虎皮鱼热激蛋白PtHsp70基因序列与低温表达分析  

Sequence Cloning and Cold-induced Tissue-specific Expression Analysis of PtHsp70 in Tiger Barb Puntius tetrazona

作　　者：刘丽丽 王晓雯[1] 朱建亚 张蓉 朱华[1] 田照辉[1] LIU Lili;WANG Xiaowen;ZHU Jianya;ZHANG Rong;ZHU Hua;TIAN Zhaohui(Beijing Key Laboratory of Fishery Biotechnology,Beijing Fisheries Research Institute,Beijing 100068,China)

机构地区：[1]北京市水产科学研究所渔业生物技术北京市重点实验室

出　　处：《水产科学》2019年第6期774-782,共9页Fisheries Science

基　　金：国家大宗淡水鱼类产业技术体系项目(CARS-45-31);北京市自然科学基金资助项目(6194032);北京市博士后工作经费资助项目(2018ZZ058);现代农业产业技术体系北京市观赏鱼创新团队项目(BAIC03-2019);(北京市财政)观赏水族养殖关键技术研究项目

摘　　要：通过同源克隆和cDNA末端快速克隆技术扩增获得狭温热带鱼类虎皮鱼热激蛋白70(PtHsp70)基因的cDNA序列,BLAST比对分析基因序列同源性,预测其编码氨基酸序列和结构,实时荧光定量PCR技术分析虎皮鱼PtHsp70基因组织特异性表达谱和低温胁迫下PtHsp70基因的表达模式。研究结果表明,PtHsp70基因cDNA序列全长2317 bp,其中5′非编码区120 bp,3′非编码区266 bp,编码区1932 bp,对应的开放阅读框为121~2052 bp,预测编码643个氨基酸。PtHsp70基因mRNA在成年虎皮鱼不同组织中表达水平依次为肝脏>肌肉>鳃>脑,其中肝脏PtHsp70基因本底表达水平显著高于其他组织(P<0.01),表明其在肝脏中具有重要的生物学功能。而随温度降低,肝脏PtHsp70基因表达水平呈先降后升的趋势,表明肝脏PtHsp70基因参与虎皮鱼应对低温胁迫的过程;随温度降低,虎皮鱼脑、鳃和肌肉组织PtHsp70基因mRNA水平显著上调(P<0.01),说明低温胁迫诱导PtHsp70基因mRNA表达。本研究克隆并鉴定了PtHsp70基因序列和组织表达谱,分析了低温胁迫下PtHsp70基因表达模式,研究结果显示,狭温热带鱼类虎皮鱼的PtHsp70基因具有组织特异性和温度诱导型表达特征。研究发现,PtHsp70基因在响应低温胁迫过程中具有重要作用,具有作为虎皮鱼低温胁迫分子标志物的潜能。cDNA sequence of heat shock protein 70 gene(PtHsp70)was cloned in tiger barb(Puntius tetrazona)by homologous clone strategy and rapid-amplification of cDNA ends(RACE)technology,and then the sequence homology was analyzed and the encoded amino acid sequence was predicted.Particularly,the expression analysis of PtHsp70 in various tissues and expression of PtHsp70 under gradient cold temperature stress were conducted by quantitative real-time PCR(qPCR).It was found that the full length of PtHsp70 cDNA was 2317 bp,including 120 bp in the 5′untranslated region,266 bp in the 3′untranslated region,and 1932 bp in coding region,with open reading frame from 121 to 2052 bp and a predicted sequence of 643 amino acids.The PtHsp70 coding sequence was highly similar to the Hsp70 sequence of fish in Cyprinidae(>90%),especially to zebrafish homologue with the identity of 94%,indicating that PtHsp70 was highly conservative in the process of evolution.The descending order of tissue-specific expression levels of PtHsp70 mRNA in adult tiger barb was described as liver>muscle>gill>brain,with the minimal expression level of PtHsp70 mRNA in brain tissues,and the relative PtHsp70 mRNA expression levels of 2.1 times in gill,19.8 times in muscle and 78.8 times in liver to that in brain(P<0.01),indicating that PtHsp70 gene played a vital biological function in the liver.The fluctuant mRNA levels of liver PtHsp70 under different temperatures indicated that the expression of liver PtHsp70 gene was induced by cold stress.There were significantly upregulated PtHsp70 mRNA levels in the brain,gills and muscle of the fish exposed to gradient cold temperature stress,15.3 times at 15℃and 18.6 times at 13℃as that in the brain in the control group(P<0.01).The PtHsp70 mRNA levels in the gill were 2.8 times at 15℃and 5.3 times at 13℃as in the gill in the control group(P<0.01),in the muscle 8.8 times at 19℃,10.9 times at 15℃,and 15 times at 13℃as in the muscle in the control group(P<0.01).The expression levels of PtHsp70 gene in the bra

关 键 词：狭温热带鱼 PtHsp70 诱导型表达 组织特异性表达 低温胁迫 

分 类 号：Q786[生物学—分子生物学]