水稻抗纹枯病重要育种价值基因qSB-9TQ的克隆与功能解析

Sheath blight (SB) one of the most serious diseases in rice worldwide, causing significant grain losses annually. Rice resistance to SB is controlled by polygenes, namely quantitative trait locus (QTL). Utilization of SB resistance QTLs is the most important approach to develop SB resistance variety. Previous studies have showed that qSB-9TQ, a major quantitative resistance gene from Teqing (TQ) with moderate resistance, is of great breeding potential in rice breeding against SB disease. For finemapping qSB-9TQ, we developed a set of chromosome single segment substitution lines (CSSLs), in which the substituted TQ segments are contiguous overlapping the preliminary mapping interval of qSB-9TQ with high resolution. After phenotyping, we finally narrowed the gene down to a 21kb region including 3 expressed genes. . To further investigate the resistant mechanism of qSB-9TQ, in this project we will 1) to clone qSB-9TQ by sequencing and transgene verification experiments; and 2) to analyze its resistant mechanism and signaling pathway associated with SB resistance by the methods like histopathology, metabonomics, qRT-PCR, subcellular localization, RNA-seq and phytohormone treatments; and 3) to identifiy its interaction proteins and determine the roles of interaction proteins in qSB-9TQ medidated SB resistance; and 4) to mine its elite allelic variants in rice natural germplasms as well as developing functional molecular markers for breeding. Results in this study will accelerate the ultization of qSB-9TQ in developing SB resistance variety, and provide new knowledge for understanding the mechanism of rice resistance to SB disease.

纹枯病是水稻最重要的病害之一，水稻对该病的抗性受多基因控制，抗病育种中只能应用数量抗病基因。研究表明中抗品种特青（TQ）第9染色体上存在一个主效抗纹枯病数量基因qSB-9TQ，具有较高育种应用价值。通过构建高精度染色体单片段重叠代换系，我们将该基因精细定位在21kb区间内，包含3个表达的基因。. 在此基础上，本项目将通过测序、转基因功能验证，克隆qSB-9TQ；通过组织病理学、代谢物分析、表达模式、亚细胞定位、RNA-seq和激素处理等方法，明确其抗病机制和信号调控途径；筛选其互作蛋白，明确互作蛋白的生物学功能及与qSB-9TQ间的相互调控关系；分析其在水稻自然品种中的优异复等位变异及分布特征，开发功能标记服务于抗病育种。结果将加速qSB-9TQ的育种应用，丰富水稻抗纹枯病分子机理的理论知识。

国内外多个研究团队此前均在水稻第9染色体长臂上检测到一个主效抗纹枯病QTL，但彼此间的定位结果并不完全一致。本项目之前通过构建染色体片段代换系，将中抗品种特青（TQ）第9染色体上的qSB-9TQ定位在21kb区间内。在此基础上，本项目首先通过转基因敲除和超表达实验，证实该定位区间内的3个基因及外侧的1个可能基因均不参与水稻的纹枯病抗性，不是qSB9TQ的目标基因。进而通过筛选遗传背景更一致的部分染色体片段代换系并进行重复表型鉴定，证实qSB-9TQ的下方还存在一个抗纹枯病QTL（命名为qSB9TQ-2），而原qSB-9TQ（更名为qSB9TQ-1）仍位于前期发表的146kb精细定位区间内，包含上述21kb区间。通过转录组分析，发现qSB9TQ-2可能参与调控苯丙烷信号途径，而qSB9TQ-1可能参与调控MAPK信号途径。结合转录组和重测序数据，对qSB9TQ-1和qSB9TQ-2中的所有基因进行了整合分析，最终分别筛选出12和27个候选基因，对各候选基因均进行了敲除研究，发现了4个参与调控纹枯病抗性的基因；进一步通过转基因互补试验，证实其中1个（命名为SBR9-2）编码具有DUF结构域的未知功能蛋白是qSB9TQ-2；亚细胞定位实验显示其在细胞质和细胞核中均有定位。SBR9-2受纹枯病菌诱导表达水平显著高于其感病等位基因sbr9-2，两者之间在基因内存在多个SNP和InDel变异，基于这些变异开发的基因内标记未来可直接用于抗病育种。SBR9-2在水稻中共存在4种单倍型，其中包含SBR9-2的Hap1主要分布在籼稻亚群中。研究过程中，还在第12染色体上鉴定到了1个新的抗纹枯病数量基因座qSB12YSBR1，并对其进行了精细定位；同时，在候选基因批量分析中还鉴定到1个具有育种应用价值的负调控水稻纹枯病抗性的ERF转录因子基因OsERF65。以上进展为进一步解析水稻抗纹枯病分子机理奠定了重要基础，同时将加速水稻抗纹枯病分子育种进程。