Genome-wide identification of the cystatin gene family and functional characterization of cystatin B in flathead mullet (Mugil cephalus).

The cystatin superfamily is a class of endogenous cysteine protease inhibitors that play a key role in immune regulation and host defense. However, there remains a lack of genome-wide identification and functional studies of this gene family in the flathead mullet. Therefore, this study conducted a systematic identification of the cystatin gene family in flathead mullet at the genome-wide level and further characterized the function of McCysB (Mugil cephalus Cystatin B). The results revealed that seven cystatin genes were identified in the flathead mullet genome, which were classified into three subfamilies, namely stefins (type I), cystatins (type II), and kininogens (type III), based on phylogenetic analysis. based on phylogenetic analysis. Structural and conserved motif analysis indicated that all members contained a highly conserved cystatin domain, suggesting functional conservation during evolution. Chromosomal mapping revealed that these genes were unevenly distributed across four chromosomes and exhibited tandem duplication events. Collinearity analysis further revealed the evolutionary conservation of the cystatin gene locus in teleosts. Tissue expression profiling indicated that most McCys genes were highly expressed in immune-related tissues, with the most significant expression levels observed in the kidneys. Following infection with Vibrio harveyi, the expression level of McCysB was significantly upregulated, suggesting its potential involvement in the antimicrobial immune response. Furthermore, this study successfully expressed and purified the recombinant McCysB (rMcCysB), and the in vitro functional assays confirmed that this protein exhibited dose-dependent antibacterial activity against Staphylococcus aureus, Nocardia seriolae, Escherichia coli and V. harveyi, with a more pronounced inhibitory effect against Gram-negative bacteria. In summary, this study systematically elucidated the molecular characteristics, evolutionary patterns, and potential immunological roles of the cystatin gene family in the flathead mullet and revealed McCysB as a potential immune effector molecule, providing a candidate target for the development of antimicrobial strategies in aquaculture.