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

摘要:

摘要:【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

Abstract:

Abstract:［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．