Cloning and Functional Analysis of the Porcine Growth Hormone Gene Promoter

猪生长激素启动子的克隆及功能分析(英文)

[Objective] This study aimed to clone the porcine growth hormone gene promoter and determine the core promoter sequences and the cis-acting elements. [Method] Sequence of the 5＇flanking region of porcine growth hormone gene was searched out and downloaded from the NCBI website. According to the targeted se- quence, primers were designed and synthesized for the PCR amplification. The 1 882 bp （-1 821 bp-＋61 bp） fragment was amplified by PCR. Nine promoter frag- ments with different lengths were obtained by genome-walking deletion method and then cloned into luciferase reporter vectors. Relative transcriptional activities of these 5＇ terminal-deleted plasmids in pituitary and non-pituitary cells were determined by transient transfection of the rat pituitary adenoma cell （GH3）, porcine lilac endotheli- um cell （PIEC） and porcrne Kidney-15 （PK15） with the constructed dual-luciferase vectors. [Result] Result of DNA sequencing showed that the 1 882 bp fragment of GH 5＇ promoter was successfully cloned. Nine luciferase reporter gene plasmids were constructed. DuaI-Luciferase reporter assay indicated that the promoter inserted into reporter gene vector had very strong cell specificity. [Conclusion] Porcine growth hormone gene specifically expresses in pituitary cells. The minimal promoter of the porcine growth hormone gene is mapped at the region -110 bp-＋61 bp. Promoter regions 218 bp--110 bp and -429 bp--218 bp contain positive regulatory elements.

[目的]克隆猪生长激素启动子,确定其启动子核心序列和主要的顺式作用元件。[方法]根据NCBI上公布的序列设计引物,PCR扩增了猪生长激素5’端-1821^+61bp的序列,并通过移步缺失的方法,获得9段长短不一的启动子序列,将其分别构建到双荧光素酶表达载体pGL3-basic上。通过重组质粒瞬时转染大鼠垂体瘤细胞(GH3)、猪髋动脉血管内皮细胞(PIEC)和猪肾细胞(PK15)和转染后细胞荧光素酶活性的测定,检测这些5’末端缺失质粒在垂体及非垂体细胞中的相对转录活性。[结果]成功扩增了猪GH基因5’上游启动区1882bp的片段并构建了9个pGL3-mGHpromoter报告基因载体;双荧光素酶报告基因检测系统证实插入报告基因载体中的启动子具有非常强的细胞特异性。[结论]猪生长激素特异性在垂体细胞中表达,其最小启动子位于-110bp以内,启动子区-218^-110bp和-429^-218bp间存在正向调控元件。