中国野生毛葡萄转录因子VqWRKY6与VqbZIP1互作调控抗白粉病功能分析

doi:10.3864/j.issn.0578-1752.2022.23.005

Abstract

Abstract:

【Objective】 As the world’s main cultured varieties, Vitis vinifera has the advantages of high yield and good quality, however, the resistance of V. vinifera to disease is poor. Powdery mildew is a fungal disease that seriously endangers viticulture. The resources of Chinese wild grape are abundant, which can provide sufficient germplasm resources for disease-resistant breeding. This objective of this study is to screen grape transcription factor genes that regulate the resistance to powdery mildew, and to explore the mechanism of these genes regulating the resistance to powdery mildew, which can provide high-quality genetic resources for selection and breeding of grape disease-resistant varieties.【Method】 VqWRKY6 was cloned from Chinese wild Vitis quinquangularis ‘Shang-24’. The sequences were analyzed using DANMAN and MEGA-X software and subcellular localization analysis was adopted using Arabidopsis thaliana protoplasts by PEG-mediated transformation, which was performed for the location of transcriptional regulation. The yeast two-hybrid and bimolecular fluorescence complementation were adopted to prove that VqWRKY6 can interact with the transcription factor VqbZIP1 to form a transcriptional complex. Taking the leaves of the disease-susceptible grape ‘Cabernet Sauvignon’ as the material, the transient transformation mediated by Agrobacterium was performed in the leaves of ‘Cabernet Sauvignon’. After Uncinula necator inoculation on the leaves, the pathogenesis symptoms were observed, hyphal development progression was microscopically visualized with trypan blue staining, and reactive oxygen species (ROS) accumulation was visualized using DAB staining. The differences between grape leaves co-overexpressing of VqWRKY6 and VqbZIP1, overexpressing of VqWRKY6 alone, overexpressing of VqbZIP1 alone, and the control group were compared. The qRT-PCR was used to analyze the expression level of anti-disease genes under U. necator induction.【Result】 VqWRKY6 is located on grape chromosome 2, encodes 342 amino acids and belongs to the group Ⅲ subfamily of the WRKY family. VqWRKY6 exerts a transcriptional regulatory function in nucleus. The propagation rate of U. necator on the surface of ‘Cabernet Sauvignon’ leaves after co-overexpressing of VqWRKY6 and VqbZIP1 was significantly slower than that of leaves overexpressing of VqWRKY6 alone and overexpressing of VqbZIP1 alone, and the ROS content in the leaf tissues co-overexpressing of VqWRKY6 and VqbZIP1 was significantly higher than that of leaves overexpressing of VqWRKY6 alone and overexpressing of VqbZIP1 alone. In addition, the synergistic regulation of VqWRKY6 and VqbZIP1 could activate PR3 and PR4 of the jasmonate (JA) pathway, and the gene expression levels were significantly upregulated.【Conclusion】 The synergistic effect of VqWRKY6 and VqbZIP1 may improve the disease resistance of grape to powdery mildew by activating the JA pathway, promoting the production of ROS, enhancing the expression of disease-resistant genes to inhibit the growth of U. necator. VqWRKY6 and VqbZIP1 are important disease-resistant genetic resources, and ‘Shang-24’ is an important disease-resistant germplasm resource.

Key words: Chinese wild Vitis quinquangularis, WRKY transcription factor, powdery mildew, transcriptional regulation

ZHANG Jie, JIANG ChangYue, WANG YueJin. Functional Analysis of the Interaction Between Transcription Factors VqWRKY6 and VqbZIP1 in Regulating the Resistance to Powdery Mildew in Chinese Wild Vitis quinquangularis[J].Scientia Agricultura Sinica, 2022, 55(23): 4626-4639.

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References 54

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引物名称 Primer name	引物序列 Primer sequence (5′-3′)	用途 Use
VqWRKY6-GFP-F-Kpn I	GGGGACGAGCTCGGTACCATGGACACTGGTTTGAAATGGCAG	pC2300
VqWRKY6-GFP-R-Sal I	GCTCACCATGGTGTCGACGGAGAAAAATCCTGGGGTAT AAA	pC2300
VqbZIP1-GFP-F-Kpn I	GGGGACGAGCTCGGTACCTGGCGTCGTCGAAGGTGAT	pC2300
VqbZIP1-GFP-R-Sal I	GCTCACCATGGTGTCGACCCACAGAACAGACTGCACT	pC2300
VqWRKY6-BD-F-EcoR I	CATATGGCCATGGAGGCGAATTCATGGACACTGGTTTGAAATGGCAG	pGBKT7
VqWRKY6-BD-R-Sal I	ATGCGGCCGCTGCAGGTCGACGGAGAAAAATCCTGGGGTATTAAA	pGBKT7
VqbZIP1-AD-F-EcoR I	GCCATGGAGGCCAGTGAATTCATGGCGTCGTCGAAGGTGATG	pGADT7
VqbZIP1-AD-R-BamH I	CTGCAGCTCGAGCTCGATGGATCCCCACAGAACAGACTGCACT	pGADT7
VqbZIP1-pSPYNE-F-Kpn I	TCCGTCGACCTCGAGGGTACCATGGCGTCGTCGAAGGTGATG	pSPYNE
VqbZIP1-pSPYNE-R-Kpn I	CTCCTACCCGGGAGCGGTACCCCACAGAACAGACTGCACT	pSPYNE
VqWRKY6-pSPYCE-F-Xho I	GGGACTCTAGAGGATCTCGAGATGGACACTGGTTTGAAATGGCAG	pSPYCE
VqWRKY6-pSPYCE-R-Kpn I	ATCGTATGGGTACATGGTACCGGAGAAAAATCCTGGGGTATTAAA	pSPYCE

Functional Analysis of the Interaction Between Transcription Factors VqWRKY6 and VqbZIP1 in Regulating the Resistance to Powdery Mildew in Chinese Wild Vitis quinquangularis

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