簇毛麦HvCMPG互作蛋白基因HvFP3在小麦抗白粉病中的功能及作用机制

The broad spectrum powdery mildew resistance gene Pm21 from Haynaldia villosa, has been proven to be one of the most important gene resources in wheat improvement for durable resistance. The Pm21-Blumeria graminis f.sp. tritici also privides a study system for elucidating the molecular mechanisms underlying different levels of plant innate immunity system, such as Non-host resistance, Pattern-triggered Innate Immunity and Effector-triggered Innate Immunity. Supported by the previous NSFC grants, we initiated the projects for cloning the Pm21 candidates and elucidating the mechanism of Pm21-mediated powdery mildew resistance. An E3-ligase gene participating the ubiquitination degradation pathway, HvCMPG, was cloned from H.villosa and proven playing important roles in improving broad spectrum powdery mildew resistance.A Yeast Two Hybrid cDNA library of H. villosa was constructed and used to screen the interaction protein of HvCMPG, and an HvFP3 gene encoding farnesylation protein with heavey-metal association domain was identified and cloned from H. villosa.In the proposed research, the function of HvFP3 in powdery mildew resistance will be analyzed by over-expression via transformation and gene silencing using VIGS,etc. The potential value of the HvFP3 in improvement for powdery mildew resistance will be evaluated; The mechanism of HvFP3 and HvCMPG interaction and the relationship of the interaction with powdery mildew resistance will be clarified by point mutation, in vitro ubiquitination assay and Pulldown techniques, etc; The relationship of the gene structure, evolution and function will be studied by cloning of the HvFP3 homologs from different species; The regulation of heavy metal association and distribution and their roles in powdery mildew resistance will be elucidated.The outcomes of the research will not only facilitate our prehensive understanding of plant innate immunity, but also provide new gene resource for crop improvement aiming disease resistance.

来自簇毛麦的抗白粉病基因Pm21抗谱广、抗性强，是提高白粉病持久抗性的重要抗源，也是解析植物广谱抗性分子机制的重要系统。为解析其抗性机制，前期研究中克隆了一个簇毛麦E3连接酶基因HvCMPG，证明其过量表达可提高广谱白粉病抗性；通过构建和筛选酵母双杂交文库，克隆了与HvCMPG互作的法尼化蛋白基因HvFP3。本项目在此基础上，利用转基因等研究HvFP3的功能及其参与的信号通路，评价其在抗白粉病基因工程中的价值；利用点突变、泛素化分析等研究HvFP3和HvCMPG的互作机制，探明其互作在Pm21介导的抗白粉病中的功能；利用同源克隆、重金属结合分析等研究Hv-FP3的结构、进化与功能的关系，探明其在调控重金属离子分布中的作用及与抗白粉病的联系。通过研究，丰富植物抗病免疫分子机制理论，并为抗病育种提供新的基因资源。

小麦近缘属簇毛麦广谱、高抗小麦白粉病，是提高小麦白粉病持久抗性的重要抗源，也是解析植物广谱抗性分子机制的重要系统。在前期研究中克隆了一个广谱抗小麦白粉病的E3连接酶基因CMPG1-V，为了进一步解析其抗病机理，构建和筛选酵母双杂交文库，筛选并克隆了与CMPG1-V互作的法尼化蛋白基因HIPP1-V。本项目在此基础上，利用转基因等验证HIPP1-V的功能，解析其介导的抗病信号通路，评价其在抗白粉病基因工程中的价值；利用点突变、泛素化分析等研究HIPP1-V和CMPG1-V的互作机制；利用同源克隆、重金属结合分析等研究HIPP1-V的结构、进化与功能的关系，探明其在调控重金属离子分布中的作用及与抗白粉病的联系。结果表明，HIPP1-V具有抗小麦白粉病功能，与活性氧和水杨酸信号通路有关，获得稳定的过量表达HIPP1-V的小麦转基因材料，评价了该基因在抗白粉病基因工程育种中的应用价值，为抗病育种提供新的基因资源；明确了法尼化修饰即影响HIPP1-V的膜系统定位，也影响其与其它蛋白如CMPG1-V或自身的互作；明确了HIPP1-V和CMPG1-V之间的互作机制，HIPP1-V可被CMPG1-V体外泛素化但不被降解，探明了二者互作共同介导的广谱白粉病抗性的关系；明确了HIPP1-V的结构、进化与功能之间的关系，阐明了其在小麦重金属耐受性方面的功能，丰富了小麦抗病免疫分子机制理论，为抗病育种提供新的基因资源。