日本沼虾过氧化物还原酶基因的克隆及其表达分析  

Molecular cloning and gene expression of peroxiredoxin(Prx) in oriental river pawn(Macrobrachium nipponense) in response to environmental hypoxia and reoxygenation

作　　者：孙盛明[1] 戈贤平[1,2] 傅洪拓[1,2] 朱健[1] 张世勇[2] 

机构地区：[1]中国水产科学研究院淡水渔业研究中心、农业部淡水渔业与种质资源利用重点实验室,江苏无锡214081 [2]南京农业大学无锡渔业学院,江苏无锡214081

出　　处：《中国水产科学》2014年第3期474-483,共10页Journal of Fishery Sciences of China

基　　金：国家“十二五”科技支撑计划项目(2012BAD26B04,2012BAD25B07)

摘　　要：利用 cDNA 末端快速克隆方法获得了青虾（Macrobrachium nipponense）的过氧化物还原酶基因（Prx）全长cDNA序列。该基因cDNA全长878 bp,包括72 bp的5′末端非翻译区,594 bp的开放阅读框（ORF）,212 bp的3′末端非翻译区,开放阅读框编码198个氨基酸。氨基酸相似度比对显示,所分离的青虾过氧化物还原酶基因包括两个半胱氨酸残基的区域“FYPLDFTFVCPTEI”和“GEVCPA”。系统进化树分析表明,青虾过氧化物还原酶基因与南美白对虾（Litopenaeus vannamei）过氧化物还原酶聚在一起,具有最近的亲缘关系。荧光定量 PCR 检测显示,过氧化物还原酶基因在青虾不同组织中均有表达,其表达量由低到高依次为肠道、心脏、卵巢、肌肉、鳃、肝胰腺。使用荧光定量PCR检测青虾在低氧胁迫和复氧条件下肝胰腺中的过氧化物还原酶基因mRNA的时空表达情况,结果显示,与对照组相比,实验组青虾过氧化物还原酶在肝胰腺和鳃组织中的表达量分别在低氧胁迫12~24 h 和复氧6 h出现了3次明显上调,由此推测过氧化物还原酶基因参与低氧应激分子过程。本研究结果可为进一步了解青虾低氧应激分子机制提供参考。Peroxiredoxins （Prxs） are a family of ubiquitous proteins that minimize the harmful effects of oxidative stress by catalyzing the reduction of hydrogen peroxide （H2O2） and organic hydroperoxides to less harmful forms. A full-length cDNA corresponding to a 2-Cys Prx gene was isolated from the oriental river pawn Macrobrachium nippo-nense and designated as MnPrx （GenBank accession no. KC866353）. The full-length cDNA was 998 bp, containing a 72 bp 5′untranslated region （UTR）, a 212 bp 3′UTR with a poly （A） tail, and a 594 bp open reading frame （ORF） en-coding a polypeptide of 198 amino acids with a molecular mass of 22.131 Da. Like other 2-Cys Prxs, the MnPrx protein possesses two conserved cysteine residues that play an essential role in the antioxidant activity of this proteins. The MnPrx protein, as deduced from the cDNA sequence, has a high level （87%） of sequence similarity to the 2-Cys Prxs from Pacific white shrimp Litopenaeus vannamei. Quantitative real-time RT-PCR analysis revealed that the Prx gene was expressed in the ovary, hepatopancreas, muscle, heart, testis, and intestines, with expression being highest in the hepatopancreas. MnPrx mRNA expression was significantly higher in the hepatopancreas and gill of prawns exposed to hypoxia （12 and 24 h） and reoxygenation （6 h） stress than in the control group. This suggests a possible role in alleviat-ing oxidative stress by increasing Prx mRNA expression in response to environmental hypoxia and reoxygenation.

关 键 词：日本沼虾 过氧化物还原酶 基因克隆 低氧胁迫 定量PCR 

分 类 号：S917[农业科学—水产科学]