To explore the function of Nramp in the grass carp (Ctenopharyngodon idellus) immune system, gene expression was analyzed in different developmental stages and tissues. Four tissues of grass carp at different times after challenge with the pathogenic bacterium Aeromonas hydrophila were analyzed by real-time quantitative RT-PCR. The 642 bp Open Reading Frame (ORF) region of grass carp Nramp was expressed using prokaryotic expression technology. The fusion protein had a relative molecular mass of 40 kD. Real-time quantitative RT-PCR analysis indicated that the Nramp mRNA level in embryos gradually increased during embryogenesis from cleav-age to fry stages after fertilization, with the highest level in the fry stage. In adult fish, the Nramp mRNA level in different tissues significantly differed; Nramp transcripts were found to be highly abundant in liver; moderately abundant in the head kidney, spleen, and intestine;and least abundant in the eye and bladder. After challenge with A. hydrophil, with uninjected groups as the control, the Nramp mRNA level in the head kidney, liver, spleen, and intestine significantly increased (P<0.05). There was significant up-regulation in head kidney at 12 h post-treatment, with a maximum level at 48 h post-infection;significant down-regulation at 72 h and 96 h;and the expression regressed to the initial level by 7 d. Nramp expression in the liver was significantly increased at 12 h post-treatment;followed by a decrease at 24 h post-treatment;another increase at 48 h, with a maximum level at 72 h post-infection;and the high expression level was maintained at 96 h and 7 d post-treatment. There was sig-nificant up-regulation in the intestine at 12 h post-treatment, with a maximum level at 48 h post-treatment; sig-nificant down-regulation at 72 h and 96 h; and the expression regressed to the initial level by 7 d. In the spleen, Nramp mRNA level increased at 4 h post-treatment, with a maximum level at 12 h; then, there was significant down-regulation at 24 h and 48 h;and the expression regressed to the initial level by 7 d. Bacterial infection can increase Nramp expression in immune-related organs, which may indicate that Nramp plays an important role in the innate immune response of grass carp.%为探讨Nramp基因在草鱼(Ctenopharyngodon idellus)免疫应答中的作用,采用实时荧光定量PCR技术,对比研究了Nramp基因在草鱼不同发育阶段、不同组织及嗜水气单胞菌(Aeromonas hydrophila)攻毒感染后4个组织不同时间的差异表达,并利用原核表达技术对草鱼Nramp基因 ORF区642 bp的目的片段进行蛋白表达,获得40 kD的融合蛋白。实时荧光定量PCR分析结果表明, Nramp在草鱼胚胎发育过程中的表达量随着发育时间的增加而逐渐增强,在幼鱼期的表达量达到最大。成年健康草鱼不同组织中的表达量差异显著(P<0.05),在肝中的表达量最高,其次是在脾、头肾、肠中的表达量较高,而在眼和鳔中的表达量最低。草鱼感染嗜水气单胞菌后,在头肾、肝、脾、肠中Nramp的表达量都显著上升(P<0.05),在头肾中感染12 h后显著上升,48 h达到最大,在72 h和96 h时又显著下调,在7 d时表达量继续下调而逐渐回归到初始水平；在肝中Nramp基因的表达量在4 h时出现下调,在12 h又显著上调,随后在24 h出现下调,在48 h又上调,72 h达到最大并保持较高水平到7 d；在肠中mRNA表达量在12 h显著上调,48 h达到最大,在72 h和96 h显著下调,在7 d时表达量基本回归到初始水平；在脾中mRNA表达量在4h开始上升,12h达到最大,在24h和48h表达水平显著下调,在7d时表达量回归到初始水平。草鱼感染致病菌可引起免疫相关组织中Nramp的表达量上升,说明Nramp在硬骨鱼类的天然免疫反应过程中发挥着重要作用。
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