Genomic characterization and expression analysis of complement component 9 in rock bream (Oplegnathus fasciatus).

Abstract

The complement component 9 (C9) is a single-chain glycoprotein that mediates formation of the membrane attack complex (MAC) on the surface of target cells. Full-length C9 sequence was identified from a cDNA library of rock bream (Oplegnathus fasciatus), and its genomic sequence was obtained by screening and sequencing of a bacterial artificial chromosome (BAC) genomic DNA library of rock bream. The rock bream complement component 9 (Rb-C9) gene contains 11 exons and 10 introns and is composed of a 1782 bp complete open reading frame (ORF) that encodes a polypeptide of 593 amino acids. Sequence analysis revealed that the Rb-C9 protein contains two thrombospondin type-1domains, a low-density lipoprotein receptor domain class A, a membrane attack complex & perforin (MACPF) domain, and an epidermal growth factor (EGF)-like domain. Important putative transcription factor binding sites, including those for NF-κB, SP-1, C/EBP, AP-1 and OCT-1, were found in the 5' flanking region. Phylogenetic analysis revealed a close proximity of Rb-C9 with the orthologues in puffer fish, and Japanese flounder. Quantitative real-time RT-PCR analysis confirmed that Rb-C9 was constitutively expressed in all the examined tissues isolated from healthy rock bream, with highest expression occurring in liver. Pathogen challenge, including Edwardsiella tarda, Streptococcus iniae, lipopolysaccharide endotoxin and rock bream iridovirus led to up-regulation of Rb-C9 in liver but no change in peripheral blood cells. The observed response to bacterial and viral challenges and high degree of evolutionary relationship to respective orthologues, confirmed that Rb-C9 is an important immune gene, likely involved in the complement system lytic pathway of rock bream.