肌球蛋白Myosin-5氨基酸点突变导致禾谷镰刀菌对氰烯菌酯产生抗性的分子机制

Myosins comprise a family of ATP-dependent motor proteins and are responsible for actin-based motility. Fusarium graminearum Schwade [teleomorph=Gibberella zeae(Schweinitz) Petch] is one of the main pathogens causing Fusarium head blight (FHB) on wheat. Previous study showed that mutations in myosin-5 conferred resistance to phenamacril in F. graminearum. In order to understand the molecular mechanism of the resisitance of Fusarium graminearum to phenamacril conferred by the point mutation in Myosin-5, we are planing to do the research in the following aspects. First, the expression vectors of Myosin-5 with different point mutations are constructed. Myosin-5 with different point mutations express in E. coli and are purified. The affinity between Myosin-5 and phenamacril is measured. Second, the ability of ATP hydrolysis by Myosin-5 with different point mutations is measured. Third, actin gene is marked with Green Fluorescent Protein (GFP) in different point mutations. Motility and distribution of actin are observed. Forth, RNA-sequences of different point mutants with different resistance level are analyzed. Differentially expressed genes are knocked down and the sensitivity of deletion mutants to phenamacril are determined. The relationship between differentially expressed genes and Myosin-5 are studied by Yeast two-hybrid system and bimolecular fluorescence complementation assay. This study will help to find new targets and study the synergistic use of technology for fungicides with different mechanism of action.

肌球蛋白（Myosin）是一种马达蛋白，它可以作用于肌动蛋白丝，把储存于ATP中的化学能转变为机械能。禾谷镰刀菌是引起小麦赤霉病的主要病原菌。前期研究表明，禾谷镰刀菌肌球蛋白Myosin-5氨基酸点突变会使其对杀菌剂氰烯菌酯产生抗性，为了探明其分子机制，本项目拟从以下4个方面进行研究：一、构建Myosin-5蛋白不同点突变表达载体，原核表达和纯化，体外研究其与氰烯菌酯的亲和性；二、研究不同点突变体Myosin-5蛋白水解ATP的能力；三、GFP原位标记不同点突变体的肌动蛋白，研究不同突变体的肌动蛋白运动和分布情况；四、将具有不同抗性水平突变体进行转录组分析，构建差异表达基因敲除突变体，研究差异表达基因与氰烯菌酯抗药性及Myosin-5基因之间的关系。研究成果将有助于发现杀菌剂新的靶标位点，解释田间抗性水平进化，为基于作用靶标的新农药分子合理设计和田间抗性治理提供理论依据。

由镰刀菌（主要为禾谷镰刀菌和亚洲镰刀菌）引起的小麦赤霉病是国内外小麦生产中的重要病害之一，氰烯菌酯是我国自主研发的对小麦赤霉病有特效的氰基丙烯酸酯类杀菌剂，前期研究表明禾谷镰刀菌肌球蛋白Myosin-5氨基酸点突变会使其对杀菌剂氰烯菌酯产生抗性，为了探明其分子机制，本项目拟从以下4个方面进行研究：（1）构建Myosin-5蛋白不同点突变表达载体，原核表达和纯化，体外研究其与氰烯菌酯的亲和性。（2）研究不同点突变体Myosin-5蛋白ATP酶活性。（3）GFP原位标记不同点突变体的肌动蛋白，研究不同突变体的肌动蛋白运动和分布情况。（4）将具有不同抗性水平突变体进行转录组分析，构建差异表达基因敲除突变体，研究差异表达基因与氰烯菌酯抗药性及Myosin-5基因之间的关系。结果表明：（1）肌球蛋白 Myosin-5的135位（A135T）、217位（S217L）、418位（S418R）或420位（E420K）氨基酸突变以后，与氰烯菌酯亲和性下降，亲和性下降程度与抗药性水平没有直接关系，E420K> S217L> A135T> S418R（E420K和S217L引起高等水平抗性，S418R引起中等水平抗性，A135T引起低等水平抗性）。（2）A135T、S217L、S418R氨基酸突变的Myosin-5蛋白与野生型菌株PH-1的Myosin-5蛋白的ATP酶活性无显著性差异，但是420位（E420K）氨基酸突变的Myosin-5蛋白ATP酶活性显著下降。（3）抗性突变体和野生型菌株在氰烯菌酯处理下其F-actin的运动无显著性差异。（4）高等抗性菌株中缺失Dbf2或Smy1均会导致抗性水平下降，说明Dbf2和Smy1参与调控禾谷镰刀菌对氰烯菌酯的抗性，而Smy1是通过与Myo2B互作来调控抗药性相关基因的转录表达进而调控小麦赤霉病菌对氰烯菌酯的抗药性水平。由以上结果可知，肌球蛋白Myosin-5的135位、217位、418位和420位氨基酸是与氰烯菌酯结合的关键氨基酸位点，该结果为基于作用靶标的新农药分子合理设计和田间抗性治理提供理论依据；Dbf2和Smy1调控小麦赤霉病菌对氰烯菌酯药敏性，可作为新的药剂分子靶标。