高产纤维素酶绿色木霉基因工程菌的构建

[目的]构建CBHⅡ基因过量表达的绿色木霉工程菌。[方法]根据绿色木霉cDNA序列设计合成引物,PCR扩增CBHⅡ基因序列,将完整的目的基因与表达载体pCAMBIA1302连接,转入大肠杆菌DH5α中,获得重组质粒pCAMBIA1302-CBHⅠ。再将pCAM-BIA1302-CBHⅠ重组质粒与瑞氏木霉外切葡聚糖纤维二糖水解酶Ⅱ（CBHⅡ）PcbhⅡ启动子片段连接,将潮霉素磷酸转移酶（hyg）基因片段插入PcbhⅡ启动子下游,转入大肠杆菌DH5α中,获得重组表达质粒pCAMBIA1302-CBHⅡ。[结果]构建的重组表达质粒电转化后经SDS-PAGE电泳检测,绿色木霉纤维素酶CBHII基因在原宿主菌中成功过量表达,证实CBHⅡ基因在PcbhⅡ启动子控制下进行高效表达。[结论]为高产纤维素酶系的工业化菌株构建奠定基础。

Construction of Genetic Engineering Bacteria of Trichoderma viride with High Cellulose

[Objective]The aim was to construct genetic engineering bacteria of Trichoderma viride with high cellulose.[Method]The primers were designed by cDNA sequence of Trichoderma viride,and gene order of CBH Ⅰ was amplificated by PCR.The gene was conjugated with the carrier of pCAMBIA1302,and was shifted into DH5α,recombinant plasmid of pCAMBIA1302-CBH Ⅰ was obtained.Recombinant plasmid of pCAMBIA1302-CBH Ⅰand fragment of promoter Pcbh II was conjugated,and then the hyg gene were inserted into the Pcbh Ⅱpromoter’s downstream,the pCAMBIA1302-CBH Ⅱ expression box could be shift in DH5α.[Result] The electrophoresis detection results showed that the CBH Ⅱ gene was integrated to the transformant DNA,and CBH II gene was highly expressed under the control of Pcbh Ⅱpromoter.[Conclusion] The study lay foundation for construction of genetic engineering bacteria of Trichoderma viride with high cellulose.