Cloning And Expression Analysis Of Antimicrobial Peptide Crustin Of Swimming Crab Portunus Trituberculatus

The swimming crab, Portunus trituberculatus (Crustacea: Decapoda: Brachyura),one of the important economic crabs, widely distributies off the coasts of China,Japan and Korea. In recent years, as the development of aquaculture, various diseasesincluding bacterial, yeast and viral diseases have frequently outbroken, causingenormous economic losses in P. trituberculatus culture. In this situation, the study ofimmune defense mechanism and resistance strains breeding for crabs is veryimportant.In this study, two novel crustin genes (PtCrustin2and PtCrustin3) were identifiedbased on the eyestalk and hemocytes cDNA libraries of Portunus trituberculatus byexpressed sequence tags (EST) analysis. The full-length cDNA of PtCrustin2was1105bp in length, containing a complete open reading frame (ORF) of297bp. TheORF of PtCrustin2coded putative protein of98amino acids with calculatedmolecular weight of10.97kDa and predicted isoelectric point of5.23. The full-lengthcDNA of PtCrustin3was629bp in length, containing an complete open reading frame(ORF) of348bp. The ORF of PtCrustin3coded putative protein of115amino acidswith calculated molecular weight of12.65kDa and predicted isoelectric point of6.42.The amino acid sequences of PtCrustin2and PtCrustin3contain a putative signalpeptide at the amino terminus, a whey acidic protein (WAP) domain at the carboxylterminus and a Cys-rich region between the signal sequence and WAP domain.Sequence alignment of two genomic DNAs showed that PtCrustin2and PtCrustin3 might be encoded by the different genomic locus. Four exons and three introns areidentified in genomic DNA sequence of PtCrustin3while three exons and two intronsin PtCrustin2. Three tandem repeats were found in PtCrustin2and four in PtCrustin3.Three SNPs in the fragments of PtCrustin2gene and four SNPs in the fragments ofPtCrustin3gene were detected association with Vibrio alginolyticus-resistance bydirect sequencing. Most of these association SNPs located in introns. Only SNP locus87in PtCrustin3was in exon and was non-synonymous mutations. In addition, amicrosatellite was found in intron2of PtCrustin2associatied withsusceptibility/resistance.In healthy P. trituberculatus, PtCrustin2and PtCrustin3transcripts weredifferentially expressed in all of tested tissues. Different from the known crustins, themRNA transcripts of PtCrustin2and PtCrustin3were mainly detected in eyestalk andgills, but not in heamocytes. After challenged with Gram-negative bacteria Vibrioalginolyticus, Gram-positive bacteria Micrococcus luteus and yeast Pichia pastoris,the expression levels of two crustins in heamocytes exhibited different expressionpatterns. The expression level of PtCrustin2up-regulated and reached the highestlevel at24h post-injection (P <0.01). The expression level of PtCrustin3rapidlyincreased and the highest expression levels appeared in8h or4h post-challenge (P <0.05). This result suggested that PtCrustin3seemed to respond more quickly tomicrobial challenge than Ptcrustin2.The antimicrobial activity assay showed that the antimicrobial activity spectrawere similar for both peptides. Unlike most crustins, both recombinant PtCrustin2andPtCrustin3exhibited antibacterial activity against Gram-positive bacteria M. luteusand S. aureus and Gram-negative bacteria P. aeruginosa. In addition, rPtCrustin2ismoderately active against yeast P. pastoris and rPtCrustin3show significant activityagainst Gram-negative bacteria V. alginolyticus. Whilst, the assay of bactericidaleffect displayed that the two rPtCrustins readily killed M. luteus, S. aureus and P. aeruginosa after incubation for less than6h. Hence, the rPtCrustin2and rPtCrustin3possessed the bactericidal activity. These results will helpful in understanding theimmune defense mechanisms and guiding breeding.