摘要
为探明坝上长尾鸡的前黑素小体蛋白(Pre-melanosomal protein,Pmel)基因核心启动子区,首先构建了双荧光素酶表达载体,通过脂质体瞬时转染鸡胚成纤维细胞DF1,并利用双荧光素酶检测试剂盒进行启动活性检测。成功克隆了坝上长尾鸡pmel基因5?侧翼区片段1268bp,预测启动子区(-1200–+68)含有2个CpG岛和多种转录因子结合位点,构建了9个含有不同长度pmel基因启动子片段的表达载体及1个核心启动子区突变载体,说明鸡pmel基因启动子的核心区域为-840–+68bp,其中-840–-590bp和-525–-266bp区域为正调控区,-590–-525bp区域为负调控区,多态位点(-456、-435、-410、-374和-341)对pmel基因启动子活性有较大影响。
To explore the activity of the pmel core promoter of Bashang long-tail chickens, we constructed dual-luciferase expression vectors and transiently transfected into DF1 cells with Lipofectamine 2000. We measured the luciferase activity with the dual-luciferase detection kit. The 1 268 bp fragment in 5￠-flanking region of the pmel gene in Bashang long-tail chickens was cloned. The region from-1 200 bp to ＋68 bp included 2 CpG islands and multiple transcription factor binding sites. We constructed 9 expression vectors with different promoter regions and a mutant vector of the core promoter region of the pmel gene of Bashang long-tail chickens. The core promoter region from –840 bp to ＋68 bp was identified in the pmelgene. The region from-590 to-525 bp negatively regulated the pmel gene during the transcription process. The-840–-590 bp and-525–-266 bp regions were positive regulatory regions. The polymorphic sites（-456,-435,-410,-374 and-341） had a significant effect on the promoter activity of the pmel gene.
作者
刘小辉
周荣艳
彭永东
张传生
李兰会
李祥龙
Xiaohui Liu;Rongyan Zhou;Yongdong Peng;Chuansheng Zhang;Lanhui Li;Xianglong Li(College of Animal Science and Technology,HebeiAgricultural University,Baoding 071001,Hebei,China;College of Animal Science and Technology,Hebei Normal University of Science ＆ Technology,Qinhuangdao 066004,Hebei,China)
出处
《生物工程学报》
CAS
CSCD
北大核心
2018年第11期1750-1759,共10页
Chinese Journal of Biotechnology
基金
河北省现代农业产业技术体系蛋鸡产业创新团队项目(No.HBCT2013090206)
河北省科技计划项目(No.15226302D)资助~~
关键词
坝上长尾鸡
pmel基因
启动子
转录因子
Bashang long-tail chicken
pmel gene
promoter
transcription factor
