牛CART基因启动子片段重组载体构建  

Construction of Recombinant Vector for the Promoter Fragment of Bovine CART Gene

作　　者：郭跃荣 任静 李鹏飞[2] GUO Yue-rong;REN Jing;LI Peng-fei(Service Center of Modern Agricultural Industry Cluster Zone in Fanshi County,Xinzhou City,Fanshi,Shanxi 034300,China;College of Life Science,Shanxi Agricultural University,Taigu,Shanxi 030801,China)

机构地区：[1]繁峙县现代农业产业集聚区服务中心,山西繁峙034300 [2]山西农业大学生命科学学院,山西太谷030801

出　　处：《中国牛业科学》2023年第3期40-44,共5页China Cattle Science

基　　金：山西省应用基础研究计划面上项目(20210302123380);山西农业大学校地合作项目(2021HX23,2022HX010);山西省现代农业产业技术体系建设专项。

摘　　要：[目的]构建以及鉴定可卡因—苯丙胺调节转录肽(cocaine-and amphetamine regulated transcript peptides,CART)基因启动子片段双荧光素酶报告载体,为后续筛选CART基因核心启动子奠定试验基础。[方法]美国国家生物技术信息中心(national center for biotechnology information,NCBI)基因数据库(登录号:NC_037347.1)获取牛CART基因序列,经聚合酶链式反应(PCR)扩增启动子片段,经双酶切后连接pGL3-Basic载体。将pGL3-1222载体、pGL3-Control载体和pRL-TK载体转染293T细胞,检测细胞相对荧光活性。[结果]pGL3-1222载体测序结果显示与目标序列一致,证明载体构建成功;转染293T细胞后,细胞状态良好且绿色荧光分布均匀、强度适中,则转染操作无误;检测相对荧光活性显示pGL3-1222组相对荧光活性显著高于pGL3-Basic,则证明CART基因启动子片段具有活性。[结论]CART基因启动子-1200 bp—+22 bp区域双荧光素酶载体构建成功。The double luciferase reporter vector to identify the promoters of the cocaine and amphetamine regulated transcript peptides(CART)gene was constructed,which would lay the experimental foundation for the subsequent screening of the core promoter of CART gene.The bovine CART gene sequence was obtained from the national center for biotechnology information(NCBI)Gene Database(Accession No.:NC_037347.1),and the promoter fragment was amplified by polymerase chain reaction(PCR).After double enzyme digestion,the pGL3—Basic vector was ligated.The pGL3—1222 vector,pGL3—Control vector and pRL—TK vector were transfected into 293T cells,,and the relative fluorescence activity of the cells was detected.The sequencing results of pGL3—1222 vector were consistent with the target sequence,indicating the successful construction of the vector.After transfection,the 293T cells were in good condition with uniform green fluorescence distribution and moderate intensity,indicating that the transfection operation was correct.The relative fluorescence activity of pGL3—1222 group was significantly higher than that of pGL3—Basic,which proved that the CART gene promoter fragment was active.The dual luciferase vector in the-1200 bp—+22 bp region of CART gene promoter was successfully constructed.This experiment will create experimental conditions for screening the core promoter of CART gene in the future.

关 键 词：CART 启动子 双荧光素酶报告基因 

分 类 号：S823[农业科学—畜牧学]