文章摘要
团头鲂tf和tfr1a基因启动子克隆及转录调控分析
Cloning and transcriptional regulation of tf and tfr1a promoter in Megalobrama amblycephala
投稿时间:2019-03-25  修订日期:2019-06-02
DOI:
中文关键词: 团头鲂  tf基因  tfr1a基因  启动子  转录调控
英文关键词: Megalobrama amblycephala  tf  tfr1a  promoter  Transcriptional regulation
基金项目:国家自然科学基金(31572613),中央高校基本科研业务费专项资金(2662015PY134)
作者单位E-mail
王济秀 华中农业大学 408197151@qq.com 
刘红 华中农业大学 liuhong59@mail.hzau.edu.cn 
张锋 华中农业大学  
王卫民 华中农业大学  
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中文摘要:
      转铁蛋白(Transferrin, TF)和转铁蛋白受体(Transferrin Receptor, TFR)在维持机体铁稳态中起着重要作用。为探索鱼类tf和tfr1a基因转录调控机制,以团头鲂(Megalobrama amblycephala)为研究对象,在公共数据库中获取tf和tfr1a基因序列,对两基因启动子候选区转录因子结合位点及CpG岛进行预测,通过PCR法克隆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,双荧光素酶报告基因检测系统进行检测。结果表明,团头鲂tf基因启动子区无CpG岛位点,而tfr1a基因启动子区有两个CpG岛位点。成功构建9个团头鲂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-1308~-1102 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,-1229~-1219 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。
英文摘要:
      Transferrin (TF) and Transferrin Receptor (TFR) play vital roles in iron homeostasis. In order to explore the transcriptional regulation mechanism of tf and tfr1a genes in Megalobrama amblycephala, the genomic sequences of tf and tfr1a were obtained from public database. Transcription factor binding sites and CpG islands in the promoter regions of tf and tfr1a genes were predicted by using bioinformatics methods. Fragments of different length of the predicted promoter region of tf and tfr1a were cloned of 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 -1308~-1102 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 -1229~-1219 bp region might contain positive regulatory transcription factor binding sites that promote tfr1a gene expression.
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