Characterization, promoter analysis and expression of the interleukin-6 gene in blunt snout bream, Megalobrama amblycephala

Abstract

Interleukin-6 (IL-6) is one of the most important multifunctional cytokines, playing essential roles in mediating the innate and adaptive immune responses. In this study, il-6 gene and its promoter from blunt snout bream, Megalobrama amblycephala, were characterized, and its expression at the transcript level in healthy fish and after bacterial infection was determined by quantitative real-time PCR. The results showed that the M. amblycephala il-6 (Mamil-6) cDNA had an ORF of 699 bp, encoding 232 amino acids, and contained 9 instable motifs in the 3' UTR. The deduced MamIL-6 possessed a 24-amino acid signal peptide and was located in the cytoplasm. Although sequence alignment and phylogenetic analysis revealed that IL-6 is poorly conserved in vertebrates, the protein and genomic structure of il-6 gene was well conserved. Analysis of the Mamil-6 promoter revealed the presence of a conserved TATA box and six major cis-regulatory elements, including C/EBPβ (NF-IL6), AP-1, CRE, GRE, GATA and NF-κB binding sites. In healthy fish, Mamil-6 was the most abundant in the spleen. After Aeromonas hydrophila infection, Mamil-6 was significantly up-regulated in all 6 immune-related tissues examined, suggesting that Mamil-6 plays an important role in the blunt snout bream immune system.

Characterization of tree shrew (Tupaia belangeri) interleukin-6 and its expression pattern in response to exogenous challenge

“…The comparison of functional cis-regulatory elements from human and tsIL-6 promoter regions is shown in Fig. 3B ( 24 ).…”

Identification and Characterization of MicroRNAs in the Liver of Blunt Snout Bream (Megalobrama amblycephala) Infected by Aeromonas hydrophila

“…Previous studies in our lab indicated that some immune-related genes are activated at an early stage of A. hydrophila infection in M. amblycephala [31,32,33,34,35]. Therefore, three small RNA libraries were analyzed from M. amblycephala liver at 0 (non-infection), 4, and 24 h post infection (poi) using the Illumina Hiseq 2000 sequencing platform.…”

Classical Signaling and Trans-Signaling Pathways Stimulated by Megalobrama amblycephala IL-6 and IL-6R

“…The il-6 gene has been identified in several teleost, such as zebrafish (Danio rerio, Hamilton 1822) [30], fugu (Takifugu rubripes, Temminck & Schlegel 1850) [31], Paralichthys olivaceus (Temminck & Schlegel, 1846) [32], Sparus aurata (Linnaeus, 1758) [33], Larimichthys crocea (Richardson, 1846) [34], rainbow trout (Oncorhynchus mykiss, Walbaum 1792) [35], and blunt snout bream (Megalobrama amblycephala, Yi Bolu 1955) [36]. In teleost, IL-6 has been proved to play vital roles in the immune response, inducing the expression of hamp and regulating cytokines genes (il-1β and il-6) and socs1-3 [33,37,38].…”

“…The blunt snout bream il-6 gene could be strongly induced in Aeromonas hydrophila (Kluyver & Van Niel, 1936) in many tissues, indicating that il-6 plays an active role in the defense against pathogens [36,42]. IL-6 amino acid sequence in blunt snout bream (maIL-6) shares 91% identity with that of grass carp (Ctenopharyngodon idellus, Cuvier et Valenciennes 1844) (ciIL-6), but only 23-30% identity with those of mammals, birds, and amphibians [36].…”

“…The blunt snout bream il-6 gene could be strongly induced in Aeromonas hydrophila (Kluyver & Van Niel, 1936) in many tissues, indicating that il-6 plays an active role in the defense against pathogens [36,42]. IL-6 amino acid sequence in blunt snout bream (maIL-6) shares 91% identity with that of grass carp (Ctenopharyngodon idellus, Cuvier et Valenciennes 1844) (ciIL-6), but only 23-30% identity with those of mammals, birds, and amphibians [36]. Therefore, to explore the regulation mechanisms of IL-6 classical signaling and transsignaling in fish, we first verified whether L8824 cells (grass carp hepatocyte cells with IL-6R) can receive signals from recombinant IL-6 and whether recombinant maIL-6 (rmaIL-6) has biological activity.…”