水貂PMEL基因启动子活性及转录调控元件分析

发布时间：2018-03-28 05:00

  本文选题：前黑素小体蛋白基因(PMEL)　切入点：启动子　出处：《农业生物技术学报》2017年06期

【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理论依据。
[Abstract]:Mink color is an important quality trait of mink skin and an important factor affecting its price. Premelanosome protein gene PMEL is an important candidate gene to influence the color variation of animal hair. In recent years, the aim of this study was to screen the active region of the PMEL promoter and transcription factor binding sites of the coat color gene of mink Mustela visonon, so as to provide a theoretical basis for exploring the regulation mechanism of the expression of this gene. The primer design was based on the sequence of M. putorius furo)PMEL gene (GenBank: NW004569320.1), which had high homology with mink. The promoter fragment of PMEL gene was amplified from the genomic DNA of black mink fur and cloned into pMD19 vector. The positive plasmid and pGL3-Basic vector were digested by restriction endonuclease Kpn 鈪，

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