水稻稻瘟病是最严重的水稻病害之一，稻瘟病是一种由真菌Magnaporthe oryzae引起的水稻病害，严重影响水稻产量，每年可导致水稻减产约30％。为了减少农业损失，研究水稻与稻瘟病菌之间的相互作用已成为急需解决的问题。因此越来越多的科学家开始研究水稻与稻瘟病之间相互作用的机制，稻瘟病已经成为研究植物与真菌之间相互作用的一种模式菌种。至今，培育广谱、持久稻瘟病抗性的水稻品种是防治稻瘟病菌最有效的方法之一。 特特普是一种具有持久稻瘟病抗性的高抗水稻品种。首先我们完成了特特普的全基因组测序，预测出37054个基因，包含455个核苷酸结合位点和富亮氨酸重复蛋白的基因(NBS-LRR类型基因，简称NLR基因)。我们在特特普品种中利用高通量克隆方法成功克隆了219个NLR基因，分别转基因至两个感病品种TP309和Shin2中，并用12个不同的稻瘟病菌株进行鉴定。研究表明，其中90个克隆的NLR基因能够对抗至少1种稻瘟病菌株，并且每一个稻瘟病菌株都能够被很多个NLR基因识别，然而有很少的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。 为了验证NLR基因是否存在互作，本研究从最简单的两个基因，即成对NLR基因入手，提出了一种快速鉴定成对NLR抗性基因的方法，其中每个基因都作为独立的个体行使不同功能，只有当两个NLR基因同时存在时才能引起植株的免疫反应。我们分别在特特普和日本晴、明恢63、R498其他三种基因组中发现大于20％的NLR基因都是成对基因，在特特普基因组成功鉴定出43对成对NLR基因，在日本晴基因组成功鉴定出47对成对NLR基因。并且选取25对成对NLR基因进行基因敲除，成功得到7对成对NLR基因敲除突变体植株，得到与其功能相对应的表型。 综上，本研究深入研究了高抗稻瘟病品种Tetep的持久、广谱抗性的潜在抗性机制，通过对该抗性基因组进行长序列组装、NLR基因注释、并对其中大部分NLR基因进行克隆和抗性鉴定工作，揭示了其具有高度的抗性冗余与复杂的抗性互作网络，同时进一步利用敲除实验验证了成对基因的频繁互作，在此基础上构建了能够快速鉴定NLR基因簇和基因对的分子标记数据库，从而推进新的高抗品种育种的进程。 关键词：特特普；NLR基因；高通量克隆；基因敲除；抗性鉴定

 Rice blast, caused by Magnaporthe grisea, is one of the most serious disease threatening rice production, which casuse about 30% annual yield loss. To reduce the loss of agricultural yield, studying the interaction between rice and rice blast fungus has become an urgent problem to be solved. Therefore, more and more scientist study the mechanism of interaction between rice and rice blast fungus. Meanwhile, rice blast has become a model strain for studying the interaction between plants and fungi, so far, one of the best strategies for rice blast disease management is to develop broad-spectrum and durable resistance rice cultivars. Tetep is a rice cultivar highly resistant to blast, a devastating fungal disease. We sequenced its genome and annotated 37,054 genes, including 455 nucleotide-binding site leucine-rich repeat (NLR) genes. We cloned and tested 219 NLR genes in two susceptible cultivars using 12 diversified pathogen isolates. Ninety cloned NLRs showed resistance to ≧1 of the 12 pathogen isolates and each pathogen was recognized by many NLRs. However, very few NLRs showed resistance to >6 isolates, suggesting that high resistance redundancy in the Tetep genome and multiple NLRs are required for Tetep’s broad-spectrum resistance to blast. Developing a method to identify NLR pairs each of which functions as a single unit, we found that >20% of the NLRs in the Tetep and three other genomes are paired. In total, we found 43 pairs in Tetep, 47 in Nipponbare genome . we randomly picked 25 NLR pairs to do single NLR gene knockout using the CRISPR/Cas9 system . Of the 7 pairs for which both sensor and helper knockouts were available . This study increased our understanding of the genetic basis of broad-spectrum blast resistance, developed NLR cloning and pair identification methods and provided an NLR gene database with molecular markers to facilitate breeding for new resistant cultivars. In this study we assembled the complete genome of Tetep and identified 455 NLR genes, we conducted a large-scale cloning, transformation and functional study of Tetep NLRs in two susceptible rice cultivars to identify Tetep NLR genes that are potentially blast resistant, suggesting high resistance redundancy and multiple NLR genes interaction in the Tetep genome. By identification of paired NLRs and functional evaluation by CRISPR knockout, we tried to decipher the NLR defense networks in Tetep. Finally, we designed and tested an extensive set of molecular breeding markers that will facilitate the breeding of new blast resistance cultivars. Key words: Tetep; NLR genes, Large-scale cloning, Knockouts of genes, Blast resistant