猪CuZnSOD基因启动子的克隆鉴定及分析

Cloning and analysis of promoter of pig copper zinc superoxide dismutase gene (CuZnSOD)

摘要:

猪铜锌超氧化物歧化酶 (CuZnSOD) 是一种重要的抗氧化酶，其功能已被广泛研究，但CuZnSOD基因的转录调控尚不明确。为了研究猪CuZnSOD基因的核心启动子区域，并对其转录调控机制进行探讨，运用PCR方法从猪基因组克隆CuZnSOD基因5′上游调控区853 bp的片段，然后通过巢式PCR方法获得5′末端逐渐缺失的启动子系列片段，并将这些片段定向插入到荧光素酶报告基因表达载体 (pGL3-Basic) 中。瞬时转染小鼠胚胎细胞 (NIH/3T3)，利用双荧光素酶报告基因检测不同长度启动子活性。检测结果显示，在CuZnSOD基因5′上游调控区-87 bp和-266 bp处分别存在2个潜在转录起始位点，-383 bp~+67 bp启动区活性最强，进一步缺失分析发现-75 bp~-32 bp区域内含有猪CuZnSOD基因转录所必需的基础启动子序列，其中存在多个潜在的转录因子结合位点，研究结果提示这些转录因子结合位点可能是参与CuZnSOD基因转录的重要调控序列。

Abstract:

Pig copper zinc superoxide dismutase (CuZnSOD) is an important antioxidant enzyme. Some studies focused on the function of CuZnSOD gene, but the transcriptional regulation of the CuZnSOD gene is not yet fully elucidated. Therefore, the aims of the study were to determine the core promoter region and to explore its mechanism of transcriptional regulation. The 853 bp DNA sequence of 5′-flanking promoter was amplified by performing PCR. A series of CuZnSOD promoter fragments with gradually truncated 5′-end were produced by nested PCR and inserted into pGL3-Basic vector. The activities of the promoters were measured by the dual-luciferase assay system after transient transfection into the NIH/3T3 cells. The results demonstrated that there were 2 potential transcription start sites in the regions from initiation codon to -87 bp and -266 bp, respectively. The region from -383 bp to +67 bp in CuZnSOD gene promoter showed higher activity than other regions, and further deletion analysis demonstrated that the region from -75 bp to -32 bp contained an essential promoter sequence for pig CuZnSOD gene transcription. In addition, several potential transcription factor binding sites were predicted with bioinformatics method. These results suggest that these transcription factor binding sites may be involved in the transcriptional regulation of CuZnSOD gene.