北极狐PMEL基因启动子活性及转录调控区域研究  

Activity and Regulatory Region Analysis of PMEL Gene Promoter in Vulpes lagopus

作　　者：郑晓宁 王亚琪 王瑞宁 郭敏[1] 巩元芳[1] 刘铮铸[1] 彭永东[1] 李祥龙[1,2] ZHENGXiao-Ning;WANGYa-Qi;WANGRui-Ning;GUO Min;GONG Yuan-Fang;LIU Zheng-Zhu;PENG Yong-Dong;LI Xiang-Long(College of Animal Science and Technology,Hebei Normal University of Science ＆ Technology,Qinhuangdao 066004,China;College of Animal Science and Technology,Hebei Agricultural University,Baoding 071000,China)

机构地区：[1]河北科技师范学院动物科技学院,秦皇岛066004 [2]河北农业大学动物科技学院,保定071000

出　　处：《农业生物技术学报》2018年第8期1351-1360,共10页Journal of Agricultural Biotechnology

基　　金：河北省自然基金重点项目(No.C2016407114);河北省自然科学基金项目(No.C2017407037);河北省高校创新团队领军人才培育计划项目(No.LJRC004);河北省高等学校青年拔尖人才计划项目(No.BJ2016026);国家自然科学基金(No.31272412)

摘　　要：前黑素小体蛋白基因(premelanosome protein gene,PMEL)可以直接启动黑素小体内纤维的形成,促进黑素小体合成,是调控动物毛色的主要候选基因之一。目前关于北极狐(Vulpes lagopus)毛色差异表达基因的相关研究相对较少,本研究旨在筛选调控北极狐毛色的PMEL基因的启动子核心区域及转录因子。研究通过基因组测序技术,获得了北极狐PMEL基因启动子序列,利用生物信息学方法对其核心启动子区域和转录因子结合位点进行预测;以北极狐基因组DNA为模板,扩增出该基因不同长度的启动子缺失片段,并连接至p GL3-Basic载体,将重组质粒瞬时转染到A375和293T细胞,利用基因检测仪进行活性验证。结果表明,成功构建了8个含有不同长度启动子片段的重组质粒,经双荧光素酶活性检测发现北极狐PMEL基因的-1 162/+8区域为核心启动子区域;生物信息学预测分析发现在北极狐PMEL基因的-1 162/-655区域存在5个转录因子结合位点,分别为Sp1(-966/-952)、Sp1(-912/-900)、Sp1(-750/-736)、NF-1(-712/-699)和NF-1(-682/-673);利用重叠延伸PCR技术成功构建了5个突变载体,经双荧光素酶活性检测发现该5个突变载体活性均显著下降(P<0.05),表明这5个转录因子是北极狐PMEL基因转录调控的正调控元件。本研究为探究该基因的表达调控机制提供了科学依据,并为北极狐毛皮品质分子育种和彩色毛皮材料的创制提供了思路。The premelanosome protein gene （PMEL） can directly initiate the formation of fibers in the melanin small body and promote the melanosome synthesis, which is one of the main candidate genes to regulate animal coat color. At present, there are relatively few researches on the differentially expressed genes of the coat color of the Vulpes lagopus. This research was designed to study the activity region and transcription factors of PMEL gene in Vulpes lagopus. The gene promoter sequence of Vulpes lagopus PMEL gene was obtained through transcriptome sequencing technology, and the method of bioinformatics was used to predict the core promoter region of PMEL gene and the transcription factor binding site. Using Vulpes lagopus DNA as template, promoter deletion fragments of different lengths of the gene were amplified and ligated to pGL3-Basic vector. The recombinant plasmid was transiently transfected into A375 and 293T cells, and the activity was verified by the dual-luciferase assays system. The results showed that 8 fragments with different lengths of promoter regions were amplified and cloned into the vector pGL3-Basic.The region -1 162/＋8 of PMEL gene in Vulpes lagopus was detected by dual-luciferase assays system as the core promoter region. There were some positive regulatory elements in the region from -1 162/-655. The bioinformatics prediction analysis revealed that there were 5 transcription factor binding sites in the region. Using the overlap extension PCR technology successfully constructed 5 mutation vectors. Their activity was significantly decreased by dual luciferase assay. It was suggested that these 5 transcription factors were the positive regulatory elements in Vulpes lagopus PMEL gene transcription. In this study, the core region of PMEL gene promoter were identified that 1 162/＋8. Sp1 （-966/-952）, Sp1 （-912/-900）, Sp1 （-750/-736）, NF-1 （-712/-699） and NF-1 （-682/-673） binding sites were the positive regulatory elements of Vulpes lagopus PMEL gene.This study provides a s

关 键 词：北极狐 前黑素小体蛋白基因(PMEL) 启动子 点突变 转录调控 

分 类 号：S865.23[农业科学—野生动物驯养] Q78[农业科学—畜牧兽医]