水稻白叶枯病抗性基因Xa2的克隆和功能分析

Bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the major destructive diseases in rice worldwide. Cloning resistance genes for breeding resistant cultivars is the most effective approach to control BB. Our previous results have revealed that the transcription activator-like (TAL) effector TAL6a from Xoo stimulated IRBB2 plants to produce defense response. The resistant and susceptible varieties ratio of the F2 generation plants derived from a cross between IR24 and IRBB2 was three to one. We try to isolate the Xa2 gene by SSR markers, map-based cloning, combining with 2b RAD-seq ( 2b restriction-site associated DNA-sequencing), analysis of the expression profiles of the IR24 and IRBB2 plants inoculated or not inoculated by TAL6a using RNA-seq, as well as prediction EBE through TAL6a DNA sequence. The Xa2 gene can be identified by GUS staining, EMSA, dTALE and TALEN technology. Simultaneously, the functional elements of Xa2 gene can be confirmed by analyzing its homology in different rice varieties, combining with motif and domain. The TAL6a-Xa2 resistance mechanism will be defined through comparing the hormone levels of the rice carried Xa2 gene, EBE modified rice with IR24. The results from this project will not only highlight our scientific understandings on mechanism of resistance of rice, but also provide resistant rice lines for using Xa2 gene breeding highly resistant rice cultivars.

稻黄单胞菌Xoo引起的白叶枯病(BB)是我国水稻上重要的细菌病害，克隆抗性基因培育抗性品种是防治该病害的最有效措施。前期研究发现，Xoo的转录类似因子TAL6a可激发IRBB2产生抗病反应，并且感病IR24与抗病IRBB2杂交后F2代植株抗感比为3:1。我们拟通过SSR分子标记、图位克隆、并结合2b-RAD深度重测序、RNA-seq技术分析TAL6a存在与不存在状态下的表达谱以及TAL6a预测的EBE，分离Xa2基因；借助GUS报道法、EMSA、dTALE以及TALEN技术，鉴定Xa2基因；同时分析Xa2在不同水稻上的同源性，结合motif和domain分析，确定Xa2功能所需元件；亚细胞定位Xa2；比较Xa2水稻、EBE修饰水稻与IR24 的激素水平变化，明确TAL6a-Xa2抗性机理。这不仅对揭示水稻抗病机理具有科学意义，而且还为有效利用Xa2基因培育抗性品种具有实践意义。

本项目围绕着水稻白叶枯病抗性基因Xa2的克隆，对前期引进的IR24、IRBB2以及其杂交后代IR24/IRBB2的F2群体进行白叶枯病抗感鉴定，结果发现F2代植株全生育期抗或感白叶枯病菌株的比例接近3:1，表明抗病亲本IRBB2的全生育期抗白叶枯病能力由Xa2单基因控制。通过研究，项目组得到TAL6a菌株可以用于鉴别水稻是否含有Xa2基因这一功能，该结果为筛选水稻是否含有Xa2抗病基因奠定了技术方法。通过SSR标记定位分析，将Xa2定位在RM3687和RM5473之间，遗传距离约为192 kb。根据最新公布的籼稻R498基因组数据对RM3687和RM5473标记之间DNA序列进行Blast分析，项目组筛选了7个含有NBS-LRR结构的候选基因，分别命名为Q12、Q23、Q29、Q42、Q47、Q51及Q57。PCR扩增后，只有Q57基因在抗病和感病亲本中的扩增产物不同，且Q57与连锁分子标记A1的位置最近，推测Q57基因为Xa2的候选基因。为此，本项目选择Q57构建植物表达载体，经农杆菌转化，将候选基因转入感病水稻材料02428，于2017年11月获得转基因再生植株，其功能验证及TAL6a-Xa2抗性机理将在今后工作中进一步的探索，期望得到好的结果。