畜牧兽医学报2018，Vol.49Issue(4)：667-674,8.DOI:10.11843/j.issn.0366-6964.2018.04.002
猪Nhlh2基因启动子活性及其表达调控的初步研究
Analysis of the Promoter Activity and Expression Regulation of Porcine Nhlh2 Gene
摘要
Abstract
The present study aimed to explore the promoter activity of Nhlh2 gene and the relationship between the promoter activity and transcription factors in cells,and further provide a theoretical basis for the regulating mechanism of Nhlh2 gene in pigs.Based on the template of porcine ovarian genomic DNA,PCR was used to amplify different length deletion fragments of Nhlh2 gene promoter.These fragments were cloned into pGL3-basic to build the promoter recombinant plasmid,and then were transfected into the porcine ovarian granulosa cells.The relative luciferase activities of these plasmids were further measured by using the dual luciferase assay system.Chromatin immunoprecipitation assay (ChIP) was used to confirm the interaction between YY1,C/EBPβ and the promoter of Nhlh2.Finally,the overexpression and mutant vectors,small interfering RNA (siRNA) of YY1 and C/EBPβ were constructed,the luciferase activity was detected by dual luciferase reporter assay system.Results showed that transcription activity of P7 (-238-+129) region was the highest,-654--238 was the potential region containing the negative regulatory element,and-238--20 was the potential region containing the positive regulatory element;After the bioinformatic analysis and ChIP test,we found the binding sites of YY1 and C/EBPβ at Nhlh2 gene promoter were-101--85 and-153--140,respectively.After mutating the binding sites of YYl and C/EBPβ,the transcription activity of P7 was significantly up-regulated;The overexpression of YY1 and C/EBPβ significantly reduced the transcription activity of P7,and the transcription activity was significantly up-regulated by YY1-siRNA and C/EBPβ-siRNA.These results indicate that the core promoter region of Nhlh2 is-238-+129.Furthermore,YY1 and C/EBPβ respectively bound at-101--85 and-153--140 of Nhlh2 gene promoter to inhibit the transcription of Nhlh2 gene in pigs.关键词
猪/Nhlh2基因/启动子活性/转录调控元件Key words
pigs/Nhlh2 gene/promoter activity/transcription regulatory elements分类
农业科技引用本文复制引用
温丽娟,刘发伟,林长光,陈子韬,张哲,张豪,李加琪,袁晓龙..猪Nhlh2基因启动子活性及其表达调控的初步研究[J].畜牧兽医学报,2018,49(4):667-674,8.基金项目
国家现代农业产业技术体系(CARS-35) （CARS-35）
福建省科技重大专项(2012NZ0003-3) （2012NZ0003-3）
国家科技基础性工作专项(2014FY120800633) （2014FY120800633）
华南农业大学大学生创新训练项目 （）
