抗玉米南方锈病基因RppK22的克隆及其抗病分子机理的研究

Maize southern rust (MSR) is one of major diseases in China. Cloning of major resistance genes and revealing their resistant mechanisms are important for scientific and effective disease resistance breeding. After great efforts of many years, a dominant QTL against MSR (designated as RppK22) was fine mapped on chromosome 10 and delimited to an estimated length of 12kb region, which contains two candidate genes: R1 and R2, based on the genomic sequence of B73. In this project, we will clone RppK22 and confirm its disease-resistant function mainly through screening BAC library and genetic transformation assay. Next, the RppK22-interacting proteins will be identified using yeast-two hybrid assay or immunoprecipitation-MS and validated by bimolecular fluorescence complementation (BiFC) and Co-immunoprecipitation. The biochemistry and defense function of the RppK22-interacting proteins will also be evaluated by mutants and RNAi transgenic plants. Moreover, we will identify other genes participating in RppK22-mediated signal pathway using RNA-seq assay and other methods to discover RppK22 disease-resistant molecular network and to explore resistance mechanism thoroughly. This study will not only enrich the understanding of resistance mechanisms against MSR, but also provide novel defense genes and ideas for molecular disease resistance breeding.

玉米南方锈病是我国重要病害之一，克隆主效抗病基因并揭示其抗病分子机理是指导科学有效抗病育种的重要基础。通过多年努力，申请人课题组已将一个新的玉米抗南方锈病主效QTL（命名为RppK22）精细定位于第十条染色体上约12kb区段内，根据B73的基因组序列，该区段含有2个候选基因: R1和R2。本项目拟结合筛选BAC库和转基因功能分析等分子生物学和遗传学方法, 克隆抗病基因RppK22并明确其抗病功能；然后通过酵母双杂和免疫沉淀-质谱联用筛选互作因子，再用双分子荧光互补和免疫共沉淀、突变体或RNAi转基因等方法对互作蛋白的生化和抗病功能进行验证。此外，通过RNA-seq等方法鉴定参与RppK22抗病信号途径的成分，阐明以RppK22为核心的抗病分子网络，从而全面揭示RppK22抗病分子机理。本研究的完成不但可以丰富对玉米抗南方锈病分子机制的认识，而且为抗病分子育种提供新的基因资源和新思路。

玉米南方锈病是我国玉米产区的重要病害之一，克隆主效抗南方锈病基因并揭示其抗病分子机制是指导科学有效的抗病分子育种的重要基础。本研究前期工作在第十条染色体上定位到一个主效抗南方锈病基因RppK22。本研究通过图位克隆和转基因功能验证成功克隆了RppK22基因，并证实其抗病功能。该基因编码一个CC-NB-LRR的蛋白；基于烟草瞬时表达和玉米原生质体表达系统，确定了RppK22蛋白定位在细胞质中。为了揭示RppK22蛋白的抗病分子机制，本研究通过酵母双杂交系统鉴定到一个与RppK22蛋白互作的蛋白CR3P4。通过Split-Luc实验证实了CR3P4蛋白与Rppk22蛋白的互作关系。此外，利用遗传转化获得了CR3P4基因的过量表达和CRISPR转基因玉米植株。田间接种实验发现过量表达CR3P4基因的转基因阳性植株比阴性植株更抗玉米南方锈病，而CRISPR转基因植株比阴性植株更感南方锈病。由此可知，CR3P4基因正向调控玉米对南方锈病的抗病性。以此，本研究揭示了玉米抗病基因RppK22与CR3P4协调抵抗玉米南方锈病的分子机制。本研究的完成不仅丰富了对玉米抗南方锈病的分子机制的认识，而且为玉米抗病分子育种提供了新的抗病基因资源和新的抗病材料。