粗糙脉孢菌erg11特异性响应唑类药物的调控机制研究

麦角甾醇为真菌细胞膜的必需组分，当前的抗真菌药物以其合成抑制剂为主。为维持麦角甾醇含量的稳定，参与其合成的关键基因（如erg11），在唑类等药物胁迫下，会增强表达以缓解药物对甾醇合成的抑制，这是真菌耐受唑类药物的生理基础。目前已知的麦角甾醇合成调控以终产物麦角甾醇的反馈调控为主。而我们前期发现erg11等特异性响应唑类药物，该响应和积累特定甾醇中间代谢物直接相关，暗示麦角甾醇的合成存在精细调控机制以使真菌更高效地应对甾醇合成过程所受的干扰。本项目将以粗糙脉孢菌erg11的启动子关键调控区域为靶，通过DNA亲和层析、蛋白质谱、突变体分析和ChIP-PCR，鉴定参与上述调控的关键转录因子；通过RNA-seq、ChIP-seq等构建转录因子的调控网络；通过蛋白磷酸化、剪切和定位分析，明确转录因子对唑类药物的特异性响应。研究结果将加深我们对麦角甾醇合成调控的理解，并为病原真菌防控提供更充实的参考。

Ergosterol is the major constituent of fungal cell membrane and is critical for many cellular activities. The majority of currently used antifungals are ergosterol biosynthesis inhibitors. Fungi could maintain the content stability of ergosterol in membrane. Under the stresses of ergosterol biosynthesis inhibitors like azoles, fungi could upregulate the expression of key genes in the biosynthesis of ergosterol, such as erg11, to alleviate the inhibition. This response is the physiological basis of azole resistance in fungi. It has been highly accepted that fungal ergosterol biosynthesis is transcriptionally controlled by a feedback regulation of ergosterol, the end product of ergosterol biosynthesis. However, based on our preliminary results, the transcript levels of erg11 response to azole stress specifically, and this is mainly due to the accumulation of sterol intermediates. This may imply the existence of more elaborate mechanism for the regulation of ergosterol biosynthesis, thus making fungi more efficient in dealing with perturbations in ergosterol biosynthesis. Thus, in this proposal, by using the key regulatory region of erg11 promoter as a bait, we will identify the key transcription factor(s) directly regulating expression of erg11 through DNA affinity chromatography, protein identification via mass spectrometry and phenotypic analysis of deletion mutants. And the specific response of the identified transcription factors to azoles will be verified by western blotting and subcellular location analysis. We will also investigate the regulatory mechanism of the identified transcription factor through RNA-seq and ChIP-seq. Collectively we will give a glance on the elaborate regulatory pathway of ergosterol biosynthesis governed by sterol intermediates, deepen our understanding on the regulation of ergosterol biosynthesis and provide more comprehensive reference on the control of infections caused by fungal pathogens.