樟芝深层发酵过程中节孢子产生及萌发的分子生物学机制

Antrodia camphorata, a medicinal mushroom that distributed only in Taiwan, has received tremendous attention from the public due to its specific biological feature and potent bioactivity. The fermentation of Antrodia camphorata is widely recognized as a complex process with numerous problems, such as long culture time and difficulties in controling the inoculum size, mycelium morphology and metabolite biosynthesis. In our previous study, asexual sporulation of Antrodia camphorata were observed, and a large number of arthroconidia produced at the end of submerged culture. With the produced arthroconidia as the inoculum, fermetation of Antrodia camphorata were repeated. It is considered that arthroconidia were beneficial to the easy control of inoculum size, growth and fermentation course of Antrodia camphorata. However, information about the molecular mechanism of arthroconidia germination and sporulation is scarce. In this study, proteomic analysis is performed to study differentially expressed proteins during the arthroconidia germination and sporulation. Meanwhile, transcriptomic analyses of differentially expressed genes during arthroconidia germination and sporulation are performed. And express levels of key functional genes during the germination and sporulation are analyzed. Furthermore, effects of nutritional and environmental factors on express levels of the key genes are extensively studied. This study will elucidate the molecular mechanisms of germination and sporulation of Antrodia camphorata arthroconidia, and the results will be useful to the further study on physiology and metabolism of Antrodia camphorata. Also, this study will be of importance to the development of novel fermentation method to solve technical problem that existed in the submerged culture of Antrodia camphorata.

樟芝由于其特殊微生物学特性和良好药理活性已成为目前大型药食用真菌研究领域的热点之一。前期工作中发现樟芝深层液态发酵后期大量形成无性节孢子，并建立了基于节孢子接种的樟芝快速循环发酵工艺，有效解决了深层液态发酵樟芝周期长等瓶颈问题，但还缺乏对节孢子发生过程及机制的研究。本项目拟应用蛋白质组学技术分析对樟芝节孢子产孢和萌发过程中差异性表达的蛋白，继而采用转录组学技术分析该过程中差异性表达的基因及其关键调控基因的表达水平差异，进一步解析与樟芝节孢子发生过程相关的分子机制并探讨其对环境和营养条件的应答机制。本项目实施可对樟芝发酵过程中无性繁殖途径进行较为系统的研究，是樟芝生理与代谢研究的重要补充，同时为孢子产生和萌发过程可控的高生产强度樟芝快速循环发酵工艺提供坚实的理论基础。

樟芝由于其特殊微生物学特性和良好药理活性已成为目前大型药食用真菌研究领域的热点之一。前期工作中首次发现樟芝深层液态发酵后期大量形成无性节孢子，但还缺乏对节孢子发生过程及机制的研究。本项目建立了基于节孢子接种的樟芝快速循环发酵工艺，与传统批次发酵相比，将8个批次的总生产时间由80 d缩短到58 d，将平均生产周期由10 d缩短到7 d。同时，胞内多糖批次生产力提高了40.3%，总三萜批次生产力提高了43.2%，Atrodin A批次生产力提高了42.5%，Atrodin B批次生产力提高了58.6%。有效解决了深层液态发酵樟芝周期长等瓶颈问题；联合应用iTRAQ、二维电泳-质谱鉴定等蛋白质组学技术以及RNA-seq等转录组学技术，对樟芝无性孢子产生和萌发过程的蛋白质组和转录组进行了分析，鉴定了樟芝节孢子产孢和萌发过程中差异性表达的蛋白和基因，预测了樟芝节孢子产生和萌发过程的信号通路模型图，解析了与樟芝节孢子发生过程相关的分子机制；进一步考察了不同寄主（牛樟树）生长因子、环境和营养条件对樟芝无性孢子产生和萌发过程的影响，探讨了樟芝无性产孢和萌发过程对上述生长因子、环境和营养条件的分子应答机制。本项目实施可对樟芝发酵过程中无性繁殖途径进行较为系统的研究，是樟芝生理与代谢研究的重要补充，同时为孢子产生和萌发过程可控的高生产强度樟芝快速循环发酵工艺提供坚实的理论基础。