Studies On Cloning, Sequence Analysis Of Piscidin-like Gene From Two Species Of Marine Fish And Phylogenetic Relationship Of Piscidins Family

As possessing broad-spectrum bactericidal activity,antimicrobial peptides(AMPs) play the crucial roles in innate immune system in tested animals.Piscidin is initially described as a peptide antibiotic derived from mast cell of hybrid-striped bass,and the later researches revealed that pleurocidins from flat fishes,moronecidin from hybrid-striped bass,dicentracin from sea bass,epinecidin-1 from orange-spotted grouper,shared great similarity and identity in the properties of amino acid sequence and peptide secondary structure,representing asα-helix cationic amphipathic AMPs,which could properly be classified as piscidin family.In this study,two species of commercial maricultured fish in China,red-spotted grouper(Epinephelus akaara) and large yellow croaker (Pseudosciaena crocea),were taken as samples to be used to study on piscidin cDNA cloning,deduced amino acid sequence alignment,secondary structure analysis,and phylogenetic relationship.The results are mainly described as follows:1.By using RT-PCR,3'RACE,5' RACE and sequencing,we amplified piscidin-like AMP cDNA from the kidney and spleen of red-spotted grouper.After the analysis on amino acid sequence and protein secondary structure,the peptide was identified as aα-helix cationic amphipathic propeptide,which strongly matched the main character of piscidin family.The RRRH motif in the C-terminal of mature peptide was found as that of epinecidin-1 and predicted to develop the electrostatic binding activity to negatively charged bacterial phospholipids.The similarity and identity between this peptide and other piscidin family members ranged respectively from 59%-79%and 45%-75%.Experimental evidence above demonstrated that the cloned cDNA is probably a new member in piscidin family. 2.Piscidin-like AMP cDNA from large yellow croaker was amplified by RT-PCR,3'RACE,5'RACE and sequencing.After the analysis on amino acid sequence and protein secondary structure,the peptide was identified as aα-helix cationic amphipathic propeptide,which strongly matched the main character of piscidin family.The shortages of RRRH motif which presented in the mature peptide C-terminal of epinecidin-1 as well as the XQQ motif in the prodomain C-terminal of moronecidin maked the piscidin of large yellow croaker related more closely to pleurocidins rather than other two above.The similarity and identity between this peptide and other piscidin family members ranged respectively from 50%-77%and 41%-70%.Experimental evidence above demonstrats that the cloned cDNA is probably a new member in piscidin family.3.Sequence alignment via the biosoft CLUSTALW confirmed the remarkable homology among piscidin family,the conserved domain extended from signal peptide through the mature peptide,which also indicated the harmony between structure and function in organisms.Phylogenetics relationship analysis via MEGA 4.0 supported the wide evolutionary conservation of piscidins in fish species,including both red-spotted grouper and large yellow croaker.The cluster of piscidin family clearly separated from hepcidin family in the agreement with the fact that of piscidins that are linear peptide whereas hepcidins are characterized by 4 intra chain bonds giving totally different 3D structures.