抗稻瘟病基因富集簇的挖掘及其抗性机制的研究

Rice blast is one of the most serious diseases of rice. Although many rice blast resistance genes have been cloned, there are few genes with high resistance and broad-spectrum and none has been successfully applied in production. It indicates that the resistance of the resistant cultivar is not determined by a single resistance gene. In our previous study, based on the feature of plant-pathogen co-evolution, we cloned plenty of rice blast resistance gene having different resistance spectrum and we found some rice blast resistance genes reside in a gene cluster, meaning the rice blast resistance gene cluster. We predict that the rice blast resistance gene clusters are supposed to be vital to the resistance of the highly resistant cultivars. So, how many the resistance gene clusters are included in a resistant cultivar? Do the genes in the cluster interact to each other or have no interaction? Or both cases are included? In this project, we select highly rice blast resistant cultivars as the donor materials and survey the candidate rice blast resistance gene clusters in genome-wide by re-sequencing. Then we estimate the function of these candidate gene clusters by gene cloning, transformation and rice blast screening. The functional gene clusters are chosen for testing the interaction of members in the gene cluster by different means, such as yeast two-hybrid system and comparison of the resistance spectrum. The discovery and digging of the disease resistance gene clusters supply the new materials for studying the resistance mechanism. Meanwhile, these resistance gene clusters can be directly applied in the disease resistance breeding.

稻瘟病是水稻最严重的病害之一。虽然目前已克隆了不少抗稻瘟病基因，但广谱高抗的基因较少，且很少有单个基因成功应用于实际生产，说明抗性品种的抗性不是简单的由单个抗性基因所决定。在我们前期的研究中，以植物-病原共进化为基础，克隆了大量的、抗谱不一的抗稻瘟病基因，初步发现了抗稻瘟病基因成簇分布的现象，即抗病基因富集簇。我们推断抗病基因富集簇对高抗品种的抗性起到了重要的作用。那么，一个抗性品种究竟包含多少抗病基因富集簇？富集簇内的各基因是通过简单的抗性累加还是相互作用行使抗病功能？还是两种机制兼有？本项目以高抗稻瘟病品系为材料，在全基因组水平筛选潜在的抗病基因富集簇，通过基因克隆、遗传转化、病原菌鉴定等流程进行功能确证，并采用酵母双杂、抗谱抗性比对等手段来检验抗病基因富集簇内各功能基因之间的相互关系，进而探讨抗性品系广谱高抗的根源，为抗病机制的研究提供新素材，同时也为抗病育种的直接利用提供新途径。

水稻稻瘟病是最严重的水稻病害之一，每年可导致水稻减产约30%。虽然目前已经克隆了不少稻瘟病抗性基因，但广谱高抗的基因非常少，且很少有单个基因能成功应用于实际生产。那具有持久、广谱高抗的品系的抗性是由单个高抗基因决定的还是多个抗性基因决定的呢？如果多个基因对抗性起贡献，是简单的叠加还是组合互作呢？前期研究表明抗病基因存在成簇分布的现象，这种富集簇对高抗品系的抗性有多大作用？本项目以高抗稻瘟病的水稻品系为材料，利用重测序，结合比较基因组学和分子生物学手段，在全基因组水平筛选潜在的抗病基因及抗病基因富集簇，进一步检验抗病基因间及富集簇内各功能基因之间的相互关系，进而探讨抗性品系广谱高抗的根源。我们以特特普为主要研究材料，完成基因组重测序，筛选出455个NBS-LRR（NLR）类型的基因并成功克隆获得了219个NLR基因的转基因株系。通过抗性鉴定发现，其中90个基因能够抗至少1种稻瘟病菌株，但很少NLR基因能够对抗6种以及以上的稻瘟病菌株，表明特特普NLR基因表现出比较高的抗性冗余现象，同时特特普广谱、持久抗性可能需要多个NLR基因共同作用。同时为了验证NLR基因（簇）的互作，我们以最简单的成对基因为研究对象，在特特普和日本晴基因组分别鉴定出43对和47对成对NLR基因。通过基因敲除实验验证了成对基因间存在互作。本项目的研究加深了我们对高抗品种持久、广谱抗性机制的了解，揭示了其高度的抗性冗余与复杂的抗性互作网络。

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