HM47介导的水稻白叶枯病抗性分子机理研究

In our breeding practice, disease resistance to rice bacterial blight (BB) pathogens is largely depends on a few well-known major resistance genes such as Xa4, Xa21 and Xa23. It is vulnerable and would lead to the breakout of severe disease, therefore, it is necessary to look for new sources or germplasm of disease resistance which could possibly avoid severe damage from the pathogens. In our mutant bank containing 17899 EMS-induced IR64 mutants, we identified a mutant HM47 which shows broad-resistance to rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) and also enhanced resistance to several blast pathogens (isolates) tested in our preliminary studies. HM47 shows hypersensitive response-like symptom that is controlled by a single recessive nuclear gene located on the rice chromosome 4. Thus，we consider HM47 is an ideal genotype for the studies of rice bacterial blight resistance. We have already completed map-based cloning of the candidate gene, while the mechanism involved for the resistance is barely known. Therefore, in this proposal, we aim to confirm the candidate gene by complementary study, and identify the basic principal or pathway through which the mutant shows enhanced disease resistance to Xoo. We also aim to generate a couple of rice materials useful in our future rice breeding programs for BB resistance.

在水稻育种以及生产实践中，水稻抗白叶枯病品种的培育过分依赖于Xa4、Xa21和Xa23等抗性主基因，存在突发抗性丧失的风险，因此有必要寻找新的广谱抗性基因源来避免这类风险。我们在17899份EMS诱导的IR64突变体中筛选到HM47突变体，该突变体与对照品种IR64相比表现为广谱抗白叶枯病，并且对多个稻瘟病小种的抵抗力也得到增强；HM47所表现的性状类似于过敏性反应，而且该性状是由一对隐性核基因控制的，因此该突变体材料是一个难得的用来研究植物系统性获得抗性的好材料。通过前期的研究工作，我们完成了HM47基因的定位工作，并找到了目标基因，其抗病机理是否有别于传统的基因-基因假说，有待于我们去探索。本研究旨在阐明HM47基因介导的水稻白叶枯病抗性分子机理，明确其抗病途径，并以此来指导我们的育种实践，希望通过本研究获得若干份广谱抗白叶枯病的水稻新品系。

本实验室前期已经报道过HM47斑点叶突变体对百叶枯病菌和稻瘟病菌的明显的抗性。通过图位克隆，我们克隆了导致该表型的基因OsPELOTA，该定位区间内含有三个开放阅读框。经测序分析发现，突变体中蛋白编码基因LOC_Os04g56480（OsPELOTA）编码区CDS序列中为556位T突变为A(T556A)，致使其所编码的蛋白序列第186位的苯丙氨酸(Phe)突变成异亮氨酸(Ile)。通过构建splHM47功能互补载体进行转化试验，结果表明splHM47基因可以互补突变表型，是控制斑点叶性状的目标基因。激素测定分析结果显示HM47突变体叶片中脱落酸、茉莉酸甲酯和水杨酸分别是野生型对照的0.62±0.09（p<0.05），0.53±0.05（p<0.01），1.69±0.08（p<0.01）。随后的转录组测序结果显示HM47突变体在能量代谢、氨基酸代谢及次生代谢产物的生物合成等方面产生了巨大变化。HM47斑点叶突变体对病原微生物抗性的增强极有可能是植物内源激素SA的升高、蛋白磷酸化途径和萜类代谢途径中大量基因表达模式的改变等因素的综合作用结果。酵母双杂交实验表明，OsPELOTA可以与SPL33，OsGEF，OSEX2及SUMO E1a互作。