摘 要: 为了后续研究里氏木霉(Trichoderma reesei)纤维素酶基因的表达与调控, 利用overlap PCR及分子克隆技术构建了含有ColE1原核复制起始位点、氨苄青霉素抗性、里氏木霉的丙酮酸脱羧酶启动子、丙酮酸脱羧酶终止子、潮霉素B抗性的筛选标记并能表达增强型绿色荧光蛋白(ZsGreen)的表达载体pLXT-ZsGreen。将该载体转化里氏木霉QM9414原生质细胞, 使用潮霉素B筛选平板得到阳性转化子, 随后使用荧光显微镜在488 nm激发光下观察菌丝, 并随机挑取4个转化菌株进行Western-blot验证。结果显示, 里氏木霉菌丝体可发出明亮的绿色荧光, 而且Western-blot验证了该载体能够在里氏木霉中有效地表达增强型绿色荧光蛋白。上述研究表明, 载体pLXT-ZsGreen在里氏木霉中能够稳定高效地表达外源基因, 为研究里氏木霉的基因表达调控奠定了实验基础。

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.