cDNA sequence and expression analysis of an antimicrobial peptide, theromacin, in the triangle-shell pearl mussel...

Bivalve molluscs rely on the interaction between cellular and humoral factors for protection against potential pathogens. Antimicrobial peptides (AMPs) have been proven to be one of the most important humoral components that afford resistance to pathogen infection. The AMP gene to be identified was that encoding theromacin in the triangle-shell pearl mussel Hyriopsis cumingii (Hc theromacin); this gene was identified from a suppression subtractive hybridization library, and subsequently cloned by 3' and 5' rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR). The full-length theromacin cDNA contains 547 bp, with a 294-bp open reading frame that encodes a 97-amino acid peptide, and the deduced peptide sequence contains a 61-amino acid putative mature peptide. The sequence also contains 10 cysteine residues. Reverse transcriptase (RT)-PCR analysis showed that Hc theromacin transcripts were constitutively expressed in the liver, foot, gill, adductor muscle, heart, mantle, intestine, and hemocytes, with the highest level in hemocytes. Theromacin mRNA levels were found to increase after challenge with gram-positive and gram-negative bacteria. After injection of the gram-positive bacteria Staphylococcus aureus and Bifidobacterium bifidum, Hc theromacin expression showed the highest fold-change at 48 and 36 h after infection, respectively, and its levels decreased gradually thereafter.