灵芝中钙调磷脂酶/Crz信号对灵芝酸生物合成的影响及其机制研究

Ganoderic acids (GAs), important bioactive constituents of the famous medicinal mushroom Ganoderma lucidum, have received wide attention due to extraordinarily pharmacological functions. Ganoderic acid (GA) production is enhanced in the liquid static culture of G. lucidum by addition of calcium ion, while its molecular regulatory mechanism is unclear. Our previous data demonstrated that transcription levels of the key genes encoding calcineurin signaling pathway components, calcineurin gene (CnA) and calcineurin-responsive zinc finger gene (Crz), were up-regulated in liquid static culture of G. lucidum under calcium ion induction conditions. Bioinformatic analysis also reveals that putative calcineurin-dependent response element (CDRE) motifs exist in the promoter region of key GA biosynthetic genes. Based on our preliminary data, we propose that calcium ion regulates GA biosynthesis through calcineurin/Crz signal pathway in G. lucidum. In order to prove our hypothesis, the interaction between the key GA biosynthetic gene promoter and Crz will be detected by electrophoretic mobility shift assays (EMSA) in vivo. Moreover, the roles of Crz and CnA in regulation of GA biosynthesis will be characterized by knock-down of those genes individually in G. lucidum. To further elucidate whether the function of CnA is mediated by Crz, the localization of Crz-eyfp and different phosphorylated forms of Crz will be detected in wild-type and CnA-silenced G. lucidum strains in presence of calcium ion. This work will allow us to comprehensively understand the regulatory mechanism of the calcineurin/Crz signal pathway in GA biosynthesis in G. lucidum. The related information will be helpful for the further research on secondary metabolite biosynthesis in other mushrooms.

灵芝酸是药用真菌灵芝中的重要活性成分。灵芝细胞培养过程中添加钙离子可提高灵芝酸的产量，但是对于钙离子是如何进一步调控灵芝酸生物合成的仍不清楚。我们前期的研究显示钙离子可以诱导灵芝中钙调磷脂酶信号途径中关键因子Crz和CnA转录水平的提高，灵芝酸生物合成基因的启动子上也具有Crz的结合位点。结合初步数据，本课题假设：钙调磷脂酶/Crz信号介导了钙离子对灵芝酸生物合成的调控。为了验证该假设，计划采用凝胶阻滞实验(EMSA)开展Crz与灵芝酸合成途径关键基因启动子的互作研究。在灵芝中分别沉默Crz和CnA基因，分析不同条件下基因沉默对灵芝酸合成的影响，鉴定它们在合成调控中的作用。通过比较野生型和CnA沉默菌株中Crz的磷酸化和细胞定位，确定CnA的作用是否通过Crz介导。本研究以期揭示钙调磷脂酶/Crz信号对灵芝酸生物合成的调控机制，为利用药用真菌生产次级代谢产物提供坚实的理论基础。

灵芝酸是药用真菌灵芝中的重要活性成分。灵芝细胞培养过程中添加钙离子提高了灵芝酸的产量，但是钙离子是如何进一步调控灵芝酸生物合成的仍不清楚。我们早期的实验结果显示钙离子可以诱导灵芝中钙调磷脂酶信号途径中关键因子Crz和CnA转录水平的提高。本研究通过Crz基因沉默实验发现Crz 调控了灵芝酸的生物合成。凝胶阻滞实验表明钙离子诱导条件下Crz与灵芝酸生物合成基因FPS的启动子结合，进而调控FPS基因的表达。CnA基因沉默和CnA免疫抑制剂CsA添加实验揭示了CnA通过调控灵芝酸生物合成基因的表达量和中间代谢产物积累，进而影响灵芝酸的生物合成。Crz1-GFP融合蛋白的细胞定位和磷酸化状态分析表明CnA 通过调控Crz蛋白的磷酸化，导致去磷酸化后的Crz蛋白进入细胞核中，进而调节灵芝酸的生物合成。本研究揭示了钙调磷脂酶/Crz信号对灵芝酸生物合成的调控机制，加深了我们对灵芝酸生物合成调控机制的理解。

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