中文摘要：

随着草鱼养殖规模的扩大,草鱼的病毒性疾病极大地影响着草鱼的产量。开展鱼类病毒免疫反应相关功能基因的研究意义重大。研究首先通过同源克隆的方法从草鱼中克隆到了一段Prkrip1基因的EST序列,进一步通过RACE、长片段PCR和Genome walking的方法获得了该基因的全长cDNA序列、基因组DNA序列和启动子区序列。氨基酸序列分析显示,Prkrip1含有3个核定位信号和一个双链RNA结合区,并具有与PKR结合的保守N端区;荧光报告基因的表达证实我们所克隆到的启动子区是有活性的,可用于后续该基因的转录调控分析;Real-time PCR分析发现,Prkrip1基因在草鱼的肝和血中表达量最高,GCRV感染后在大部分免疫组织中均上调表达,说明该基因确实与病毒感染相关。研究结果为Prkrip1基因在硬骨鱼类的功能研究提供了线索,也为鱼类天然免疫反应中调控PKR信号通路的系统研究提供了理论依据。

英文摘要：

With the expansion of the scale of grass carp,the viral disease of grass carp greatly affeated the yield of grass carp.To carry out fish virus immune response-related functional genes research,Partial cDNA sequence of Prkrip1 in grass carp was isolated from head kidney cDNA library by the method of homology cloning.The full length cDNA of grass carp Prkrip1 was obtained by means of 3′ RACE and 5′ RACE,respectively.The full length cDNA of grass carp Prkrip1 was 1057 bp,consisting of a 5′-terminal untranslated region（UTR） of 39 bp,a 3′-terminal UTR of 472 bp,and an open reading frame of 546 bp.Sequence alignment showed that the deduced amino acid sequence of grass carp Prkrip1 had an overall similarity of 69%—87% to that of other species homologues.Amino acid sequence analysis indicated the existence of three nuclear localization signals,a dsRNA binding region and an N-terminal conserved region as binding element of PKR.Then we used PCR to obtain a genomic DNA which covers the entire coding region of grass carp Prkrip1.In the 8.5 k-long genomic sequence,six exons and five introns were identified.The promoter region-driven GFP eukaryotic expression plasmid was constructed.Expression of the GFP reporter gene confirmed the promoter cloned in this study was active,could be used for the analysis of the transcriptional regulation.Real-time RT-PCR results showed that grass carp Prkrip1 was expressed predominantly in liver and PBL,medium in gill,spleen,intestine,skin,brain,muscle,kidney and lower in head kidney.Whereas under GCRV-infection condition,the expression of Prkrip1 gene was significantly up-regulated in most immune-related tissues in a time-dependent manner,in which Prkrip1 expression was increased to the highest value in four days post-infection,and then began to decline five days post-infection.In the fourth day,the two tissues with the highest expression level were intestine and liver,and higher expression in the spleen,kidney and head kidney.Compared with the expression level before infection,th