钙离子调控樟芝深层发酵无性产孢及其分子机制

摘要

Taiwanofungus camphoratus is a valuable and rare medicinal mushroom with various bioactivities,such as liver protection and anti-cancer.T.camphoratus can produce many arthroconidia at the end of submerged fermentation,but molecular mechanism underlying this submerged conidiation remains unknown.In this study,we found that Ca2+ concentration in culture medium significantly affected the arthroconidium production of T.camphoratus.Then,we identified two proteins (CaM and HSP90) involved in Ca2+/calmodulin signaling pathway and one protein (AbaA) involved in FluG-mediated conidiation pathway by two-dimensional etectrophoresis analyses.Furthermore,we proposed a Ca2+/calmodulin-and FluG-mediated signaling pathway by bioinformatics analysis.By real-time quantitative PCR analyses of 23 key genes in the Ca2+/calmodulin-and FluG-mediated conidiation pathway,we found that expression levels of 7 genes (crz1,hsp90,flbB,brlA,abaA,wetA andfadA) showed significant responses to Ca2+ concentration in fermentation medium.Our research is beneficial for elucidating the underlying mechanism of submerged fermentation conidiation for T.camphoratus.%樟芝是原产于台湾的珍稀药用真菌,具有保肝、抗癌等活性.樟芝在深层发酵过程中能够产生大量无性孢子,但是目前对樟芝无性产孢的影响因素及其分子机制尚不明确.本研究报道了樟芝发酵培养基中Ca2+浓度能够有效调控樟芝无性产孢的现象;采用双向电泳(2-DE)技术,对不添加Ca2+培养(对照)的菌丝体、添加1 mmol/L Ca2+培养(促进产孢)的菌丝体及添加200 mmol/L Ca2+培养(抑制产孢)的菌丝体进行差异蛋白质组学分析,鉴定了参与Ca2+/钙调素信号通路的CaM蛋白和HSP90蛋白,以及参与FluG调控产孢信号通路的AbaA蛋白;进一步通过生物信息学分析,预测了Ca2+/钙调素和FluG介导的樟芝无性产孢信号通路模型图;采用实时定量PCR (RT-qPCR)技术,对该通路上23个功能基因的转录水平进行了分析,发现了受Ca2+调控最为灵敏的7个产孢相关功能基因:crzl、hsp90、flbB、brlA、abaA、wetA及fadA.本研究结果为解析樟芝无性产孢机制提供了实验依据.