Molecular characterization and functional analysis of DIGIRR from golden pompano (Trachinotus ovatus)

Abstract

Mammalian single immunoglobulin (Ig) interleukin-1 receptor related molecule (SIGIRR), an important member of the Toll/interleukin-1 receptor (TIR) family, plays important balancing roles in the inflammatory responses. In the present study, the double Ig interleukin-1 receptor related molecule (DIGIRR), the homologous of SIGIRR, was characterized in golden pompano (Trachinotus ovatus) (termed as trDIGIRR). The full-length cDNA of trDIGIRR was 2,167 bp with an open reading frame (ORF) of 1,572 bp encoding 523 amino acids. The trDIGIRR contained several conserved domains including a signal peptide, two Ig domains, a transmembrane domain and a TIR domain, and shared high sequence identities with its teleost counterparts. Realtime qPCR analysis revealed that the trDIGIRR was distributed in all tissues examined, with high expressions in intestine, liver and head kidney. The expressions of trDIGIRR were induced by Vibrio alginolyticus, lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid (poly I:C) challenge. Further analysis revealed that trDIGIRR was mainly located in the cytoplasm. In addition, the co-immunoprecipitation (co-IP) assay identified that trDIGIRR could interact with myeloid differentiation factor 88 (MyD88), but not interact with TIR domain containing adaptor protein inducing interferon-β (TRIF). Our results provide basis for studying the immune role of fish DIGIRR.

Molecular Characterization and Expression Analysis of Four Janus Kinases (JAK1, JAK2a, JAK3 and TYK2) from Golden Pompano (Trachinotus ovatus)

“…The qPCR was performed using a LightCycler480 (Roche, Germany). The qPCR reaction volume and qPCR program were consistent with those of a previous study [35] except for the primers. All samples were analyzed in triplicate.…”

“…Total RNA extraction, total RNA quality assessment and cDNA synthesis were conducted in line with the methods described in our previous studies [35]. First-strand cDNA was synthesized from 2 µL of total RNA (1.19 µg/µL), and the cDNA was diluted 50-fold for gene cloning.…”

“…Nine tissues, including spleen, head kidney (HK), skin, muscle, gill, heart, liver, brain and intestine, were separately sampled from five healthy golden pompanos for basal expression analysis. The methods for total RNA extraction and cDNA synthesis followed those of a previous study [35]. The expression levels of trJAK1, trJAK2a, trJAK3 and trTYK2 in each tissue were detected using the qPCR method.…”

“…The method for determining the transcription changes for trJAK1, trJAK2a, trJAK3 and trTYK2 followed those of our previous study [35]. Briefly, the fish in four groups were intraperitoneally (i.p.)…”

“…In 2021, the production of golden pompano reached 243,908 tons in China [34]. However, the annual increase of pathogenic diseases caused huge economic losses to the golden pompano culture industry [35]. Until now, although one study based on transcriptome analysis has revealed that the JAK-STAT pathway of golden pompano was activated in response to Streptococcus agalactiae infection [32], the sequence characteristics and specific expression levels of JAKs in golden pompano challenged with different pathogens have not been determined.…”