为探索鱼类转铁蛋白基因tf 和转铁蛋白受体基因tfr1a的转录调控机制，本实验以团头鲂为研究对象，在其全基因组数据库中获取tf和tfr1a基因序列，对2个基因候选启动子区转录因子结合位点及CpG岛进行预测，通过PCR方法克隆得到tf和tfr1a基因近端启动子区不同长度片段，连接至pGL3-Basic/pEGFP-1载体，瞬时转染入Hela细胞，并采用双荧光素酶报告基因检测系统进行检测。结果发现，团头鲂tf基因启动子区无CpG岛位点，而tfr1a基因启动子区有2个CpG岛位点。成功构建9个tf和10个tfr1a不同长度启动子片段的重组质粒，经双荧光素酶报告基因系统检测发现，tf启动子核心区域为−268~+56 bp，且−1 308~−1 102 bp片段可能存在正调控该基因表达的转录因子结合位点；tfr1a启动子核心区域为−224~+48 bp，且+48~+92 bp可能存在抑制该基因转录的负调控元件，而−1 229~−1 219 bp区域可能存在促进tfr1a基因表达的正调控转录因子结合位点。

In order to explore the transcriptional regulation mechanism of the tf and tfr1a genes in Megalobrama amblycephala, the genomic sequences of tf and tfr1a were obtained from whole genome sequence database. Transcription factor binding sites and CpG islands in the promoter regions of tf and tfr1a genes were predicted by bioinformatics methods. Fragments of different length of the predicted promoter region of tf and tfr1a were cloned by PCR amplification. The amplified different fragments were ligated to the pGL3-Basic/pEGFP-1 vector. Subsequently, the recombinant plasmids were transiently transfected into Hela cells for fluorescence detection by the Dual-Luciferase Reporter System. Bioinformatics analysis showed that there was no CpG island site in the tf promoter, and there were two CpG island sites in the tfr1a promoter. A total of 9 tf and 10 tfr1a recombinant plasmids containing promoter fragments of different lengths were successfully constructed. The detection of Dual-Luciferase Reporter System showed that the core region of the tf promoter was -268—+56 bp, and the -1 308—-1 102 bp fragment may have a transcription factor binding site that positively regulates the gene expression. The core region of the tfr1a promoter was -224—+48 bp, and the +48—+92 bp region may contain negative regulatory elements that inhibit the transcription of this gene, while the -1 229—-1 219 bp region might contain positive regulatory transcription factor binding sites that promote the tfr1a gene expression.

Q785;S917.4

国家自然科学基金(31572613)；湖北省协同创新中心建设专项资金(2016ZXPY04)