白粉病诱导的中国野生华东葡萄VpPR10蛋白基因定位表达研究

Powdery mildew, caused by the Erysiphe necator, is a serious fungal disease in the cultivated European grapevines (Vitis vinifera L.).PR10 proteins from many plant species have been reported with important roles in plant defense in response to fungal invasion and other biotic or abiotic stresses.The objective of this study is to clone VpPR10 gene and its promoter from Vitis pseudoreticulata, and screen target proteins interaction with VpPR10 protein using yeast two hybrid method.The VpPR10 coding region with the native promoter and 35S promoter respectively were amplified and further sub-cloned into the plant expression vector with or without YFP tag.Localized expression of the VpPR10 gene was further analyzed in the transferred Arabidopsis and European grapevine plants.Our research involves analysis of function of target genes in host plant in response to pathogen stress, and also identified the interaction between VpPR10 protein and the pathogen. The goals are to elucidate the mechanism of disease resistance of host plant in response to the pathogen stress and to identify key components that can potentially be engineered for improvement of European grapvines to encounter pathogen stress, finally in detail to provide preferably theoretical basis for disease resistant gene studies and guidance for technical references.

葡萄白粉病是危害葡萄生产的主要真菌病害之一，病程相关蛋白PR10在植物抗病防御以及逆境胁迫中发挥重要作用。本研究是在获得中国野生华东葡萄抗白粉病株系白河-35-1VpPR10基因的基础上，克隆分离该基因上游启动子序列,通过体外及体内的蛋白互作研究VpPR10与其互作蛋白的关系。进一步将该VpPR10蛋白基因与我们前期克隆获得的VpPR10蛋白基因启动子和35S启动子融合分别构建YFP标签或无YFP标签的植物表达载体，转化获得转基因植物。在白粉病病原菌诱导下研究模式植物拟南芥及欧洲葡萄中VpPR10基因定位表达分析，研究VpPR10基因在寄主植物中的抗病功能；揭示VpPR10蛋白与病原菌互作关系，阐明其调控增强寄主植物的抗病机理，识别增强植物抗病能力的关键作用元件，为葡萄抗病基因研究和育种提供理论依据与技术体系。

用真空渗透瞬时转化法将VpPR10.1基因转入欧洲葡萄叶片，该基因过量表达能够增强葡萄白粉病抗性。用100 μg/mL浓度的VpPR10.1重组蛋白处理烟草BY-2悬浮细胞，在24 h之内，细胞死亡的数目随诱导时间延长而增多，诱导后的烟草悬浮细胞DNA降解，为PR10诱导细胞程序性死亡提供了依据。克隆中国野生华东葡萄株系‘白河-35-1’不同结构域的PR10基因4个：VpPR10.4、VpPR10.6、VpPR10.7、VpPR10.9； VpPR10.4、VpPR10.7氨基酸序列中第89-121位氨基酸含有Betv 1结构域，VpPR10.6和VpPR10.9氨基酸序列中不包含Bet v 1结构域。纯化的VpPR10.4、VpPR10.7重组蛋白具有核酸酶活性，进一步验证了PR10氨基酸序列中保守结构域P-loop和Bet v 1以及核酸酶特征性氨基酸位点与蛋白体外核酸酶活性之间对应关系。.利用酵母双杂交技术筛选到与VpPR10.1互作蛋白VDAC3，通过BiFC、Pull-Down和CoIP实验证明了VpPR10.1与VDAC3蛋白互作关系。细胞定位分析表明：PR10.1蛋白在细胞质和线粒体中均能表达，而VDAC3仅在线粒体中表达。通过农杆菌介导法将VpPR10.1基因转入欧洲葡萄品种无核白中，共计筛选42个转基因株系。对筛选的无核白葡萄转基因植株接种霜霉菌，转基因葡萄叶片接种霜霉菌后48h出现了气孔阻塞物，接种后转基因葡萄叶片胞质蛋白中能够检测到细胞色素C。相比于野生型植株，转基因株系在接菌后菌丝生长均受到抑制，转基因株系离体叶盘接菌后72h和96h，在霜霉菌菌丝生长处有明显活性氧积累；转基因株系叶肉细胞内霜霉菌菌丝生长受到抑制。.研究结果表明：霜霉菌入侵到葡萄叶片后，VpPR10.1蛋白受到诱导表达，促进与VvVDAC3形成复合体，诱导ROS合成通路上RbOH和Aox基因和蛋白表达，增强了胞内产生ROS，从而引起寄主细胞凋亡。另外，VpPR10.1与VvVDAC3蛋白相互作用诱导线粒体膜孔通道变大，使细胞色素C从线粒体释放到胞质中，从而激活了胞质中Meta-caspases基因表达，诱导病原菌入侵部位细胞死亡，阻止霜霉菌进一步侵染。本研究主要创新点是筛选到新的与VpPR10.1互作蛋白 VvVDAC3，揭示了中国野生华东葡萄病程相关蛋白VpPR10.1与V