水稻小种非专化性抗稻瘟病新基因Pi2-A35的克隆和功能鉴定

Rice blast is one of the most widespread and destructive rice diseases worldwide. Utilization of host resistance (R) genes is the most effective and environment-friendly strategy for tackling rice blast disease. Contrary to race specific R genes, race non-specific R genes usually function independently of avirulence genes in the pathogen and thus are durable, which are of high value for breeding resistant varieties. However, Only a very few race non-specific R genes have been cloned so far. Recently, we found that the derived isogenic line from a novel Pi2/9 allele from a rice line CIRAD, designated Pi2-A35, displayed partial resistance to all tested blast isolates，suggest that Pi2-A35 is most likely a race non-specific R gene. In this study, we will obtain the full-length clone of Pi2-A35 and generate the transgenic plants via gene complementation test. The resistance and spectrum of the transgenic plants will be assessed to diverse blast isolates. Meanwhile, the resistance level of the Pi2-A35 isogenic line will be evaluated in natural disease nursery. After the confirmation of the function of Pi2-A35 in race non-specific resistance, a set of Pi2-A35 variants will be constructed by either domain swapping or amino acid substitutions based on the sequence differences from other known Pi2/9 alleles. The transgenic rice plants with individual Pi2-A35 variants will be evaluated for disease resistance and spectrum. The critical domains or amino acid residues controlling the race specificities of Pi2-A35 and other Pi2/9 alleles will be analyzed and deduced. Phylogenetic analysis and detection of amino acid sites under positive selection will be conducted to investigate the evolutionary dynamics and variation mechanisms of the Pi2-A35 and the Pi2/9 locus. The cloning and functional characterization of Pi2-A35 from this study will not only provide a novel race non-specific R gene for breeding blast resistant varieties but also provide new insight into the understanding of the mechanisms of durable resistance to blast.

稻瘟病是威胁水稻生产的最主要病害之一。利用抗病基因是防控稻瘟病最为有效、环保的策略。相对于小种专化性抗病基因，小种非专化性抗病基因不依赖稻瘟菌的无毒基因，往往表现持久抗性，在抗病育种中有很好的利用价值。然而，目前已克隆的小种非专化性抗稻瘟病基因还非常有限。前期工作中通过等位基因挖掘和标记辅助筛选技术，鉴定了一个来源于水稻品种CIRAD的Pi2/9等位基因Pi2-A35，接种结果表明该基因很可能是一个小种非专化性抗稻瘟病基因。本项目将通过候选基因的克隆、功能互补、抗谱分析和抗性鉴定，完成该基因的克隆。在此基础上，通过结构域互换和点突变技术，鉴定控制Pi2-A35小种非专化抗性的关键结构域或氨基酸序列。利用多重序列比较和氨基酸位点选择压力的分析，研究Pi2/9遗传座位的进化动力学和变异机制。研究成果不仅可为水稻抗稻瘟病分子育种提供持久抗病新基因资源，还能为进一步阐明稻瘟病持久抗病机理提供参考。

稻瘟病是威胁水稻生产的最主要病害之一。利用抗病基因是防控稻瘟病最为有效、环保的策略。相对于小种专化性抗病基因，小种非专化性抗病基因不依赖稻瘟菌的无毒基因，往往表现持久抗性，在抗病育种中有很好的利用价值。然而，目前已克隆的小种非专化性抗稻瘟病基因还非常有限。并且小种非专化性基因往往抗病力较弱，单一基因难以持续抵抗病菌侵袭，聚合多个小种非专化性抗病基因是解决这一问题的首选方法。本项目在前期工作基础上构建了Pi2-A35单基因系BAC文库，通过序列分析确定了候选基因；完成了候选功能基因全长序列克隆、表达载体构建与功能互补转化；补充了对Pi2-A35基因进行精细遗传定位分析的工作；创制了Pi2-A35等3个小种非专化抗性基因的聚合系，明确其高抗效果，为广谱持久抗病提供了新的思路；调查了Pi2/9位点基因及其它主效抗稻瘟病基因在中国主推品种分布情况。研究成果为水稻持久抗稻瘟病分子育种提供了新基因资源和新思路，为抗病基因在育种中的布局利用提供参考。