柑橘绿霉病菌对DMI杀菌剂抗性的调控机制研究

Green mold, caused by Penicillium digitatum, is the most important diseases of postharvest citrus. The control effect of green mold using DMI fungicides is seriously compromised due to the emergence of DMI-resistant strains. In previous investigation we found that the resistance of P. digitatum to DMi fungicides was associated with the overexpression of DMI target genes CYP51A and CYP51B, which was caused by the duplication of a 126 bp transcriptional enhancer or the insertion of a powerful promoter PdMLE1 in the promoter regions upstream the genes. However, the mechanisms that drive the target gene overexpression are largely unknown. In this project, we propose to analyze the differential expression profiles of DMI-sensitive P. digitatum under the DMI-treatment and non DMI-treatment, thus to understand the genes and metabolic pathways that response to DMI stress. After the identification of cis-element that responses to the inducing of DMI in promoter regions of CYP51A and CYP51B, as well as in the promoter PdMLE1, we will used these identified elements to isolate the trans elements (transcriptional factors) that intact with the cis-elements by using the method of yeast one-hybrid technology. Thereafter, we will construct the disruption and complement mutants of the obtained transcriptional factors, and characterize their biological functions, and the association of these transcriptional factors with the DMI-sensitivity of P. digitatum. The downstream genes as well as their DNA binding sites of the transcriptional factors will be identified by comparing the expression profiles of disrupted mutants of transcriptional factors explored to DMI and not at genome wide scale using RNA-seq as well as ChIP-seq. Based on these experiments, the regulation mechanisms of P. digititum to resist DMI fungicides will hopefully be clarified. The results we expected will not only deepen our insight into the fungicide resistance, but also shine the future for new disease control strategy development in the more cost-effective way.

绿霉病是柑橘贮运期最主要病害，DMI药剂防治效果常因病菌的抗药性而深受影响。前期研究表明,DMI药物靶基因CYP51A和B启动子区转录增强子拷贝数增加和强启动子PdMLE1插入均可引起CYP51A和B基因过量表达和病菌对DMI药物的抗性，但其调控机理尚不明确。本研究首先通过分析敏感菌株DMI药剂诱导表达谱，明确病菌响应DMI胁迫的基因和代谢途径;同时在确定敏感菌株CYP51A和B基因启动子区响应DMI诱导作用的顺式元件、抗性菌株PdMLE 1启动子核心序列基础上，应用酵母单杂交等技术分离鉴定与之互作的转录因子；然后构建基因缺失和互补突变体，比较表型，明确转录因子的功能及其与DMI药物抗性的关系；最后通过转录因子缺失突变体响应DMI诱导表达谱和ChIP-seq分析，明确转录因子调控病菌对DMI药物抗性机制。研究结果不仅将深化对病菌抗药性机理的认识，还为防治柑橘绿霉病的新型剂研发提供理论依据。

柑橘绿霉病菌 (Penicillium digitatum) 引起的柑橘绿霉病是柑橘采后腐烂的主要原因之一。目前对柑橘绿霉病的防治主要采取采后杀菌剂处理的措施，使用最多的是甾醇脱甲基酶抑制剂类 (DMI) 杀菌剂。本实验室前期工作表明，柑橘绿霉病菌DMI抗性的产生主要是由药剂靶标基因PdCYP51A或PdCYP51B启动子区的插入突变导致基因过表达引起的。甾醇调控元件结合蛋白(SREBPs)是真核生物中甾醇合成途径的主要调控因子，在子囊菌中SREBP蛋白前体利用Dsc E3连接酶复合体进行剪切激活。本文通过基因敲除、生物学表型分析、表达谱测序分析、凝胶迁移、酵母双杂交等技术手段，获得了以下三方面的结果：1）明确了两个SREBP转录因子基因PdsrbA和PdsrbB都是麦角甾醇合成途径及DMI抗性的主要调控因子，共同调控该途径中大部分的基因，但ERG2只受PdsrbA调控，而PdCYP51B只受PdsrbB调控；2）柑橘绿霉病菌Dsc E3连接酶复合体与PdSrbA蛋白的剪切激活相关，且PddscB为复合体核心亚基；3）利用酵母单杂交技术，从柑橘绿霉病菌cDNA文库中筛选到了一个与CYP51s启动子区域的插入序列结合的转录因子，确定了该转录因子DMI抗性调控中的作用。本文的研究结果为解释柑橘绿霉病菌DMI杀菌剂的抗性调控机制提供了依据，也为新型杀菌剂靶标的筛选提供了理论基础。