Cloning, Identification And Characterization Of Transferrin And CCL4 In Grass Carp (Ctenopharyngodon Idellus)

In this study,we had preliminary screened and conserved the Poly I:C-induced liver and kidney grass carp(Ctenopharyngodon idellus) cDNA library which had been constructed.In the library the full-length cDNA sequences of grass carp transferrin and chemokine genes had been cloned.Transferrin has diverse functions that it not only participates in the transportation and metabolism of organism iron ions,but also plays key effect in cellular respiration, cellular growth and proliferation,antimicrobial bactericidal bioactivity and related to innate immunity.The full-lenghth Grass carp transferrin gene(CtTf) contained 5 'non-coding region of 31bp,the largest open reading frame for 2025bp which encoding 674 amino acids,and contained 3' non-coding region of 266bp.N-lobe of grass carp transferrin was a signal peptide of 15 amino acides.CtTf was composed of two similar domains,which lie in 24-334 and338-665aa respectively,and the identity of the two domains was 31%.Similarly with the transferrin in other speciens,CtTf has four iron ion binding sites in each domain,and positions of them were relatively conservative.In the domains there were several other conservative sites besides the eight iron ion binding sites.CtTf had the high homology with human and other species,such as it shared the identity of about 65%-73%with other Cyprinid fishes, 43%-50%with marine fishes,and 40%-42%with amphibians,reptiles,aves, mammals.Phylogenetic tree analysis revealed CtTf shared the highest homology with zebrafish and cyprinoid.The result of RT-PCR experiment indicted that in the normal liver,intestine,kidney,spleen,heart,muscle,gill and brain of grass carp,the transferrin gene had the highest expressional level in liver,the spleen and intestine ranked secondly,and trace amount in gill and brain.After Poly I:C-inducing, expression levels in liver,spleen,gill significantly increased,the expression in the spleen more than the liver,and almost unchanged in intestines and brain.Chemokine which was an important part of congenital immune system also was a large family of cytokine.The grass carp CCL4 that full-length was 854bp,ORF was located in 104-397bp,encoded 97 amino acids.Its conservative CC chemokine superfamily domain was located at 32-73aa,and there was a signal peptide of 24 amino acids in N-terminal.Grass carp CCL4 shared the the highest homology of 69% with zebrafish,and 68%with CCL-C25y which positioned in chromosome 25 of zebrafish.In addition,the homology with other fish(Catfish,Atlantic salmon, rainbow trout and so on) less than 50%.Phylogenetic analysis also showed that it had the closest relationship with zebrafish CCL4.The results of adaptive evolutionary analysis indicated that amino acid sites of 38D,43V,47A were suffered the positive selection effect.In the normal tissues of grass carp,CCL4 gene had the highest expressional level in spleen,followed by heart,muscle,gill,liver,and little amount in intestine and kidney,trace amount in brain.The expression in liver,spleen,intestine, kidney and gill increased at different degrees after infecting by Poly I:C,while there were no significant change in heart,muscle and brain.