畜牧兽医学报2017，Vol.48Issue(5)：826-835,10.DOI:10.11843/j.issn.0366-6964.2017.05.006
山羊PMEL基因启动子活性及转录调控元件分析
Analysis of the Promoter Activity and Transcriptional Regulatory Elements of Goat PMEL Gene
摘要
Abstract
The research was designed to screen the promoter activity region and transcription factors of goat PMEL gene,and to provide a theoretical basis for exploring the mechanism of expression regulation of PMEL gene and provide ideas for breeding and improvement of colored goats.Using goat genomic DNA as template,PCR was used to amplify PMEL gene promoter sequence of different length fragments,and the fragment was cloned into pGL3-basic vector.The recombinant plasmid was transfected into 293T and A375 cells,the relative luciferase activity was measured by dual luciferase assay system,and the transcription factor binding sites in the core promoter region were predicted by bioinformatics.The transcription factor binding sites on the pGL3-327 plasmid were mutated by overlap-extension PCR,and the activity of mutants was verified.The results showed that 7 promoter fragments of different lengths were constructed successfully,and 6 of them had obvious promoter activity.-251/+76 region was detected by dual luciferase activity as the core promoter region.By comparing the activity of PMEL gene promoter sequence of different length fragments,the deletion of-251/-62 region made the promoter activity from the highest to none,which indicated that the region had important influence on the transcriptional regulation of goat PMEL gene.Bioinformatics analysis found that the-251/-62 region had 5 transcription factor binding sites,the 5 mutation vectors were constructed by point mutation,their activity were significantly decreased by double luciferase assay.It is suggested that these 5 transcription factors were the positive regulatory elements in goat PMEL gene transcription.The results indicate that the core promoter of goat PMEL gene is-251/+76 region,NF-1 (-206/-197),Sp1 (-186/-174),Sp1 (-151/-139),CREB (-91/-82) and Sp1 (-82/-71) binding sites are positive regulatory elements of goat PMEL gene.关键词
PMEL基因/启动子/瞬时表达/点突变/转录调控元件Key words
PMEL gene/promoter/transient expression/point mutation/transcriptional regulatory elements分类
农业科技引用本文复制引用
李丽莎,彭永东,郑晓宁,李祥龙..山羊PMEL基因启动子活性及转录调控元件分析[J].畜牧兽医学报,2017,48(5):826-835,10.基金项目
河北省高校创新团队领军人才培育计划项目(LJRC004) （LJRC004）
河北省应用基础研究计划重点基础研究项目(15962901D) （15962901D）
河北省高等学校青年拔尖人才计划项目(BJ2016026) （BJ2016026）
秦皇岛市科技支撑计划项目(201502A058) （201502A058）
