摘要
利用改进的锚定PCR方法克隆了团头鲂(Megalobrama amblycephala)促性腺激素Ⅱβ(GtHⅡβ)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GtHⅡβ亚基基因5′端侧翼序列长度为1354bp,其中包括TATA盒、ERE、ARE、PRE、GRE、LHX3、SF-1、Sp1、Pit-1、NF-Y和AP1等可能对GtHⅡβ亚基基因转录调控起重要作用的功能转录因子结合位点;转录起始位点位于-40～10bp。进一步利用PCR方法扩增得到了811bp(-771～+40bp)、601bp(-561～+40bp)、386bp(-346～+40bp)、239bp(-199～+40bp)和98bp(-58～+40bp)的5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GtHⅡβ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依据。
In teleosts,as in other vertebrates,the pituitary gonadotropic hormones,GtH I and GtHⅡ,play an important role in regulating gametogenesis. However,the mechanism of gonadotropinⅡβ-subunit gene transcriptional regulation has not been thoroughly understood yet. The present study was to get basic information of the possible cis-acting elements that involved in transcriptional regulation of the GtHⅡβ gene expression of blunt snout bream （Megalobrama amblycephala）,and provide preconditions for further research on the molecular mechanism of these cis-acting elements in the GtHⅡβ gene transcriptional regulation. According to cDNA sequence information of blunt snout bream gonadotropinⅡβ-subunit（ bGtHⅡβ）gene,the 5′ region of bGtHⅡβ 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 bGtHⅡβ gene is 1 354 bp and the region contains some potential transcription factors which may have important functions for the transcriptional regulation of the gene,such as ERE,ARE,PRE,GRE,LHX3,SF-1,Sp1,Pit-1,NF-Y,AP1 etc. And the promoter sequence is located on - 40 ～ 10 bp. Based on the above information,five 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 811bp（ - 771- ＋ 40 bp）,601 bp（ - 561- ＋ 40 bp）,386 bp（ - 346 - ＋ 40 bp）, 239 bp（ - 199 - ＋ 40 bp） and 98 bp（ - 58 - ＋ 40 bp）,respectively. It lays foundation for further research on mechanism of GtH Ⅱβ subunit gene transcriptional regulation.
出处
《中国水产科学》
CAS
CSCD
北大核心
2010年第4期649-658,共10页
Journal of Fishery Sciences of China
基金
上海市重点学科建设项目(S30701)
