高渗介导的AoHog1调控赭曲霉毒素A合成的分子机制

Ochratoxin A (OTA) is a strongly toxic and carcinogenic secondary metabolites which is produced by Aspergillus ochraceus naturally occurring in various food products, which is a serious threat to food safety and human health. Previous studies have shown that the osmotic pressure would affect the growth and OTA biosynthesis in Aspergillus ochraceus, and the elements AoHog1 and AoMsn2 involved in the hyperosmotic regulation pathway are existed in Aspergillus ochraceus genome. Thereinto, the transcriptional binding sites are existed between AoMsn2 and OTA biosynthetic regulatory gene AoOTA1, AoOTA2. So we propose a hypothesis “OTA biosynthesis is regulated by AoHog1 under high osmotic environment”. In order to deduce the mechanism of OTA synthesis regulated by AoHog1, the relationship between the OTA biosynthesis and AoHog1 phosphorylation is elucidated under different kind of high osmotic environment in this project, by detecting the osmotic pressure sensitivity, OTA content and phosphorylation. Yeast two-hybrid system will be used to study the interaction between AoMsn2 and AoOTA1, AoOTA2. The function of AoHog1 downstream transcription factor AoMsn2 involved in regulating OTA synthesis is further studied by constructing gene-knockout mutant ΔAoMsn2 and binding sites-knockout mutantΔAoOTA1、ΔAoOTA. The study will reveal the molecular mechanism of OTA synthesis regulated by AoHog1 under hyperosmotic stress, which will provide clues to OTA prevention and control during food processing and storage.

赭曲霉毒素A(OTA)主要由赭曲霉产生，具有强毒性、致癌性，在食品中污染广泛，严重威胁食品安全和人类健康。前期研究发现渗透压影响赭曲霉生长和OTA产生，基因组分析测序赭曲霉中存在高渗应激元件AoHog1及下游元件AoMsn2，其中AoMsn2与OTA合成调控基因AoOTA1、AoOTA2存在转录结合位点。据此，提出了“高渗介导的AoHog1调控OTA合成”的假说。本项目通过检测渗透压敏感性、OTA含量及磷酸化等，明确不同高渗环境下AoHog1磷酸化与OTA合成的关系；通过酵母双杂交等明确AoMsn2与AoOTA1、AoOTA2的蛋白相互作用；进一步构建ΔAoMsn2突变株、ΔAoOTA1、ΔAoOTA2结合位点缺失突变株，揭示AoMsn2调控OTA合成的功能特性。研究将阐明高渗介导的AoHog1调控OTA合成的分子机制，为食品加工储藏过程中OTA的防控提供线索。

赭曲霉毒素A(OTA)是由曲霉属和青霉属在谷物、水果、咖啡、干腌肉等食品中产生的具有强毒性、致癌性的次级代谢产物，严重威胁食品安全和人类健康。高渗透性甘油促分裂原活化蛋白激酶(HOG-MAPK)途径是调节高渗应激的主要机制，参与调控真菌毒素的合成。本项目确证赭曲霉中存在一个赭曲霉毒素的合成基因簇，包含4个合成基因（OtaA-D）和1个调控基因（OtaR1）。序列同源比对分析，赭曲霉存在HOG-MAPK途径中元件AoHog1、AoSho1、AoMsn2。本项目通过构建⊿AoHog1基因敲除突变株，发现AoHog1基因参与调控赭曲霉菌的生长、产毒与致病。进一步研究AoHog1对OTA合成基因的调控作用，发现AoHog1正向调控OTA合成基因。高渗透压下AoHog1磷酸化，NaCl处理在低浓度（20 g/L）时磷酸化水平高于高浓度（70 g/L），与OTA合成量变化趋势一致。通过比较蛋白质组学研究在不同渗透压环境下赭曲霉蛋白质表达情况，共鉴定蛋白2646个，其中有功能描述的蛋白1919个，未鉴定功能的蛋白727个。差异倍数大于1.5、FDR<0.05的差异表达蛋白126个，主要参与碳水化合物代谢、胁迫应激、氧化应激等生物进程。渗透压调控基因Aohog1，Aosho1和全局调控转录因子AoleaA, AoVeA, AoVelB在添加70 g/L NaCl的培养基中均下调表达。上述研究将揭示AoHog1调控OTA合成的分子机制，为食品加工储藏过程中OTA的防控提供线索，为保证食品安全奠定理论基础。