灰葡萄孢菌（Botrytis cinerea）对氟啶胺抗药性分子机制

Fluazinam is a high efficiency, broad spectrum, and environmental friendly pyridine-amine fungicide with great application prospect. It has excellent inhibitory activity against plant pathogens of six phyla and was registered in China in 2013. Serious fluazinam-resistance cases however, have been reported in Japan and the Netherlands after its application of 1-6 years. Unfortunately, the molecular resistance mechanism to fluazinam is not well understood nowadays. In this project, the genome-sequenced and assembled Botrytis cinerea strain B05.10 will be used to conduct the following studies: (1) recovery of fluazinam-resistant mutants domesticated by the fungicide, and determining their biochemical characteristic, including mycelial radial growth, sporulation, pathogenicity and oxidative phosphorylation of mitochondria; (2) re-sequencing resistant mutants and bioinformatics analysis to find the mutated genes, such as SNPs and InDels of resistant mutants; (3) cloning of resistance candidate genes from other fluazinam-resistant mutants to confirm whether the traits were repeated and establishing relationship between mutation patterns of the certain gene(designated as the resistance gene to be confirmed) and sensitivities of mutants to fluazinam; (4) constructing a series of mutants via DNA manipulation to verify this resistance gene and to analyze its biological functions in the important stages of the live cycle of the pathogen. The results will reveal the molecular resistance mechanism of B. cinerea to fluazinam and lay the foundation to build platforms for early warning and forecasting resistance to the fungicide, to re-innovate chemical structure and to provide scientific usage guidance. Three papers will be published in journals of SCI source and three postgraduate students will be brought up.

氟啶胺是高效、广谱、与环境友好的吡啶胺类杀菌剂，对植物病原菌六个门的菌物均具有优异的抑菌活性，应用前景良好。2013年在我国获得登记。但是，日本、荷兰等国在使用1-6年后，相继报道了病原菌对其产生严重抗药性的事件。目前，关于该药剂抗药性机制的研究仍为空白！本项目拟以核基因组已经测序组装的灰霉病菌（Botrytis cinerea）B05.10菌株为靶标生物，研究：(1)以该菌株及其药剂驯服抗药性突变体单胞菌株为材料，明确抗药性突变体的生物学及氧化磷酸化等理化特征；(2）对抗药性突变体进行重测序，采用生物信息学分析该突变体的SNP位点等变异基因；（3）利用其他抗药性突变体对抗药性候选基因核实，建立基因突变与药敏性之间对应关系；（4）构建系列突变体，对抗药性基因验证和功能分析。该研究将揭示氟啶胺的抗药性分子机制，为构建抗药性早期预警、测报和科学用药奠定基础。SCI 3篇，培硕3名。

氟啶胺是高效、广谱、与环境友好的吡啶胺类杀菌剂，对植物病原菌六个门的菌物均具有优异的抑菌活性，应用前景良好。2013年在我国获得登记。但是，日本、荷兰等国在使用1-6年后，相继报道了病原菌对其产生严重抗药性的事件。本研究为揭示氟啶胺毒理学机制，筛选了由6032个突变体组成的酿酒酵母突变体库，共获得对氟啶胺表现敏感性升高的酵母突变体133株；对氟啶胺表现敏感性下降的突变体106株。根据酵母突变体缺失的基因编码的蛋白氨基酸序列，采用BLASTP分析方法，在灰葡萄孢核基因组数据库中钓取相对应的同源基因；239个基因的功能分析表明：这些基因主要参与调控菌体氨基酸代谢、能量代谢、核酸转录、跨膜转运等途径。甲硫氨酸在生物体蛋白质合成过程中具有重要的作用，在哺乳动物线粒体中谷胱甘肽能够显著调控氟啶胺的毒理作用，另外在真菌中，谷胱甘肽对甲硫氨酸合成具有重要的调控作用，并且γ-胱硫醚合成酶是甲硫氨酸合成途径的关键调控酶，具有将胱硫醚转化成同型半胱氨酸的活性，然后菌体利用同型半胱氨酸合成甲硫氨酸。另外，在线粒体中，氟啶胺可能通过调控线粒体的转运蛋白，降低线粒体基质与膜间隙的质粒梯度差，解偶联线粒体内膜呼吸链的氧化磷酸化，抑制ATP的产生进而干扰线粒体的能量平衡。筛选酿酒酵母突变体库的结果显示，Str2、Elp4、Mtg2 和Mtp1可能与氟啶胺毒理作用相关。在酿酒酵母中，Str2编码的γ-胱硫醚合成酶直接参与胱硫醚的合成；Elp4编码的组氨酸乙酰转移酶复合体亚基具有tRNA转运活性并能够调控菌体对ATP的利用；Mtg2编码的线粒体内膜GTPase水解酶，能够调控菌体中核糖体的组装、核酸转录和能量平衡；Mtp1编码线粒体内膜转运蛋白具有跨膜转运活性，能够转运多种生化物质。所以，在灰葡萄孢鉴定出BcStr2、BcElp4、BcMtg2 和BcMtp1。研究这些基因在调控灰葡萄孢对氟啶胺药敏性中的贡献，并分析其生物学功能。结果表明，灰霉病菌对氟啶胺抗药性风险对低到中等，其药敏性涉及多个基因，即非单个主效基因控制的，可以利用其进行灰霉病菌对其他抗药性的治理。发表标注论文6篇，其中5篇SCI论文；获得江苏省科技进步二等奖1项。研究技术运用于田间抗药性治理，取得了良好的效果。