-a-2-2-2, and Xintai Mici) and one tobacco germplasm (Nicotiana benthamiana) were used in this study. B21-a-2-1-2 and B21-a-2-2-2 were used for gene cloning and expression pattern analysis, while Xintai Mici was used for gene transformation. Nicotiana benthamiana was used for transient expression analysis. All plants were grown at 25°C under a photoperiod of 16h (light):8h (dark).

[0030] II. Inoculation with powdery mildew fungus

[0031] Powdery mildew fungus was collected from infected cucumber leaves in a greenhouse and purified in the laboratory. The leaves infected with powdery mildew were soaked in distilled water to prepare a concentration of 2 x 10⁻⁶. 5A suspension of 1 spore / mL was then sprayed evenly onto the cotyledons of 9-day-old (overexpressed) / 2-week-old (silenced) Xintai Micizi, as well as the cotyledons of 9-day-old B21-a-2-1-2 and B21-a-2-2-2.

[0032] III. Gene Cloning and Expression Analysis

[0033] Genomic DNA was extracted from the sample using the "SteadyPure Plant Genomic DNA Extraction Kit"; Genomic DNA was extracted using the "SteadyPure Plant Genomic DNA Extraction Kit"; Total RNA was extracted from the samples using the "Super Total RNA Extraction Kit"; reverse transcription was performed using the "EvoM-MLV RT for PCR Kit". Gene-specific primers were used to clone the corresponding sequences, and the PCR reaction was performed according to the "Hieff" protocol. The "Gold High-Fidelity DNA Polymerase" kit was used. Gene expression analysis was performed by RT-qPCR, and the reaction program followed the "SYBR Green Premix Pro Taq HS qPCR Kit" procedure. The procedure was performed on a 480II instrument. CsActin was used as an internal control. The primers used in this invention are listed in Table 1.

[0034] Table 1 List of primers used in this invention

[0035]

[0036]

[0037]

[0038] Example 2: Transcriptional Regulation of CsCSE1 by CsMYB82 and CsODO1

[0039] I. Yeast one-hybrid screening for CsMYB that bind to the CsCSE1 promoter

[0040] In Arabidopsis thaliana, the AtCSE gene promoter can be activated by AtMYB transcription factors (including AtMYB46, AtMYB83, AtMYB63, and AtMYB85) that specifically activate lignin biosynthesis or participate in plant secondary cell wall biosynthesis. In this invention, promoter analysis using the PlantCARE database revealed that the CsCSE1 promoter contains two MYB binding sites (Table 2). To determine whether the CsCSE1 promoter can bind to CsMYB, phylogenetic analysis was performed using the MEGA7 Neighbor-Joining method, and homologous sequence alignment was performed using DNAMAN software. Corresponding CsMYB transcription factors, namely CsMYB82, CsMYB46, CsMYB58, and CsODO1, were obtained in cucumber (see [link to relevant documentation]). Figure 1 (a) and Figure 1 (b). Based on the cis-acting element, the promoter of CsCSE1 is divided into 5 segments (P CsCSE1-1 To P CsCSE1-5 ),See Figure 2 (a) These fragments were inserted into the pAbAi vector to form bait vectors. The bait vectors were transformed into Y1H Gold competent cells and plated on SD / -Ura medium. PCR identification was then performed, and the obtained positive clones were prepared as competent cells. The CDS of CsMYB46, CsMYB58, CsMYB82, and CsODO1 were recombined into the pGADT7 vector to construct prey vectors. The prey vectors and the empty pGADT7 vector were introduced into the self-made competent cells and plated on SD / -Leu(-AbA / +AbA) medium for interaction verification. The results showed that P CsCSE1-4 It can bind to CsMYB82 and CsODO1. The nucleotide sequence of the CsODO1 gene is shown in SEQ ID NO.4, the amino acid sequence of the encoded protein is shown in SEQ ID NO.5, and the nucleotide sequence of the specific sequence is shown in SEQ ID NO.6. When P... CsCSE1-4 When the presumed MYB recognition site “CAACCA” in the region mutated to “AAAAAA”, no binding was observed. (See [link to relevant documentation]). Figure 2 (b) In the figure, the recombinant vector pAbAi-P CsCSE1-4 and pAbAi-mP CsCSE1-4 They carried wild-type (WT) and mutant CsCSE1 promoters, respectively; the mutant was generated by changing the MYB recognition site from "CAACCA" to "AAAAAA". (300 ng / mL) -1 In the presence of gold basidiomycin A (AbA), pGADT7-CsMYB82 / pGADT7-CsODO1 and pAbAi-P CsCSE1-4The co-transformed yeast grew extensively on SD / -Leu medium, indicating that CsMYB82 / CsODO1 positively binds to the WT promoter of CsCSE1.

[0041] Table 2. Prediction of cis-regulatory elements in the CsCSE1 gene promoter region.

[0042]

[0043]

[0044] II. EMSA identification of the binding of CsMYB82 and CsODO1 to the "CAACCA" sequence in the CsCSE1 promoter.

[0045] The coding sequences of CsMYB82 and CsODO1 were constructed into the pET30a vector to generate recombinant proteins with His tags. The recombinant proteins were expressed in *E. coli* Rosetta (DE3) and purified using the His-tag Protein Purification Kit. DNA probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and biotin-tagged the 3' end. EMSA was performed using the Chemiluminescent EMSA Kit, and the biotin signal was detected. The results showed that CsMYB82 and CsODO1 could bind to the biotin-tagged P... CsCSE1-4 (Biotin-P CsCSE1-4 Probe binding causes band shift. Competitive probes containing non-biotin-labeled probes (Cold-P) bind to the target band. CsCSE1-4 ) Reduced CsMYB82 and CsODO1 with Biotin-P CsCSE1-4 The binding of the probe is strong, and the binding band weakens with the addition of more competing probes. When Biotin-P CsCSE1-4 When “CAACCA” was mutated, no binding band was observed. Figure 2 (c) In the experiment, the 3' end of the probe was labeled with biotin. A non-biotin-labeled competing probe, Cold-P, was also used. CsCSE1-4 (50x, 100x (and 150x)) competing CsMYB82 and CsODO1 with the biotin-labeled probe Biotin-P CsCSE1-4 The combination of Biotin-mP CsCSE1-4 It is by using Biotin-P CsCSE1-4 The MYB recognition site is generated by a mutation from "CAACCA" to "AAAAAA". Figure 2 (c) indicates that CsMYB82 and CsODO1 can specifically bind to “CAACCA” in the CsCSE1 promoter.

[0046] III. GUS Reporting System Analysis: Regulation of CsCSE1 Expression by CsMYB82 and CsODO1

[0047] CsMYB82 and CsODO1 were constructed as effectors into GFP-tagged vectors driven by a 35S promoter. CsCSE1 and P CsCSE1-4 They were recombined separately onto vectors containing GUS tags, and as report sub-sub ... Figure 3 (a) The above-described vector was used to transform tobacco leaves for GUS histochemical staining and activity analysis. For histochemical staining, tobacco samples were immersed in GUS staining buffer (39 mM disodium hydrogen phosphate, 61 mM sodium dihydrogen phosphate, 0.05 mM potassium ferricyanide, 0.05 mM potassium ferrocyanide, 1 mM EDTA, 10% (w / v) X-Gluc, 0.1% (v / v) Triton X-100) under vacuum for 10 min and then protected from light at 37°C for 24 h. Destaining was performed using a destaining solution (ethanol:acetic acid = 7:3, v / v). For GUS activity analysis, 100 mg of tobacco sample was ground into powder in liquid nitrogen, and 1 mL of GUS extraction buffer (39 mM disodium hydrogen phosphate, 61 mM sodium dihydrogen phosphate, 0.01% (w / v) SDS, 0.1% (v / v) Triton X-100, 1 mM EDTA, 0.1% (v / v) β-mercaptoethanol) was added to extract total protein. The total protein concentration was determined according to Bradford (1976)'s method, and a standard curve was constructed using BSA as a standard. 100 μL of the protein supernatant was added to 900 μL of GUS reaction buffer containing 2 mM 4-MUG, and the reaction was carried out at 37 °C for 30 min. Then, 200 μL of the reaction solution was added to 800 μL of 0.2 M Na₂CO₃ to terminate the reaction. The fluorescence intensity at an excitation wavelength of 365 nm and an emission wavelength of 455 nm was measured using a fluorescence spectrophotometer, and the corresponding 4-MU concentration was calculated based on the fluorescence standard curve. The results showed that, compared with the control, CsMYB82 / CsODO1 and P CsCSE1 / P CsCSE1-4 The tobacco leaves co-expressing GUS showed light staining and weak GUS enzyme activity, see Figure 3 (b)

[0048] IV. Dual-luciferase reporter system analysis of the regulation of CsCSE1 expression by CsMYB82 and CsODO1

[0049] The promoter of CsCSE1 (P CsCSE1 and P CsCSE1-4 (Clone into pGreenII 0800-LUC as a reporter vector, see...) Figure 3(a) The previously constructed effector vectors GFP:CsMYB82 / GFP:CsODO1 and the reporter vector were transiently co-expressed in tobacco. Three days later, tobacco leaves were sprayed with 0.2 mM luciferin solution, and LUC fluorescence was detected using a plant in vivo molecular imaging system. Simultaneously, LUC activity was detected using the "Dual Luciferase Reporter Gene Assay Kit". The co-expression of the pRI101-GFP empty vector and the reporter vector was used as a control. The results showed that both CsMYB82 and CsODO1 (but not the GFP empty vector control) significantly inhibited P... CsCSE1 / P CsCSE1-4 Driven expression of LUC reporter genes, see Figure 3 (c) In the figure, the data are the mean ± SD of three biological replicates for each treatment, and significance was assessed by LSD multiple comparisons (*P≤0.05, **P≤0.01).

[0050] Example 3: The role of CsMYB82 and CsODO1 in cucumber's resistance to powdery mildew.

[0051] I. Analysis of the expression patterns of CsMYB82 and CsODO1 genes under powdery mildew stress

[0052] Gene expression analysis was performed on leaves of B21-a-2-1-2 and B21-a-2-2-2 cucumbers inoculated with powdery mildew at 0, 3, 6, 9, 12, and 24 h using RT-qPCR. The results showed that CsMYB82 and CsODO1 were induced by powdery mildew in both B21-a-2-1-2 and B21-a-2-2-2 cucumber germplasms, and their expression levels in B21-a-2-1-2 were generally lower than those in B21-a-2-2-2. (See [link to relevant documentation]). Figure 4 In the figure, cotyledons were examined at 0, 3, 6, 9, 12 and 24 h after infection with powdery mildew. The data are the mean ± SD of three biological replicates for each treatment. Significance was assessed by LSD multiple comparisons (*P≤0.05, **P≤0.01). Figure 4 This indicates that both CsMYB82 and CsODO1 are involved in the regulation of powdery mildew resistance.

[0053] II. Regulation of powdery mildew resistance by silencing CsMYB82 and CsODO1 genes

[0054] Virus-induced gene silencing (VIGS) experiments were performed using pTRV1 and pTRV2 vectors. CDS region-specific fragments of CsMYB82 (289 bp) and CsODO1 (287 bp) were recombined into the pTRV2 vector, see [link to pTRV2 vector]. Figure 5(a) Recombinant vectors, pTRV1, and pTRV2 were introduced into Agrobacterium tumefaciens strain EHA105. Equal volumes of pTRV1 bacterial suspension were mixed with pTRV2 and the recombinant vector to generate TRV:00, TRV:CsMYB82, and TRV:CsODO1, which were then infiltrated into the cotyledons of 7-day-old cucumber seedlings. Ten days after Agrobacterium infiltration, cotyledons were collected for phenotypic observation and expression analysis. The results showed that the chlorotic spots on the cotyledons of cucumbers with TRV:00, TRV:CsMYB82, and TRV:CsODO1 indicated that TRV had successfully invaded the plant. This phenomenon was not observed in untreated plants or plants injected only with EHA105. Figure 5 (b) The expression levels of CsMYB82 and CsODO1 were significantly reduced in silent plants, see [reference needed]. Figure 5 (c) In the figure, data represent the mean ± SD of three biological replicates for each treatment. Significance was assessed using LSD multiple comparisons (*P≤0.05, **P≤0.01). Powdery mildew was inoculated 7 days after Agrobacterium infiltration, and resistance was assessed 7 days later. Results showed that, compared to the control, CsMYB82 and CsODO1 silenced plants exhibited milder lesions, fewer mycelia, and lower DI (Distribution Indication). Figure 5 (d) In the figure, the scale bar is 100 μm, indicating that the silencing of CsMYB82 and CsODO1 enhances the defense against powdery mildew.

[0055] III. Regulation of powdery mildew resistance by overexpression of CsMYB82 and CsODO1 genes

[0056] The vectors GFP:00 (control), GFP:CsMYB82, and GFP:CsODO1 were introduced into the cotyledons of 7-day-old cucumber seedlings via Agrobacterium-mediated transformation. Five days after Agrobacterium infiltration, PCR identification was performed using primers pRI101-GFP-CsMYB82-F / pRI101-GFP-CsODO1-F and GFP-R to identify the CsMYB82 / CsODO1 and GFP fusion genes. Corresponding transgenic positive bands were detected in the overexpressing plants. (See attached image). Figure 6 (a) In the figure, arrows indicate fragments amplified by recombinant structures identified by PCR. PCR analysis was performed using cucumber cotyledons transformed with GFP:00, GFP:CsMYB82, and GFP:CsODO1, as well as the recombinant plasmid (Vector). Vector and GFP:00 served as positive and negative controls, respectively. Simultaneously, RT-qPCR results showed that the expression levels of CsMYB82 and CsODO1 were significantly increased in overexpressing plants. Figure 6(b) In the figure, data represent the mean ± SD of three biological replicates for each treatment. Significance was assessed using LSD multiple comparisons (*P≤0.05, **P≤0.01). Two days after Agrobacterium infiltration, powdery mildew was inoculated. Resistance assessment seven days after inoculation revealed that, compared to the control, plants overexpressing CsMYB82 and CsODO1 infected with powdery mildew exhibited higher susceptibility, i.e., more severe lesions, more hyphae, and a higher DI (Distribution Indication). See [Figure Number]. Figure 6 (c), scale bar 100 μm, further supports the view that CsMYB82 and CsODO1 negatively regulate powdery mildew resistance.

[0057] IV. Regulation of lignin by CsMYB82 and CsODO1 genes

[0058] Following existing methods, lignin content and histochemical staining analysis were performed on cucumber leaves transiently expressing CsMYB82 and CsODO1 for 10 days of silence and 5 days of overexpression. The results showed that lignin accumulation in CsMYB82 / CsODO1-silenced plants was significantly increased compared to the control; conversely, lignin accumulation in CsMYB82 / CsODO1-overexpressing plants was significantly lower than the control. Figure 7 (a) and Figure 7 (b) In the figure, FW represents fresh weight, and the data are the mean ± SD of three biological replicates for each treatment. Significance was assessed by LSD multiple comparisons (*P≤0.05, **P≤0.01), scale bar 100 μm. The results indicate that CsMYB82 and CsODO1 play a negative regulatory role in lignin biosynthesis.

Claims

1. Cucumber CsMYB82 The application of genes is characterized by, The CsMYB82 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the application is: silencing. CsMYB82 Genes or their specific sequences are used to enhance cucumbers' defense against powdery mildew. CsMYB82 The specific sequence of the gene is shown in SEQ ID NO.

3.

2. The application according to claim 1, characterized in that, The powdery mildew fungus is a biotrophic fungal pathogen, with the Latin name [blank]. Podosphaera xanthii .

3. Cucumber CsMYB82 The application of genes is characterized by, CsMYB82 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the application is silencing. CsMYB82 Genes are used to increase the accumulation of lignin in cucumber plants.

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