柑橘绿霉病菌转录因子PdMbp1调控DMI抗性的机制研究

Green mold is the major cause of citrus postharvest decay. Currently, chemical control, based on the fungicide treatment of sterol demethylation inhibitor (DMI), is still the main method to control the citrus postharvest disease. However, as the extensive and continuous use of these fungicides, the fungicide-resistance problem in the pathogen Penicillium digitatum is growing. Previous studies demonstrated that the insertion of a transposable element PdMLE1 within the promoter region of the target gene PdCYP51B is one of the major reasons that cause DMI fungicide resistance in P. digitatum. However, how this element is recognized and regulated remains unknown. Recently, we screened out one transcription factor that can bind to the PdMLE1 element, designated PdMbp1. In this project, we plan to a) figure out the biological roles of PdMbp1 in DMI resistance by constructing gene deletion and complementary mutants in DMI-resistant strains of P. digitatum, b) analyze the regulatory effect of PdMbp1 on its target genes in response to DMI stress using ChIP-seq and RNA-seq, c) and utilize affinity capture assay to identify the interacting proteins that also take part in the regulation of DMI resistance. This project aims to reveal the regulatory mechanism of DMI resistance mediated by PdMbp1. The results would not only deepen the understanding of the regulatory mechanism of fungicide resistance resulting from the insertion mutation of transposable elements but also provide a scientific basis for improving the chemical control strategies and developing novel fungicides.

绿霉病是柑橘采后腐烂的主要诱因，以甾醇脱甲基抑制剂类(DMI)杀菌剂处理为主的化学防治是目前主要的防治措施。但随着杀菌剂的连续广泛使用，抗药性问题与日俱增。药靶基因PdCYP51B启动子区一个转座元件PdMLE1的插入是导致柑橘绿霉病菌对DMI产生抗药性的主要原因之一，但该元件是如何被识别调控的尚不清楚。我们前期在柑橘绿霉病菌中筛选到一个与PdMLE1结合的转录因子PdMbp1。本项目拟在DMI抗性菌株中构建PdMbp1基因的缺失和回补突变体，通过表型和表达分析明确其在DMI抗性中的作用；利用ChIP-seq和RNA-seq技术解析其在DMI胁迫条件下对靶标基因的调控作用；并通过免疫亲和捕获筛选互作蛋白，明确共同参与调控的基因，最终阐明由PdMbp1介导的DMI抗性调控机制。研究结果将深入揭示由转座元件插入引起的杀菌剂抗性调控机制，为制定新的化学防治策略和开发新型杀菌剂提供科学依据。

由指状青霉(Penicillium digitatum)引起的柑橘绿霉病是造成柑橘采后腐烂的主要原因。甾醇脱甲基抑制剂类(sterol demethylation inhibitors, DMIs)杀菌剂是目前防治柑橘绿霉病的有效药剂。前期研究表明，PdCYP51B基因启动子区199bp转座元件PdMLE1的插入是导致柑橘绿霉病菌对DMIs杀菌剂产生抗性的主要原因之一。本研究针对PdMLE1的结合蛋白Pdmbp1，通过生物信息学分析、基因敲除、凝胶迁移、Chip-seq和RNA-Seq等技术和方法得到了以下结果：1）获得了菌株PdW03染色体级别的完整基因组，为项目后期的数据比对和分析提供了高质量的数据库；2）明确了Pdmbp1基因的生物学功能以及在DMI抗性中的作用；3）获得了Pdmbp1可能结合的顺式作用元件；4）分析了Pdmbp1基因在抑霉唑药剂处理条件下对基因表达的影响；5）明确了Pdmbp1下游基因Pdy1h15在DMI抗性中的作用。本项目的研究结果为研究柑橘绿霉病菌对DMI杀菌剂的抗性调控机制提供了理论参考。