Genome-wide identification of the cystatin gene family in Japanese flounder (Paralichthys olivaceus) and functional characterization of PoCystatin B and PoCystatin C.

Members of the cystatin superfamily serve as key regulators of cysteine protease activity that play critical roles in immune defense and inflammatory regulation, yet systematic studies on this family in Japanese flounder (Paralichthys olivaceus) remain limited. In this study, five cystatin family members were identified from the P. olivaceus genome, designated as Pocystatin B, Pocystatin C, Pocystatin, Pocystatin F, and Pokininogen 1. Phylogenetic analysis, gene structure characterization, conserved motif identification, and multiple sequence alignment collectively revealed the highly conserved structural features of this family. Ka/Ks analysis demonstrated that all members were subject to purifying selection, while chromosomal collinearity analysis reflected the functional divergence pattern following whole-genome duplication in teleosts. Tissue expression analysis showed that Pocystatin B and Pocystatin C were predominantly expressed in gills and liver, respectively, suggesting their specialized physiological roles in distinct immune organs. Following Edwardsiella tarda challenge, both genes exhibited differential dynamic expression patterns in blood, kidney, and gill tissues, indicating that they cooperatively participated in innate immune responses through distinct regulatory pathways. In vitro functional assays confirmed that both rPoCystatin B and rPoCystatin C possessed broad-spectrum antibacterial activity, papain inhibitory activity, Ca2+-dependent bacterial agglutination activity, robust thermostability, and specific binding capacity against multiple bacterial pathogens. These findings provide an important theoretical foundation for understanding innate immune mechanisms in P. olivaceus; however, as the current evidence is primarily based on expression profiles and in vitro assays, further in vivo studies are required to fully evaluate their potential as molecular targets for disease-resistant breeding and precise disease prevention in aquaculture.