cDNA cloning and expression analysis of prophenoloxidase in Cherax quadricarinatus

The Australian redclaw crayfish,Cherax quadricarinatus,is a crustacean belonging to the order Decapoda,family Parastacidae.In recent years,the cultivation of redclaw crayfish(Cherax quadricarinatus)is developing,and the disease of C.quadricarinatus was one of the major factors in its culture and even caused redclaw crayfish to die.The viral disease was found in polyculture of redclaw crayfish with Penaeus vannamei.To explore the pathogenic mechanism of C.quadricarinatus infected by WSSV(white spot syndrome virus),a prophenoloxidase gene(CqproPO)was cloned from haemocytes of C.quadricarinatus by Rapid Amplification Complementary DNA Ends(RACE)method,the proPO gene expression patterns in different tissues and the mRNA expression of proPO gene in hemocyte,hepatopancreas and gill tissues of C.quadricarinatus artificially infected by WSSV were studied.The results indicated that the full length cDNA of CqproPO consisted of 2 962 bp with a 1 998 bp Open Reading Frame(ORF),which encoded 665 amino acids,and the predicted molecular mass was 75.86 ku.Sequence analysis showed CqproPO contained two conserved copperbinding sites;The secondary and tertiary structure assay also showed that the CqproPO has α-helices and β-strands,which is delimiting a cavity where the hydrophobic ligands are bound just as other HCs.ORF contains two tyrosine kinase phosphorylation sites,13 casein kinase Ⅱ phosphorylation sites,7 protein kinase C phosphorylation sites,one dependent on cAMP-and cGMP protein kinase phosphorylation sites and three N-glycosylation sites,and these sites were structural basis of the physiological functions completed.The deduced amino acids sequence of CqproPO shared 79% homology with Procambarus clarkii and 74%,69%,67%,67% with Pacifastacus leniusculus,Nephrops norvegicus,Homarus americanus,Homarus gammarus respectively;Phylogenetic analysis revealed that CqproPO and prophenoloxidase from P.clarkii,P.leniusculus,N.norvegicus,H.americanus,H.gammarus and Panulirus longipes were in the same phylogenetic branch;The Realtime-PCR results showed that CqproPO was widely distributed,with the highest expression level in haemocytes,small amount of expression in intestine,antennal gland,gills,ovary and hepatopancreas,detectable expression level in stomach and muscle,while expression was almost undetectable in testis;The expression levels of prophenoloxidase(proPO)in haemocytes,hepatopancreas and gills from C.quadricarinatus were studied and compared by means of artificial WSSV infection.The results indicated that the expression level of CqproPO in the non-immunized infected group(group Ⅱ)and immunized infected group(group Ⅲ)reached the maximum at 12 h and 24h,which was 1.3-2.55 times higher than that in the control group,and was noticeably higher than the controls(P0.05).But the expression level of prophenoloxidase gene had sharply declined with the time extending of the infection.The expression level of proPO gene from crayfishes injected by immune polysaccharides before an infected virus(groupⅢ)were higher than the directly affected groups(groupⅡ)in haemocytes,hepatopancreas and gills,showing an immunoprotective rate of 51.86% 7 days after exposure.These observations indicated that the immunopotentiator could improve the innate anti-viral ability of the crustaceans against WSSV.