猪NOX1基因启动子区转录因子的筛选与鉴定

Screening and identification of pig NOX1 gene promoter transcriptional elements

为了寻找调控猪NADPH氧化酶1(NOX1)基因转录的转录因子,本研究采用3′端固定,5′端逐渐截短的方法,扩增了NOX1基因的启动子区序列,并将它们分别连入双荧光素酶报告基因载体,转染PK15细胞后,检测双荧光素酶的相对活性。根据活性高低判定转录因子可能结合的位置,利用重叠PCR技术定点缺失转录因子的结合序列,再次连入报告载体后,根据荧光素酶活性的变化判定是否为NOX1基因真正的转录调控因子。结果发现,NOX1基因启动子区的-1618~-1 bp区间存在转录调控因子的结合位点,对该区间开展2轮截短试验后,初步判定-1149~-1091 bp和-208~-115 bp区间存在正调控元件,-1091~-1052 bp区间存在负调控元件。针对上述3个区间,同时结合在线软件的预测结果,定向缺失了-1104~-1092 bp,-1051~-1043 bp,-1043~-1033 bp和-126~-116 bp小片段后,确定-1104~-1092 bp和-126~-116 bp区间存在正调控该基因转录的元件结合位点,分别为ZNF384、ELF-1和TBR1。本研究克隆了猪NOX1基因的启动子序列,对可能结合到该区间的转录因子进行了筛选与分析,为后续分析NOX1的转录调控机制,探讨其生物学功能奠定了基础。

NOX1 is related to the content of oxygen free radicals in cells and is a gene related to cell oxidative stress.In order to find transcription factors that regulate the transcription of pig NOX1 gene,the promoter region of NOX1 gene was amplified by means of 3′-end fixation and 5′-end truncation.Then,these fragments were inserted into the dual luciferase reporter vectors and were transfected into PK15 cells respectively.The relative activity of dual luciferase was detected.The possible binding sites of transcription factors were determined according to the activity level.The binding sequences of transcription factors in the promoter were deleted by the overlapping PCR technology.Then the mutant promoter was inserted into the reporter vector again,the real transcription regulator of NOX1 gene was determined according to the change of luciferase activity.The results showed that there were binding sites for transcriptional regulatory factors in the region of-1618 to-1 bp of NOX1 gene.After two rounds of truncated tests,it was preliminarily determined that there were positive regulatory elements in the-1149 to-1091 bp and-208 to-115 bp,and negative regulatory elements in the-1091 to-1052 bp.Based on these three regions and combined with the prediction results of online software,the-1104 to-1092 bp,-1051 to-1043 bp,-1043 to-1033 bp and-126 to-116 bp fragments were directionally deleted.The-1104 to-1092 bp and-126 to-116 bp regions were identified as the element binding sites positively regulating the transcription of the gene at last.The ZNF384,ELF-1 and TBR1 were considered as the positive regulatory elements.In this study,the promoter sequence of pig NOX1 gene was cloned,and the possible transcriptional regulators were screened and analyzed,which provide the basis for the subsequent analysis of the transcription regulation mechanism of NOX1 and the discussion of its biological function.