抗稻瘟病基因Pid3等位基因间抗性特异识别位点鉴定研究

Rice blast is one of the major diseases that drastically damage rice production. The use of plant varieties with resistance (R) genes is the most cost-effective and environmentally-friendly way to control this disease. The relationship between Rice and Magnaporthe oryzae is a good example of the classical “gene for gene” hypothesis. It is important to identify sites in resistance genes, especially, alleles that determine resistance specificity, in view of the large number and rapid variation of M. oryzae isolates. Two alleles of the rice blast resistance gene Pid3, Pid3-A4 and Pid3-38 were cloned in our preliminary work. Pid3-A4 is resist to the largest number of the tested isolates, followed by Pid3. It is the CC-NBS but not the usual LRR domain was found determining the resistance specificity between Pid3-38 and Pid3, Pid3-A4, respectively, after functional analysis in transgenic plants of LRR domain swapping constructed in vitro. Taking into account the very few different amino acid residues, our goal of this project is identification of specific sites in the 3 alleles of Pid3 that determine resistance specificity, by using LRR domain swapping and point mutation. We believe that our research project will elucidate the evolution mechanism of the rice blast resistance Pid3 locus, and it is probable that we could create new rice blast resistance genes with broader resistance spectrum.

稻瘟病是水稻最严重的病害，应用含有抗病基因的水稻品种是一种有效防治稻瘟病危害的手段。水稻与稻瘟病菌之间符合经典的“基因对基因”假说，由于稻瘟病菌小种数量众多且变异迅速，因此研究抗稻瘟病基因间，特别是等位基因间对稻瘟病菌的抗性特异识别机制具有重要意义。在前期工作中，我们克隆了抗稻瘟病基因Pid3的两个等位基因Pid3-A4和Pid3-38，在检测过的稻瘟病菌小种中，Pid3-A4抗谱最广，Pid3次之，Pid3-38最窄。通过Pid3-38与Pid3、Pid3-A4的LRR区域互换重组，发现它们之间决定抗谱差异的区域并不是通常认为的LRR，而是CC-NBS结构域。由于这三个基因编码氨基酸序列差异极少，本项目拟继续通过LRR互换重组以及对鉴定出的识别区段内差异位点突变研究，来鉴定等位间抗性特异识别具体位点,以此揭示Pid3位点上抗性基因进化机制，并尝试人工创制抗谱更广的抗稻瘟病基因。

利用抗性基因是防治稻瘟病的有效手段。到目前为止已经克隆了超过30个抗稻瘟病基因，其中大多数都是某一抗性位点上的等位基因。水稻与稻瘟病菌间符合经典的“基因对基因”假说，由于稻瘟病抗性等位基因间序列差异不大，而对稻瘟病具有小种特异抗性，因此研究等位间抗性特异识别位点对理解抗稻瘟病基因进化机制及生产应用均具有重要价值。本项目在前期克隆了Pid3位点上三个具有不同抗谱的等位基因Pid3、Pid3-38、Pid3-A4基础上，利用LRR结构域互换重组，明确了Pid3-38和Pid3间，Pid3-38和Pid3-A4间是由CC-NBS结构域负责抗性特异识别，进一步对CC-NBS区域内存在的差异位点进行点突变发现，22位的氨基酸变异位点决定了这两对等位基因间的抗谱特异性。同时，LRR结构域互换重组发现Pid3和Pid3-A4间的抗性特异识别是由CC-NBS和LRR结构域相互作用决定的，单一结构域并不能完全决定抗谱特异性。利用抗谱Pid3-A4>Pid3>Pid3-38这一特点，本项目还将Pid3-A4位于CC-NBS区域中两个与Pid3-38相同的氨基酸突变为同Pid3一致，进行了更广谱等位基因的创制。最后，本项目对栽培稻中Pid3所有等位类型进行了完整分析，明确了Pid3在进化上相对保守，其功能等位在我国籼稻育种中已经得到了广泛利用，而在粳稻中，有功能的等位基因比例还不到10%，为进一步有针对性利用Pid3位点奠定了基础。