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

Cloning of Blakeslea trispora carRA gene by PCR-driven overlap extension and construction of an activity detection system

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

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 pAC-LYC△(crtB) carrying crtI/crtE gene clusters, then co-transtormed them with pET28a-carRA to BL21(DE3) to validate phytoene synthase activity. Based on color complementary, and HPLC analysis of metabolites, we confirmed that the CarRA protein activity detection system was reliable. Our study provides a screening model for specific mutation of lycopene cyclase without affecting phytoene synthase activity.