水稻WRKY80转录因子基因在抗病防御过程中的调节功能与分子机制

WRKY transcription factors play important regulatory roles in plant response to pathogens. So far, several rice WRKY genes have been identified to be involved in disease resistance, and most of them are associated with salicylic acid (SA)-dependent defense signal pathway. However, the knowledge on the function of jasmonic acid (JA) in the rice WRKYs-mediated defense signaling is rather limited. Previously, we confirmed by WRKY cDNA array analysis and Northern blot that rice WRKY80 was induced by blast and sheath blight causing pathogens and JA application, but not by SA application, suggesting that it might be involved in the defense response to these pathogens through JA-dependent signal pathway. To confirm this hypothesis, we focus on systematic functional analysis for the WRKY80 gene. First, we will analyze its expression profiles including expression specificity in different organs and development stages and induced expression patterns subject to various biotic, abiotic stresses and chemical treatments. Second, we will define the relationships between structural and biochemical characteristics of WRKY80 protein, including subcellular localization, DNA-binding ability and transcriptional activation or suppression. Third, to obtain the biological functions for the WRKY80 gene, we will investigate the resistance to pathogens in over-expression, RNAi transgenic lines or insertion mutants compared with wild-type plants. Moreover, to understand the regulatory mechanisms of the WRKY80 functioning in defense response to pathogens, we will focus on analysis for expression of defense-related genes and marker genes in signal pathways and identification of interacting components of rice WRKY80. The research will attach importance to elucidate the function and molecular regulatory mechanisms for rice WRKYs in plant disease resistance, and may provide a promising candidate gene for molecular breeding of crop broad resistance.

WRKY转录因子的重要功能是调控植物抗病反应。从已鉴定功能的几个水稻WRKY基因来看，主要通过水杨酸（SA）抗病信号途径发挥调节作用，而茉莉酸（JA）在WRKY介导的抗病信号网络中的作用仍不清楚。前期，cDNA阵列和Northern杂交分析表明，水稻WRKY80受稻瘟病菌、纹枯病菌和JA诱导，而不受SA诱导，提示其可能通过JA信号途径介导水稻抗病防御。为验证此假说，本项目在分离基因cDNA序列基础上，分析基因的器官与发育阶段特异性及诱导表达模式；研究蛋白核定位、DNA元件结合和转录激活或抑制活性，以明确结构与生化功能的关系；创建超表达、RNAi转基因突变株，分析抗病性表型，以明确其生物学功能；重点分析突变株中防卫相关基因、信号通路标志基因的表达变化，鉴定互作组分，以明确其调节抗病反应的分子机理，对深入阐明水稻WRKY参与抗病防御调节及分子机制具有意义，同时可为作物广谱抗病育种提供候选基因。

WRKY转录因子最重要的功能是通过SA等信号途径介导植物的基础免疫和诱导抗性。但JA信号途径在WRKY的抗病调节中的作用仍不清楚。.前期实验发现，水稻WRKY80受JA和病原真菌快速而显著诱导，可能参与JA信号途径参与水稻对真菌病原的防御反应。本项目分析了WRKY80表达模式；研究了WRKY80与DNA元件的特异结合、转录激活以及亚细胞定位等生化特征；创建了超表达和RNAi突变株，分析了抗性表现；分析了转基因植株中重要抗病相关基因的表达，提出了WRKY80调控水稻对真菌病原抗性的分子机制。.1、WRKY80 cDNA长1392 bp，含一个ORF（1164 bp）。含1个WRKY结构域，锌指为CCHH，属WRKY第II组。2、WRKY80在叶、根和穗中表达较高，花次之，茎和颖果低；成熟叶、根中的表达分别高于幼叶和幼根。受JA、ET、稻瘟病菌和纹枯病菌强烈而瞬间诱导。提示，WRKY80具有发育阶段特异性，可能通过JA/ET抗病信号途径参与水稻抗病。3、WRKY80定位于细胞核，有转录激活特性。N端（1-61 aa）和C端（301-387aa）为转录激活所必需，S6和Q8在转录激活活性维持中发挥作用。4、获得了10个超表达T2代株系、12个RNAi T2代株系，并获表达分析验证。超表达株系植株矮小，冠根变少，而RNAi株系的表型无明显变化。超表达植株对纹枯病抗性增强，RNAi植株的抗性减弱，表明WRKY80是水稻抗纹枯病的正调子。5、在超表达和RNAi株系中，WRKY80与先前鉴定的WRKY4在一些抗病相关基因的表达上有相似的表现：超表达植株中PR1a、R1b、PR5和PR10/PBZ1表达显著增强，而RNAi植株中这些基因的表达显著下降。同时，WRKY80超表达株系中，WRKY4的表达显著增强，而RNAi株系中则显著降低。提示，WRKY80位于WRKY4的上游，可能通过JA/ET抗病信号途径，实现水稻对纹枯病的抗性。6、WRKY80在酵母中能特异地与WRKY4启动子中的W盒结合，且在烟草中得到证实，进一步证明了WRKY4是WRKY80作用靶点。.总之，WRKY80-WRKY4通过JA/ET防御信号途径调节水稻纹枯病菌的抗性，对阐明WRKY介导水稻抗病分子机制具有重要意义，也可作为候选基因应用于作物抗病基因工程。