猪脂肪酸结合蛋白4启动子的克隆及转录活性分析  

Cloning and Transcriptional Activity Analysis of Porcine Fatty Acid Binding Protein 4 Promoter

作　　者：刘可可 钟洁 李红强 张瑾[2] 东方阳 毋文静 LIU Keke;ZHONG Jie;LI Hongqiang;ZHANG Jin;DONGFANG Yang;WU Wenjing(College of Agronomy and Biotechnology,Hebei Normal University of Science and Technology,Qinhuangdao 066000,China;College of Biological Chemical Sciences and Engineering,Jiaxing University,Jiaxing 314000,China)

机构地区：[1]河北科技师范学院农学与生物科技学院,秦皇岛066000 [2]嘉兴学院生物与化学工程学院,嘉兴314000

出　　处：《中国细胞生物学学报》2022年第2期283-289,共7页Chinese Journal of Cell Biology

基　　金：浙江省自然科学基金(批准号:LY20C170003);国家自然科学基金(批准号:32102506、32172708);嘉兴市公益性研究计划(批准号:2021AY10046)资助的课题。

摘　　要：该研究旨在解析猪(Sus scrofa)脂肪酸结合蛋白4(FABP4)基因的转录调控机制,并获得猪脂肪组织特异性启动子元件。以6月龄大白猪为材料,通过荧光定量PCR结合Western blot证明了猪FABP4为脂肪组织特异性表达较强的基因,并分析推断其核心启动子区位于-2115 bp—-2066 bp;克隆得到从起始密码子开始到其上游的870 bp、2 914 bp、5 060 bp片段,分别构建了双荧光素酶报告载体并将其命名为pGL3-F-1 Kb/3 Kb/5 Kb,分析发现pGL3-F-3 Kb在脂肪细胞中的活性最高,而在非脂肪细胞中无活性;通过与脂代谢密切相关转录因子(C/EBPα、KLF4和C/EBPβ)共转染发现,C/EBPα正调控FABP4转录活性,而KLF4和C/EBPβ负调控FABP4的转录活性。研究表明,猪FABP4基因的起始密码子上游3 Kb片段具有脂肪组织特异启动子活性,可以用于构建脂肪组织特异性基因修饰猪。The purpose of this study was to analyze the transcriptional regulation mechanism of porcine FABP4 (fatty acid binding protein 4) gene and obtain porcine adipose tissue specific promoter elements.Using 6-month-old large-white pigs as materials,it was proved that porcine FABP4 was a gene specifically expressed in adipose tissue by fluorescence quantitative PCR and Western blot analysis.The core promoter region of FABP4 was located between -2115 bp—–2066 bp.The truncated fragments 870 bp,2 914 bp,5 060 bp upstream of the start codon were cloned,and dual luciferase reporter vectors were constructed naming as pGL3-F-1 Kb/3 Kb/5 Kb.It was found that pGL3-F-3 Kb had the highest activity in adipocytes,but had no activity in non-adipocytes.By co-transfection of transcription factors related to lipid metabolism,it was found that C/EBPα positively regulated FABP4 transcriptional activity,while KLF4 and C/EBPβ negatively regulated the transcriptional activity of FABP4.This study showed that the 3 Kb fragment upstream of the start codon of porcine FABP4 gene had adipose tissue-specific promoter activity,which could be used to construct adipose tissue-specific gene modified pigs.

关 键 词：猪 脂肪沉积 启动子 脂肪组织特异性 转录调控 

分 类 号：S828[农业科学—畜牧学]