尖孢镰刀菌四个同源CYP51基因的生物学功能研究与利用

The demethylation inhibitors (DMIs) target 14α-demethylase (encoded by CYP51 gene) in the ergosterol biosynthesis pathway. Most fungi contain only single CYP51 gene, however, our preliminary study showed for the first time that Fusarium oxysporum genome contains four paralogous CYP51. All four CYP51 genes are transcribed and upregulate in the response to triadimefon treatment. FoCYP51A/B/C deletion mutants showed significantly difference in sensitivity to variety DMI fungicides. FoCYP51A gene deletion mutant, but not FoCYP51B and C mutants, exhibited noticeably increased sensitivity to DMI fungicides including triadimenfon, tebuconazole, propiconazole, epoxiconazole and prochloraz. However, FoCYP51B mutant showed increased sensitivity to fluconazole and diniconazole. Based on these preliminary results, this project will further explore the biological function of these four paralogous CYP51 genes in F. oxysporum. Expected results from this project will be significant for management of F. oxysporum with DMI fungicides, and providing scientific basis for novel drug development against Fusarium wilt.

14α-脱甲基酶（CYP51）是脱甲基抑制剂（DMI类杀菌剂）的靶标，多数真菌仅有一个CYP51基因，课题组前期研究发现，尖孢镰刀菌有4个同源且正常转录表达的CYP51，其表达量响应DMI类药剂三唑酮的诱导显著上升，这是目前在真菌中发现的数量最多的CYP51同源基因。靶向敲除实验表明FoCYP51A、B、C缺失对多种DMI类药剂的敏感性变化存在显著差异。FoCYP51A缺失突变体对三唑酮、戊唑醇等变得更加敏感，而FoCYP51B和C缺失突变体对这些药剂的敏感性水平与野生菌株相当；然而FoCYP51B缺失突变体对氟康唑和烯唑醇的敏感性增加。在此基础上，本项目将进一步阐明尖孢镰刀菌4个同源CYP51基因的生物学功能及其与DMI类杀菌剂的敏感性关系；分析现有DMI类药剂联用对枯萎病的防治效果。本项目预期研究成果将为DMI类杀菌剂的合理使用、新型杀菌剂的研制和枯萎病综合防控策略的制定提供理论依据。

14α-脱甲基酶（CYP51）是脱甲基抑制剂（DMI类杀菌剂）的靶标，多数真菌仅有一个CYP51基因，本项目研究发现，尖孢镰刀菌有4个同源且正常转录表达的CYP51基因（以下称为FoCYP51A, -B, -C 和 -D），这4个同源基因功能具有保守性，转化到酿酒酵母ERG11缺失突变体后可部分恢复14α-脱甲基酶的功能。这是目前在真菌中发现的数量最多的CYP51同源基因。靶向敲除实验获得了这4个基因的单、双基因缺失突变体（FoCYP51A,-B, -C, -D, -AC, -AD, -BC, -BD, -CD），FoCYP51AB缺失突变体未获得。结果表明尖孢镰刀菌FoCYP51B和C参与调控菌丝色素的合成，其中FoCYP51B还调控菌丝的生长。FoCYP51A缺失突变体（包括单基因缺失突变体ΔFoCyp51A和双基因缺失突变体 ΔFoCyp51AC和ΔFoCyp51AD）对三唑酮、戊唑醇、氟康唑和咪酰胺变得更加敏感，而FoCYP51B缺失突变体（包括单基因缺失突变体ΔFoCyp51B和双基因缺失突变体 ΔFoCyp51BC和ΔFoCyp51BD）对以上4种药剂中的2种药剂的敏感性增加。FoCYP51C和D的单、双基因缺失突变体对以上所有DMI类药剂均无明显敏感性变化。将不同的DMI类杀菌剂进行复配对尖孢镰刀菌生长的抑制作用增强。尖孢镰刀菌FoCYP51A、B、C、D与致病力调控不相关。本项目明确了尖孢镰刀菌4个同源CYP51基因与菌株的生长、色素合成等生物学功能相关，并且阐明了DMI类杀菌剂在尖孢镰刀菌中的作用靶标，其中FoCYP51A、B调控不同种类的DMI类杀菌剂的敏感性变化，将不同种类的DMI类药剂联用可提高对尖孢镰刀菌的生长抑制。本项目的研究成果将为DMI类杀菌剂的合理使用、新型杀菌剂的研制和尖孢镰刀菌引起的枯萎病综合防控策略的制定提供理论依据。