转录因子UstR调控黄曲霉毒素的合成及其生物学功能研究

Study on transcription factor UstR regulating aflatoxin biosynthesis and its biological function

中文关键词:  黄曲霉  稻曲菌素B调控蛋白R  转录因子  黄曲霉毒素  代谢产物

英文关键词:Aspergillus flavus  ustiloxin B biosynthesis protein R  transcription factor  aflatoxin  metabolites

中文摘要:

      目的 明确黄曲霉中Zn(II)2Cys6转录因子稻曲菌素B调控蛋白R (ustiloxin B biosynthesis protein R, UstR)功能, 解析UstR对黄曲霉生长、次级代谢和黄曲霉毒素(aflatoxin, AF)合成的调控。方法 本研究利用聚乙二醇介导的同源重组构建ustR基因缺失突变体; 比较敲除突变体和野生型菌株在黄曲霉生长发育、胁迫响应、侵染能力、AF合成及次级代谢产物合成等方面的差异, 并通过酶联免疫吸附测定(enzyme-linked immunosorbent assay, ELISA)试剂盒测定AF合成底物变化, 利用实时聚合酶链式反应(real-time polymerase chain reaction, RT-qPCR)验证AF合成相关基因的转录水平改变, 明确转录因子UstR对AF合成的调控作用。结果 基因敲除突变体的分生孢子形成受抑制, 孢子产量降低, 但菌核形成能力显著提高; ustR缺失不影响黄曲霉细胞壁完整性、胁迫响应能力以及侵染能力; UstR能够影响黄曲霉多种次级代谢产物产量, 其中UstR通过正调控AF合成基因簇表达影响AF生物合成。结论 UstR是黄曲霉重要的Zn(II)2Cys6转录因子, 调控黄曲霉生长发育及AF产生, 研究进一步完善了AF合成调控网络, 为AF污染防控提供了新的防控潜在靶点。

英文摘要:

      Objective To clarify the function of Zn(II)2Cys6 transcription factor ustiloxin B biosynthesis protein R (UstR) in Aspergillus flavus, and analyze the function of UstR on the growth, secondary metabolism and aflatoxin (AF) synthesis. Methods In this study, ustR gene knockout mutant was constructed by polyethylene glycol-mediated homologous recombination; the growth and development, stress response, infection ability, and AF biosynthesis of mutant and wild type strain were compared, the changes of AF biosynthesis substrates were determined by enzyme-linked immunosorbent assay (ELISA) kit, and transcription changes of AF biosynthesis-related genes were verified by real-time polymerase chain reaction (RT-qPCR) to clarify the regulatory of UstR on AF synthesis. Results The conidia formation of ustR knockout mutant was inhibited and conidia yield was reduced, but the sclerotia formation ability was significantly improved; the deletion of ustR did not affect the cell wall integrity, stress response ability and infection ability of mutant. UstR could affect the production of multiple secondary metabolites in A. flavus, and it affected AF biosynthesis by positively regulating expression of AF synthesis gene cluster. Conclusion UstR is an important Zn(II)2Cys6 transcription factor which regulated the growth, development and AF biosynthesis in A. flavus. This study further complement the AF biosynthesis regulatory network. UstR may be a new potential target for prevention and control of AF pollution.