Molecular cloning and immune function study of an oyster IκB gene in the NF-κB signaling pathway

Mass mortality caused by pathogens has severely affected the oyster cultivation industry, but our knowledge about the innate immune system of oysters is still limited. The nuclear factor-kappa B (NF-kappa B) signaling pathway plays a vital role in many cellular, developmental, and biological processes. And inhibitor of NF-kappa B (I kappa B) proteins can prevent NF-kappa B nuclear localization and DNA binding, thereby inhibiting NF-kappa B functions. However, little is known about their existence and function in invertebrates. In this study, a novel homolog of the I kappa B gene was identified in the Pacific oyster Crassostrea gigas (designated as CgI kappa B4). The open reading frame of CgI kappa B4 is 1029 bp and encodes a putative protein of 342 amino acids. Comparative and phylogenetic analyses revealed that the CgI kappa B4 belongs to the invertebrate I kappa B family. The results of quantitative real-time PCR showed that CgI kappa B4 transcripts are expressed in all test tissues, and with the highest expression level in the stomach and labial palpus, and that the expression is significantly induced after LPS, PGN, and poly(I:C) challenges. Importantly, Co-IP assays showed that CgI kappa B4 can interact with oyster NF-kappa B family proteins (i.e., CgRel1 and CgRel2) directly. The results of dual-luciferase reporter assays showed an inhibition of CgI kappa B4 on CgRel1-dependent NF-kappa B and TNFa activation. These findings demonstrate that CgI kappa B4 may play an important role in the innate immunity of C. gigas through regulation of NF-kappa B activity. And the research results will be helpful for deciphering the mechanisms of innate immunity in invertebrates and provide theoretical basis for molecular breeding of disease-resistance oysters.