CAJ | 학술논문

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。
수착초어양식규모적확대，초어적병독성질병겁대지영향착초어적산량。개전어류병독면역반응상관공능기인적연구의의중대。연구수선통과동원극륭적방법종초어중극륭도료일단 Prkrip1기인적 EST 서렬，진일보통과RACE、장편단PCR화Genome walking적방법획득료해기인적전장cDNA서렬、기인조DNA서렬화계동자구서렬。안기산서렬분석현시， Prkrip1함유3개핵정위신호화일개쌍련RNA결합구，병구유여 PKR결합적보수 N단구；형광보고기인적표체증실아문소극륭도적계동자구시유활성적，가용우후속해기인적전록조공분석；Real-time PCR분석발현， Prkrip1기인재초어적간화혈중표체량최고， GCRV감염후재대부분면역조직중균상조표체，설명해기인학실여병독감염상관。연구결과위Prkrip1기인재경골어류적공능연구제공료선색，야위어류천연면역반응중조공PKR신호통로적계통연구제공료이론의거。
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 in-dicated 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 re-gion-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 expres-sion 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, the mag-nitude of up-regulated expression in intestine, kidney and head kidney was the most significant. This result showed that Prkrip1 was indeed associated with viral infection, played a role in inhibition of PKR function and prevent the excessive damage of the host cell suicide. In summary, our researches have shown that the cDNA sequence cloned from grass carp in Prkrip1 and genomic DNA structure were conserved during evolution, verified the activity of the promoter region obtained, demonstrated the distribution and response of Prkrip1 gene in various tissues of grass carp before and after GCRV-infection. This article provides clues for the function studies of Prkrip1 gene in teleost, also provides a theoreti-cal basis for the regulation and control system of the PKR signaling pathway in the innate immune response of fish.