广谱抗瘟基因Pi50（t）的克隆及其基因簇的分子进化研究

Rice blast, is one of the devastating rice diseases in the worldwide, and broad-spectrum resistance genes are highly desirable for disease control and prevention. We previously identified a novel broad-spectrum blast resistance gene Pi50 (t) which mapped to Pi2/9 loci, from a resistance donor Er-ba-zhan (EBZ) in South China. We further revealed that the resistance spectrum and race specificity of Pi50 (t) allele were different from other known Pi2/9 carriers. We sequenced the region of Pi50 (t) loci in EBZ which containing 7 NBS-LRR like R gene candidates (Nbs 1-7) clustered in this locus. Phylogenetic analysis of the multiple alleles based on the amino acid sequence revealed that 2 out of 7 Pi50 (t) candidates are grouped into the same clade which contains the functional Pi2, Pi9 and Piz-t but they shared very low similarity. It is suggesting a high variation in that clade, may also explaining the difference of their race specificity. To probe the characterization of these 2 specific Pi50 (t) candidates in blast resistant, both of forward- and reverse-genetics methods will be used to in this study. We will perform the disease resistance screening on the complementary and RNAi transgenic plants which carrier single or 2 candidates combination, and clarify the function and their cooperation in rice blast response. Further, By using qPCR, we will study the transcriptional profiling of Pi50 (t) candidates in the response to rice blast and its relationship with the PR genes regulation. Meanwhile, target region re-sequence among 6 different rice germplasm will be performed in searching the key variation of the NBS-LRR genes. Knowledge of this work on exploring the benefit of multiple gene coexistence in EBZ may prove a foundation for the mechanism studying on the broad-spectrum resistance, and open a way on the resistance genes deployment.

全球稻瘟病连年爆发,迫切需要挖掘和合理利用广谱抗瘟基因。本课题组前期从华南抗源稻种28占中鉴定出一个位于Pi2/9座位而抗谱却明显不同的广谱抗瘟基因Pi50(t)。基因组测序、预测和进化分析表明，Pi50(t)座含有7个NBS-LRRs抗性基因，其中2个基因与Pi2/9的功能组份聚于同一进化分枝，而其间氨基酸序列的相似度却很低。为探明Pi50(t)的功能基因及功效，本研究拟运用正、反向遗传学的手段，通过克隆前期发现的2个候选基因，对其及其组合的互补或沉默型转化系的抗性鉴定，探明各基因的功效及组合的协同效应；利用qPCR摸清病原物诱导过程中各抗性候选基因的表达模式及其对病原诱导基因表达的调控；通过对6个不同抗源品种间目的区域重测序以及NBS-LRR变异热点及其进化的分析，寻找多基因成簇存在与水稻广谱久抗性的关系。为多基因成簇存在及其互作机制的研究提供理论基础，为合理开发抗瘟基因开创一个思路。

本课题组前期从华南抗源稻种28占中鉴定出一个位于Pi2/9座位而抗谱却明显不同的广谱抗瘟基因Pi50(t)。以日本晴基因组序列为参考的预测分析表明，Pi50(t)座含有7个NBS-LRRs抗性基因，其中2个基因与Pi2/9的功能组份聚于同一进化分枝，而其间氨基酸序列的相似度却很低。本项目的研究就是运用遗传学的手段，通过克隆Pi50(t)候选基因，探明其功效及特性；进一步通过对6个不同抗源品种间目的区域重测序以及NBS-LRR变异热点及其进化的分析，寻找多基因成簇存在与水稻广谱持久抗性的关系。至此，我们探明了Pi2/9位点中，广谱抗稻瘟病基因Pi50的特点。它存在于一个含有12个NBS-LRR结构域的串联重复基因簇中。与其他PI2/9位点不同的是，Pi50基因簇中包含四个核苷酸序列极其相似的重复基因（Pi50_NBS4_1至_4）。此外，这四个复制的基因编码了两种蛋白质：Pi50_NBS4_1/ 2和Pi50_NBS4_3/ 4，它们彼此仅相差4个氨基酸。转基因互补试验和稻瘟病抗谱分析表明，是Pi50_NBS4_1/2而不是Pi50_NBS4_3/4控制着与Pi50近等基因系（NIL-e1）一致的抗瘟特性。Pi50与其他三个Pi2/9位点的R蛋白，如Pi9，Piz-t和Pi2，分享96％以上的氨基酸序列同源性，而且其间的氨基酸变化主要发生在LRR区域内。Pi50这种新抗病基因特异性的鉴别，将有助于剖析Pi2/9位点各基因不同抗性特异性的分化及其进化背后的机制。