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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Ca~(2+)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Ca~(2+)培养(对照)的菌丝体、添加1 mmol/L Ca~(2+)培养(促进产孢)的菌丝体及添加200 mmol/L Ca~(2+)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Ca~(2+)/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Ca~(2+)/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Ca~(2+)调控最为灵敏的7个产孢相关功能基因:crz1、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。
Antrodia camphorata is a rare medicinal fungus native to Taiwan, with liver and anti-cancer activity. Antrodia camphorata can produce a large amount of asexual spores in deep fermentation, but the influencing factors and molecular mechanisms of asexual spores of Antrodia camphorata are not clear at present. This study reported that the Ca 2+ concentration in the fermentation medium of Antrodia camphorata could effectively control the sporulation of A. camphorata. The two-dimensional electrophoresis (2-DE) ) Mycelia were cultured in the presence of 1 mmol / L Ca2 + (to promote sporulation) mycelium and 200 mmol / L Ca2 + culture (to inhibit sporulation) mycelium Differential proteomics analysis identified CaM and HSP90 proteins involved in the Ca2 + / calmodulin signaling pathway as well as AbaA proteins involved in the sporulation signal transduction by FluG regulation. Further bioinformatics analysis predicted Ca ~ (2 +) / calmodulin and FluG-mediated model of asexual sporulation of Antrodia camphorata. The transcription level of 23 functional genes in this pathway was analyzed by real-time quantitative PCR (RT-qPCR) Seven sporulation-related functional genes most sensitive to Ca 2+ were found: crz1, hsp90, flbB, brlA, abaA, wetA and fadA. The results of this study provide an experimental basis for the analysis of asexual sporulation mechanism of Antrodia camphorata.