Molecular cloning, expression analyses and functional characterization of a goose-type lysozyme gene from Bostrychus sinensis (family: Eleotridae).

ABSTRACT

In this study, we sequenced and characterized the goose-type lysozyme gene, termed as BsLysG, from the Chinese black sleeper (Bostrychus sinensis). The BsLysG encodes 196 amino acids and contains a soluble bacterial lytic transglycosylases domain, three catalytic residues (Glu72, Asp85 and Asp102) and the GLMQ motif (Gly97, Leu98, Met99 and Gln100). No signal peptide was observed in the BsLysG protein. The genomic DNA of BsLysG contains five exons and four introns. The sequence analyses showed that the BsLysG exhibits high similarity with LysG from other fishes. Phylogenetic analyses showed that the BsLysG is clustered together with its counterparts from other teleost fishes. The Real-time PCR analyses showed that the BsLysG was found to be ubiquitously expressed in ten examined organs in Chinese black sleeper, with predominant expression in spleen, followed by head kidney and peripheral blood. Expression analyses showed that the BsLysG was significantly upregulated in vivo after either pathogen Vibrio parahemolyticus infection or poly (IC) challenge in peripheral blood, head kidney, liver and spleen organs. The purified recombinant BsLysG (rBsLysG) has optimal activity at 35 °C and pH 5.5. The rBsLysG exhibited antimicrobial activity against two Gram-positive bacteria (Micrococcus lysodeikticus and Staphylococcus aureus) and two Gram-negative bacteria (Escherichia coli and V. parahemolyticus). The Scanning electron microscope (SEM) imaging analyses showed that the rBsLysG-treated V. parahemolyticus cells displayed morphological deformation. These results indicate that the BsLysG is involved in host immune defense against bacterial infection.