Expression pattern, promoter activity and bactericidal property of ß-defensin from the mandarin fish Siniperca chuatsi.

ABSTRACT

ß-Defensin (BD) are cysteine-rich, cationic antimicrobial peptides which play an important role in innate immune system against invading microbes. In the present study, the cDNA cloning, expression analysis, transcriptional regulation and antimicrobial activity of ß-defensin (ScBD) from mandarin fish (Siniperca chuatsi) were characterized. The cDNA sequence of ScBD is 596 bp which encodes a protein of 63 amino acids (aa). The ScBD gene comprises three exons and two introns. The signal peptide is located in the first exon. ScBD contains 6 cysteines, and belongs to fish defensin 2 group based on phylogenetic analysis. Real-time quantitative PCR results showed that the mRNA transcripts of ScBD were distributed mainly in mucosal and lymphoid organs/tissues including intestine, gill, head kidney, kidney and spleen, with the highest level observed in spleen. Western blotting analysis revealed that the ScBD protein was abundant in head kidney, gill and spleen. A total of 3268 bp 5' flanking region of the ScBD gene promoter was sequenced, which contained a number of putative transcriptional binding sites for transcription factors. These transcription factors were analyzed using in vitro luciferase assay. The DNA region from position of -705 to -498 bp contains positive regulatory elements and that of -227 to +54 bp harbors the TATA which is essential for initiating gene expression. In addition, the ScBD peptide showed antibacterial activity against Escherichia coli M15, Staphylococcus aureus and Aeromonas hydrophila, whilst no effect on Edwardsiella tarda. These data suggest that the ScBD is importantly involved in host immune responses to invasion of bacterial pathogens.