杨树SnRK2基因调控叶枯病抗性分子机制研究

The blight disease of Poplar (Populus L.) caused by Alternaria (alternata(Fr.)Keissl). is a serious threat to woody plants. However, there is little research about molecular mechanisms resistance to disease so far. Our group firstly discovered that the SnRK2 family gene of poplar can respond to the infection of Alternaria fungi, disease resistance was detected by Agrobacterium injection of leaves to overexpress SnRK2 or inhibit SnRK2 expression, it was found that SnRK2.8 was able to improve poplar leaf disease resistance. Meanwhile, we used SnRK2.8 as a bait to screen of Poplar yeast cDNA library and obtain a number of interaction factors. Based on this research, we constructed SnRK2.8 transgenic poplar to detect the resistance of SnRK2.8 and the effects on the phenolic metabolites. Next, the transcription factors that were phosphorylated by SnRK2 were screened out from the obtained interacting factors by yeast co-transformation, BiFC interaction analysis, PHA hybridization and protein spectrum detection. While Agrobacterium tumefaciens injection method was used to screen out transcription factors with disease-resistant effect. Finally, the downstream functional genes regulated by transcription factors were screened by group analysis. The molecular mechanism of SnRK2.8 mediated resistance to leaf blight was established.

杨树叶枯病是由链格孢属真菌引起的严重病害，对包括杨树在内的众多木本植物危害极大，但尚未有深入的抗病分子机制研究。本课题组前期研究发现杨树SnRK2家族基因能够响应链格孢属真菌侵染，农杆菌注射叶片过表达及抑制SnRK2检测抗病性发现SnRK2.8能提高杨树叶片抗病能力。同时，利用SnRK2.8筛选杨树酵母cDNA文库获得一批互作因子。本项目在此基础上，首先通过SnRK2.8转基因杨树，确定其抗病性及对酚类代谢物的影响；其次，通过酵母共转化和BiFC互作分析，磷酸泛抗体杂交与蛋白质谱检测等方法从已获得的互作子中筛选被SnRK2磷酸化的转录因子，同时利用农杆菌注射方法筛选具有抗病作用的转录因子；第三，通过转基因杨树及接菌实验确定该转录因子的抗病能力；最后，采用组学分析等方法筛选转录因子调控的下游功能基因，最终解析杨树SnRK2.8介导的抗叶枯病分子作用机制。

本项目针对由链格孢属真菌引起的杨树叶枯病开展抗病分子机制的研究工作，主要完成以下研究内容：首先，通过杨树转基因和代谢物分析等实验确认了SnRK2能够通过提高部分黄酮和酚类物质的含量提高杨树叶片的抗病性；其次，利用酵母文库筛选、蛋白体内体外互作和蛋白磷酸化分析等验证了SnRK2能够磷酸化bHLH48转录因子并提高杨树的抗病性；第三，通过杨树过表达bHLH48和WRKY25等，确认了其对杨树代谢物积累是抗性提高的重要原因，同时筛选了下游的抗病功能基因如：CERK1、CIPK14和CBL1等，绘制了杨树抗叶枯病的分子通路图。重要研究结果包括：解析了杨树通过SnRK2磷酸化bHLH48调控代谢物积累抵抗叶枯病的分子机制，筛选了多个与调控代谢物合成或抗病性提高的核心基因，以及建立了包括杨树在内的具有广谱适用性的林木基因Hairy Root转化系统等。本项目的重要科学意义在于，深入解析了杨树叶枯病的抗病分子机制并筛选了抗病基因，为有效防治包括杨树在内的链格孢属真菌病害提供了理论基础，同时，为创制广谱抗病的杨树新种质提供了手段，也为其他木本植物的链格孢属真菌病害的抗病基因挖掘和分子改良提供了参考。