在不同养殖背景下鳜mc1r组织表达差异和启动子转录调控分析  

TISSUE EXPRESSION DIFFERENCES OF MELANOCORTIN 1 RECEPTOR(MC1R) IN DIFFERENT TANK BACKGROUNDS AND PROMOTERREGULATORY ELEMENTS ANALYSIS OF CHINESEPERCH (SINIPERCA CHUATSI)

作　　者：贾晓丹 梁旭方[1,2] 何珊 JIA Xiao-Dan;LIANG Xu-Fang;HE Shan(College of Fisheries,Chinese Perch Research Center,Huazhong Agricultural University,Wuhan 430070,China;Key Lab of Freshwater Animal Breeding,Ministry of Agriculture and Rural Affair/Key Lab of Agricultural Animal Genetics,Breeding and Reproduction,Ministry of Education,Wuhan 430070,China)

机构地区：[1]华中农业大学水产学院,华中农业大学鳜鱼研究中心,武汉430070 [2]农业农村部淡水生物繁育重点实验室/农业动物遗传育种与繁殖教育部重点实验室,武汉430070

出　　处：《水生生物学报》2024年第1期149-156,共8页Acta Hydrobiologica Sinica

基　　金：国家自然科学基金(31972809)资助。

摘　　要：为探究鳜(Siniperca chuatsi)黑素皮质素1受体基因mc1r在不同养殖背景下的组织表达差异及转录调控机制,研究以在黑白养殖背景下产生的深、浅色体表鳜为研究对象,通过实时荧光定量PCR测定mc1r基因的组织表达差异;其次以鳜基因组DNA为模板,通过PCR扩增mc1r基因5′侧翼区不同长度的缺失片段并克隆至pGL3-Basic载体,之后将重组质粒转染到HEK 293T细胞,检测双荧光素酶的相对活性并预测其存在潜在的转录因子。结果显示,黑色背景养殖所产生的深色体表鳜与野生型更为相似;RT-qPCR检测显示深色体表鳜mc1r在背部皮肤和腹部皮肤表达量最高,而浅色体表鳜脑中表达量最高;双荧光素酶活性检测发现,构建的5个缺失片段启动子活性之间存在显著差异,且−159∽−+292启动子活性最高,推测其为mc1r核心启动子区域,并且通过在线软件预测出存在MITF、SP1等转录因子。研究克隆了鳜mc1r基因启动子区域并进行了转录活性分析,并且对潜在的转录因子进行了预测,为鱼类mc1r基因分子机制表达调控及表皮颜色可控性研究提供了理论依据。In order to explore the tissue expression difference and transcriptional regulation mechanism of mc1r gene in Chinese perch(Siniperca siniperca)under different culture backgrounds,the dark and light surface Chinese perch under black and white tank backgrounds were used as the research objects,and the tissue expression differences of mc1r gene were determined by Quantitative real-time PCR(qRT-PCR).In addition,the researchers amplified different lengths of the promoter deletion fragments at the 5′flanking region of the mc1r gene by PCR,and cloned them into pGL3-Basic plasmids,then transfected the recombinant plasmids into human embryonic kidney(HEK293T)cells and measured the relative activity of dual luciferase to predict its potential transcription factors.The results showed that the Chinese perch cultured in the black background had a deeper body color similar to that of the wild Chinese perch.The expression of mc1r gene in dark-colored Chinese perch was the highest in the dorsal and ventral skin,while that of light-colored Chinese perch was the highest in brain.The double luciferase activity detection revealed that the promoter activities of the constructed five deleted fragments were significantly different,with the−159∽+292 bp promoter fragment had the highest activity,which was speculated to be the core region of the mc1r promoter.The online software predicted the existence of transcription factors such as MITF and SP1 in the core promoter region.In this study,the promoter region of the Chinese perch mc1r gene was cloned for the first time and the transcriptional activity was analyzed,and then the potential transcription factors were predicted.This study provides a theoretical basis for further research on the expression and regulation of the molecular mechanism of the mc1r gene in fish.

关 键 词：MC1R 组织表达 启动子活性 转录因子 鳜 

分 类 号：Q344.1[生物学—遗传学]