云南元江普通野生稻Pid2单倍型基因广谱高抗稻瘟病的分子机制研究

The gene Pid2 encodes a transmembrane receptor-like protein kinase including two signaling proteins extracellular recognition domains, B-lectin and PAN. In our pre-research work, the resistance analysis of transgenic lines with the different Pid2 haplotypes cloned from rice resources in Yunnan showed that the Pid2-YP haplotype from the common wild rice in Yuanjiang had the characteristics with the broad-spectrum and the high-resistance, and the characteristics was related to the variation of the 363th amino acid in PAN domain. In this study, the Pid2-YP haplotype transgenic line will be used to inoculate non-affinity races to construct cDNA libraries for the different periods of time, and the candidate proteins that interacting with the extracellular domain of Pid2-YP screened by the DUAL membrane Yeast two-hybrid technology, and the function of candidate genes verified in rice or pathogen through the high expression technology and the site-specific knockout technology to candidate genes, and the effect of candidate proteins on the interaction between Pid2-YP and substrate proteins verified by the techniques, the yeast three-hybrid experiments, transient expression analysis and co-immunoprecipitation. This study will reveal the mutual recognition pattern between the extracellular domain of Pid2-YP and the rice blast signal-related protein molecules, and further analyze the molecular mechanism of interaction between the receptor class R protein and the rice blast fungus.

Pid2编码一个跨膜受体类蛋白激酶，胞外有B-lectin和PAN两个信号蛋白识别结构域。前期对日本晴遗传背景下的不同Pid2单倍型转基因系稻瘟病抗性分析发现，元江普通野生稻的Pid2-YP单倍型具有广谱高抗特性，且与PAN结构域第363位氨基酸的变异相关。本研究拟用非亲和稻瘟病菌小种接种Pid2-YP转基因系并构建不同时段的cDNA文库，利用膜体系DUAL-membrane酵母双杂交技术筛选与Pid2-YP胞外结构域互作的候选蛋白；应用候选基因高表达技术和基因定点敲除技术验证水稻或病菌候选基因的功能；通过酵母三杂交实验、瞬时表达分析、免疫共沉淀等技术验证候选蛋白对Pid2-YP与底物蛋白的互作影响。本研究将揭示Pid2-YP胞外结构域与稻瘟病信号相关蛋白分子的相互识别模式，进一步解析受体类激酶R蛋白与稻瘟病菌互作的分子机制。

前期对日本晴遗传背景下的不同Pid2单倍型转基因系稻瘟病抗性分析发现，元江普通野生稻的Pid2-YP单倍型具有广谱高抗特性，为探究其原因，本研究构建了受稻瘟病诱导后不同时间段的含抗性基因OsPid2YP水稻材料的cDNA文库，并通过GATEWAY技术将该文库转化为酵母双杂交核体系和膜体系文库；利用来自于云南普通野生稻PID2基因全长及胞内结构域编码片段构建了诱饵载体分别进行膜体系及核体系文库筛选；结果获得了proline-rich protein、chromatin structure- remodeling complex protein、acyl-protein thioesterase等一系列的互作蛋白；定位于膜上的E3酶PUB4在两个筛库过程中都被鉴定到，其表达量受到稻瘟病菌的诱导，在OsPid2YP高表达背景下敲除PUB4可以降低植株抗病性，以此推测OsPUB4是PID2介导的抗病性信号转导通路的重要组成部分。这些结果为进一步揭示水稻抗稻瘟病的信号转导机制奠定了基础。