中国农业科学 ›› 2022, Vol. 55 ›› Issue (23): 4626-4639.doi: 10.3864/j.issn.0578-1752.2022.23.005

• 植物保护 • 上一篇    下一篇

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

张洁(),姜长岳,王跃进*()   

  1. 西北农林科技大学园艺学院/旱区作物逆境生物学国家重点实验室/农业农村部西北地区园艺作物生物与种质创制重点实验室,陕西杨凌 712100
  • 收稿日期:2022-06-28 接受日期:2022-08-08 出版日期:2022-12-01 发布日期:2022-12-06
  • 联系方式: 张洁,E-mail:jiee@nwafu.edu.cn。
  • 基金资助:
    国家自然科学基金面上项目(31872055)

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

ZHANG Jie(),JIANG ChangYue,WANG YueJin*()   

  1. College of Horticulture, Northwest Agriculture and Forestry University/State Key Laboratory of Crop Stress Biology in Arid Areas/Key Laboratory of Horticultural Plant Germplasm Resource Utilization in Northwest China, Ministry of Agriculture and Rural Affairs, Yangling 712100, Shaanxi
  • Received:2022-06-28 Accepted:2022-08-08 Published:2022-12-01 Online:2022-12-06

摘要: 目的 欧洲葡萄作为世界葡萄主栽品种,具有产量高、品质佳的优点,但对病害抵抗能力差。白粉病是严重危害葡萄栽培的一种真菌性病害,中国野生葡萄资源丰富,可为抗病育种提供充足种质资源。论文旨在筛选调控抗白粉病的葡萄转录因子基因,探究转录因子基因调控抗白粉病的作用机理,为选育葡萄抗病品种提供优质的基因资源。方法 从中国野生毛葡萄‘商-24’中克隆得到转录因子基因VqWRKY6,使用DANMAN和MEGA-X软件对序列进行分析。利用PEG介导转化拟南芥原生质体进行亚细胞定位分析发挥转录调控作用的位置,利用酵母双杂交和双分子荧光互补试验证明VqWRKY6能够和转录因子VqbZIP1互作形成转录复合体。以感病葡萄‘赤霞珠’叶片为试材,通过农杆菌介导法瞬时转化到‘赤霞珠’葡萄叶片,叶片进行白粉菌(Uncinula necator)接种后,观察发病症状,用台盼蓝染色在显微镜下观察菌丝发育进程,使用DAB染色观察活性氧积累,比较共同过表达VqWRKY6VqbZIP1的葡萄叶片、单独过表达VqWRKY6的葡萄叶片、单独过表达VqbZIP1的葡萄叶片和对照组叶片的差异。使用实时荧光定量PCR技术对抗病基因在白粉菌诱导下的表达水平进行分析。结果 VqWRKY6定位于葡萄2号染色体,编码342个氨基酸,属于WRKY家族的group Ⅲ亚家族。亚细胞定位和酵母转录激活试验证明,VqWRKY6在细胞核内发挥转录调控功能且在酵母中有转录激活活性。‘赤霞珠’叶片共同过表达VqWRKY6VqbZIP1后,叶片表面白粉菌菌丝扩繁速率显著慢于单独过表达VqWRKY6和单独过表达VqbZIP1的叶片,共同过表达VqWRKY6VqbZIP1的叶片组织中活性氧含量显著高于单独过表达VqWRKY6和单独过表达VqbZIP1的叶片;此外,VqWRKY6和VqbZIP1的协同调控能够激活茉莉酸(JA)途径的PR3、PR4,基因表达量显著上调。结论 VqWRKY6和VqbZIP1协同作用可能通过激活JA抗病途径,促进活性氧产生,增强抗病基因表达,抑制白粉菌的生长,从而提高葡萄对白粉病的抗性。因此,中国野生毛葡萄‘商-24’是重要的抗病种质资源,而VqWRKY6VqbZIP1可作为重要的抗病基因资源。

关键词: 中国野生毛葡萄, WRKY转录因子, 白粉病, 转录调控

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