Characterization, Genomic Organization And Expression Analysis Of A Novel Anionic Antimicrobial Peptide, Scygonadin, Isolated From Scylla Serrata

Scylla serrata (Forsk?l, 1775) is one of the most important economic marine aquaculture species in China. A novel antibacterial peptide named scygonadin has been isolated and purified from the seminal plasma in Scylla serrata using liquid chromatography. The full-length cDNA nucleotide sequence and amino acid sequence of scygonadin were characterized using PCR and RACE. The gene organization of scygonadin was clarified by the way of Bioinformatics analysis. The expression of the gene in the different tissues and organs was analyzed using Dot-blot and RT-PCR assay. The present study provides the basic knowledge for further understanding the innate and reproductive immunity mechanism of Scylla serrata. The main results of this study were shown below.1. A novel protein was isolated from the seminal plasma of the mud crab, Scylla serrata (Forsk?l, 1775). It exhibited an antibacterial activity against the Gram-positive bacterium Micrococcus leteus with IC90 of 125μg/mL. The extraction procedure for the protein included techniques of acid extraction, ion-exchange chromatography on SP-Sepharose Fast Flow and reverse-phase liquid chromatography on Source 5R RPC. It showed a molecular mass of 10.8 kDa by SDS-PAGE. A partial 20 residues NH2-terminal sequence was determined by Edman degradation and MS-fingerprint of the protein was conducted. Similarity search (BLAST) in the protein databases revealed that the protein exhibited no significant homology to any other reported antimicrobial peptides. The name Scygonadin (from the gonad of Scylla serrata) for this antibacterial protein was proposed.2. Full-length cDNA sequence of scygonadin has been obtained. Based on the partial 20 residues NH2-terminal sequence of the peptide H-GQALNKLMPKIVSAIIYMVG-OH determined by Edman degradation, the gene sequence of scygonadin from the male gonad of Scylla serrata was cloned using a degenerated reverse transcriptase (RT)-PCR and rapid amplification of cDNA end (RACE). The full-length cDNA of scygonadin is 539 bp including a 5' untranslated region of 56 bp and a 3' untranslated region of 127 bp with the putative...