摘要
为了构建以PiggyBac(PB)转座子为载体元件,绿色荧光蛋白(GFP)为报告基因,含UCP3基因启动子的PiggyBac转座子二元系统PB-513B-UCP3,并验证其转座活性,试验采用PCR技术扩增获得UCP3基因启动子,分别通过T4DNA连接酶与质粒PB-513B-1和pEGFP-N3连接,构建重组质粒PB-513B-1-UCP3和pEGFP-N3-UCP3-pro;用脂质体法将质粒pEGFP-N3、重组质粒pEGFP-N3-UCP3-pro和PB-513B-1-UCP3以及PiggyBac转座子二元系统分别转染至小鼠成肌细胞C2C12中,检测其在小鼠成肌细胞C2C12中的表达情况。结果表明:PCR扩增得到大小为1080bp的单一目的条带;经酶切和测序鉴定,成功构建重组质粒PB-513B-1-UCP3和pEGFP-N3-UCP3-pro,PiggyBac转座子二元系统的阳性克隆数明显多于重组质粒PB-513B-1-UCP3和pEGFP-N3-UCP3-pro的克隆数。说明试验成功构建了PiggyBac转座子介导的重组质粒PB-513B-1-UCP3及pEGFP-N3-UCP3-pro,且证实PiggyBac转座子介导的牛UCP3基因启动子在小鼠成肌细胞C2C12中具有较高的转座活性。
In order to construct PiggyBac transposon binary system PB-513B-UCP3 with PiggyBac (PB) transposon as vector component,green fluorescence protein (GFP) as reporter gene,and to verify its transposition activity,PCR method was adopted for the amplification of UCP3 promoter which was ligated to plasmids PB-513B-1 and pEGFP-N3 by T4 DNA ligase for the construction of recombinant plasmids PB-513B-1-UCP3 and pEGFP-N3-UCP3-pro,respectively. Liposome method was used for the transfection of the plasmid pEGFP -N3,recombinant plasmid pEGFP-N3-UCP3-pro,PB-513B-1-UCP3 and PiggyBac transposon binary system respectively in mouse myoblast C2C12 cells,and the expression in mouse myoblast C2C12 cells was detected. The results showed that PCR amplification resulted in a single target band with a size of 1080 bp;the recombinant plasmids PB-513B-1-UCP3 and pEGFP-N3-UCP3-pro were successfully constructed by restriction analysis and sequencing. The number of positive clones of PiggyBacPB transposon binary system was significantly higher than that of the recombinant plasmids PB-513B-1-UCP3 and pEGFP-N3-UCP3-pro. The results suggested that PiggyBac transposon mediated recombinant plasmids PB-513B-1-UCP3 and pEGFP-N3-UCP3-pro were successfully constructed,and it was confirmed that PiggyBac transposon mediated bovine UCP3 gene promoter had high transposable activity in mouse myoblast C2C12 cells.
作者
陈伟
许厚强
陈祥
杨涛
吴雨
黄明捷
卢贤君
CHEN Wei;XU Houqiang;CHEN Xiang;YANG Tao;WU Yu;HUANG Mingjie;LU Xianjun(Ministry of Education Key Laboratory of Animal Genetics,Breeding and Reproduction of Plateau Mountainous Region,Guizhou University,Guiyang 550025,China;Guizhou Province Key Laboratory of Animal Genetics,Breeding and Reproduction,Guiyang 550025,China;College of Animal Science,Guizhou University,Guiyang 550025,China)
出处
《黑龙江畜牧兽医》
CAS
北大核心
2019年第15期1-5,175,共6页
Heilongjiang Animal Science And veterinary Medicine
基金
国家自然科学基金青年科学项目(31401054)
贵州省重大专项(黔科合NY字[2013]6008号)
贵州省农业领域重点项目(黔科合NY[2015]3002)
作者简介
陈伟(1980-),女(锡伯族),高级实验师,博士,研究方向为遗传学,chenweigzu@163.com;通信作者:许厚强(1957-),男,教授,博士,研究方向为分子细胞生物学,gzdxxhq@163.com.
