适用于粉红粘帚霉绿色荧光标记的重组质粒的构建及应用

Construction of a recombination plasmid labelled with green fluorescence and its application in Clonostachys rosea

【目的】构建含有完整的绿色荧光蛋白(Green Fluorescence Protein,GFP)和潮霉素抗性基因的表达质粒,观察该质粒在丝状真菌粉红粘帚霉中的表达。【方法】采用PCR、酶切、去磷酸化、酶连、转化等多种分子生物学手段,利用原生质体制备及转化的方法将其转入机会真菌粉红粘帚霉中,并在荧光显微镜下观察表达情况。【结果】成功构建了用于真菌标记的真核表达载体pANGH3,将其转化粉红粘帚霉后,在荧光显微镜下观察到菌丝有绿色荧光产生。【结论】重组质粒pANGH3的成功建立及其在染粉红粘帚霉中的表达,为研究真菌侵染机理的模型奠定了实验基础。

[Objective] We constructed a recombinant eukaryotic expression vector harboring green fluorescent protein（GFP） and the hygromycin resistance gene hph,and observed its expression in Clonostachys rosea.[Methods] We used PCR,enzyme digestion,phosphorylation,ligation and transformation to construct the plasmid.Using protoplast preparation and transformation technologies,we expressed the plasmid in the fungi C.rosea.[Results] We created the eukaryotic expression vector,transformed it into C.rosea and observed green fluorescence with fluorescence microscope.[Conclusion] The successful construction of the pANGH3 recombinant plasmid and its expression in C.rosea establishes a new model for studying fungal infection mechanisms.