芸薹生链格孢AbFus3-MAPK信号通路三级激酶的锚定互作研究

Mitogen-activated protein (MAP) kinase signaling pathways are ubiquitous and evolutionarily conserved in eukaryotic organisms.With the deep insight into its function and mechanism, the research on its application also experienced rapid development. The components of signal transduction pathways have been identified as an important molecular target of development of antitumor and antipsoriasis drugs. Moreover,the docking site in the protein kinases of the MAPK cascade can be a new target for drug development. Like in mammal and yeast,MAPK cascades also play very important roles in plant pathogenic fungi,especially in the pathogenicity ,which makes them potential targets of future fungicides.But the mechanism of docking interaction of protein kinases in MAPK cascades in plant pathogenic fungi is seldom reported except in Magnaporthe grisea. In our previous works, we found that the AbFus3-MAPK signalling pathway was closely related with the pathogenicity of Alternaria brassicicola, which means it being a good target of fungicide for this fungus. In this topic, we try to explore the mechanisms of docking interaction in MAPK signalling pathway in A. brassicicola . By using gene disruption method and GST-pull down assays, we aim to determine the docking site in AbFus3 and AbSte7. All these works will widen our knowledge of the mechanism of AbFus3 MAPK signaling, and contribute to our understanding of the molecular mechanism of the pathogenicity of A. brassicicola. Also this work will lay a foundation of the new fungicides targeted AbFus3 MAPK signaling pathway.

MAPK信号通路是真核生物体内非常保守的信号转导途径之一，随着对其功能和作用机理认识的逐步深入，相应的应用研究也迅猛发展，以该信号通路激酶为作用靶标的药物如抗肿瘤药和抗银屑病药已经上市，而激酶间的锚定互作位点也成为了药物发展的一个新作用靶点。 同样在植物病原真菌中MAPK通路也起着非常重要的作用，尤其是在致病过程中。这也使得其有可能成为未来杀菌剂的作用靶标，而激酶间锚定作用机理的研究很少，只在稻瘟菌中有过报道。我们前期工作表明AbFus3-MAPK信号通路与芸薹生链格孢的致病性有紧密联系。因此在以上工作的基础上，本项目拟运用基因缺失突变法和GST-pull down技术，进一步深入研究该信号途径激酶三级激酶的锚定互作机制，探索相互之间的锚定作用位点。工作的开展将有助于我们全面认识芸薹生链格孢MAPK 信号通路激酶的互作机制，也可为未来开发以AbFus3途径为靶标的新型杀菌剂奠定基础。

MAPK信号通路是真菌体内非常保守的信号转导途径之一，目前该信号通路的功能被逐渐认识，其中Fus3-like途径广泛参与调控植物病原真菌的致病性，这种功能也让其信号通路的激酶成为新杀菌剂的作用靶标，而激酶间的锚定互作位点也成为了药物发展的一个新作用靶点。在本项目中我们以芸薹生链格孢为研究对象，在前期研究了AbFus3信号途径功能的基础上，进一步研究了三级激酶之间的锚定互作位点，明确了MAPK在MAPKK上的锚定作用位点，即两个特征序列均起到了锚定功能；同时在研究MAPKK在MAPK上的锚定位点时，我们发现其除了通用位点外，C端322-352氨基酸序列段同样也起到了锚定作用；而MAPKKK在MAPKK上的作用位点为C端的344-451序列段。这些研究结果有助于我们全面认识芸薹生链格孢MAPK 信号通路激酶的互作机制，也可为未来开发以AbFus3途径为靶标的新型杀菌剂奠定基础。