鳜胰蛋白酶基因进化及启动子功能分析  

Analysis of the evolution and promoter function of trypsin genes in mandarin fish(Siniperca chuatsi)

作　　者：胡子俊 卢宏亮 赵晨雨 何珊[1,2] HU Zijun;LU Hongliang;ZHAO Chenyu;HE Shan(College of Fisheries,Huazhong Agricultural University,Wuhan 430070,China;Engineering Research Center of Green Development for Conventional Aquatic Biological Industry in the Yangtze River Economic Belt,Ministry of Education,Wuhan 430070,China)

机构地区：[1]华中农业大学水产学院,湖北武汉430070 [2]长江经济带大宗水生生物产业绿色发展教育部工程研究中心,湖北武汉430070

出　　处：《中国水产科学》2024年第5期524-536,共13页Journal of Fishery Sciences of China

基　　金：国家自然科学基金项目(32172951);设施渔业教育部重点实验(大连海洋大学)开放课题项目(202317).

摘　　要：为探究胰蛋白酶与鳜(Siniperca chuatsi)开口时期活鱼食性之间的联系及鳜关键胰蛋白酶基因的转录调控机制,利用生物信息学方法对鳜等11种不同食性的鱼类胰蛋白酶基因进行数量、序列特征及系统发育分析,并结合qPCR、双荧光素酶报告及胰蛋白酶活性检测分析鳜关键胰蛋白酶基因的转录调控机制,及其与鳜早期胰蛋白酶活性间的联系。结果显示,鳜基因组中共存在分布在4条染色体的6个基因座上的13个胰蛋白酶基因拷贝,多于杂食性及草食性鱼类,表明肉食性鱼类胰蛋白酶基因的扩增与其食物环境需求的高胰蛋白酶相适应。鳜在与人关键胰蛋白酶基因PRSS1直系同源的基因所处关键基因座2(包含SC7-LG17-21898及SC7-LG17-21899)和基因座3(包含SC7-LG17-21428、SC7-LG17-21429-1、SC7-LG17-21429-2、SC7-LG17-21430-1及SC7-LG17-21430-2)上出现基因扩增,且基因座3上扩增更为显著,其中鳜胰蛋白酶基因SC7-LG17-21430-2亚型的表达水平显著高于其他亚型,推测其为鳜关键胰蛋白酶基因亚型。双荧光素酶报告结果显示,构建的SC7-LG17-21430-2亚型启动区域的3个逐段缺失片段启动子活性均存在显著差异,且−593 bp~+20 bp区域启动子活性最高,推测为启动子核心区域。此外,在该核心区域,发现PDX1这一与早期胰腺发育相关且在鳜仔鱼期几乎不表达的转录因子结合位点,同时PDX1结合位点点突变后导致核心区域启动子活性显著下降。胰蛋白酶活性检测显示3 dph鳜仔鱼的胰蛋白酶活性显著性低于同时期斑马鱼(Danio rerio)仔鱼,表明转录因子pdx1的低表达可能导致了鳜胰蛋白酶基因关键亚型SC7-LG17-21430-2的低表达以及胰蛋白酶的低酶活,因而鳜通过摄食活鱼苗来补偿内源性胰蛋白酶低活性,形成自开口起只食活鱼的奇特食性,为鳜活鱼食性形成机制的深入解析提供了理论依据。This study aimed to investigate the relationship between trypsin and the feeding habits of live prey fish in Siniperca chuatsi and to elucidate the transcriptional regulatory mechanism of the key trypsin gene in S.chuatsi.In this study,we used bioinformatics methods to conduct quantitative,structural,and phylogenetic analyses of trypsin genes in 11 fish species with different feeding habits,combining qPCR,dual-luciferase reporter,and trypsin activity assays to analyze the transcriptional regulation mechanism of the key trypsin gene subtype in S.chuatsi and its correlation with trypsin activity at the first feeding stage.The results showed that a total of 13 trypsin genes distributed at six loci on four chromosomes were identified in S.chuatsi,which was more than that in omnivorous and herbivorous fish,indicating that the amplification of trypsin genes in carnivorous fish is highly adapted to the food environment.Carnivorous S.chuatsi was amplified at key gene loci 2(SC7-LG17-21898 and SC7-LG17-21899)and 3(SC7-LG17-21428,SC7-LG17-21429-1,SC7-LG17-21429-2,SC7-LG17-21430-1,and SC7-LG17-21430-2),which are homologous to the key trypsin gene PRSS1 in humans.More significant gene amplification occurred at locus 3,where SC7-LG17-21430-2 had a significantly higher expression level than the other subtypes,indicating that it was the key trypsin subtype in S.chuatsi.The dual-luciferase reporter assay results showed that the promoter activity of three constructed deleted fragments in the 5'flanking region of the SC7-LG17-21430-2 subtype had significant differences,and the region of–593 bp−+20 bp had the highest promoter activity,suggesting it as the core promoter region.Furthermore,PDX1,a transcription factor associated with early pancreatic development,was barely expressed in S.chuatsi at 3 dph and was observed in the core promoter region.Moreover,point mutations in the binding site of PDX1 result in a significant decrease in the promoter activity of the core region.The trypsin activity of S.chuatsi at 3 dph was signific

关 键 词：鳜 胰蛋白酶 食性 酶活性 启动子活性 

分 类 号：S917[农业科学—水产科学]