生菜质体表达载体构建及gfp基因原核表达分析  

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

作　　者：周丽英[1] 李虎[1] 丁玉梅[2,3] 卜璐璐[1] 侯思明[4] 许俊强[1] 张应华[1] 杨正安[1] 

机构地区：[1]云南农业大学园林园艺学院,云南昆明650201 [2]云南省农业科学院生物技术与种质资源研究所,云南昆明650223 [3]云南省农业生物技术重点实验室,云南昆明650223 [4]昆明学院生命科学与技术系,云南昆明650118

出　　处：《西南农业学报》2015年第6期2644-2648,共5页Southwest China Journal of Agricultural Sciences

基　　金：国家自然科学基金(31260481;30972012);云南省自然科学基金(2011FB049);云南省重点实验室开放基金(2014005)

摘　　要：以四季耐抽薹生菜为材料,PCR扩增并克隆了生菜质体rpo A基因的两侧翼的基因片段,利用该两基因区段作为定点整合外源基因的同源重组片段,构建了生菜质体定点转化载体p Brpo A-GFP,并进行了原核表达分析。结果表明,PCR所扩增的两条基因区段大小为1.2与1.1 kb。进行序列分析后,经酶切、连接和转化,构建成含Prrn-gfp-aad A-Tpsb A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Tpsb A的调控下高效表达,表达量约占总可溶性蛋白的45.6%,包涵体占菌体蛋白的47.5%,该载体的构建为后期生菜质体转化体系的建立和其它功能基因导入生菜质体进行性状改良奠定了基础。We amplified and cloned lettuce plastid rpo A gene two flanking gene fragments from the Lactuca line of‘Four seasons lettuce＇by PCR,and using the two gene segments as fixed-point integration of heterologous gene homologous recombination fragment,constructed the lettuce plastid expression vector p Brpo A-GFP,which carried the expression cassette of Prrn-gfp – aad A-Tpsb A. The results showed that two genes section size amplified by PCK was 1. 2 and 1. 1 kb. After sequence analysis,the enzyme cut,connection and transformation,enzyme identification showed that the construct carrier conformed to the expected design. The analysis results of SDS-PAGE electrophoresis indicated that GFP protein expression level was up to 45. 6 % of the total soluble protein and 47. 5 % of inclusion body protein. The plastid expression vector p Brpo A-GFP was very useful and suitable to apply in trait improvement of lactuca via plastid genetic transformation.

关 键 词：生菜 质体 表达载体 GFP基因 高水平表达 

分 类 号：S636.2[农业科学—蔬菜学]