基于T-DNA插入突变体库及生命组学的镰刀菌多重耐药性研究

Fusarium is a large genus of fungus widely distributed in soil and in association with plants. They can cause superficial and systemic infections of human. As a new, global epidemic fungal infection, the incidence of fusaridiosis increased over these years, and the mortality rate was extremely high, which had seriously threatened the health and life of human. They are also important plant pathogens caused significant economic impacts. At present, Fusarium has a significant multidrug resistance to almost all clinical antifungal drugs, making treatment extremely difficult. And its resistance to agricultural fungicide is also serious. In this study, T-DNA insertion mutant library will be established by reverse genetics technology. Drug susceptibility changed mutants will be selected to find out the unknown genes, known genes with new function, or non-coding sequences associated with drug resistance. And the function of related genes will be verified and annotated. Based on omics technologies, the regulatory pathways related to drug resistance will be identified, which will help us to discover the critical genes regulated different drug targets, analyze the phylogenesis of these genes, understand the relationship between intrinsic and acquired resistance, and determine potential new targets for antifungal drugs and blockers. The findings may also provide a valuable reference for the study of drug resistance in other fungi.

镰刀菌属广泛分布于土壤和植物中。它们可引起人类浅部和深部感染，作为新现、全球流行的真菌感染，发病率逐年增高，且死亡率极高，已严重地威胁人类的健康和生命。它们亦为重要的植物病原菌，可造成巨大的经济损失。目前，镰刀菌对几乎所有的临床抗真菌药物呈现显著的多重耐药性，使得抗真菌治疗变得异常艰难。且其对农用杀真菌剂的耐药状况亦日趋严重。本项目拟以茄病镰刀菌和尖孢镰刀菌的多重耐药株为研究对象，利用反向遗传学技术建立T-DNA插入突变体库，筛选药物敏感性异常突变体，寻找与耐药相关的未知基因、已知基因新功能或非编码序列，验证并注释其功能。结合生命组学数据，解析耐药相关基因的作用方式及调控网络，寻找对不同药物靶点起整体调控作用的关键基因；分析其系统进化关系；探讨固有耐药与获得性耐药的关系；寻找新的、潜在的抗真菌药物及阻断剂作用靶点。本研究亦可为其他真菌的耐药相关研究提供非常有价值的参考资料。

镰刀菌是一类广泛分布于土壤和植物中的丝状真菌。该菌可引起人类浅部和深部感染，作为新现、全球流行的真菌感染，其发病率逐年增高，且病死率极高，已严重地威胁人类的健康和生命。它们亦为重要的植物病原菌，可造成农业上巨大的经济损失。目前，镰刀菌对几乎所有的临床抗真菌药物呈现显著的多重耐药性，使得抗真菌治疗变得异常艰难。且其对农用杀真菌剂的耐药状况亦日趋严重。镰刀菌耐药性产生是一个复杂的、多基因互相作用的过程，目前仍有许多未解之谜。本项目以多重耐药尖孢镰刀菌和茄病镰刀菌为研究对象，利用优化的根癌农杆菌介导的遗传转化技术建立了T-DNA插入突变体库，筛选获得了一些药物敏感性改变的突变体，以寻找与耐药相关的未知基因、已知基因新功能或非编码序列。利用TAIL-PCR技术结合比较基因组学、表观遗传学等技术，定位T-DNA插入位点，明确打断基因，获得耐药相关基因信息，并对其功能进行了验证和注释。研究表明细胞色素还原酶、转录因子以及一些假定蛋白与镰刀菌的耐药性产生有关。其中假定的锌指蛋白可通过调节糖酵解途径，影响菌株的生长发育及耐药性；NADPH依赖的细胞色素P450还原酶CPR1通过调节麦角甾醇合成，影响下游cyp51基因表达和麦角甾醇含量，导致菌株对唑类药物耐药；推测的锌簇转录因子可能通过调节麦角甾醇合成，影响菌株耐药性。这些发现目前尚未见报道。研究结果提示相关基因有望成为潜在的抗真菌治疗靶标，对于揭示镰刀菌耐药机制、提高临床疗效、防控耐药性发生具有重要意义。