Molecular cloning, functional characterization and expression analysis of p65 subunit of golden pompano (Trachinotus ovatus) and response to high fat diet and LPS administration

Abstract

p65 is an important component of NF-κB signaling pathways. However, information about p65 subunit in golden pompano (Trachinotus ovatus) is quiet limit. Thus, the full length of p65 subunit was cloned from T. ovatus in this study. The full-length cDNA sequence was 3162 bp in length and comprised of a 1886 bp open reading frame (ORF) preceded by 153 bp 5′-untranslated region (UTR), and followed by a 1143 bp 3′UTR. The p65 of T. ovatus contains a conserved N-terminal REL-homology domain (RHD) with 273 amino acids. The phylogenetic tree showed that p65 in T. ovatus was clustered in the clade of that in marine fish. mRNA expression of p65 was detected in 12 different tissues. The results showed that expression of p65 in liver was higher than that in other tissues (P < 0.05). Immune-related tissues, including kidney and spleen, were moderately highly expressed. In 72 h after LPS administration, mRNA expression level of p65 and IL1β in liver and head kidney showed significantly differently higher than that in control group (P < 0.05), while in 24 h there was no significant difference between the two groups in expression level of both p65 and IL1β(P > 0.05). After 8-weeks feeding, the hepatic expression level of p65 and IL1β in fish fed diet with high fat showed significant difference with that in control diet (P < 0.05). This work showed that p65 subunit played a pivotal role in inflammation caused by LPS and high fat diet, which provides information about response to inflammation on the basis of the NF-κB pathway in T. ovatus.