团头鲂PGPH α亚基基因5’端侧翼序列克隆及其表达载体的构建  

Molecular cloning of 5’flanking region of blunt snout bream (Megalobrama amblycephala) pituitary glycoprotein hormone α subunit gene and construction of expression vector

作　　者：曲宪成[1] 周正峰[1] 崔严慧[1] 刘颖[1] 胡萍华[1] 金一春[1] 尚婧瑨[1] 

机构地区：[1]上海海洋大学生命与科学技术学院,上海200090

出　　处：《中国水产科学》2008年第4期550-558,共9页Journal of Fishery Sciences of China

基　　金：回国留学人员科研启动基金(科01-113);上海市重点学科建设项目资助(Y1101);上海水产大学博士启动基金(科00-119)

摘　　要：首先运用PCR-末端加尾法克隆了团头鲂（Megalobrama amblycephala）脑下垂体糖蛋白激素α亚基（Pituitary glycoprotein hormone α subunit,PGPH α）基因5’端侧翼序列;然后利用生物信息学理论对该序列进行了序列分析;最后在序列分析结果的基础上,构建了荧光素酶质粒表达载体。序列分析结果显示,克隆所得到的团头鲂PGPH α亚基基因5’端侧翼序列长1399bp;其序列中包含潜在的TATA盒、GC盒,以及ERE、TRE、CRE、PGBE等对PGPH α亚基基因转录调控起重要作用的转录因子结合位点;启动子区域位于-40-10bp处。利用PCR方法扩增得到了全长1439bp以及1120bp、532bp、173bp均包含转录起始位点下游40bp序列的缺失片段,并将其连接至pGL3-Basic报告基因载体,成功构建了团头鲂PGPH α亚基基因5’端侧翼序列的表达载体,为进一步研究、分析其转录调控机制提供了基础。The pituitary follicle-stimulating hormone （FSH）, luteinizing-hormone （LH）, thyroid-stimulating hormone （TSH） and human chorionic gonadotropin （HCG） are members of glycoprotein family. In teleosts, as in other vertebrates, each glycoprotein hormone is non-covalently binded to form bioactive dimmer molecules by α and β subunits. The α subunit is common and highly conserved： whereas β subunit is specific to each hormone and determines the specificity of hormone physiological processes. Nowadays, researches on the transcriptional molecular mechanism of mammiferous PGPH α have been reported comprehensively, but not in fishes. At present, genome walking and methods based on PCR technologies, such as inverse PCR, panhandle PCR, thermal asymmetric interlaced PCR etc, are usually used to clone the gene flanking region, but these methods are used restrictedly because of the low specificity, complexity to manipulation and some other vulnerabilities. In this study, the 5＇ flanking region of blunt snout bream （Megalobrama amblycephala） pituitary glycoprotein hormone α subunit （PGPHα） gene was cloned with an improved end tailed-PCR strategy, which was simple and not dependent on restriction cutting or mapping. In the first step of this method, a library of single-stranded flanking sequences was generated by linear amplification with one primer in the known region. A homooligomeric cytosine tail was added to each of the single-stranded fragments by a terminal transferase catalyzed reaction. The tailed fragments were then amplified by PCR with a nested primer in the known region and a poly-guanine primer complementary to the cytosine tail in the unknown region. Finally, the different fragments were separated by cloning and identified by sequencing. The cloned sequence was analyzed using bioinformatics tools. Then, on the basis of bioinformatics analysis, the luciferase expression vectors were constructed. Sequence analysis revealed that 5＇ flanking region of blunt snout bream PGPH α subunit

关 键 词：团头鲂 垂体糖蛋白激素α亚基 5’侧翼序列 启动子 表达载体 

分 类 号：Q959[生物学—动物学]