粳稻地方品种抗性基因Pi-hk1介导的广谱抗稻瘟病分子机制解析

Taihu lake area is one origin of japonica landrace. We previously mapped and cloned broad spectrum resistance gene Pi-hk1 from Taihu lake japonica landrace Heikezijing, and Pi-hk1 was a special resistance gene encoding NBS-LRR-WRKY-WRKY protein. The preliminary results showed that Pi-hk1 was related with penetration resistance and WRKY domains played important roles in the resistance. In our further research project, the microscopy assay will be carried out in the full length Pi-hk1 and truncated forms transgenic plants for resistance mechanism analysis. Two yeast hybrid system, BiFC and Co-IP will be used to identify the interactions between WRKY and NBS-LRR, and CHIP-seq method will be used to identify the WRKY regulated downstream genes. The transgenic plants of WRKY and downstream genes will be constructed by CRISPR/Cas9 method to analyze theses genes’ functions in the resistance pathway. The Pi-hk1 sequences in 3000 sequenced varieties and 200 cultivated rice varieties will be used to trace the evolutionary origin and distribution of Pi-hk1. Finally, the molecular mechanism and signal pathways of broad spectrum mediated by Pi-hk1 will be clarified.

太湖流域是我国粳稻起源地之一。本项目前期经过多年积累从太湖流域粳稻地方品种黑壳子粳中定位和克隆了广谱抗性基因Pi-hk1，编码蛋白含NBS-LRR-WRKY-WRKY特殊结构域组合，初步抗性机理研究结果表明Pi-hk1与稻瘟病的侵入抗性有关，WRKY结构域在抗性中起重要作用。拟在现有研究基础上，通过显微观察方法分析全长Pi-hk1和不同截短基因参与侵入抗性的机理；通过酵母双杂交、BiFC和Co-IP等方法分析WRKY结构域和NBS-LRR结构域之间是否存在互作，并通过ChIP-seq的方法分析WRKY结合的下游基因，并采用CRISPR/Cas9的方法构建WRKY和下游基因的转基因材料分析这些基因在抗病过程中的功能；分析3000份已测序品种和200份主栽品种中Pi-hk1序列来研究其起源进化及分布情况。最终阐明Pi-hk1介导的广谱抗性分子机制和信号途径。

本项目在前期从太湖流域粳稻地方品种黑壳子粳中鉴定到一个主效抗稻瘟病基因位点Pi-hk1的基础上，进一步对该位点候选基因进行转基因功能验证，发现Pi-hk1的稻瘟病抗性是由候选基因P13和P15共同贡献的，P13编码一个Myb转录因子，P15编码一个NBS-LRR-WRKY-WRKY蛋白，P13可以结合到P15基因的启动子上实现稻瘟病抗性。单独超量表达P13基因显示转基因水稻分蘖角度的增大有匍匐生长的趋势，而P15基因抗性水平与表达量呈现正相关关系。在病菌侵染水稻时，P15在水稻细胞菌丝入侵处聚集以阻止稻瘟病菌的侵染。通过对P15FL-GFP，P15-W-GFP，P15-WW-GFP三种转基因材料的稻瘟病抗性表型鉴定，发现WRKY结构域的有无对于P15的抗性具有重要影响。对P15FL-GFP，P15-W-GFP，P15-WW-GFP三种蛋白进行水稻原生质体亚细胞定位观察显示，WRKY结构域会影响P15在亚细胞中的作用位置。与对照相比，P13/P15聚合互补转基因材料稻瘟病抗性显著增强，而分蘖角度，株型，株高，分蘖数，粒长，粒宽和百粒重等农艺性状与对照没有显著差异。