里氏木霉中小分子RNA对纤维素酶表达调控的作用

摘要

Objective: By over-expressing the small RNAs and inhibiting the small RNAs function, investigating the regulation function of small non-coding RNAs on cellulase expression in Trichoderma reesei QM9414. Meth⁃ods: For studying the effect of over-expressing 3 small RNAs(milR5, milR7 and milR10) and inhibiting its func⁃tion on cellulase expression in T.reesei QM9414, the plasmid p-Ppdc'-Tcbh1 which contained a T.reesei strong pro⁃moter Ppdc was used to constructed over-expression cassettes and Tough Decoy(TuD) technology. The expression quantity of small RNAs was tested by real-time PCR, and the the cellulase expression was tested by filter paper activity(FPA). Results: The expression quantity of small RNAs showed that over-expression cassette enhanced the expression of the small RNAs, and the TuD technology inhibited the function of the small RNAs, respectively. The FPA showed that the recombinant strains OE-milR7 improved FPA distinctly, and TuD7 decreased FPA. Con⁃clusion: One of the small RNAs, milR7, might participate in the regulation of cellulase expression, which demon⁃strates a new strategy for increasing cellulase activities in T.reesei.%目的：通过过表达小分子RNA和抑制小分子RNA功能，研究小分子RNA对里氏木霉QM9414纤维素酶表达的影响。方法：利用含里氏木霉强启动子Ppdc的质粒p-Ppdc'-Tcbh1，分别构建过表达载体和Tough Decoy（TuD）序列，对里氏木霉QM9414的3个小分子RNA（milR5、milR7和milR10）进行过表达和抑制，过表达和抑制后的小分子RNA表达量通过荧光定量PCR检测，最终测定转化菌株的滤纸酶活，研究过表达和抑制小分子RNA后对纤维素酶表达的影响。结果：过表达载体能提高小分子RNA的表达量，过表达重组菌株OE-milR7的滤纸酶活明显提高；TuD序列表达载体可抑制小分子RNA的功能，抑制小分子RNA的重组菌株TuD7酶活略有下降。结论：milR7可能参与纤维素酶表达调控，为对里氏木霉的产纤维素酶能力进行改造提供了新的靶点。