摘要
旨在构建脂肪生成素基因(adipohenin,ADIG)在牛不同组织中的表达谱,鉴定其核心启动子区域及关键转录因子,以期阐明其转录调控的分子机制。本研究采集3头((24±2)月龄)健康南阳公牛的心、肝、脾、肺、肾、肌肉和皮下脂肪组织,通过逆转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)检测各组织中ADIG基因的相对表达量;克隆获得牛ADIG基因上游启动子区2245 bp序列,构建6个pGL3-WT,与pRL-TK共转染293T细胞、原代脂肪细胞和3T3-L1细胞,进一步利用生物信息学预测其核心启动子区的关键转录因子。结果发现,ADIG基因在南阳牛肺(P<0.05)、肾(P<0.05)和皮下脂肪(P<0.05)组织中高表达;克隆得到2245 bp及其6段缺失片段序列的ADIG基因的启动子序列,成功构建了pADIG-1621/+59、pADIG-1288/+59、pADIG-850/+59、pADIG-589/+59、pADIG-321/+59和pADIG-79/+59双荧光素酶报告载体,并检测到牛ADIG基因启动子核心区域在-321/-79;生物信息学初步预测牛ADIG基因核心区域含有C/EBPα、PPARδ、CREB、STAT5和AP-2α等转录因子结合位点。综上,牛ADIG基因在皮下脂肪组织中表达量最高,其启动子核心区位于-321/-79,C/EBPα、PPARδ、CREB、STAT5和AP-2α等转录因子对ADIG基因转录活性具有调控作用。本研究结果为探究ADIG基因在肉牛分子水平上的改良培育提供理论依据。
The aim of this study was to construct the expression profile of adipohenin(ADIG)in different tissues of cattle and to identify its core promoter region and key transcription factors in order to elucidate the molecular mechanism of its transcriptional regulation.Heart,liver,spleen,lung,kidney,muscle and subcutaneous adipose tissues were collected from 3((24±2)months old)healthy Nanyang bulls,and the relative expression levels of ADIG gene in each tissue was detected by reverse transcription-polymerase chain reaction(RT-PCR).The 2245 bp sequence of the upstream promoter region of ADIG gene was cloned,6 pGL3-WT were constructed and cotransfected with pRL-TK into 293T cells,primary adipocytes and 3T3-L1 cells,and further bioinformatics was used to predict the key transcription factors of their core promoter region.The results showed that ADIG gene was highly expressed in lung(P<0.05),kidney(P<0.05)and subcutaneous fat(P<0.05)tissues of Nanyang cattle;the promoter sequence of ADIG gene with 2245 bp and its 6 missing fragment sequences were cloned.pADIG-1621/+59,pADIG-1288/+59,pADIG-850/+59,pADIG-589/+59,pADIG-321/+59 and pADIG-79/+59 dual luciferase reporter vectors were successfully constructed,and the core region of bovine ADIG gene promoter was detected at-321/-79.Bioinformatics preliminarily predicted that the core region of bovine ADIG gene contained C/EBPα,PPARδ,CREB,STAT5 and AP-2αand other transcription factor binding sites.In summary,the bovine ADIG gene is most highly expressed in subcutaneous adipose tissue with a promoter core region located at-321/-79.Transcription factors such as C/EBPα,PPARδ,CREB,STAT5 and AP-2αhave regulatory roles on the transcriptional activity of the ADIG gene.The results of this study provide a theoretical basis for exploring the improved breeding of ADIG gene at the molecular level of beef cattle.
作者
唐林
魏大为
汪书哲
雷召雄
高晓茜
王兴平
马燕芬
马云
TANG Lin;WEI Dawei;WANG Shuzhe;LEI Zhaoxiong;GAO Xiaoqian;WANG Xingping;MA Yanfen;MA Yun(Ningxia Key Laboratory of Ruminant Molecular and Cellular Breeding, School of Agriculture, Ningxia University, Yinchuan 750021, China)
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2022年第3期722-730,共9页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家自然科学基金(32072720,31672403)
宁夏回族自治区重点研发引才专项(2019YCZX0068)
宁夏回族自治区科技创新团队计划项目(03010360052)
现代农业产业技术体系建设项目(CARS-36)。
