破囊壶菌Δ~4-脂肪酸脱饱和酶基因5'端侧翼区域的克隆与鉴定  

Cloning and Functional Analysis of 5' Flanking Region of Δ~4-Desaturase Gene from Thraustochytrium

作　　者：江贤章[1] 陈金卿[1] 田宝玉[1] 高媛媛[1] 舒正玉[1] 李欣[1] 蓝灿华[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,福建福州350108

出　　处：《福建师范大学学报（自然科学版）》2008年第6期89-94,共6页Journal of Fujian Normal University：Natural Science Edition

基　　金：国家自然科学基金资助项目(30370028);福建省自然科学基金重大项目(2003F005);福建省教育厅基金资助项目(JB07079);福建省发改委重大项目([2004]477);福建省科技厅重大项目(2005Q007);福建省青年人才基金资助项目(2008F3036)

摘　　要：应用衔接头PCR(LA-PCR)技术,扩增得到约1000 bp的海洋破囊壶菌Δ4-脱饱和酶基因5’端侧翼区域的单一产物.对该产物测序,经启动子软件与序列比对分析,发现该序列(Genkbank登录号EU074209)具有真核启动子序列的基本结构特征,含有TATA盒、CAAT盒、INR等元件.将扩增得到的脱饱和酶基因5’端侧翼序列亚克隆到含有绿色荧光蛋白(GFP)报告基因的质粒pGlow-TOPO中,构建重组表达质粒,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.The 5＇ flanking region DNA encoding △^4-desaturase from Thraustochytrium sp. FJN-10 with about 1000 bp was specially amplified by LA-PCR, which was cloned into pMD-18T vector and sequenced. Computational analysis results showed that the sequence （GenBank accession No. EU074209） contained a novel promoter region with TATA box, CAAT box and INR elements. The amplified product of 5＇ flanking region was ligated into the pGlow-TOPO vector which contained the green fluorescence protein （GFP） gene as a reporter gene to construct the expression plasmid. The rec6mbinant plasmid was transferred into the Escherichia coil TOP10. Many E. coli TOP10 with the recombinant plasmid could emit fluorescence. The result indicated that the 5＇ flanking region had the promoter gene segment, which could promote the expression of GFP gene in E. coli TOP10.

关 键 词：破囊壶菌 脱饱和酶 衔接头PCR 启动子 绿色荧光蛋白 

分 类 号：Q78[生物学—分子生物学]