真菌cel3D内含子保留调控基因功能的机制研究

内含子保留是调控生理活动的重要因素，但真菌内含子保留对基因功能的调控机制尚不清晰。本项目以里氏木霉中负调控纤维素酶合成的β-葡萄糖苷酶cel3D的内含子保留为例，从mRNA、蛋白和调控功能三个层次进行研究：1）定量分析内含子保留对cel3D不同mRNA表达水平的变化，解析其对cel3D mRNA降解和剪切的影响；2）通过酶活检测，研究内含子保留对cel3D不同蛋白变体的表达量和酶活的定量影响, 并通过荧光定位研究不同蛋白变体的时空分布；3）在mRNA、蛋白和酶活水平上，定量解析纤维素酶受cel3D内含子保留的影响，揭示cel3D内含子保留对纤维素酶生产的调控作用。综合分析以上三个层次的研究结果，深刻剖析cel3D内含子保留对基因功能的调控机制。本研究为真菌内含子的作用机制提供新的研究策略，并为研究纤维素酶复杂的调控机理提供新思路。

Intron retention plays a key role in important biological functions, but the mechanism study of intron retention in fungi is lack，leading to the knowledge on the function and regulation of intron retention is unclear. Thus, taking the intron retention of beta-glucosidase cel3D (a key regulator of cellulase biosynthesis) as an example, we’d like to study the function of intron retention on mRNA, protein and regulation levels. First, RT-qPCR and western blot will be utilized to measure the levels of cel3D mRNA variants and protein variants respectively to demonstrate the regulation of intron retention on mRNA degradation and splicing. Second, enzymatic study will be performed to study the effect of intron retention on protein expression and enzyme activity, and fluorescent labeling will be employed to track the subcellular localization of cel3D protein variants and quantitate their in vivo abundance. Third, the regulation of cel3D intron retention on cellulase production will be studied in terms of mRNA level, protein expression level and enzyme activity. These research results will lead to the detailed understanding on regulation mechanism of cel3D intron retention, which will provide a new platform for study of fungal retained intron, and a new strategy to reveal the regulation of cellulase biosynthesis.