银黑狐MC1R基因核心启动子区的鉴定

Identification of the Core Promoter Region of MC1R Gene in Silver Fox

【目的】确定银黑狐黑素皮质激素受体1(melanocortin 1 receptor,MC1R)基因的核心启动子区,为探究该基因的表达调控机制提供理论依据。【方法】以银黑狐基因组DNA为模板,PCR扩增获得MC1R基因5′-UTR区序列,利用3个在线生物学软件综合预测MC1R基因启动子活性区;PCR扩增获得该基因5′-UTR区不同长度的缺失片段,并克隆至pMD19-T载体,将重组质粒转染至黑色素B16细胞中,利用双荧光素酶检测技术对10个缺失片段进行荧光素酶活性检测。【结果】成功获得银黑狐MC1R基因5′-UTR区序列,软件预测显示-596/+73 bp可能为启动子活性区。双荧光素酶活性检测发现,构建的10个缺失片段的荧光素酶表达载体均具有启动子活性,其中pGL3-MC1RP8(-520/+73 bp)有较强的活性,提示其为核心启动子区,与软件预测结果基本一致。【结论】试验确定了银黑狐MC1R基因的核心启动子区为―520/+73 bp,为深入研究该基因的毛色调控机制奠定了理论基础。

【Objective】This study was aimed to screen the core promoter region of melanocortin 1 receptor(MC1R)gene of silver fox,and provide a theoretical basis for exploring the expression regulatory mechanism of MC1R gene.【Method】Using genomic DNA of silver fox as template,the sequence of the 5′-UTR region of MC1R gene was obtained by PCR amplification.The active region of the MC1R gene promoter of silver fox was predicted comprehensively using 3 online biology softwares.The deletion fragments of different lengths in 5′-UTR region of MC1R gene were amplified by PCR,which were cloned into pMD19-T vector,and the recombinant plasmid was transfected into melanin B16 cells.The luciferase activities of 10 missing fragments were detected by dual luciferase detection technique.【Result】The 5′-UTR region sequence of MC1R gene in silver fox was successfully obtained,and―596/+73 bp might be the active region of the promoter using biology softwares prediction.The detection of dual luciferase activity revealed that the constructed luciferase expression vectors of 10 different deletion fragments all had promoter activity.Among them,pGL3-MC1RP8(―520/+73 bp)had strong activity,indicating that it was the core promoter region,which was basically consistent with the software prediction results.【Conclusion】The core promoter region of MC1R gene in silver fox was determined to be―520/+73 bp,which laid a theoretical foundation for the further study on coat color regulation mechanism of MC1R gene.