丝状真菌瑞氏木霉纤维素酶基因转录激活模式分子机制的研究

Eukaryotic gene expression is a process involving complex interactions among transcription regulators, general transcription factors and chromatin occuring on both a time and a spatial scale. Exquisite regulation of eukaryotic gene transcription is key to assuring its normal life in response to environmental changes. Filamentous fungi represented by Trichoderma reesei can rapidly initiate the launching of cellulase synthesis in the presence of cellulose substrate which otherwise are kept silent. Unfortunately, the precise mechanism underlying this transcription control is not clear yet. The present application aims to carry out systematic study on the relative transcription factors including the essential transcription activator Xyr1 involved in the induced transcription of cellulase genes, to elucidate the mechanisms by which they act concertedly to achieve the regulatory control. In-depth functional characterization of different regions including DNA binding domain and transcriptional activation domain of Xyr1 would be firstly carried out both in vitro and in vivo to understand how different structural regions and other factors act and contribute to its transcriptional activities. By fusing fluorescent protein tags to these factors and taking advantage of bimolecular fluorescence complementation (BiFC) assisted by co-immunoprecipitation, their intracellular distribution and putative interactions in response to different carbon sources as well as the functional meaning would be carefully dissected. Further in combination with analysis and comparison of regulatory DNA-protein complexes mediated by relative transcription factors under different conditions, complex interactions between these transcription factors and the effect of changes in these interactions on transcriptional activation would be analyzed systematically. Attempts would also be made to isolate other unknown factor(s) that may involve in the regulatory process. Importantly, the pattern of dynamic binding to specific chromatin regions by these factors under noninducing and inducing conditions would be directly measured using chromatin immunoprecipitation (Chip). Based on these research, we expect to uncover the regulatory mechanism underlying the stringent control of the induced expression of cellulase genes. The results were also anticipated not only to expand our knowledge about eukaryotic gene regulation, but may also to help engineer Trichoderma reesei strains to improve their productivity of cellulases/hemicellulases, thus lowering the cost of biomass conversion.

真核生物基因转录的精确调控是细胞实现正常生命活动的重要保障。丝状真菌瑞氏木霉在纤维素底物存在的条件下可以迅速地启动纤维素酶基因的高水平转录表达，但其精确的转录调控分子机制并不清楚。本项申请拟利用实验室已经建立的丝状真菌遗传操作平台，针对瑞氏木霉中与纤维素酶基因诱导转录相关的转录调控因子，尤其是围绕重要转录激活因子Xyr1的活性调控机制开展系统研究。在对其DNA结合域和转录激活域在内的结构域功能及作用特点精细解析的基础上，研究分析Xyr1等相关转录调控因子在不同碳源条件下的细胞定位变化及其在特定染色质序列上的动态相互作用模式；结合系列突变株在阻遏和诱导条件下特定启动子序列-蛋白复合物的变化分析，系统阐述Xyr1等转录因子间相互作用及此作用变化对转录活性的影响，从而更深入地揭示丝状真菌纤维素酶基因的表达调控机制，丰富真核生物基因表达调控理论知识，并为将来构建或改良高产纤维素酶菌株提供理论依据。

真核生物基因转录的精确调控是细胞实现正常生命活动的重要保障。丝状真菌瑞氏木霉在纤维素存在条件下可以迅速启动纤维素酶基因的高水平转录表达，但其精确的调控机制并不清楚。本项目围绕与纤维素酶基因诱导转录相关的重要转录激活因子Xyr1的活性调控机制开展系统研究，取得了系列研究结果：1）对Xyr1 的相关结构域结构功能进行了系统分析，发现Xyr1 DNA结合结构域（DBD）与cbh1启动子多个区域均能结合，对-800 及-200 区域有较强的结合能力；体内活性分析表明，Xyr1 DBD的单独表达并不能与cbh1启动子进行有效的结合。Xyr1 MHR区域具有一定的介导转录激活的能力，而Xyr1的C-端转录激活区（AD）在维持蛋白整体稳定性方面更为重要；2）细胞定位分析表明，Xyr1和Ace2主要定位于细胞核中，而作为介导碳代谢阻遏的主要转录因子Cre1同样主要存在与细胞核中，但其核定位在纤维素诱导条件下存在一定的胞质转移现象；3）通过在瑞氏木霉中构建一个基于铜离子可调控的Xyr1的表达体系，发现Xyr1的高水平表达可实现纤维素酶系组分在葡萄糖等非诱导条件下的形成，且产生的纤维素酶水平与亲本菌在纤维素诱导条件下的表达水平相当。同时发现以该体系单独过表达Cre1可以消除纤维素酶基因的诱导表达，但Xyr1的同时过表达可以消除Cre1的这一抑制；4）分离鉴定了至少三个影响纤维素酶诱导表达的新调控因子。发现其中一个调控因子主要通过竞争拮抗Xyr1对纤维素酶基因的结合来抑制纤维素酶基因的表达，而另外一个调控因子可能通过翻译后修饰来实现对纤维素酶基因表达的控制；5）初步研究了染色质修饰变化在纤维素酶基因诱导表达过程中的作用。发现组蛋白乙酰转移酶复合物SAGA介导的乙酰化修饰是纤维素酶基因表达过程的一个重要方面，而Xyr1可通过募集SAGA复合物来实现这一过程控制；6）系统研究了瑞氏木霉胞内主要β-葡萄糖苷酶在乳糖诱导纤维素酶基因表达中的作用，同时分离鉴定了一个纤维素酶诱导所必须的糖转运受体蛋白。上述创新型研究结果极大得丰富了纤维素酶基因表达调控机制研究。