Genomic organization and functional diversification of two warm-temperature-acclimation-associated 65-kDa protein genes in rockbream (Oplegnathus fasciatus; Perciformes).

Two paralogue genes of warm-temperature-acclimation-associated 65-kDa protein were characterized and their mRNA expression patterns during various experimental stimulations were examined in the rockbream (Oplegnathus fasciatus; Perciformes). Rockbream Wap65 isoforms (rbWap65-1 and rbWap65-2) share basically common structural features with other teleostean orthologues and human hemopexin (HPX) at both amino acid (conserved cysteine and histidine residues) and genomic levels (ten-exon structure), although the rbWap65-2 reveals more homologous characteristics to human HPX than does rbWap65-1 isoform. Southern blot analysis indicates that each rbWap65 isoform exists as a single copy gene in the rockbream genome. Both rbWap65 genes were predicted to possess various transcription factor (TF) binding motifs related with stress and innate immunity in their 5ʹ-upstream regions, in which inflammation-related motifs were more highlighted in the rbWap65-2 than in rbWap65-1. Based on the RT-PCR assay, the liver-predominant expression pattern was more apparent in rbWap65-1 than rbWap65-2 isoform. During thermal elevation, clear upregulation was found only for the rbWap65-1. In contrast, immune stimulations (bacterial challenges, viral infection and iron overload) activated more preferentially the rbWap65-2 isoform in overall, although the inducibility was affected by the kinds of stimulators and tissue types. Taken together, our data suggest that the two paralogue rbWap65 isoforms have experienced subfunctionalization and/or neofunctionalization during their evolutionary history, in which the rbWap65-2 has retained closer, functional orthology to the human HPX while the rbWap65-1 have been diversified to be more related with thermal acclimation physiology.