蓝狐MITF-M基因序列扩增及生物信息学分析  

Sequence Amplification and Bioinformatics Analysis of MITF-M Gene in Blue Fox(Alopex lagopus)

作　　者：郭敏[1] 张东林 刘艳军 彭永东[1] 王建涛 付志新[1] 董淑珍[1] 刘铮铸[1] 巩元芳[1] 李祥龙[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]河北省唐山市畜牧工作站,唐山063000

出　　处：《中国畜牧兽医》2018年第1期47-56,共10页China Animal Husbandry & Veterinary Medicine

基　　金：河北省自然科学基金项目(C2017407040;C2016407114)

摘　　要：试验旨在研究蓝狐MITF-M基因核心启动子活性区、转录因子结合位点、基因编码蛋白结构及功能,为探究该基因的表达调控机制提供理论依据。从成年蓝狐背部皮肤组织中采集1cm×1cm样品,采用RT-PCR扩增及测序技术首次获得MITF-M基因序列3 880bp(包括5′侧翼区2 262bp、CDS区1 260bp、3′侧翼区358bp),编码419个氨基酸残基。生物信息学分析结果显示,蓝狐MITF-M基因5′侧翼区存在潜在的启动子区域(-86^-336bp),且发现TATA框、CAAT框、GC框和CRE等调控元件及CREB、LSF和Sp1等多种转录因子。MITFM基因编码的蛋白是定位于细胞核和细胞质的不稳定、可溶性亲水蛋白质。亚细胞定位结果提示该蛋白在细胞核、细胞质和线粒体的概率较高,分别为65.2%、17.4%和8.7%。DNAMAN软件对MITF-M基因编码蛋白的二级结构预测发现,该蛋白由α螺旋(33.66%)、β折叠(16.66%)和无规卷曲(49.68%)组成,且不存在跨膜区域和信号肽。蓝狐与其他物种MITF-M基因编码的氨基酸序列同源性对比和系统进化树分析均提示蓝狐与犬的遗传亲缘关系最近,与哺乳动物和禽类均具有较高的同源性(>89.1%),说明MITF-M基因编码区在物种进化过程中较为保守。本研究为深入研究MITF-M基因调控狐狸被毛颜色的分子遗传机制奠定了理论基础。In this study,to investigate the core promoter activity region,transcription factor binding site,the structure and function of encoding protein and the expression and regulation mechanism of Blue fox MITF-M gene,the skin tissue samples（1 cm ×1 cm）was collected from adult Blue fox,the partial sequence of MITF-M gene was obtained by RT-PCR and sequencing technique for the first time.The length of the Blue fox MITF-M gene was 3 880 bp（including2 262 bp 5′flanking region,1 260 bp CDS region and 358 bp 3′flanking region）,and encoding419 amino acid residues.The bioinformatics analysis showed that there were potential promoter regions at-86 to-336 bp in the 5′flanking region of MITF-M gene,and some regulatory elements（TATA box,CAAT box,GC box and CRE）and transcription factors（CREB,LSF and Sp1）were found.The protein encoded by MITF-M gene was unstable and hydrophilic which mainly located in nucleus and cytoplasm.Subcellular localization results showed that the proteinhad a higher probability in nucleus（65.2%）,cytoplasm（17.4%）and mitochondrion（8.7%）,the results of secondary structure prediction by DNAMAN software found that the protein was consist ofαhelix（33.66%）,βsheet（16.66%）and random coil（49.68%）,and there was no transmembrane region and signal peptide.Multiple sequence alignment and phylogenetic tree analysis showed that Blue fox had the closet relationship with dog and a high homology with mammal and poultry（89.1%）indicated that the MITF-M gene coding region was conservative in the course of evolution.This study laid the theoretical foundation for the in-depth study of the molecular genetic mechanism of the MITF-M gene regulating the hair color of the fox.

关 键 词：蓝狐 MITF-M基因 5′侧翼区 编码区 生物信息学分析 

分 类 号：Q786[生物学—分子生物学]