Study On The Role Of ProPO And Chst11 Genes In Anti-WSSV In Procambarus Clarkii

The red swamp crayfish,Procambarus clarkii,which belongs to Arthropoda,Crustacea,Decapoda,Procambarus,is an important freshwater economic crayfish in China.Procambarus clarkii also has been employed as an important crustacean model organism.P.clarkii has been widely cultured in China due to its delicious taste and rich nutritional value in recent years.However,with the increase in the production of the crayfish,white spot syndrome virus(WSSV)in crayfish farming have caused serious economic losses.Therefore,it is necessary to study the antiviral mechanism of P.clarkii and to cultivate its antiviral strains.In this study,based on a previous analyses of the P.clarkii transcriptome,carbohydrate sulfotransferase11(Chst11)and prophenoloxidase(proPO)may play an important role in the resistance of P.clarkii to WSSV.In the present study,the role of proPO and Chst11 genes in anti-WSSV in P.clarkii was investigated,and the SNPs associated with WSSV traits in proPO gene were identified.The results would provide a theoretical basis for the crustacean’s antiviral mechanism,and provide theoretical and technical guidance for the cultivation of antiviral crayfish.The main research contents and results are as follows:1.Cloning and expression pattern analyses of Chst11 gene from P.clarkiiThe full length of Chst11 was 1536 bp with an 831-bp open reading frame(ORF),which encoding 276 amino acids residues with a calculated molecular mass of 33.1 k Da.Chst11 contains a Sulfotransfer＿2 domain,one N-glycosylation site and three O-glycosylation sites.Phylogenetic analysis results showed that Chst11 had the highest similarity to Penaeus vannamei(79.93%).The expression profile of Chst11 in different tissues indicated that Chst11 was expressed highest in gut,gill and hypodermis,lowest in testicular duct,periesophageal nerve and hemocytes.The expression of Chst11 had different expression profile in different tissues when the crayfish was challenged by WSSV,Aeromonas hydrophila and Cp G ODN.2.The role of Chst11 recombinant protein in anti-WSSV processThe results showed that recombinant Chst11 significantly reduced the amount of WSSV copy number in hepatopancreas after 6 and 12 h post injection compared to thecontrol group injected with bovine serum albumin(BSA).It was found that Chst11 enhanced the expression of peroxinectin,proPO in hepatopancreas and midgut,and the C-type lectin(ctl)in hemocytes,hepatopancreas and midgut.Intramuscularly injection of juvenile crayfish with Chst11 could significantly decrease 60% mortality of WSSV-infected crayfish at 10 days as compared to the control group with BSA.3.The role of proPO in P.clarkii anti-WSSV processAfter the purified proPO recombinant protein injection,the copies of WSSV in the hepatopancreas and gill of P.clarkii was examined.The results showed that the recombinant proPO significantly reduced the amount of WSSV copy number in hepatopancreas and gill compared to the control group injected with bovine serum albumin(BSA).It was found that proPO enhanced the expression of proPO in hepatopancreas and midgut,peroxinectin in hepatopancreas,gill and midgut,and the C-type lectin(ctl)in hemocytes,hepatopancreas,gill and midgut.Intramuscularly injection of juvenile crayfish with proPO could significantly decrease 65% mortality of WSSV-infected crayfish at 10 days as compared to the control group with BSA.4.Identification of SNPs associated with anti-WSSV traits in proPO of P.clarkiiThe proPO genomic DNA sequence is 9085 bp in length,which includes five exons interrupted by four introns,an 82 bp 5’-flanking region,and a 1671 bp 3’-untranslated region.Two TATA boxes and three repeat sequences were predicted in the intron region of proPO.Subsequently,the single nucleotide polymorphisms(SNPs)in proPO were identified and further genotyped in the crayfish population.The association between SNPs and anti-WSSV traits was investigated,and three SNPs in intron regions were identified,and the SNP 7081T>C,the TT genotype crayfish in resistance group were significantly higher than those in sensitive group(P < 0.05).In SNP 5057 T>C and SNP5061 T>C,the TT genotype crayfish in resistance group were also higher than those in sensitive group,but the differences were not significant.These results showed that the SNP 7081T>C significantly affected the anti-WSSV traits,indicating that TT genotype had positive effect on anti-WSSV.