禾谷镰刀菌对氰烯菌酯药敏性调控机制研究

Phenamacril, 2-cyano-3-amino-3-phenylancryic acetate, is a cyanoacrylate fungicide whose intellectual property rights belong to China and provide excellent control against Fusarium head blight (FHB) or scab mainly caused by Fusarium graminearum. Previous study showed that Myo5 in F. graminearum was the molecular target of phenamacril and mutations in Myo5 conferred resistance to the fungicide. Fim、Myo2B and Smy1 could regulate the sensitivity of F. graminearum against phenamacril by interacting directly or indirectly with Myo5. In order to completely understand regulatory system on sensitivity of F.graminearum against phenamacril, we are planning to identify the interaction proteins of Myo5 by immunoprecipitation and mass-spectrometric technique. The interaction proteins of Myo5 are furthered confirmed by Yeast Two Hybrid (Y2H), Co-Immunoprecipitation (CoIP) and Bimolecular fluorescence complementation (BiFC) assays. The mutants of deletion, complement and over-expression of the interaction proteins are constructed. The sensitivities and biological characteristics of mutants are studied. Whether there are interactions between the interaction proteins of Myo5 are determined by Y2H, CoIP and BiFC assays and the expression patterns of Myo5 interaction proteins are studied. According to above results, the regulatory network on sensitivity of F. graminearum against phenamacril are constructed. The results of the study will be very useful for finding the new targets of fungicides and providing theoretical foundation for designing new chemicals based on targets.

氰烯菌酯是我国创制的具有自主知识产权的氰基丙烯酸酯类杀菌剂，对禾谷镰刀菌引起的小麦赤霉病有优异的防效。前期研究表明，肌球蛋白Myo5是氰烯菌酯对禾谷镰刀菌作用的分子靶标，Fim、Myo2B和Smy1等蛋白通过直接或间接与Myo5互作调控氰烯菌酯药敏性。为了揭示禾谷镰刀菌对氰烯菌酯药敏性调控网络，本项目拟采用免疫沉淀和质谱的方法寻找和鉴定Myo5的互作蛋白；利用酵母双杂交、免疫共沉淀和双分子荧光互补技术进行蛋白互作验证；构建Myo5互作蛋白基因的敲除、回复和过表达突变体，研究突变体对氰烯菌酯药敏性的调控作用及其生物学特征；研究氰烯菌酯药敏性调控蛋白间的互作关系及其在转录水平上的时空表达模式，构建禾谷镰刀菌对氰烯菌酯药敏性调控网络。旨在发掘药物新靶标，为基于作用靶标的新农药分子合理设计提供理论依据。

氰烯菌酯是我国创制的具有自主知识产权的氰基丙烯酸酯类杀菌剂，对禾谷镰刀菌引起的小麦赤霉病有优异的防效。前期研究表明，肌球蛋白（Myosin5）是氰烯菌酯对禾谷镰刀菌作用的分子靶标，本项目拟从Myosin5的互作蛋白入手揭示禾谷镰刀菌对氰烯菌酯药敏性调控机制，拟从以下4个方面进行研究：（1）采用免疫沉淀和质谱的方法寻找和鉴定Myosin5的互作蛋白。（2）利用酵母双杂交、免疫共沉淀和双分子荧光互补技术等进行蛋白互作验证。（3）构建Myosin5互作蛋白基因的敲除、回复和过表达突变体，研究突变体对氰烯菌酯药敏性的调控作用及其生物学特征。（4）研究氰烯菌酯药敏性调控蛋白间的互作关系及其在转录水平上的时空表达模式，构建禾谷镰刀菌对氰烯菌酯药敏性调控网络。结果表明：（1）质谱鉴定出FgMyosin5互作蛋白1140个，做进一步鉴定和研究。（2）细胞质膜蛋白FgPMA1、FgBmh2和滑行蛋白FgAglZ均负调控禾谷镰刀菌对氰烯菌酯的药敏性而正调控其抗药性水平，FgPMA1是通过与FgMyosin5和FgBmh2互作形成复合体，从而调控禾谷镰刀菌对氰烯菌酯药敏性及其菌丝生长、致病力、有性生殖等；FgAglZ与FgMyosin5不互作，但是可以通过影响FgMyosin5转录水平而调控禾谷镰刀菌对氰烯菌酯的药敏性。（3）糖原分支酶FgGlc3正调控禾谷镰刀菌对氰烯菌酯的药敏性而负调控其抗药性水平，FgGlc3缺失突变体的菌丝生长、无性繁殖能力、有性生殖能力等均显著下降，FgGlc3与FgMyosin5不互作，也不调控FgMyosin5的转录水平，推测可能与翻译后修饰有关。（4）以上基因均为组成型表达，在各阶段FgPMA1的表达量都显著高于FgPMA2；FgPMA1、FgAglZ、FgGlc3分别定位于细胞质膜、细胞质、细胞质和细胞核上。以上结果为基于作用靶标的新农药分子合理设计提供理论依据。