团头鲂促性腺激素GtH Iβ亚基基因5′端启动子区克隆及表达载体构建  

MOLECULAR CLONING AND CONSTRUCTION OF EXPRESSION VECTOR OF THE 5′FLANKING REGION OF BLUNT SNOUT BREAM(MEGALOBRAMA AMBLYCEPHALA GONADOTROPIN Iβ-SUBUNIT GENE

作　　者：曲宪成[1] 崔严慧[1] 周正峰[1] 刘颖[1] 金一春[1] 胡萍华[1] 薛婷君[1] 王琼[1] 

机构地区：[1]上海水产大学生命与科学技术学院,上海200090

出　　处：《水生生物学报》2008年第4期558-567,共10页Acta Hydrobiologica Sinica

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)资助

摘　　要：本研究利用改进的锚定PCR方法克隆了团头鲂(Megalobrama amblycephala)促性腺激素Iβ(GtH Iβ)亚基基因5′端侧翼序列,并在生物信息学方法分析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:克隆得到的GtHIβ亚基基因5′端侧翼序列长度为479bp,其中包括TATA盒、ARE、PRE、ERE、SF-1、Ptx1等可能对GtH Iβ亚基基因转录调控起重要作用的功能转录因子结合位点。利用PCR方法在基因组中扩增得到了3个缺失片段,并同全长片段一起分别连接至pGL3-Basic报告基因载体,成功构建了团头鲂GtH Iβ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。In teleosts, as in other vertebrates, the pituitary gonadotropic hormones,GtH I and GtH II, play an important role in regulating gametogenesis. Nowadays, the mechanism of gonadotropin Iβ-subunit gene transcriptional regulation has not been thoroughly understood yet. The objective of the current study was to get basic information of the possible cis-acting elements that involved in transcriptional regulation of the GtH Iβ gene expression of blunt snout bream （ Megalobrama amblyccphala） ,and provide preconditions for further research on the molecular mechanism of these cis-acting elements in the GtH Iβ gene transcriptional regulation. According to cDNA sequence information of blunt snout bream gonadotropin Iβ-subunit （bGtH Iβ）gene, the 5＇ region of bGtH Iβ was cloned by a simple method for cloning genomic DNA segments outside the boundaries of known sequences. In the first step of the method,a library of single-stranded flanking sequences is generated by linear amplification with one primer in the known region. Then a homooligomeric cytosine tail is added to each of the single-stranded fragments by a terminal transferase catalyzed reaction. The tailed fragments are 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 are separated by cloning and characterized by sequencing. Sequence analysis reveals that the length of the 5＇ flanking region of bGtH Iβ gene is 479 bp,and the region contains some potential transcription factors which may have important functions for the transcriptional regulation of the gene,such as TATA box, ARE, PRE, ERE, SF-1 and Ptxl, etc. Based on the above information, three partial deletion fragments as well as the full length of the 5＇ flanking region were cloned from the genome by PCR and linked to a luciferase reporter gene. These partial fragments contained 365bp （ -345 to ＋20）,230 bp （ -210 to ＋ 20）,and 150 bp （ -130 to ＋20）,res

关 键 词：团头鲂 促性腺激素Iβ亚基 5’侧翼序列 启动子 表达载体 

分 类 号：Q344.1[生物学—遗传学]