尼罗罗非鱼p38MAPK基因克隆与表达分析

为了探究p38MAPK与尼罗罗非鱼(Oreochromis niloticus)免疫功能的相关性,本实验运用cDNA末端快速扩增技术(rapid amplification of cDNA ends,RACE)获得尼罗罗非鱼埃及品系ntp38MAPK的cDNA全长序列,结果显示,该序列全长1789 bp,开放阅读框(Open Reading Frame,ORF)长1086 bp,5′非编码区(Untranslated Region,UTR)长342 bp,3′UTR为361 bp。ORF编码361个氨基酸,预测分子量为41.598 kD,理论等电点为5.10。序列含有p38家族典型保守的Thr-Gly-Tyr(TGY)双磷酸化位点以及紧密相连的底物结合位点Ala-Thr-Arg-Trp(ATRW)。同源性以及系统进化树分析结果显示,尼罗罗非鱼的p38MAPK与大黄鱼(Larimichthys crocea)和鲈(Dicentrarchus labrax)的相似性最高。采用qRT-PCR的方法研究了ntp38MAPK在各组织以及无乳链球菌感染过程中的表达差异情况,结果显示,ntp38MAPK在各组织均有表达,其中在肌肉的表达量最高,心脏、肝脏、脾脏次之,头肾中的表达量最低。无乳链球菌感染过程中,脾脏、头肾中ntp38MAPK的表达量在2 h开始上调,肝脏中4 h开始上调,8 h后肝脏、脾脏和头肾中的表达量都有所下降,24~72 h趋于平稳,表明ntp38MAPK在尼罗罗非鱼抵抗链球菌侵染过程中发挥重要作用。

Molecular cloning and expression analysis of p38MAPK in Oreochromis niloticus

The full-length cDNA sequence of Egyptian strain of Oreochromis niloticus(NE)ntp38MAPK gene was obtained by rapid amplification of cDNA ends(RACE)technology to explore the correlation between ntp38MAPK and the immune function of O.niloticus.The full-length cDNA sequence was 1789 bp,including a 342 bp 5′untranslated region(UTR),a 361 bp 3′UTR,and one 816 bp open reading frame(ORF).It encoded 361 amino acids with a predicted molecular weight of 41.598 kD and the theoretical isoelectric point of 5.10,containing the Thr-Gly-Tyr(TGY)double phosphorylation site which was typically conserved in p38 family and the closely linked substrate-binding site Ala-Thr-Arg-Trp(ATRW).Through multiple sequence alignment and phylogenetic tree analysis,it concluded that ntp38MAPK showed the highest similarity with Larimichthys crocea and Dicentrarchus labrax.Analysis of the tissue expression pattern of ntp38MAPK by quantitative real-time PCR showed that ntp38MAPK mRNA was expressed in all tissues.The highest levels were found in muscle,followed by heart,liver,spleen.The lowest levels was in head kidney.The liver,spleen and head kidney were sampled at different times after exposure to Streptococcus agalactiae for the expression of ntp38MAPK under S.agalactiae infection.It showed that the expression levels in spleen and head kidney were up-regulated at 2 h respectively,while that in the liver was up-regulated at 4 h,down-regulated to the lowest at 8 h,and levelled off at 24-72 h,suggesting that ntp38MAPK might play an essential role in the resistance of S.agalactiae infection in Nile tilapia.