Molecular Cloning And Expression Analysis Of Crustin Genes From Mud Crab Scylla Paramamosain

Crustins are cationic,cysreines-rich AMPs with a molecular weight of 7-14 kDa,widely distributed in the whole crustaceans,and play an important role in the immunological defense especially in antibacterial and antiviral processes.Crustins are currently classified into five major groups(typeI-V).The basic structures of the Crustins contain a signal peptide at the N-terminus and a WAP domain at the C-terminus.WAP domain are characterized by a conserved arrangement of 8 cysteines that form a 4-disulphide core.In this study,two type I Crustins(SpCrus1a and SpCrus1b)and two type II Crustins(SpCrus2a and SpCrus2b)were cloned from mud crab Scylla paramamosain by degenerate PCR.After analyzing their sequences,we further investigated into their characteristics of genemic organization,tissues distribution in normally cultured crabs and their expression profiles post challenged with LPS and PolyI:C at different time-points.These results will enrich and extend the basic knowledge of Crustins and will be helpful for clarifying the functions of Crustins family in the future.1.Two novel type I Crustins,named SpCrus1 a and SpCrus1 b,were cloned from mud crab Scylla paramamosain.The full-length cDNA sequence was 746 bp and 675 bp containing an open reading frame(ORF)encoding a putative protein of 113 and 109 amino acid residues,respectively.The deduced amino acid sequences of SpCrus1 a and SpCrus1 b consist of 20 and 18 amino acis residues of signal peptides at the N-terminus and 93 and 91 residues of mature peptides at the C-terminus.The predicted molecular mass of mature peptide was 10030.47 Da and 10091.60 Da with an estimated pI(Isoelectric point)of 8.30 and 8.49,respectively.The NJ phylogenetic analysis showed that SpCrus1 a and SpCrus1 b were clustered into type I Crustins.Four exons and three introns were identified within the genomic DNA sequence of SpCrus1 a and SpCrus1 b.Tissues distribution analysis revealed that transcripts of SpCrus1 a and SpCrus1 b were mainly detected in gills.In vivo,the results of time-course analysis demonstrated that expression of SpCrus1 a and SpCrus1 b were significantly increased at 6h and 24 h after LPS challenge,On the other hand,injection of PolyI:C caused up-rgulated the expression of SpCrus1 a and SpCrus1 b,which were peaked at 12 h post infection,and SpCrus1 a may have a more enduring response time(12-24h).These results suggested that SpCrus1 a and SpCrus1 b may be involved in both antibacterial and antiviral immune response of mud crab Scylla paramamosain.2.Two novel type II Crustins,calld SpCrus2 a and SpCrus2 b,were identified from the mud crab Scylla paramamosain.The full-length cDNA of SpCrus2 a and SpCrus2 b was 1373 bp and1497 bp and encodes a polypeptide of 131 and 225 amino acid residues,respectively,which contained a signal peptide,gly-rich region,cys-rich region and a WAP domain.The predicted molecular mass of mature peptide was 11791.14 Da and 20586.00 Da with an estimated pI(Isoelectric point)of 7.66 and 7.77,respectively.Two exons and one intron were identified within the their genomic DNA sequences.The architecture and phylogenetiic analysis suggested that they were two new members of type II Crustins subfamily.And SpCrus2 a and SpCrus2 b were highly expression in gills and were significantly induced by LPS and PolyI:C.The results collectively suggested that SpCrus2 a and SpCrus2 b were potential antimicrobial peptides,which palyed an important role in the defense system of Scylla paramamosain against bacterial and viral infections.