稻曲病菌T-DNA插入突变体B857对丙环唑产生抗性的分子机制分析

Propiconazole is a kind of triazole fungicide to control rice false smut, caused by Ustilaginoidea virens. While the resistance molecular mechanism of U. virens to this kind of fungicides is not clear at present. The mutant B857, which showed high resistance to propiconazole was screened from a T-DNA insertional mutant library of U. virens. And a single copy of T-DNA in B857 was confirmed, as well as multidrug-resistance in the preliminary study. Based on this, we intend to clone T-DNA insertional mutated gene of B857. The knockout mutants and the complementary mutants will be obtained by agrobacterium tumefaciens mediated transformation (ATMT) based gene deletion and complementation methods. Sensitivity to propiconazole and other fungicides with different modes of action will be detected. Relative expression values of genes that may contribute to fungicide resistance in the mutants and the wild strain will be tested by Real Time quantitive PCR (RT-qPCR) for validating the function of the mutated gene in fungicide resistance. Moreover, regulatory pathway of the mutated gene on resistance will be explored by transcriptional expression profile comparison between the knockout mutants and the wild strain using transcriptome sequencing. The expected results will clarify resistance molecular mechanism of U. virens to propiconazole. At the meantime, it is useful in providing theoretical basis for resistance warning of U. virens to triazole fungicides and development of new fungicides.

丙环唑是防治稻曲病的一种三唑类杀菌剂，目前稻曲病菌对该类杀菌剂的抗性分子机制尚不明确。申请人前期筛选获得一株对丙环唑高抗的T-DNA单拷贝插入突变体B857，初步分析其具有多药抗药性。本项目拟在此基础上，克隆突变基因，通过基因敲除及回复突变技术获得突变体，检测突变体对丙环唑和其他不同作用机制杀菌剂的敏感性变化，利用RT-qPCR方法检测突变体与野生型菌株中抗药性相关基因的表达水平，鉴定突变基因在抗药性产生中的功能；借助转录组测序，比较野生型菌株和敲除突变体在有药及无药条件下的转录表达谱，探索突变基因对抗药性产生的调控路径，解析稻曲病菌对丙环唑产生抗性的分子机制，为稻曲病菌对三唑类杀菌剂的抗药性预警及新型杀菌剂的研发提供理论依据。

稻曲病的发生严重影响稻米品质、威胁粮食安全，病害由绿核菌属Ustilaginoidea virens侵染引起，丙环唑是生产上防治稻曲病的主打药剂。本项目建立了稻曲病菌对丙环唑和氟环唑的敏感基线，其中，稻曲病菌对丙环唑的EC50值为0.0022-0.1069 µg/mL，平均EC50为0.0485±0.0183 µg/mL；对氟环唑的EC50值为0.0076-0.0458 µg/mL，平均EC50为0.0208±0.0082 µg/mL。首次筛选到田间抗丙环唑的稻曲病菌菌株，测定了抗性菌株和敏感菌株的温度敏感性、产孢量、细胞壁完整性、致病性等生物学指标，结果表明，抗性菌株88适合度较敏感菌株高；田间接种该抗性菌株，防效均下降；交互抗药性研究结果表明，田间抗丙环唑的稻曲病菌菌株对其它DMI药剂具有正交互抗药性。初步进行了抗药性分子机制的研究，分别扩增了抗性菌株和敏感菌株的cyp51基因及上游1000bp的序列，比对分析发现，抗性菌株88和82的cyp51基因中并未发现与抗性相关的突变位点，而抗性菌株88在cyp51基因的上游154碱基处多出两个碱基CC，推测其可能与抗药性相关；荧光定量PCR研究结果表明田间抗丙环唑的稻曲病菌菌株产生抗性与cyp51基因的诱导表达量相关，经过丙环唑处理后，敏感菌株的甾醇生物合成被抑制，而抗性菌株的甾醇生物合成显著升高。本项目初步探明了田间稻曲病菌抗性菌株对丙环唑的抗性分子机制，研究结果表明稻曲病菌对丙环唑的抗性风险较高，在田间应合理轮换和交替使用不同作用机制的杀菌剂，以避免和延缓稻曲病菌抗药性的发生和发展。本项目为深入研究稻曲病菌对三唑类杀菌剂的抗性机制提供了方法支持和相关数据，同时为田间稻曲病的抗药性治理提供理论依据。