甘蓝型油菜菌核病感病基因的鉴定与功能分析

Stem rot caused by Sclerotinia sclerotiorum is one of the main diseases of oilseed rape, there is no effective control method or resistant resource up to now. Neither key resistance genes have been identified, nor high resicstant cultivar were breed, on account of resistance to Sclerotinia sclerotiorum is controled by multiple genes in brassica napus. Research results confirmed that broad-spectrum resistance to pathogens can be acquired by knocking out or silencing sussceptiblity genes in host plants. We have found several genes expressed specially or higher in the sussceptible cultiva through RNA-Seq analysis of brasscia napus nearly isogenic which have remarkable difference in resistance to Sclerotinia sclerotiorum, and deduced that they may be related to susceptibilty of Sclerotinia sclerotiorum. This study will firstly verify the function of these genes through tobacco instantaneous conversion technology and screen out candidate susceptibilty genes, and then clone full sequences from canola with different resistance to Sclerotinia sclerotiorum. Futher function study and mechanisms analysis of candidate genes will be carried out through overexpression and RNAi strategy. This study not only provides the theory basis for the research on revealing the susceptiblity/resistance mechanism and function of calona susceptiblity genes, but also leads to a new approach to improve the resistant ability of rape and promote the sclerotinia sclerotiorum resistance breeding.

油菜菌核病是油菜上的主要病害之一，至今尚无有效的防治方法或抗源，也没有找到关键的抗性基因。而且油菜对菌核菌的抗性受多基因控制，很难通过单一的抗性基因提高油菜的抗病能力。很多研究表明，感病基因的敲除或沉默可以提高寄主的广谱抗病能力。本实验室通过对油菜感/抗菌核菌近等基因系接种菌核菌后不同时间点的RNA-Seq结果分析，筛选到多个在感病品种特异表达或高表达、并且受菌核菌诱导上调表达的基因，推测其与油菜和菌核菌的亲和互作相关。本研究拟通过烟草瞬时转化技术和接种进行初步验证，筛选出油菜菌核病感病基因的候选基因。从不同抗性品种进行克隆，分析基因的序列特征和差异、以及组织表达和诱导表达特性，再通过干扰表达和超表达进一步验证其功能，分析感病基因的调控网络和作用机理。研究结果能为揭示油菜的感/抗菌核病机理提供信息，同时也能通过基因工程技术应用感病基因提高油菜的抗菌核病能力，为油菜抗菌核病育种开辟新的途径。

油菜菌核病是油菜上的主要病害之一，至今尚无有效的防治方法或抗源。本研究通过感/抗菌核菌近等基因系RNA-Seq结果与实时荧光定量分析，筛选到3个在抗病品种中表达量明显高于感病品种，且早期表达受到菌核菌抑制的基因。克隆了BnBRCA2A的全长序列，该基因与拟南芥BRCA2B的同源性为82%，与BRCA2A的同源性为80%。在油菜根、茎、叶、花、种子中都有表达，但是在叶中的表达量最高，超表达该基因的拟南芥植株菌核病抗病能力明显提高。构建了BnaC09g13100D、BnaA03g09060D两个基因的超表达载体，获得了这两个基因的转基因植株。分析发现BnaC09g13100D通过激活水杨酸和氧爆发途径，诱导下游的防御相关基因和病程相关蛋白的表达提高植株的抗病能力。BnaA03g09060D则主要通过激活茉莉酸和氧爆发途径，诱导下游相关防御基因的表达。筛选到3个在感病品种特异表达或高表达，并且受菌核菌诱导上调表达的基因（BnaC06g05340D、BnaA03g53180D和BnaA06g10010D），推测其与油菜和菌核菌的亲和互作相关，构建了3个基因的基因编辑载体，获得了基因编辑阳性植株，其功能和分子机理有待进一步研究。分析了以上基因在不同抗性油菜叶片中的表达情况。结果发现抗病相关基因（BnaC09g13100D、BnaA03g09060D）的在油菜叶片中的表达量抗性的高低呈正相关，高抗>中抗>高感。而感病基因（BnaA03g53180D和BnaA06g10010D）则与之相反。进一步证实筛选基因与油菜菌核病的抗性相关，可以作为油菜菌核病抗性分子育种的候选基因，也可作为利用分子生物学手段对油菜菌核病抗性进行鉴定的靶标基因。