牛骨骼肌α-actin基因5′上游调控元件及内含子功能的初步分析  

Functional analysis in the upstream cis-regulatory elements and introns of bovine skeletal α-actin gene

作　　者：胡倩[1] 曹允考[1] 佟慧丽[1] 张伟伟[1] 赵丹丹[1] 王彬[1] 高朋[1] 严云勤[1] 

机构地区：[1]东北农业大学生命科学学院,黑龙江哈尔滨150030

出　　处：《中国兽医学报》2015年第7期1136-1142,共7页Chinese Journal of Veterinary Science

基　　金：国家转基因专项"高产优质转基因肉牛新品种培育"项目(2011ZX08007-002)

摘　　要：采用PCR定点突变分析法确定牛骨骼肌α-actin启动子-361^-462bp区域的调控元件Sp1/KLFs、ZF5F和Pax3结合位点的功能;利用基因重组技术,将内含子Ⅰ和内含子Ⅱ分别连在启动子缺失片段的上游和下游,构建双荧光素酶表达载体,使其转染牛骨骼肌卫星细胞和胎儿成纤维细胞,进行活性检测。结果显示,Sp1/KLFs降低牛骨骼肌α-actin基因启动子活性,ZF5F和Pax3均能增强启动子活性。内含子在启动子上游增强了启动子活性,在其下游降低了启动子活性,且顺式调控元件和内含子均具有肌肉特异性。结果表明,初步确定了参与牛骨骼肌α-actin基因转录调控的负顺式调控元件Sp1/KLFs,正顺式调控元件ZF5F和Pax3,发现了内含子插入启动子的位置很重要,为以后构建牛骨骼肌α-actin高活性的启动子奠定了基础。Bovine skeletal muscleα-actin mutation promoters were constructed by PCR site-specific mutagenesis to determine the functions of Sp1/KLFs,ZF5 F,Pax3binding sites located at-361--462bp;the reformed promoters containing introns were constructed by gene recombination technology.The dual luciferase expression vector were transfected to skeletal muscle satellite cells and bovine fetal fibroblast cells for activity detection.The results showed that,Sp1/KLFs could decrease promoter activity,ZF5 Fand Pax3could increase promoter activity.Introns located at the upstream of promoter increased the promoter activity,located at the downstream of promoter decreased the promoter activity.Cis-regulatory elements and introns had muscle-specificity.Analysis by PCR site-specific mutagenesis showed that cis-acting element Pax3 possessed as transcriptional repressor,Sp1/KLFs and ZF5 Fpossessed as transcriptional activators.It is found that the infulence of intron on bovine skeletalα-actin promoter activity is related to its location,which has laid a foundation for construction of bovine skeletalα-actin efficient promoter.

关 键 词：牛骨骼肌α-actin基因 启动子活性 调控元件 内含子 肌肉特异性 

分 类 号：S852.23[农业科学—基础兽医学]