Application of CpWRKY71-related biological products in regulating powdery mildew resistance in zucchini

Abstract

This invention belongs to the field of genetic engineering technology, specifically relating to a... CpWRKY71 The application of related biological products in regulating the resistance of zucchini to powdery mildew, the aforementioned CpWRKY71 It can be any one of the following: 1) the nucleotide sequence shown in SEQ ID NO.29; 2) the sequence obtained by truncating SEQ ID NO.29; the truncated sequence is shown in SEQ ID NO.31. This invention is the first to clearly define... CpWRKY71 In terms of the regulatory role of zucchini in powdery mildew resistance, this gene can specifically act on the signaling pathway of zucchini in response to powdery mildew stress, and enhance plant resistance in a targeted manner by regulating the expression of downstream disease resistance-related genes.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a... CpWRKY71 Application of related biological products in regulating the resistance of zucchini to powdery mildew. Background Technology

[0002] Powdery mildew is a serious plant disease caused by a fungus. It primarily affects the leaves of zucchini, but can also influence the stems and petioles, though the fruit is less affected. In the early stages, small, nearly circular white powdery spots appear on the upper or lower surfaces of zucchini leaves. These spots rapidly expand and merge, eventually covering the entire leaf, resembling a layer of flour. In later stages, the powdery spots turn grayish-white or grayish-brown, and small black granules appear on them. Infected leaves turn yellow, wither, or curl, but usually do not fall off. This severely impairs photosynthesis, leading to weakened plant growth, reduced fruit production, and decreased fruit quality.

[0003] Currently, the main methods for controlling powdery mildew in zucchini include: 1) spraying with chemical fungicides such as sulfur preparations and triazoles; and 2) applying microbial preparations containing biocontrol bacteria or antibiotics such as Bacillus subtilis, polyoxin, and ningnanmycin. These methods primarily focus on the pathogen and the environment, rather than on modifying the host, i.e., the zucchini itself. This leads to uncertain and unsustainable control effects, while also posing environmental and safety risks. Against this backdrop, exploring and utilizing technologies related to zucchini's own resistance to powdery mildew provides a new direction for the green control of the disease. Summary of the Invention

[0004] This invention provides CpWRKY71 The application of related biological products in regulating the resistance of zucchini to powdery mildew directly regulates the immune response system of zucchini at the molecular level, thereby activating or enhancing its intrinsic resistance to powdery mildew pathogens.

[0005] The technical solution adopted in this invention is:

[0006] This invention provides CpWRKY71 The application of related biological products in regulating the resistance of zucchini to powdery mildew, the aforementioned CpWRKY71 It can be any of the following:

[0007] 1) The nucleotide sequence shown in SEQ ID NO.29;

[0008] 2) The truncated sequence of SEQ ID NO.29; the truncated sequence is shown in SEQ ID NO.31.

[0009] Preferably, the biological product is any one of the following:

[0010] A: Includes CpWRKY71 Recombinant overexpression vectors;

[0011] B: Includes CpWRKY71 A silent carrier;

[0012] C: containing CpWRKY71 Agrobacterium.

[0013] Preferably, the starting vector of the recombinant overexpression vector is ZYMV-eGFP;

[0014] The starting vector for the silencing vector is pTRV2.

[0015] Preferably, the recombinant overexpression vector is used to enhance... CpWRKY71 The expression level of [a specific substance] can be increased to improve the resistance of zucchini to powdery mildew, thereby breeding zucchini varieties resistant to powdery mildew.

[0016] Using the silencing carrier to reduce CpWRKY71 The expression level of [a specific substance] was increased to reduce the resistance of zucchini to powdery mildew, and powdery mildew-sensitive zucchini varieties were bred, which could then be used as research material to analyze the resistance mechanism of zucchini to powdery mildew.

[0017] Preferably, the method for improving the resistance of zucchini to powdery mildew is as follows:

[0018] RNA was extracted from zucchini and reverse transcribed into cDNA;

[0019] Using cDNA as a template, the primers shown in SEQ ID NO.1 and SEQ ID NO.2 were used to clone the sample. CpWRKY71 ;

[0020] Will CpWRKY71 The vector was ligated into the recombinant overexpression vector;

[0021] The recombinant overexpression vector was transformed into Agrobacterium to obtain recombinant Agrobacterium;

[0022] By infecting zucchini with recombinant Agrobacterium, overexpressing plants were obtained, thus enhancing the resistance of zucchini to powdery mildew.

[0023] Preferably, the cDNA is derived from zucchini leaves.

[0024] Preferably, the process of recombinant Agrobacterium infecting zucchini is as follows:

[0025] The recombinant Agrobacterium was cultured on a large scale, and the bacterial cells were collected.

[0026] Resuspend the bacterial cells and transfer the OD 600 Adjust to 1.0 to obtain the inoculum;

[0027] Inject the infection solution into the zucchini leaves.

[0028] Preferably, the conditions for extended culture are: culture at 28℃ for 16 hours.

[0029] Compared with the prior art, the beneficial effects of the present invention are:

[0030] This invention provides CpWRKY71 The application of related biological products in regulating the resistance of zucchini to powdery mildew, the aforementioned CpWRKY71 It can be any one of the following: 1) the nucleotide sequence shown in SEQ ID NO. 29; 2) the sequence obtained by truncating SEQ ID NO. 29; the truncated sequence is shown in SEQ ID NO. 31. This invention clarifies for the first time... CpWRKY71 This invention relates to the regulatory role of a gene in zucchini resistance to powdery mildew. This gene specifically acts on the signaling pathways in zucchini responding to powdery mildew stress, enhancing plant resistance by regulating the expression of downstream resistance-related genes. Furthermore, this invention also constructed silent plants based on the sequence shown in SEQ ID NO. 31, cultivating powdery mildew-susceptible zucchini varieties, which were then used as research material to elucidate the mechanism of zucchini resistance to powdery mildew. This invention enhances zucchini resistance to powdery mildew at the gene level, offering significant advantages over existing technologies in terms of root cause, precision, persistence, and environmental friendliness, representing a fundamental shift in zucchini powdery mildew control from external intervention to internal enhancement.

[0031] In addition to being directly applied to the breeding of disease-resistant zucchini varieties, CpWRKY71 The discovery also provides a reference for research on powdery mildew resistance in other cucurbitaceous crops such as cucumber and pumpkin; through gene homology analysis and functional verification, its regulatory mechanism can be extended to disease resistance research in other crops. CpWRKY71 With potential for cross-crop and cross-scenario applications, this invention provides a new technical approach for the integrated control of powdery mildew in cucurbitaceous crops. Attached Figure Description

[0032] Figure 1 for CpWRKY71 Expression and sequence analysis. A: CpWRKY71 Phylogenetic analysis of its homologous genes. B: Inoculation of susceptible plant S1 with powdery mildew. CpWRKY71 Real-time quantitative PCR analysis. C: resistant plant R1 after inoculation with powdery mildew. CpWRKY71 Real-time quantitative PCR analysis.

[0033] Figure 2 for CpWRKY71 Expression patterns of R1 in different tissues and under plant hormone treatments. A: CpWRKY71 Levels of expression in different organizations. B: CpWRKY71Expression levels under salicylic acid treatment. C: CpWRKY71 Expression levels under jasmonic acid treatment.

[0034] Figure 3 for CpWRKY71 Subcellular localization and transcriptional activation analysis. A: CpWRKY71 Subcellular localization in *Nicotiana benthamiana*, scale bar = 20 μm. B: CpWRKY71 Detection of transcriptional activation activity in yeast cells; where SD / -Trp is a culture medium lacking tryptophan, and SD / -Trp-His-Ade is a culture medium lacking tryptophan, histidine, and adenine.

[0035] Figure 4 Fluorescence and phenotypes of different zucchini plants. A: Fluorescence of different zucchini plants. B: Phenotypes of different zucchini plants.

[0036] Figure 5 for CpWRKY71 Identification of positive seedlings overexpressing the gene. "+" represents the positive control, which is the recombinant overexpression vector ZYMV-CpWRKY71; "-" represents the negative control; OE1~OE6 represent six independent vectors. CpWRKY71 Overexpression lines, M represents a 2000bp DNA molecular weight marker.

[0037] Figure 6 To overexpress the plant CpWRKY71 qPCR analysis of the genes. CK1~CK3 in the figure represent three different ZYMV-eGFP transgenic plants.

[0038] Figure 7 for CpWRKY71 Overexpression enhances the resistance of zucchini to powdery mildew. A: Phenotypic observation of different plants 7 days after powdery mildew treatment. B: Phenotypic characteristics of different plants under a handheld fluorescent lamp. C: Mycelial growth as shown by trypan blue staining.

[0039] Figure 8 for CpWRKY71 Silence weakens zucchini's resistance to powdery mildew. A: CpWRKY71 Schematic diagram of gene silencing vector construction. B: Identification by real-time quantitative PCR. CpWRKY71 Gene silencing efficiency. C: Phenotype of cotyledons in different plants after inoculation with powdery mildew. D: Results of trypan blue staining of cotyledons in different plants after inoculation with powdery mildew.

[0040] Figure 9 The malondialdehyde (MDA) content in different plants after powdery mildew treatment.

[0041] Figure 10 The results of DAB, NBT and trypan blue staining in different plants.

[0042] Figure 11 For the analysis of physiological indicators of different plants, A~E are, in order: H2O2, O2 - The activity of CAT, SOD and POD.

[0043] Figure 12 This study analyzes the expression of disease resistance genes in different plants. A through F represent: AOS1 , LOX1 , LOX2 , PR1a , PR1b and PR1c Expression profiles. Error bars are from three biological replicates, showing the standard deviation of the mean. Asterisks indicate statistical significance.

[0044] Figure 13 Yeast one-hybrid experiments showed that CpWRKY71 and CpPR1a Promoter binding. Y1HGold[p53-AbAi+pGADT7-p53] was used as a positive control, and Y1HGold[p53-AbAi+pGADT7] was used as a negative control. In the figure, SD / -Leu-Ura is the medium lacking leucine and uracil, and SD / -Leu-Ura with AbA (500 ng / mL) is the medium lacking leucine and uracil but supplemented with 500 ng / mL adenosine A.

[0045] Figure 14 EMSA verification of CpWRKY71 and CpPR1a In vitro binding of the TTGACT promoter motif. Detailed Implementation

[0046] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0047] The inventive concept of this invention is as follows:

[0048] Current methods for controlling powdery mildew in zucchini rely heavily on chemical pesticide spraying, which not only increases planting costs but also easily leads to pesticide residues, soil and water pollution, and the development of pesticide resistance in pathogens. Therefore, this invention provides a... CpWRKY71 The application of related biological products in regulating the resistance of zucchini to powdery mildew: This invention regulates the plant's own resistance at the gene level, thereby reducing the amount of chemical pesticides used, which not only reduces farmers' planting costs, but also meets the needs of green agricultural development and reduces the negative impact on the ecological environment.

[0049] The present invention CpWRKY71 The nucleotide sequence is shown in SEQ ID NO.29, and the amino acid sequence is shown in SEQ ID NO.30.

[0050] SEQ ID NO.29:

[0051] ATGTCAGATGAAATGTTTAAAGATCTATTTTTGTCGAGCCCTTTTGGATACGGTGGGTTGGGTGATTCAGAAACAGGCTTGGATGAGTACGAGTCTCTAGCCAGAGCTTTTGAGCTGTCCTCGGATTACTCGAATGAAATTTCGGGGACTCCCATGAATTCCTCCGCTTCCTTCTCCTCCTCTGATGCTGGAGCTGACGAGGATGATTCCCTCAAAGACAAGGATAAGCAGATCAAAGACATGGATGACGGCGGAGAGAGCTCCAAGACCGCGGCTAAATCAAAGAAGAAAGTAGAGAAGAAAGAAAGAGAGCCAAGAGTTGCTTTCATGACCAAGAGCGAGGTCGATCATCTTGAAGATGGGTATCGATGGAGAAAATATGGACAGAAGGCTGTCAAGAACAGCGCTTATCCTAGGAGCTATTACAGATGCACGACGCAGAAATGCGGAGTGAAGAAACGGGTGGAGAGATCGTATGAAGATCCATCCATAGTAATTACGACGTACGAAGGGCAACACAATCACCCAATTCCCGCGACGTTAAGGGGGAACCTGTCGGCAGTGAGCGGCGCGTTTCCGCCGTCCATGTTGGCACCAATGCCGGTGGTCGGTGGCGTGAGGTATCTTCCACAGCTGATGAACAACACTTCCGTCAACAACAACCAGCCCATCGGAGGTGGTGACACCGTTTATTCACAAAGCAGCAGCTTCAATTATCCTTATAACGGACGGCAACAAGACTACGGACTTCTGCAGGACATTTTTCCGACGGCGCCGCCGTTCTTGAACCGACAACCATGA.

[0052] SEQ ID NO.30:

[0053] MSDEMFKDLFLSSPFGYGGLGDSETGLDEYESLARAFELSSDYSNEISGTPMNSSASFSSSDAGADEDDSLKDKDKQIKDMDDGGESSKTAAKSKKKVEKEREPRVAFMTKSEVDHLEDGYRWRKYGQKAVK NSAYPRSYYRCTTQKCGVKKRVERSYEDPSIVITTYEGQHNHPIPATLRGNLSAVSGAFPPSMLAPMPVVGGVRYLPQLMNNTSVNNNQPIGGGDTVYSQSSSFNYPYNGRQQDYGLLQDIFPTAPPFLNRQP.

[0054] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0055] The list of abbreviations for this invention is shown in Table 1.

[0056] Table 1. List of abbreviations for this invention

[0057]

[0058] Example 1

[0059] CpWRKY71 The application of related biological products in regulating the resistance of zucchini to powdery mildew is as follows:

[0060] 1. Materials and Methods.

[0061] 1.1 Plant materials and treatment.

[0062] This invention uses zucchini inbred lines S1 and R1. Zucchini seeds are planted in a mixed substrate of soil and sand in a 1:1 mass ratio and cultured in an artificial climate chamber with day / night temperatures maintained at 28°C / 22°C and a photoperiod of 18h / 6h.

[0063] S1 and R1 are disclosed in the reference: Xu, K., & Wang, P. (2025). Identification of the PLATZ gene family in Cucurbita pepo and functional analysis of CpPLATZ4 inpowdery mildew stress response. Functional & Integrative Genomics, 25(1), 227.

[0064] (1) Powdery mildew stress. Select zucchini seedlings at the two-leaf stage for powdery mildew stress treatment. Collect zucchini leaves 0h, 6h, 12h and 24h after inoculation with powdery mildew pathogen.

[0065] The pathogen of powdery mildew stress in this invention is the physiological race 2US of powdery mildew, which is disclosed in the reference: Xu Ke. Physiological response of seed squash to powdery mildew stress and identification of the VQ gene family of disease resistance-related transcription factors [D]. Inner Mongolia Agricultural University, 2022. DOI:10.27229 / d.cnki.gnmnu.2022.001165.

[0066] (2) Hormone treatment.

[0067] Two-leaf stage seedlings of R1 were treated with salicylic acid (0.1 mM) and jasmonic acid (500 µM), respectively. Leaf samples were collected at 0 h, 1 h, 3 h, 6 h, 9 h, and 12 h after treatment. All collected samples were immediately flash-frozen in liquid nitrogen and stored at -80°C for subsequent total RNA extraction. All samples were analyzed in triplicate (technical replicates and independent biological replicates).

[0068] 1.2 CpWRKY71 Gene cloning and expression analysis.

[0069] Amplification of R1 from zucchini by PCR CpWRKY71 The full-length coding sequence of the gene was obtained and cloned into the pMD19-T vector. After sequencing verification, the CpWRKY71 protein sequence was analyzed using DNAMAN software for bioinformatics. Furthermore, a phylogenetic tree was constructed using MEGA software to assess the evolutionary relationship between CpWRKY71 and WRKY proteins from other species. Gene expression analysis was performed using real-time quantitative PCR, and relative expression levels were calculated using a 2-1T / T ratio. -ΔΔCT The calculation method was used, and all experiments were repeated three times.

[0070] 1.3 Yeast transcription activation assay.

[0071] Will CpWRKY71 The CDS sequence was cloned into the pGBKT7 vector to construct the recombinant plasmid CpWRKY71-pGBKT7. The recombinant plasmid was transformed into yeast strain Y2H Gold, with the empty pGBKT7 vector serving as a negative control. Transformed yeast cells were plated on SD / -Trp medium and cultured at 30°C for 3 days. Successfully transformed clones were then transferred to selective SD / -Trp / -His / -Ade medium containing X-α-Gal and cultured at 28°C for 3 days to assess transcriptional activation activity.

[0072] 1.4 Gene cloning and vector construction.

[0073] (1) Construction of subcellular localization vector.

[0074] Will CpWRKY71 The coding region was inserted into pCAMBIA1300-35S-EGFP and fused with the green fluorescent protein gene to construct the fusion expression vector 35S::CpWRKY71-eGFP. The obtained 35S::CpWRKY71-eGFP and the empty vector were transformed into Agrobacterium tumefaciens GV3101 strain using a freeze-thaw method, and subcellular localization analysis was performed.

[0075] (2) Construction of recombinant overexpression vector and overexpression plants: Overexpression experiment was carried out in zucchini using ZYMV-eGFP vector.

[0076] RNA was extracted from the leaves of zucchini R1 and reverse transcribed into cDNA. RNA extraction was performed using the Tiangen RNA Extraction Kit, and cDNA reverse transcription was performed using the Total Gold Reverse Transcription Kit.

[0077] Using cDNA as a template, cloning was performed using the primers shown in SEQ ID NO.1 and SEQ ID NO.2 to obtain... CpWRKY71 The CDS sequence. CpWRKY71 The CDS sequence was cloned into the vector ZYMV-eGFP, and the eGFP in the ZYMV-eGFP vector was replaced to obtain the recombinant overexpression vector ZYMV-CpWRKY71. Recombinant Agrobacterium containing ZYMV-eGFP and ZYMV-CpWRKY71 were inoculated into LB liquid medium containing 50 μg / mL kanamycin + 50 μg / mL rifampin and cultured at 28°C for 16 h. The cells were collected by centrifugation at 6000g for 5 min. The bacterial cells were resuspended in an induction buffer containing 10 mmol / L MgCl2, 10 mmol / L MES, and 100 μmol / L acetylsyleugenone to obtain the infection solution, and the OD was adjusted. 600Adjust the concentration to 1.0, let stand at room temperature for 2 hours, then inject the infection solution into the cotyledons of zucchini using a 1 mL syringe. Repeat this process three times and culture to obtain overexpressing plants. Plants inoculated with ZYMV-eGFP were used as control plants. After inoculation, the plants were placed at 28℃ and the green fluorescence signal was observed using a handheld fluorescent lamp.

[0078] (3) Construction of silencing vector and silencing plant: Silencing experiment mediated by tobacco brittle virus.

[0079] Will CpWRKY71 A specific 300 bp fragment from the sequence was inserted into the pTRV2 vector in the antisense direction to construct the silencing vector pTRV2:CpWRKY71. The sequence of the specific fragment is shown in SEQ ID NO.31.

[0080] SEQ ID NO.31:

[0081] TGTCAAGAACAGCGCTTATCCTAGGAGCTATTACAGATGCACGACGCAGAAATGCGGAGTGAAGAAACGGGTGGAGATCGTATGAAGATCCATCCATAGTAATTACGACGTACGAAGGGCAACACAATCACCCAATTCCCGCGACGTT AAGGGGGAACCTGTCGGCAGTGAGCGGCGCGTTTCCGCCGTCCATGTTGGCACCAATGCCGTGGTCGGTGGCGTGAGGTATCTTCCACAGCTGATGAACAACACTTCCGTCAACAACAACCAGCCCATCGGAGGTGGTGACACCGTTTA.

[0082] The silencing vectors pTRV2:CpWRKY71 and pTRV2 were transformed into Agrobacterium tumefaciens strain EHA105, respectively, and the optical density OD value was adjusted using induction buffer. 600 Adjust to 1.0; simultaneously transform the pTRV1 vector into Agrobacterium tumefaciens strain EHA105, and adjust OD. 600 Adjust to version 1.0. After completion, prepare the following two sets of inoculum solutions and use them to soak the fully expanded cotyledons of zucchini.

[0083] Group 1 Infection solution: Equal volumes of EHA105 bacterial suspension carrying pTRV1 and EHA105 bacterial suspension carrying pTRV2 were mixed.

[0084] Group 2 Infection solution: Equal volumes of EHA105 bacterial suspension carrying pTRV1 and EHA105 bacterial suspension carrying pTRV2:CpWRKY71 were mixed.

[0085] Using a 1 mL needleless syringe, the two groups of infection solutions were respectively infiltrated into the fully expanded cotyledons of zucchini. The inoculated plants were then placed in an incubator to obtain control plants and silent plants. The culture conditions were: temperature 22°C, photoperiod 16h light / 8h dark.

[0086] 1.5 Measurement of various indicators.

[0087] Zucchini plants were inoculated with powdery mildew pathogens, and the plant response to the pathogens was observed 7 days after inoculation. Leaf samples were collected 72 hours after inoculation for the determination of relevant physiological indicators and histological observation.

[0088] Malondialdehyde (MDA) content, superoxide anion, hydrogen peroxide, catalase, superoxide dismutase (SOD), and peroxidase activity were determined using kits from Beijing Solarbio Science & Technology Co., Ltd. H2O2 and O2 were detected using 3,3-diaminobenzidine, nitrotetrazole blue chloride, and trypan blue staining. - The accumulation of allergic necrotic cells and the death of cells.

[0089] The formula for calculating the powdery mildew disease index of zucchini is as follows:

[0090] .

[0091] In the formula, A Disease Index (DI); B Number of leaves in disease grade; C : Representative value for disease severity; D Total number of leaves surveyed.

[0092] This formula classifies disease severity based on the area covered by powdery mildew spots on individual leaves, as detailed in Table 2. Plants to be evaluated are then selected, and the disease severity is recorded leaf by leaf. The number of diseased leaves at each level is then entered into the formula. The results of the above formula are expressed as a percentage; higher values ​​indicate more severe disease. Calculations must adhere to a unified grading standard, ensure sample representativeness, and the data must be the mean ± standard deviation of three independent experiments to ensure statistical significance.

[0093] Table 2 Classification of powdery mildew disease severity

[0094]

[0095] 1.6 Yeast one-hybrid experiment.

[0096] Will CpWRKY71The coding sequence was cloned into the pGADT7 vector. A 30 bp DNA fragment containing the target cis-acting element was tandemly repeated three times and inserted into the pABAi vector. The constructed vectors were co-transformed into Y1H Gold yeast cells according to a specific combination and cultured on SD / -Ura-Leu medium. Positive transformants were selected for genomic PCR verification and then inoculated onto SD / -Leu-Ura medium containing amoeboidin A. Growth was observed and recorded after 3 days of incubation at 28°C. Yeast strains transformed with the empty pGADT7 vector served as negative controls.

[0097] 1.7 Analysis of changes in electrophoretic mobility.

[0098] The GST-CpWRKY71 fusion protein was expressed in *E. coli* BL21 and purified according to the Clontech manual. EMSA experiments were performed using the Thermo Scientific LightShift chemiluminescent EMSA kit, strictly following the manufacturer's instructions. Chemiluminescent signals of protein-DNA interactions were detected using the ChemiDoc™ MP imaging system. Unlabeled wild-type and mutant DNA fragments were used as competitors at 100-fold and 1000-fold molar excesses, respectively, with GST protein alone serving as a negative control.

[0099] 2. Results.

[0100] 2.1 CpWRKY71 Expression patterns, phylogenetic analysis, and sequence characteristics.

[0101] This invention identified a differentially expressed [substance] between disease-resistant material R1 and disease-susceptible material S1. WRKY Genes. Phylogenetic analysis indicates that, CpWRKY71 Clustered in group IIc, with pumpkin ( Cucurbita maxima ) and Indian squash ( Cucurbita moschata )of WRKY71 The homologous gene sequences showed the highest homology. Sequence analysis showed that... CpWRKY71 It contains an 801 bp open reading frame encoding a 266-amino acid protein. This protein contains a typical WRKY domain and a highly conserved C-terminus.

[0102] qRT-PCR analysis showed that within 24 hours of powdery mildew stress, CpWRKY71 The expression patterns were significantly different in the resistant material R1 and the susceptible material S1. It is noteworthy that... CpWRKY71 Significant accumulation was observed in the disease-resistant material R1 12 hours after inoculation with the powdery mildew pathogen. CpWRKY71 The expression level reached its peak, indicating that... CpWRKY71It may play a role in the resistance response of zucchini to powdery mildew. The above results are shown in... Figure 1 Therefore, the present invention cloned from the disease-resistant material R1 CpWRKY71 The genes will be studied further.

[0103] Tissue-specific expression analysis showed that CpWRKY71 It is primarily expressed in leaves. Furthermore, CpWRKY71 The sample responded to salicylic acid treatment but not to jasmonic acid treatment, suggesting that its main function may be related to leaves and participate in the SA-mediated signaling pathway. Results are shown in […]. Figure 2 .

[0104] 2.2 CpWRKY71 Its subcellular localization and transcriptional activation characteristics.

[0105] To determine CpWRKY71 Subcellular localization, this invention constructs CpWRKY71 The fusion expression vector 35S::CpWRKY71-eGFP, containing enhanced green fluorescent protein, was co-transformed into the epidermal cells of *Nicotiana benthamiana* leaves along with a nuclear localization marker. Confocal microscopy analysis showed that the fluorescence signal of the CpWRKY71-eGFP fusion protein was localized only in the nucleus, while the fluorescence signal of the empty vector control was detected in both the cytoplasm and nucleus. These results are shown in [Figure number missing]. Figure 3 A confirms CpWRKY71 It is located in the cell nucleus.

[0106] For further research CpWRKY71 The transcriptional activation characteristics were investigated using yeast transcriptional activation assays. CpWRKY71-pGBKT7 and the empty vector pGBKT7 were transformed into the yeast strain Y2H gold, respectively. The results showed that both CpWRKY71-pGBKT7 and pGBKT7 could grow on SD / -Trp medium; however, only yeast cells containing CpWRKY71-pGBKT7 could grow on SD / -Trp-His-Ade medium. Figure 3 B indicates CpWRKY71 It possesses transcriptional activation activity. In summary, these results confirm... CpWRKY71 It is a nuclear-localized transcription activator.

[0107] 2.3 CpWRKY71 Construction of overexpression plants.

[0108] To explore CpWRKY71 Functional role, CpWRKY71 The full-length CDS sequence was inserted into the ZYMV-eGFP vector to construct... CpWRKY71The recombinant overexpression vector ZYMV-CpWRKY71 was used. Further, six overexpressing plants (OE1-OE6) were obtained through mechanical inoculation. One representative plant was selected, and fluorescence was observed using a handheld fluorescent lamp; the results showed that only the control plant containing ZYMV-eGFP exhibited green fluorescence in its leaves, confirming the effectiveness of the overexpression system. See [see details]. Figure 4 .

[0109] The successful integration of ZYMV-CpWRKY71 into plants was further verified by PCR amplification. qRT-PCR analysis showed that, compared with control plants, the overexpressing plants had significantly higher levels of ZYMV-CpWRKY71 in their leaves. CpWRKY71 The significantly increased gene expression level indicates successful transient overexpression. Results are shown below. Figure 5 and Figure 6 .

[0110] 2.4 Overexpression CpWRKY71 Enhance the resistance of zucchini to powdery mildew.

[0111] For research CpWRKY71 Whether transient overexpression enhances the disease resistance of zucchini was investigated in this invention. CpWRKY71 Further analysis was conducted on the three significantly overexpressed lines OE2, OE5, and OE6.

[0112] Phenotypic assessment of OE2, OE5, and OE6 individuals 7 days after inoculation with powdery mildew showed that, compared to wild-type WT and the control group CK, CpWRKY71 The overexpressing plants exhibited significantly enhanced disease resistance, with the white lesion symptoms in all three overexpressing lines being significantly less pronounced than those in the WT and CK lines. Furthermore, trypan blue staining of infected leaves from different plants revealed that the fungal hyphae spread at a significantly slower rate in the overexpressing plants compared to the wild-type plants. (See results below.) Figure 7 This invention was further validated in silent plants. CpWRKY71 Resistance to powdery mildew, such as Figure 8 As shown, silence CpWRKY71 The disease resistance of the plants was significantly weakened.

[0113] In addition, compared with wild-type WT and control group CK, the malondialdehyde accumulation in overexpressing plants was significantly reduced, indicating that membrane lipid peroxidation damage was significantly alleviated.

[0114] In summary, these experimental results indicate that... CpWRKY71 It plays a positive role in regulating the resistance of zucchini to powdery mildew. Results Figure 9 .

[0115] 2.5 Overexpression CpWRKY71 Enhance the defense response of zucchini against powdery mildew.

[0116] To further clarify CpWRKY71This invention investigates the mechanisms by which zucchini resists powdery mildew, focusing on early defensive responses during infection, including H2O2 and O2. - O2 accumulation and induction of allergic reactions. O2 was assessed by NBT and DAB staining, respectively. - The generation of H2O2 was observed, and allergic reactions were observed using trypan blue staining, followed by histological analysis.

[0117] The results showed that after exposure to powdery mildew pathogens, CpWRKY71 H2O2 and O2 were present at the infection sites of overexpressing plants, WT plants, and CK plants. - Accumulation of H2O2 and O2 was observed, but the production rate in overexpressing plants was significantly higher than that in WT and CK. Quantitative analysis confirmed that H2O2 and O2 were produced in overexpressing plants. - Levels were significantly elevated. Trypan blue staining showed that, compared with WT and CK, overexpression resulted in more pronounced cell death in the plants, a trend consistent with H2O2 and O2 levels. - The accumulation pattern is consistent. The above results are shown in... Figure 10 .

[0118] Furthermore, biotic stress often induces severe oxidative damage in plants, impairing growth and development. Analysis of antioxidant enzyme activity indicates that... CpWRKY71 The activities of superoxide dismutase, catalase, and peroxidase in overexpressing plants were significantly higher than those in WT and CK. See the above results. Figure 11 .

[0119] In summary, these results indicate that CpWRKY71 It enhances resistance to powdery mildew by triggering a strong allergic reaction and cell death, while upregulating antioxidant enzymes to reduce oxidative stress.

[0120] 2.6 CpWRKY71 Altering the expression of defense-related genes in zucchini.

[0121] To explore CpWRKY71 This invention investigates the molecular mechanisms underlying powdery mildew resistance in zucchini. It analyzes the expression patterns of several key resistance genes 72 hours after powdery mildew inoculation, including those involved in the salicylic acid signaling pathway. PR1a , PR1b and PR1c and related to the jasmonic acid signaling pathway AOS1 , LOX1 and LOX2 .

[0122] The results showed that after inoculation with powdery mildew, the expression levels of JA biosynthesis-related genes did not differ significantly between overexpression lines and control plants. In contrast, the expression of SA signaling marker genes was significantly upregulated in the overexpression lines. Notably, PR1aIt exhibits high basal expression levels even in the absence of pathogen infection, and this upregulation is more pronounced after powdery mildew stress. These findings suggest that... CpWRKY71 It is likely that plant resistance to powdery mildew is regulated primarily through activation of the SA signaling pathway rather than the JA pathway. The above results are shown in... Figure 12 .

[0123] 2.7, CpWRKY71 and CpPR1a Specific binding of the promoter.

[0124] Numerous studies have shown that WRKY transcription factors regulate gene expression by specifically recognizing W-box cis elements in the promoters of target genes. In this invention, CpWRKY71 Overexpression leads to PR1a Significantly upregulated, and bioinformatics analysis showed PR1a The presence of a typical W-box motif in the promoter region suggests... PR1a may be CpWRKY71 The direct target of action.

[0125] To verify this hypothesis, a yeast one-hybrid experiment was conducted under a selection pressure of 500 ng / mL basidiomycin A. The experimental group Y1HGold[CpPR1a-AbAi+pGADT7-CpWRKY71] also showed the same growth state as the positive control; the negative control group Y1HGold[p53-AbAi+pGADT7] without prey protein and Y1HGold[AbAi+pGADT7-CpWRKY71] without the target promoter showed almost no growth, indicating that the bait carrier has no self-activation activity and the prey carrier has no specific activation, demonstrating good experimental specificity. The results are shown in [Figure number missing]. Figure 13 This confirms that CpWRKY71 and CpPR1a Promoters exhibit specific interactions.

[0126] In vitro binding assays further showed that the purified GST-CpWRKY71 fusion protein could bind to biotin-labeled... PR1a The promoter probe forms a specific DNA-protein complex, and a significant shift in electrophoretic mobility is observed. Figure 14 This binding can be competitively inhibited by an excess of unlabeled wild-type probes, but is unaffected by probes with mutations in the W-box core sequence; no migration bands were observed in the GST-tagged control protein, verifying the binding of CpWRKY71 with... PR1a Sequence specificity of promoter binding. In summary, in vitro and in vivo experimental results indicate that CpWRKY71 binds to... PR1a The W-box element in the promoter directly activates its transcription.

[0127] Figure 14 The text shows the relationship with... CpPR1aThe promoter-corresponding probe sequence is shown, with red letters indicating the W-box and its mutant W-box. After incubating the purified GST-CpWRKY71 protein with the probe, the protein-DNA complex was separated by non-denaturing polyacrylamide gel electrophoresis. GST protein alone served as a negative control. "-" indicates no addition, and "+" indicates addition. Competitive binding assays were performed by adding different concentrations of unlabeled or mutant probes to the reaction system to detect binding specificity. Arrows indicate the location of the migration bands.

[0128] The primer sequences for the relevant genes in this invention are shown in Table 3.

[0129] Table 3 Primer sequences

[0130]

[0131] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0132] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. CpWRKY71 The use of a related biological agent in modulating resistance to powdery mildew in zucchini is characterized in that, The CpWRKY71 The nucleotide sequence is shown in SEQ ID NO.29; The regulation refers to: improving CpWRKY71 expression to improve the resistance of zucchini to powdery mildew.

2. Use according to claim 1, wherein The biological product is any one of the following: A: contains CpWRKY71 recombinant overexpression vector; B: Agrobacterium containing the recombinant overexpression vector in A.

3. Use according to claim 2, wherein the compound is ###0002### The starting vector of the recombinant overexpression vector is ZYMV-eGFP.

4. The use according to claim 2, wherein the compound is ###0002### The expression of the recombinant overexpression vector is used to improve the expression of CpWRKY71 Squash plants with resistance to powdery mildew are bred by improving the resistance of squash to powdery mildew.

5. The use according to claim 4, wherein the compound is ###0002### The method for improving the resistance of zucchini to powdery mildew is as follows: Extract the RNA of zucchini and reverse transcribe it into cDNA; Using cDNA as template, the primer shown in SEQ ID NO. 1 and SEQ ID NO. 2 was used to clone to obtain CpWRKY71 ; will be described in detail below. CpWRKY71 ligated into the vector to obtain a recombinant overexpression vector; Transform the recombinant overexpression vector into Agrobacterium to obtain recombinant Agrobacterium; Use the recombinant Agrobacterium to infect zucchini to obtain overexpression plants, that is, to improve the resistance of zucchini to powdery mildew.

6. The use according to claim 5, wherein the compound is ###0002### The cDNA is derived from the leaves of zucchini.

7. The use according to claim 5, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The process of using recombinant Agrobacterium to infect zucchini is as follows: Subculture the recombinant Agrobacterium and collect the bacterial cells; Resuspend the bacteria and adjust OD to 1.0 to obtain the infection solution. 600 Adjust OD to 1.0 to obtain the infection solution. Inject the infection solution into the cotyledon of zucchini.

8. Use according to claim 7, wherein the compound is ###0002### The subculture condition is: 28℃ for 16h.

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