新的小麦蛋白激酶TaPKc1的抗病功能和分子机制的研究

Wheat sharp eyespot, caused primarily by the soil-borne fungi Rhizoctonia cerealis, has been became to be one of the most devastative diseases, and a major threat in wheat production. Isolating key genes in the resistance response and developing the functional molecular markers will accelerate the speed of wheat molecular breeding with resistance. Based on comparative transcriptomic analysis, a novel protein kinase-coding gene, named TaPKc1, was isolated from resistant wheat lines Shanhongmai and CI12633. The VIGS functional analysis in wheat indicated that TaPKc1 is a key gene in wheat resistance defense to sharp eyespot, and regulates the expression of known functional resistance-related genes. In this proposal, we will use methods of genetics, molecular biology, biochemical and bioinformatics to further study the functional role and elucidate molecular mechanism of TaPKc1 in wheat defense responses. At the same time, we will develop and validate the functional molecular markers of TaPKc1 in various wheat cultivars/lines and RIL population. Thus, this potential project will elucidate a novel defense signaling pathway to sharp eyespot in wheat, which benefit for the dissecting of gene network in wheat defense to sharp eyespot and for speeding resistant wheat molecular breeding.

以小麦纹枯病为代表的土传病害，已成为小麦重要病害，难以防治。为加快抗病育种进程，分离抗纹枯病重要基因，我们前期对山红麦/温麦6重组近交系的抗纹枯病、感纹枯病高代株系进行RNA-SEQ、生物信息学、比较转录组学分析、病毒诱导的基因沉默-功能分析，分离克隆出新的小麦蛋白激酶基因TaPKc1，证明TaPKc1是小麦抗纹枯病反应的关键基因。本项目将利用遗传学、分子生物学、生物化学和生物组学等研究方法，深入研究TaPKc1的抗病功能，解析TaPKc1激酶的互作蛋白、调控的下游基因、参与的信号路径，揭示该激酶抗病作用的分子机制，同时研究该基因的等位变异、开发功能标记，分析其对纹枯病抗性的遗传贡献率，为小麦抗纹枯病分子育种提供优异基因、分子标记、理论依据和技术支撑。

利用BSR-Seq、表达抗性关联和VIGS功能分析，鉴定到小麦蛋白激酶编码基因TaMKK5-6B （申请项目时暂命名为TaPKc1，其蛋白包含具保守的[S/T]-x5-[S/T] motif 和 D(I/L/V)K活性位点的PKc结构域。TaMKK5-6B表达量受纹枯病菌诱导上调，且抗病小麦中显著高于感病小麦中。TaMKK5-6B过表达显著地提高小麦对纹枯病的抗性和6个防御相关基因转录丰度；TaMKK5-6B基因沉默后降低了小麦对纹枯病的抗性和防御相关基因表达。TaMKK5 BiFC、Y2H、Pull-down和蛋白磷酸化实验结果证明，TaMKK5与 TaMPK3直接互作，并磷酸化激活TaMPK3，TaMPK3 与TaERF3直接互作，活化的TaMPK3磷酸化激活TaERF3。TaMKK5调控TaERF3，TaCPK7-D，TaGST1， Defensin和Chitinase 2是通过与TaMPK3级联， TaMKK5-TaMPK3-TaERF3级联调控防卫基因TaGST1， Defensin和Chitinase 2的表达，由此明确了TaMKK5在小麦抗纹枯病反应中的功能和分子机制，提出了TaMKK5调控小麦纹枯病抗性的分子机制模型，TaMKK5通过TaMKK5-TaMPK3-TaERF3级联模块和激活调控多个防御相关基因表达，正向贡献于小麦纹枯病抗性。另外，鉴定出TaMKK5信号通路上游的6个蛋白激酶编码基因，VIGS功能分析表明TaWAK7D、TaWAK6D、TaWAK-2A800、TaCRK-7A、TaRLCK1B和TaPK3A均为小麦抗纹枯病反应所需的基因，通过MAPK信号通路发挥功效。该项目为我国及世界抗纹枯病小麦分子改良提供了新的基因资源；发表该学术论文7篇， 其中 SCI 论文 6 篇；申请了国家发明专利2项，获得授权的国家发明专利 1 项；培养研究生7名。