泛素化介导云南疣粒野生稻抗白叶枯病的机理研究

Oryza meyeriana Baill from Yunnan province is a high resistant wild rice ecotype to bacterial blight disease. This feature is very prominent in oryza. But the mechanism of resistance is unclear. Ubiquitination is one of the most refined regulation systems of protein degradation in plant. Its function involve in disease resistance. Until now, there is no report about the disease resistance mechanism in O. meyeriana mediated by ubiquitination. In previous research, an ubiquitin conjunction enzyme (E2) gene (ME207) was isolated after O. meyeriana infected by xanthomonas oryzae pv. oryzae (Xoo). It implied that ubiquitination regulated the bacterial blight disease resistance. In order to make resistance mechanism clear, the transcriptome database will be constructed to select the ubiquitination enzyme (E1, E2, E3) genes after O.meyeriana infected by xanthomonas oryzae pv. oryzae (Xoo). The differential expression ubiquitination enzyme genes will be identified by compared with the transcriptome database none infected by Xoo. Then, the disease response E1, E2 and E3 genes will determined using bioinformatics, ubiquitin and gene espression profile analyze. Yeast two hybrid and western blot will be used to determine the specificity of gene interaction and the self- ubiquitin ability of E2 proteins in vitro in absent E3 ligases. One or two candidate E3 will be choosen to identify the target proteins. BiFC will used to vertify interaction between the E3 and target proteins in vivo. The resistance signal transduction pathway will be established. According to these research, many diseased resistance-related genes will be isolated; the molecular mechanism of highly resistant bacterial blight disease mediated by ubiquitination will be explore in O. meyeriana.

云南疣粒野生稻高抗甚至免疫白叶枯病，在稻属中表现突出，但抗病机理还不清楚。蛋白质泛素化降解是生物体内最精细的调控体系之一，功能涉及抗生物胁迫。疣粒野生稻中泛素化调控抗病的机理值得探索。我们前期研究发现一个泛素结合酶基因ME207参与疣粒野生稻抗白叶枯病，表明泛素化系统参与抗病过程。为弄清泛素化介导的云南疣粒野生稻抗白叶枯病的机理，本项目拟开展受白叶枯病菌胁迫的云南疣粒野生稻转录组测序，筛选泛素化酶基因，与非胁迫转录组中的该类基因进行比较，经泛素化酶活检测及基因表达谱分析，确定抗病应答的泛素化酶基因E1、E2、E3，经酵母双杂交和Western 分析，弄清候选基因的互作特性和E2的自身泛素化功能特性，并挑选1-2个E3筛选其靶蛋白，建立抗病信号在泛素化系统中的转导模式。本研究将发掘一批应答白叶枯病的泛素化酶基因，揭示泛素化在疣粒野生稻抵御白叶枯病中的作用，为多角度解析植物抗病机制提供新线索。

蛋白质泛素化降解是生物体内最精细的调控体系之一，功能涉及抗多种生物胁迫，云南疣粒野生稻高抗白叶枯病，蛋白质泛素化调控体系可能是云南疣粒野生稻抗白叶枯病的途径之一，但机理还不清楚。本项目采用转录组测序、遗传转化、酵母双杂交等方法对泛素化酶在云南疣粒野生稻抗白叶枯病中的作用开展了研究。从受白叶枯病胁迫的云南疣粒野生稻转录组中，筛选出一批泛素化酶基因，经过白叶枯病胁迫的表达谱分析，明确了其中10个基因受白叶枯病菌诱导表达量提高，说明泛素化酶基因参与疣粒野生稻抵御白叶枯病的过程。对转录水平显著升高的3个抗病候选基因MEE2-17、MEE3-29、MEXB3-6进行基因功能研究，发现这3个基因能显著提高转基因水稻材料的抗白叶枯病性；通过酵母双杂交方法获得了2个E3基因（MEE3-29和MEXB3-6）的靶基因MEPBL17和MEPDH、MEPPR，转基因植株检测结果显示靶基因分别受这2个E3基因调控，说明泛素化酶基因通过调控下游靶基因的表达介导疣粒野生稻抵御白叶枯病；激素信号途径分析，发现白叶枯病菌可诱导云南疣粒野生稻内源SA、JA含量显著升高，通路中NPR1、TGA、COI1、JAZ关键基因转录水平提高，10个泛素化酶基因也分别受SA和JA诱导表达，表明云南疣粒野生稻抗白叶枯病依赖SA、JA激素信号途径。综合以上研究结果推测：泛素化介导云南疣粒野生稻抗白叶枯病的信号转导模式为，白叶枯病菌胁迫后，疣粒野生稻内源SA、JA含量增加，激活了激素依赖的信号转导途径，信号通路关键基因表达，信号得以传递，启动了泛素化介导的蛋白质水解，10个泛素化酶基因响应抗性信号转录水平显著提高，并通过负调控STK和PPR类基因、正调控PDH类基因的表达，提高云南疣粒野生稻对白叶枯病的抗性。研究结果为解析云南疣粒野生稻抗白叶枯病的机制和发掘抗病基因奠定基础。发表论文2篇，其中SCI 收录1篇，申请专利1项，获得发明专利2项。