CLONING AND EXPRESSION ANALYSIS OF GRASS CARP (CTENOPHARYNGODON IDELLUS) PRKRIP1, A NEW IMMUNE RELATED GENE

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,the magnitude 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 theoretical basis for the regulation and control system of the PKR signaling pathway in the innate immune response of fish.