毛葡萄凝集素类受体激酶基因VqLecRK1抗白粉病功能及其调控机理

Powdery mildew (Uncinula necator) is one of the important fungal diseases that seriously reduce the grape production in the world. Meanwhile, cultivated European grape cultivars are susceptible to powdery mildew; therefore directional genetic improvement is necessary for European grape resistance enhancement. In the early stage of the project, a G-type membrane protein gene VqLecRK1, which was up-regulated after the induction of powdery mildew, was screened out by analyzing the transcriptions of Chinese wild Vitis quinquangularis clone Shang-24 at different stages of infection, and its function was preliminarily analyzed. According to our previous research, the biological function of VqLecRK1 in grapevine will be analyzed by gene over-expression and knockout in susceptible Thompson seedless and disease resistant Shang-24, respectively. The upstream regulation protein will be selected by yeast one-hybrid system; Chip-qPCR will be used to verification the regulation ability, expounding the regulation mechanism. The interaction proteins will be screened by yeast two-hybrid system, BiFC and CoIP will be used for proteins interaction verification, revealing the resistance regulation system for VqLecRK1. Our project will clearly elaborate VqLecRK1 regulation system in grapevine powdery mildew resistance, which will lay a solid theoretical foundation for grape resistance molecular breeding and germplasm improvement.

葡萄白粉病是世界范围内严重危害葡萄产业重要的真菌病害之一，生产上广泛栽培的欧洲葡萄抗病性差，因此，对欧洲葡萄进行定向改良以提高抗病性成为葡萄抗病育种的重要方向。项目组前期通过对抗病中国野生毛葡萄在白粉菌侵染后不同时期的转录组分析，筛选出了一个在白粉菌诱导下显著上调表达的凝集素类受体激酶基因VqLecRK1，并初步进行功能分析。本项目在此基础上，构建VqLecRK1过量表达和基因DNA编辑载体，分别转化感病葡萄及抗病毛葡萄，明晰VqLecRK1抗病功能；通过酵母单杂交筛选VqLecRK1上游调控基因，利用Chip-qPCR验证其结合活性，阐明上游调控基因对VqLecRK1的调控机理；通过酵母双杂交筛选VqLecRK1互作蛋白，并利用双分子荧光互补、免疫共沉淀完成互作验证，揭示其参与的抗病调节机制。从而弄清VqLecRK1基因在葡萄白粉病抗性中的作用和调控机制，为葡萄抗病分子育种提供理论依据。

葡萄白粉病是危害葡萄生产重要的真菌病害，我国野生葡萄资源蕴藏着丰富的抗病基因，开展野生葡萄抗白粉病VqLecRK1功能及其作用机理研究具有重要的育种价值。本研究利用课题组拥有的优异抗病葡萄种质资源，在抑制消减杂交、高通量转录组测序的基础上，获得了中国野生毛葡萄‘商-24’葡萄抗白粉病关键VqLecRK1基因，cDNA序列全长为2412bp，编码803个氨基酸。构建了VqLecRK1植物过表达载体和干扰载体，通过遗传转化成功获得了VqLecRK1转基因葡萄植株，明确了VqLecRK1抗白粉病功能。筛选出了VqLecRK1的互作基因VqPGDH，克隆了VqPGDH的全长cDNA序列，明确了VqPGDH的亚细胞定位和响应白粉菌的表达模式，并利用瞬时表达系统转化葡萄叶片对VqPGDH基因进行了功能研究，阐明了VqLecRK1与互作基因VqPGDH之间的联系，建立了VqLecRK1在葡萄抗白粉病过程中的调控模式。进一步的研究明确了葡萄VqBAK1、VqWRKY31、VqWRKY46、VqWRKY56抗白粉病功能，并筛选验证了互作基因、上游调控基因及下游靶基因，建立了葡萄抗白粉病调控网络，创制了一批葡萄抗白粉病新种质，为抗病优质葡萄品种选育奠定基础。结合项目实施，发表研究论文7篇，获陕西省自然科学一等奖1项，获国家发明专利2项，培养博士后1人、研究生9人，其中博士生4人。