扩展青霉碱性脂肪酶基因5’端侧翼区域的克隆与鉴定  

Cloning and functional analysis of 5' flanking region of genomic DNA encoding alkaline lipase from Penicillium expansum

作　　者：杨月梅[1] 江贤章[1] 张娟梅[1] 谢必峰[1] 杨欣伟[1] 林琳[1] 黄建忠[1] 

机构地区：[1]福建师范大学工业微生物教育部工程研究中心,福州350108

出　　处：《工业微生物》2007年第1期14-19,共6页Industrial Microbiology

基　　金：国家自然科学基金(30270033);福建省自然科学基金(B0120001)资助

摘　　要：应用衔接头PCR技术,扩增得到约2000 bp的扩展青霉碱性脂肪酶基因5’端侧翼区域的单一产物。对该产物测序并提交GenBank数据库(GenBank accession DQ677520),经序列比对分析,发现该序列具有真核启动子序列的基本结构特征,含有TATA盒、CAAT盒、GC盒等元件。将扩增得到的脂肪酶基因5’端侧翼序列,连接到含有绿色荧光蛋白(GFP)报告基因的质粒中,构建了一个重组表达质粒,转化大肠杆菌细胞。荧光显微观察大肠杆菌阳性转化子发出荧光,侧翼序列含有启动子功能得到确认。A 5＇ flanking region DNA encoding alkaline lipase from Penicillium expansum with about 2000 bp was specially amplified by LA-PCR, which was cloned into pMD 1ST vector and sequenced . The sequence （GenBank accession DQ77520） contained a novel promoter region with TATA box,CAAT box and CA2 box-like elements in the identical positions common to the eucaryote promoter sequence regions in comparison with the sequences in the GenBank. The amplified product of 5＇ flanking region was ligated into the pGlow-TOPO vector which contained the green fluorescence protein （GFP） gene as the reporter gene to construct the expression plasmid The recombined plasmid was transfered to the Escherichia. coil TOP10. Many Escherichia. coil TOP10 with the recombined plasmid could emit fluorescence. The result indicated that the 5＇ flanking region had the promoter gene segment, which could promote the GFP gene expression in Escherichia. coil TOP10.

关 键 词：扩展青霉 脂肪酶 5’端侧翼区 衔接头PCR 启动子 GFP 

分 类 号：TQ465[化学工程—制药化工]