丝状真菌瑞氏木霉外源基因表达系统的构建

采用PCR技术体外扩增获得了瑞氏木霉外切葡聚糖纤维二糖水解酶Ⅰ (CBHⅠ )启动子和终止子序列 .并以大肠杆菌质粒pUC1 9为骨架 ,在该启动子和终止子序列间加入多克隆位点 ,构建了瑞氏木霉强表达整合型载体pTRIL .以质粒pAN7 1为模板 ,体外扩增了带有潮霉素磷酸转移酶(hph)基因的DNA片段 ,将hph插入pTRIL的cbh1启动子和终止子序列之间 ,构建了Pcbh1 hph Tcbh1表达盒 .用此表达盒转化瑞氏木霉C30原生质体 ,在潮霉素平板上得到 1 5株抗性转化子 .对其中的H1转化子进行了PCR和Southern印迹分析 ,证实hph基因确实整合到转化子染色体DNA上 ,并在Pcbh1 启动子控制下进行高效表达 .转化子H1对潮霉素抗性达 1 5 0mg L ,比出发菌株提高 2倍 .瑞氏木霉强表达整合型载体pTRIL的构建成功为开展瑞氏木霉分子生物学研究以及进一步的工程菌株构建工作奠定了基础

Construction of Heterogeneous Genes Expression System of Filamentous Fungus Trichoderma reesei

The strong promoter and terminator of cellobiohydrolase Ⅰ (CBHⅠ) gene from cellulase producing filamentous fungi Trichoderma reesei 9414 was isolated by PCR. The expression vector pTRIL was constructed by inserting multiple cloning sites into the place between the promoter and terminator with pUC19 as backbone. To validate the usefulness of pTRIL, the DNA fragment encoding hygromycin phosphotransferase ( hph ) gene conferring resistance to the antibiotic hygromycin B was inserted into Xho Ⅰ and Sal Ⅰ sites of pTRIL, resulting in recombinant pTRIL hph . The pTRIL hph containing the transforming Pcbh1 hph Tcbh1 cassette was introduced into the strain Rut C30 by transformation. 15 transformants were isolated in minimal medium plates containing 100 μg /ml of hygromycin B. The hygromycin resistant transformant H1 was analyzed by PCR and Southern hybridization. The results indicated that hph gene integrated into the chromosome DNA of H1 and expressed under the control of Pcbh1. The hygromycin B resistance of T. reesei H1 was 150 mg/L, which was two times higher than that of Rut C30. The construction of pTRIL is beneficial to molecular biological research on filamentous fungi and genetic modification of T. reesei .