猪CYP11A1基因编码区扩增及核心启动子区的鉴定与分析  

Amplification of porcine CYP11A1 gene coding region and identification and analysis of core promoter region

作　　者：曾强 关钦泽 王思琪 李齐发[1] 杜星[1] ZENG Qiang;GUAN Qinze;WANG Siqi;LI Qifa;DU Xing(College of Animal Science and Technology,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学动物科技学院,江苏南京210095

出　　处：《南京农业大学学报》2022年第4期743-751,共9页Journal of Nanjing Agricultural University

基　　金：江苏省自然科学基金项目(BK20180524);中国博士后科学基金项目(2018M632321)。

摘　　要：[目的]本文旨在扩增猪CYP 11A1基因编码区,鉴定并分析其核心启动子区,探究猪CYP 11A1基因的转录调控机制。[方法]通过基因克隆测序技术获得猪CYP 11A1基因编码区全序列并对其序列特征进行分析;利用荧光素酶活性鉴定猪CYP 11A1基因核心启动子区,再通过生物信息学分析猪CYP 11A1基因核心启动子区潜在的甲基化位点与转录因子结合位点;利用Western blot、RT-qPCR以及染色质免疫沉淀(ChIP)等分子生物学手段解析转录因子NR2C1对猪CYP 11A1基因的转录调控功能。[结果]猪CYP 11A1基因的编码区序列全长为1563 bp,在哺乳动物的进化过程中高度保守;猪CYP 11A1基因的核心启动子区为-398~-108 bp(转录起始位点为+1),序列特征分析发现猪CYP 11A1基因核心启动子区包含多个潜在的转录因子的结合元件,包括NR2C1、SOX10、NFIX和ELF5等;另外,转录因子NR2C1可促进猪CYP 11A1基因核心启动子区活性,同时显著上调体外培养的猪卵巢颗粒细胞中CYP 11A1基因的表达。[结论]哺乳动物CYP 11A1基因在进化过程中高度保守,转录因子NR2C1能够促进猪卵巢颗粒细胞中CYP 11A1基因的转录。[Objectives]This paper aimed to amplify the coding region of the pig CYP 11A1 gene,identify and analyze its core promoter region,and explore the transcription regulation mechanism of the pig CYP 11A1 gene.[Methods]The full sequence of the pig CYP 11A1 gene coding region was obtained through gene cloning and amplification technology,and its sequence characteristics was analyzed.Luciferase activity test was used to identify the core promoter region,and bioinformatics was used to identify the methylation sites and potential transcription factor binding sites of CYP 11A1.Western blot,RT-qPCR and Chromatin immunoprecipitation(ChIP)were used to analyze the regulatory function of transcription factor NR2C1 on porcine CYP 11A1 gene transcription.[Results]The total length of the coding region of the pig CYP 11A1 gene was 1563 bp,which was highly conserved in the evolution of mammals.The core promoter region of pig CYP 11A1 gene was located at-398 to-108 bp(transcription start site was+1).Sequence analysis revealed that there were many important transcription factor binding elements on it,including NR2C1,SOX10,NFIX and ELF5,etc.The activity of the core promoter region of the CYP 11A1 gene was promoted by NR2C1,and the expression of the CYP 11A1 gene in porcine ovarian granulosa cells cultured in vitro also was significantly up-regulated.[Conclusions]Our findings indicated that the mammalian CYP 11A1 gene was highly conserved in the evolutionary process and its transcription in porcine ovarian granulosa cells was promoted by the transcription factor NR2C1.

关 键 词：猪 CYP11A1基因 核心启动子 鉴定 分析 NR2C1 转录调控 

分 类 号：S813.52[农业科学—畜牧学]