北极狐MITF-M基因启动子活性及转录调控元件的分析  

Analysis of promoter activity and transcriptional regulatory elements of MITF-M gene in Arctic fox (Alopex lagopus)

作　　者：郭敏[1] 李寒妹 王瑞宁 郑晓宁 刘铮铸[1] 彭永东[1] 巩元芳[1] 李祥龙[1] GUO Min;LI Han-mei;WANG Rui-ning;ZHENG Xiao-ning;LIU Zheng-zhu;PENG Yong-dong;GONG Yuan-fang;LI Xiang-long(Key Laboratory of Mining and Innovation of Charac-teristic Animal Germplasm Resources in Hebei Province,College of Animal Science and Technol-ogy,Hebei Normal University of Science&Technology,Qinhuangdao,Hebei 066004,China;College of Animal Science and Technology,Hebei Agricultural University,Baoding,Hebei 071000,China)

机构地区：[1]河北科技师范学院动物科技学院河北省特色动物种质资源挖掘与创新重点实验室,河北秦皇岛066004 [2]河北农业大学动物科技学院,河北保定071000

出　　处：《中国兽医学报》2020年第10期2066-2073,共8页Chinese Journal of Veterinary Science

基　　金：河北省自然科学基金资助项目(C2017407040,C2016407114)。

摘　　要：为了探究MITF-M基因对狐狸毛色的遗传调控机制,利用PCR扩增技术对北极狐MITF-M基因启动子区6个缺失片段(2 279,1 738,1 313,1 040,524和236 bp)进行扩增,利用双荧光素酶报告基因检测技术对该基因启动子表达载体转染细胞(A375和293T)的相对活性进行检测,通过在线软件对核心启动子区转录因子结合位点进行预测,利用重叠延伸PCR和双荧光素酶报告系统检测技术对预测出来的转录因子结合位点进行点突变活性分析。结果表明,北极狐MITF-M基因启动子区524 bp(-510 bp/+13 bp)处活性最高,预测发现-510 bp/-222 bp区域存在CREB(-312 bp/-319 bp)、Sp1(-295 bp/-307 bp)和Sp1(-249 bp/-262 bp)转录因子结合位点,这3个转录因子结合位点突变后能使北极狐MITF-M基因的启动子活性上升。综上,北极狐MITF-M基因核心启动子区在-510 bp/-222 bp区域,转录因子结合位点CREB(-312 bp/-319 bp)、Sp1(-295 bp/-307 bp)和Sp1(-249 bp/-262 bp)对北极狐MITF-M基因的转录激活起到一定的调控作用。In this study,to explore the genetic regulation mechanism of MITF-M gene on fox coat color,6 deletion fragments of the Arctic fox MITF-M gene promoter region(2 279,1 738,1 313,1 040,524 and 236 bp) were amplified by PCR amplification technology.The relative activity of the gene promoter vector transfected cells(A375 and 293 T) was detected by dual luciferase reporter gene detection technology.The transcription factor binding site of the core promoter region was predicted by online software,and then the point mutation activity of the predicted transcription factor binding site was analyzed by overlap extension PCR and dual luciferase reporter system detection technology.The results showed that it had the highest activity at 524 bp(-510 bp/+13 bp) of the promoter region for Arctic fox MITF-M gene,and it is predicted that there were three transcription factor binding sites:CREB(-312 bp/-319 bp),Sp1(-295 bp/-307 bp) and Sp1(-249 bp/-262 bp).After point mutation and dual luciferase reporter system detection,it was found that the mutation of the above three transcription factor binding sites could increase the promoter activity of the Arctic fox MITF-M gene.In summary,the core promoter region of the Arctic fox MITF-M gene was in the-510 bp/-222 bp region.The transcription factor binding sites CREB(-312 bp/-319 bp),Sp1(-295 bp/-307 bp)and Sp1(-249 bp/-262 bp)had certain regulatory effects on the transcriptional activation of Arctic fox MITF-M gene.

关 键 词：北极狐 MITF-M基因 启动子活性 转录调控元件 

分 类 号：S852.23[农业科学—基础兽医学]