Chemokine receptor genes <I>CCR3</I> and <I>CCR9</I> in turbot (<I>Scophthalmus maximus</I>): Cloning and tissue distribution

Abstract

Turbot is an important aquaculture species in the eastern North Atlantic and Mediterranean regions,China, and Korea. Research on the molecular mechanisms of the turbot immune system could contribute to improving the economic performance of turbot aquaculture. In this study, we cloned and characterized the full-length cDNA of CCR3 and CCR9 in turbot(Scophthalmus maximus). The full-length cDNAs were obtained by the 5′ and3′-RACE method. The CCR9 cDNA was 1 441 bp in length, consisting of a 59 bp 5′ UTR, a 278 bp 3′ UTR, and a1 104 bp ORF encoding a 367 amino acid polypeptide. The CCR3 cDNA was 1 451 bp in length and contained a92 bp 5′ UTR, a 267 bp 3′ UTR, and a 1 083 bp ORF encoding 360 amino acids. The TMHMM(TransMembrane prediction using Hidden Markov Models) analysis confirmed that they were seven-transmembrane-spanning proteins. A phylogenetic tree based on the amino acid sequences was constructed by the neighbor-joining(NJ) method using Mega4. The results of phylogenetic analysis revealed that turbot CCR3 and CCR9 were more similar to the cDNAs of other teleosts than to each other. The relationships exhibited in the tree are consistent with their evolutionary relationships. The CCR9 and CCR3 mRNA expression levels in various tissues was measured by quantitative RT-PCR(qRT-PCR). Both gene transcripts were expressed in all of the tissues analyzed, with the highest expression being in the spleen, head kidney, and heart. The lipopolysaccharide(LPS) challenge results suggest that CCR9 expression in liver was more sensitive than in the other three tissues, while CCR3 expression in spleen and liver was more sensitive than in head kidney or blood. CCR3 and CCR9 expression levels were both high in immune-related tissues and after induction by LPS. Our results indicate that both genes play a role in the turbot immune system.This work further elucidates the functions of these genes in immune responses, which will help to better understand the important relationship between fish disease resistance and innate immunity.