Cloning And Sequence Analysing Of Full Length Prophenoloxidase CDNA In Red Swamp Crayfish, Procambarus Clarkii

In the recent years,natural defence systems of crustacean have aroused too much attention due to increasing economic loss caused by the various diseases of shrimps and crabs.The prophenoloxidase(proPO)activating system was the recognition and defence systems in crustacean.Phenoloxidase(PO)could be regarded as an index to immune power owing to its activation association with body immunity.Understanding the activating process of proPO system in the organism could lead to improve diagnostic methods to assess animal health,and to find suited treatments to augment natural defence systems.The studies on proPO gene were the basic in understanding body immunity regulation mechanism of crustacean.The total mRNA was extracted from the P.clarkia haemocytes and reverse transcribed using primer OligodT-anchor to cDNA.The proPOF and proPOR primers were designed based on the conservative region of several proPO gene cDNA sequences of crustacean in GenBank.The partial cDNA sequence of proPO gene in P.clarkia was obtained using RT-PCR.By RACE,four transcripts sequenes of proPO gene were obtained.The length of 5'UTR in the four transcripts all was 154bp,and the lengths of 3'UTR were 318bp,957bp,1497bp and 1683bp respectively.There was an open reading frame(ORF)in proPO gene,1884bp,which ecoded a protein of 627aa.The proPO contained eight potential N-glycosylation sites,one tentative thiol-ester-like motif(GCGWPQDHL).and two putative copper-binding sites(copper A,131,135,167,and copper B,301,305,341)which have a highly conserved sequence around them.No signal peptide was detected in the four transcripts sequenes of P.clarkii proPO.Though they all contained PolyA tails,no PolyA signal(AATAAA)was find in the proPO-1,one PolyA signal was contained in the proPO-2,and three PolyA signals was detected in proPO-3 and proPO-4.Sequence analysis with the BLAST algorithm showed that the proPO deduced amino acid sequence of P.clarkii has highest similarty to freshwater crayfish P.leniusculus (63.6%),and has similarity of 48.3-54.5%to prophenoloxidase deduced amino acid from other decapod crustaceans.The prophenoloxidase deduced amino acid sequence of P. clarkii has similarity of 31.9-36.5%to prophenoloxidase deduced amino acid from insecta.The frequencies of codons were analysed,which indicated that the codon usage of P. clarki proPO had its preference,but not randomness.The correlation of codons' usage frequencies of proPO gene among P.clarki and other arthropod indicated that P.clarki proPO had its special coding strategy.