水貂PMEL基因启动子活性及转录调控元件分析  

Analysis of the Promoter Activity and Transcriptional Regulatory Elements of PMEL Gene in Mink (Mustela vison)

作　　者：李丽莎[1,2] 李兰会[2] 李祥龙[1,2] 彭永东[1] 

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》2017年第6期911-920,共10页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金项目(No.31501940);河北省高校创新团队领军人才培育计划项目(No.LJRC004);河北省自然自然基金项目(No.C2015204176)

摘　　要：貂皮颜色是水貂皮重要的质量性状,也是影响其价格的重要因素,前黑素小体蛋白基因(premelanosome protein gene,PMEL)作为影响动物被毛颜色变异的重要候选基因,近年来得到广泛重视。本研究旨在筛选调控水貂(Mustela vison)毛色基因PMEL启动子活性区域及转录因子结合位点,为探究该基因的表达调控机制提供理论依据,并为彩色水貂的育种和改良提供思路。以与水貂同源性较高的雪貂(M.putorius furo)PMEL基因序列(GenBank:NW_004569320.1)为参考进行引物设计,以黑色水貂毛皮基因组DNA为模板扩增PMEL基因启动子片段并克隆到pMD19载体,通过PCR和测序鉴定为阳性克隆,利用限制性内切酶KpnⅠ和HindⅢ对阳性质粒和pGL3-Basic载体同时进行双酶切,胶回收酶切产物,将二者的酶切产物通过T4连接酶连接成环状质粒,再利用PCR、双酶切和测序鉴定为阳性克隆,然后提取无内毒素质粒,利用lip2000脂质体转染到A375细胞和293T细胞,通过双荧光素酶检测系统测定相对荧光素酶活性值,并对核心启动子区的转录因子结合位点进行预测及活性验证。成功构建了6个不同长度的启动子片段,水貂PMEL基因-671/+87为核心启动子区域,从-671缺失到-477时启动子活性由最高降低到最低,表明在-671/-477可能存在正调控元件增强其启动活性。利用生物信息学软件分析该区域的转录因子结合位点,提示有十几种转录因子结合位点的存在,综合至少2个软件的预测结果,选择出Sp1(-516/-506)、Sp1(-505/-495)和Sp1(-499/-489)结合位点,对其分别构建了3个Sp1突变体,检测结果表明,-516/-506、-505/-495和-499/-489为转录的正调控区域。确定了水貂PMEL基因启动子核心区域-671/+87,预测的3个Sp1结合位点为水貂PMEL基因转录的正调控区域。本研究结果为了解水貂PMEL基因的生物学功能提供了重要信息,为进一步研究其调控水貂被毛颜色分子遗传机制提供新的�Mink（Mustela vison） coat color, which is an important quality trait of mink, is also an extremely important factor affecting the price. Premelanosome protein gene （PMEL） is an important candidate gene that affects the variation of coat color and has been paid more and more attention in recent years. The aim of this study were to screen the mink coat color gene PMEL promoter active region and transcription factors binding sites, to provide a theoretical basis for elucidating the gene expression and regulation mechanism, and to provide ideas for color mink breeding and improvement. The specific primers were designed based on the domestic ferret （M. putorius furo） PMEL gene sequence （GenBank： NW004569320.1）, which was highly homologous to the mink. The fragment in a 5＇ flanking region was amplified and cloned into the vector pMD19 vector. The positive colonies were identified and sequenced. Six fragments with different lengths of promoter regions were amplified and cloned into the vector pMD19 vector. The positive colonies and vector pGL3-Basic were simultaneously digested with 2 restriction enzymes Kpn Ⅰ and Hind Ⅲ. The digested mixture were purified and ligated with T4 ligase to get the circular plasmid. The endo-free plamids were isolated after the positive colonies, which were identified by PCR, double enzyme digestion, sequencing. 293T and A375 cells were transiently transfected with lip2000 liposome. The dual-luciferase assay system was used to measure the luciferase activity. Transcription factor binding sites in core promoter region were predicted and verified. The length of the fragment in 5＇ flanking region of PMEL gene in mink was 1 401 bp. The predicted active region in the promoter, conserved motifs and multiple transcription factor binding sites were involved in the cloned fragment in the 5＇ flanking region. Six different lengths of fragments were obtained and ligated with luciferase reported vector. When the promoter 5＇ was truncated, luciferase transcriptional activity f

关 键 词：前黑素小体蛋白基因(PMEL) 启动子 瞬时表达 点突变 Sp1结合位点 

分 类 号：S829.9[农业科学—畜牧学]