重叠延伸PCR克隆三孢布拉霉carRA基因及其功能活.PDFVIP

生 物 工 程 学 报 Chin J Biotech 2011, July 25; 27(7): 990−997 journals.im.ac.cn Chinese Journal of Biotechnology ISSN 1000-3061 cjb@im.ac.cn ©2011 CJB, All rights reserved. 基因工程 重叠延伸PCR 克隆三孢布拉霉carRA 基因及其功能活 性检测系统的构建 汤晖，石楠，于淼，刘龙，刘静，贾颖，牛宏彦，张利平 河北大学生命科学学院 河北省微生物多样性研究与应用实验室，保定 071002 摘 要: 三孢布拉氏霉菌CarRA 蛋白，既有番茄红素环化酶功能活性又有八氢番茄红素合成酶功能活性，为了对CarRA 蛋白进行双功能活性分析，及探测CarRA 蛋白的番茄红素环化酶功能活性位点，构建了在大肠杆菌体内通过颜色互补 检测两种酶活性的系统。通过重叠延伸PCR 的方法克隆得到了carRA 基因，并构建原核表达载体pET28a-carRA ，与携 带crtI/crtB/crtE 基因簇的质粒pAC-LYC 共转化BL21(DE3) ，验证番茄红素环化酶功能活性；与以pAC-LYC 为基础构 建的携带crtI/crtE 基因簇的质粒pAC-LYC △(crtB) 共转化BL21(DE3) ，验证八氢番茄红素合成酶功能活性。通过颜色 互补的初步鉴定，再以HPLC 对代谢产物进行分析，明确了实验中的CarRA 蛋白功能活性检测系统的可靠性，为定点 突变番茄红素环化酶活性位点而不影响八氢番茄红素合成酶功能活性提供了筛选模型。 关键词: 重叠延伸 PCR ，carRA 基因，番茄红素环化酶，八氢番茄红素合成酶 Cloning of Blakeslea trispora carRA gene by PCR-driven overlap extension and construction of an activity detection system Hui Tang, Nan Shi, Miao Yu, Long Liu, Jing Liu, Ying Jia, Hongyan Niu, and Liping Zhang Key Laboratory of Microbial Diversity Research and Application of Hebei Province, College of Life Sciences , Hebei University , Baoding 071002, China Abstract: Blakeslea trispora CarRA has both lycopene cyclase and phytoene synthase activity. In order to analyze the double functional activity of CarRA proteins and to detect the active sites of lycopene cyclase, we constructed two detection systems in Escherichia coli by color complementary. Through PCR-driven overlap extension we got carRA gene cDNA, then constructed prokaryotes expression vector pET28a-carRA. pET28a-carRA with plasmid pAC-LYC carrying crtI/crtB/crtE gene clusters were co-transformed to BL21(DE3) to validate lycopene cyclase activity. We constructed the plasmid p