生命科学研究2017，Vol.21Issue(4)：306-311,6.DOI:10.16605/j.cnki.1007-7847.2017.04.005
里氏木霉绿色荧光蛋白表达载体的构建及功能鉴定
Construction of ZsGreen Expression Vector and Verification of Its Function in Trichoderma reesei
摘要
Abstract
In order to study the expression and regulation of cellulase gene in Trichoderma reesei,overlap PCR amplification and molecular cloning techniques were employed to construct a vector pLXT-ZsGreen expressing bright green fluorescence protein in T.reesei.The vector contains prokaryotic replication origin ColE1,the ampicillin resistance gene,promoter of pyruvate decarboxylase (PDC),terminator of PDC,ZsGreen gene,and the hygromycin B resistance gene.The vectors were transformed into the protoplasts of T.reesei QM9414.The hyphae of the transformants were observed using a fluorescence microscope with 488 nm excitation light,and four colonies were randomly selected for Western-blot.The results showed that the transformants were able to express bright green fluorescence under fluorescence microscope.The Western-blot analysis further verified that the constructed plasmid could express ZsGreen protein effectively in T.reesei.The vector pLXT-ZsGreen is capable of stable and efficient expression of exogenous genes in T.reesei,and this lays a foundation for the study of gene expression regulation of T.reesei.关键词
里氏木霉/增强型绿色荧光蛋白/组成型表达/载体构建/重叠PCRKey words
Trichoderma reesei/enhanced green fluorescence protein/constitutive expression/vector construction/overlap PCR分类
生物科学引用本文复制引用
高云雨,佘炜怡,董冠园,钟路遥,刘雯莉,田生礼..里氏木霉绿色荧光蛋白表达载体的构建及功能鉴定[J].生命科学研究,2017,21(4):306-311,6.基金项目
国家自然科学基金(31070044) （31070044）
深圳市科技基础研究发展计划(ZYC201105130092A) （ZYC201105130092A）
