gfP基因甘蓝质体表达载体构建及原核表达分析

Construction of the Cabbage Plastid Transformation Vector and High-level Expression of gfp Gene in E.coli

以甘蓝自交系‘03097’为试材,PCR扩增并克隆了甘蓝质体的trnI-trnA基因区段,利用该基因区段作为定点整合外源基因的同源重组片段,构建了甘蓝质体定点转化载体,并进行了原核表达分析。结果表明,所扩增的基因区段大小为2.7 kb。进行序列分析后,经酶切、连接和转化,构建成含Prrn-gfp-aadA-TpsbA表达盒的质体表达载体,酶切鉴定表明,所构建载体符合预期设计;gfp基因能够在质体特异性启动子Prrn及终止子TpsbA的调控下高水平表达,表达量约占总可溶性蛋白的41.0%,包涵体占菌体蛋白的38.0%。该载体的构建为后期甘蓝质体转化体系的建立和其它功能基因导入甘蓝质体进行性状改良奠定了基础。

Based on the highly conservative features of trnI and trnA gene during the plastid genome evolution of higher plants, we cloned the trnI-trnA gene fragment from the cabbage line of ‘03097’. After sequencing and digesting the amplifed 2.7 kb fragment,which was used as homologous targeting sequences, we constructed the cabbage plastid expression vector named pBGLIA-GFP that carries the expression cassette of Prrn-gfp-aadA-TpsbA. Having verified by digestion with restriction enzymes, the vector was transformed into the E. coli strain of DH5a. The analysis results of SDS-PAGE electrophoresis indicated that GFP protein expression level was up to 41.0% of the total soluble protein and 38, 0% of inclusion body protein. The plastid expression vector pBGLIA-GFP is very useful and suitable to apply in trait improvement of cabbage via plastid genetic transformation.