钙依赖蛋白激酶基因VpCPK1介导中国野葡萄抗白粉病机制研究

Grapevine powdery mildew is the most important fungal disease of grapes throughout the world's grape growing regions. The Chinese wild grapevine such as Vitis pseudoreticulata accession Baihe-35-1 was demonstrated to be resistant to multiple diseases, including powdery mildew and downy mildew. Recent studies have provided compelling evidence for the involvement of CDPKs playing versatile roles in plant innate immune. Based on our previous results, VpCPK1, a homologue of At CPK1, has been isolated from Chinese wild grapevine Vitis pseudoreticulata accession Baihe-35-1. In this project, we will further investigate the relationship among the characteristics, functions and resistance to powdery mildew of VpCPK1. We will express the fusion protein in Escherichia.coli and test CDPK kinase activity in vitro. To identify proteins involved in regulation of the VpCPK1 protein function, we will construct a yeast cDNA bank induced by powdery mildew pathogen by a yeast two-hybrid using VpCPK1 as bait. In addition, we will try to analysis the regulation of some hormonal signaling such as SA, JA and ET induced by VpCPK1 after inoculated by powdery mildew in European grape over-expression of VpCPK1, and in Chinese wild grapevine accession Baihe-35-1 RNAi of VpCPK1. We will also try to find the response of VpCPK1 to Ca2+ after induced by powdery mildew. The obtained results will not only provide useful information for further understanding the molecular mechanism of Chinese wild grape disease resistance to powdery mildew, but also provide more interesting evidence to identity which pathway such as SA, JA or ET involved in VpCPK1. In practice view, it can provide better gene resource of resistance to powdery mildew for improvement European grape or other fruit trees.

葡萄白粉病是一种危害最严重的世界性葡萄病害。中国野葡萄是很好的抗病基因资源库。最近的研究表明，钙依赖蛋白激酶基因CDPK在植物天然免疫中发挥重要枢纽作用。本项目是在前期获得中国野葡萄VpCPK1基因的基础上，进一步研究其结构、功能及其与白粉病抗性的关系。通过检测VpCPK1体外激酶活性和亚细胞定位；构建白粉菌诱导的酵母cDNA文库，利用酵母双杂交技术，筛选互作蛋白；在感病葡萄中超量表达VpCPK1，在抗病野葡萄中干涉VpCPK1，分析白粉菌诱导下VpCPK1对不同激素分子的调控和对Ca2+的响应。在理论方面，项目的完成不但可以揭示中国野葡萄VpCPK1基因抗白粉病的作用机制，而且可以丰富植物CDPK基因发挥抗病调控功能作用途径的判断证据；在实践方面，可以为定向改良欧洲葡萄品种或其他果树的抗病性提供优良的基因资源，最终为利用该基因培育抗病优质葡萄或其他果树新品种（系）提供理论依据和技术支撑。

葡萄白粉病是一种危害严重的世界性葡萄病害。中国野葡萄是很好的抗病基因资源库。研究表明，钙依赖蛋白激酶基因CDPK在植物天然免疫中发挥重要枢纽作用。本项目是在前期获得中国野葡萄VpCPK1基因（根据葡萄新的基因命名规则，VpCPK1基因被重新命名为VpCDPK9，所以，后面统一用VpCDPK9代替VpCPK1进行描述）的基础上，进一步研究其结构、功能及其与白粉病抗性的关系。本项目鉴定了包括VpCDPK9在内的中国野生华东葡萄19个CDPK基因，发现VpCDPK9-YFP融合蛋白主要聚集在膜系细胞器和囊泡状细胞器中；构建了华东葡萄白河-35-1的酵母双杂交文库，用VpCDPK9全长蛋白很难筛到互作蛋白，但将其钙调结构域短截后则可以筛选到互作蛋白VpACS1、VpMPK3、VpMPK6，BIFC结果证实了这个互作结果；VpCDPK9过量表达转基因拟南芥生长缓慢，成熟叶片黄化衰老，可增强拟南芥对白粉菌的抗性，但对盐胁迫耐受性无影响；乙烯、脱落酸和茉莉酸三种植物激素可明显激活该基因启动子的转录活性，而相同浓度的过氧化氢则可以更高强度地激活该启动子；VpCDPK9基因在感病葡萄“无核白”中过量表达可以提高其对白粉菌的抗性，且与过氧化氢的积累有关；VpCDPK9基因可以在葡萄中促进乙烯的合成，精准调控乙烯合成途径限速酶ACS的活性，进而通过感应和调节细胞内氧化还原态和激素水平，间接控制下游多类防御相关基因的表达，从而在葡萄响应逆境胁迫信号途径中发挥重要功能。提出了VpCDPK9的工作模型。此外，还建立并优化了葡萄原生质体分离及转化体系，并用来研究葡萄抗病基因功能。项目的完成，初步清楚了中国野葡萄VpCDPK9基因抗白粉病的作用机制，可以为定向改良欧洲葡萄品种或其他果树的抗病性提供优良的基因资源，最终为利用该基因培育抗病优质葡萄或其他果树新品种（系）提供理论依据和技术支撑。在国内外学术刊物上发表论文6篇，其中SCI源刊论文5篇；获得授权国家发明专利1项；培养硕士4名，正在培养博士研究生1名，硕士研究生4名。完成预定研究目标。