新型杀菌剂氰烯菌酯的作用机理研究

JS399-19, 2-cyano-3-amino-3-phenylancryic acetate, was the first fungicide belonged to the cyanoacrylate fungicide group and created by our country. It was put into market in 2007. Previous study showed that JS399-19 had a special mode of action compared to other fungicide and a strong selectivity and had mainly powerful inhibitory activity against Fusariun species. It had provided excellent control of Fusaruim head blight of wheat in the field. In this study, Fusarium graminearum was target organism and proteomics technology was used for studying mechanism of action of JS399-19. Mycelium of Fusarium graminearum were treated with different concentration of JS399-19 for different time and the control was mycelium treated with nothing. Total protein were extracted from mycelium and then the protein were pured and quantitatived. Two-dimensional electrophoresis was employed and gels were scanned. Protein expression profile differences were analysed. Differentially expressed proteins were separated and cut down and identified by MALDI-TOF-MS/MS. According to the position of differentially expressed proteins in biochemical pathways, Biochemical pathways of JS399-19 against to Fusarium graminearum were speculated. The effect of JS399-19 to biochemical pathways of Fusarium graminearum were analysed by some biochemical research methods(for example: respiration) and some mainly biochemical pathways were preliminarily determined. Genes coding differentially expressed proteins that had a key role in the biochemical pathways were deleted and complemented by homologous pairs exchange. The sensitivity of deletion and complementary mutation to JS399-19 was determined and physiological and biochemical characteristics of the mutations were analysed. Mechanism of action of JS399-19 was revealed. This study will provided scientific guidance for using JS399-19 safely and reasonably and theoretical basis for rational design of new pesticide based on target.

氰烯菌酯是我国创制的第一个氰基丙烯酸酯类杀菌剂，2007年进入商品化。前期研究表明，该杀菌剂结构新颖、作用方式独特、专化性强，主要对镰孢菌表现极强的抑制作用，在田间对小麦赤霉病有良好的防效。本项目拟以禾谷镰孢菌(Fusarium graminearum)为靶标生物，应用蛋白质组学技术，通过比对分析菌体经不同药剂浓度和时间处理前后蛋白质表达谱的变化，分离、鉴定差异蛋白质；根据差异蛋白质在生化途径中的位置，推定药剂作用的可能生化途径；利用生物化学研究方法，研究验证药剂对这些生化途径的影响，初步确定药剂作用的主要生化途径；利用同源双交换，构建编码在生化途径中发挥关键作用的差异蛋白质的基因敲除/回复突变体，通过测定突变体对药剂的敏感性和生理生化特征，解析药剂作用的生化途径，从而揭示药剂的作用机理。研究成果将为氰烯菌酯的安全和合理使用提供科学指导，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第一个氰基丙烯酸酯类杀菌剂，对镰孢菌表现极强的抑制作用，为了揭示其作用机理，本项目从两个方面进行了研究。蛋白质组学研究方面，分别用EC90（0.5μg/mL）浓度氰烯菌酯处理禾谷镰孢菌敏感菌株2021和抗性菌株Y2021A 12h，同时以不加药剂的作为对照，提取总蛋白，纯化、定量，进行蛋白质双向电泳，结果表明：敏感菌株经药剂处理以后蛋白表达量变化2倍以上而抗性菌株经药剂处理以后未发生显著变化的蛋白点38个，通过MALDI-TOF-MS/MS和MOSCOT，成功鉴定出33个蛋白；生理生化途径分析结果表明，在33个蛋白中，19个为代谢相关蛋白，5个为调控相关蛋白，2个为运动相关蛋白，3个为防卫相关蛋白，2个为信号传导相关蛋白，2个为未知功能蛋白；氧电极法测定了氰烯菌酯对抗敏菌株呼吸作用的影响，结果表明，氰烯菌酯对禾谷镰孢菌抗敏菌株呼吸的抑制作用均较弱，无显著性差异；抗敏菌株运动相关蛋白----纺锤体驱动蛋白1敲除体对氰烯菌酯的敏感性无显著变化。基因组学研究方面，通过基因组重测序，发现抗性菌株YP-1中132个基因上发生了氨基酸突变，不同抗性菌株测序验证发现myosin-5基因上有稳定的氨基酸点突变，突变位点为216位、217位、418位、420位或786位，通过遗传转化发现敏感菌株中导入抗性菌株myosin5基因后的突变体对氰烯菌酯表现相应的抗性，抗性菌株myosin5基因被敏感菌株myosin5基因替代后对氰烯菌酯表现敏感，证明了myosin5蛋白氨基酸突变点突变（216、217、418、420或786位）是导致小麦赤霉病菌对氰烯菌酯产生抗性的原因，肌球蛋白Myosin-5是氰烯菌酯的分子靶标。