共培养条件下灰盖鬼伞菌漆酶Lcc9表达的转录调控机制

Research on the mechanism of up-regulation of laccase genes during the coculture of two fungi will contribute significantly to not only the theory but also the application of the enzyme, as the coculture method is one of the promising methods for fungal laccase production. However, previous studies focused mainly on the effects of environmental signals in coculture system on laccase production, and rare of them concered the transcriptional regulation mechanism of the laccase production after the fungi sensing environmental signals. Previously, we constructed a coculture system containing Coprinopsis cinerea and Gongronella sp. w5 for laccase production. In this system, the expression of lcc9 from C. cinerea can be induced to a high level. Further research found that the regulation occured at the transcriptional level, in which cis-acting elements and trans-acting factors are involved. Therefore, in this proposal, we plan to use the DNA deletion technology to find out the key upstream regulatory sequence, through which further bait the transcription factor. Then,we will study on the interaction between the regulatory sequence and the transcription factor,to elucidate the mechanism by which the C. cinerea regulates the expression of laccase gene at the transcriptional level after it sensing signals in the coculture system. The results are supposed to clarify how the fungi to be induced to synthesize laccase in the coculture system.In addition,it will provide a technical model for the research on the biocommunication and biocontrol between fungi and other microbes.

真菌共培养诱导漆酶合成是具有应用前景的漆酶制备方式，对其诱导机制研究具有重要理论意义和应用价值。但目前的研究主要集中于对漆酶基因上调表达的外界诱因进行探讨，漆酶合成菌感受外界信号后，如何在转录水平调控漆酶合成的分子机理还未被深入研究。我们前期建立了球托霉菌诱导灰盖鬼伞菌漆酶lcc9上调表达的共培养体系，对调控机制的研究发现，漆酶的表达调控发生在转录水平，潜在顺式作用元件和反式作用因子可能参与了转录调控过程。因此，申请项目拟通过DNA消减获取lcc9上游调控区关键调控元件，进而通过DNA亲和层析钓取核心转录因子，并研究二者之间的作用关系，从而揭示共培养条件下，灰盖鬼伞菌感受外界信号后，通过何种转录因子，以及何种作用方式，作用于何种转录调控元件，最终调控漆酶基因上调表达的分子机制。获得的结果不仅能够揭示共培养诱导真菌漆酶合成的内在机理，而且还将为开展真菌之间相互作用和控制机理研究提供技术模型。

真菌共培养诱导漆酶合成菌大量合成漆酶现象在自然界普遍存在，但漆酶基因上调表达的分子机制目前还不清楚。前期，我们建立了球托霉菌（Gongronella sp. w5）诱导灰盖鬼伞菌（Coprinopsis cinerea）漆酶lcc9上调表达的共培养体系，本项目拟获取参与lcc9表达调控的上游调控区关键调控元件以及核心转录因子，并研究二者之间的作用关系。我们的结果显示，C. cinerea漆酶基因lcc9上游启动子区存在一个GC富集区可能参与调控了漆酶的表达；进一步，我们以该段序列钓取获得了2个潜在的转录因子蛋白CcHsp1A和CcHp；对这两个基因的体内RNA干扰结果显示，CcHsp1A干扰后菌体生长缓慢，而CcHp干扰后漆酶同工酶表达均减弱，CcHp过表达菌株中漆酶表达增强。上述结果显示，CcHp作为非特异性转录因子，参与了漆酶同工酶的转录调控。然而，EMSA和ITC实验结果显示，体外表达并纯化获得的CcHp无法与GC富集区序列直接结合，表明CcHp可能做为转录复合物组分，而非直接与DNA结合的转录因子蛋白，参与共培养条件下的漆酶转录调控。我们的结果不仅能够揭示共培养诱导真菌漆酶合成的内在机理，而且还将为开展真菌之间相互作用和控制机理研究提供技术模型。