Cloning And Functional Analysis Of ?-amylase Gene In Procambarus Clarkii

The red swap crayfish Procambarus clarkii,an important freshwater aquaculture shrimp species,is belongs to Arthropoda,Crustacea,Decapoda.Throughout its life cycle,P.clarkii molts about a dozen times and matures after ecdysis.However,ecdysis is regulated by various factors and the digestive enzymes play an important role in this process.The cloning,expression and the biological function of ?-amylasein P.clarkii were performed in this study.Oligonucleotide primers were designed by Primer premier 5.0 software to amplify the cDNA fragment according to partial known sequence.Bioinformatic analysis revealed that the ORF was 1545 bp and encoded a protein of 514 amino acid residues with a signal peptide.The theoretical isoelectric point and molecular weight of the mature polypeptide are 4.95 and 55.1 kDa,respectively.Phylogenetic analysis showed that the Pc-Amy was highly homologous to amylases from crustaceans.The ORF was ligated to pET-28 a vector for prokaryotic expression.The expression plasmid pET-28A-amy was transformed into E.coliRosetta(DE3)strain fro protein expression by different concentrations of IPTG.SDS-PAGE and western blot analysis showed that a recombinant Pc-Amy protein was expressed in E.coli and the recombinant protein was purified by nickel affinity column chromatography.In order to obtain polyclonal antibody,the purified recombinant protein was used to immunize New Zealand rabbits,the titer of the obtained antibody was measured by ELISA.The enzyme activity of the purifiedrecombinant Pc-Amy protein was 1425 ± 28 U/mg,which was determined by the 3,5-dinitrosalicylic acid(DNS)method.Proteins and total RNAs were extracted from muscle,heart,stomach,gut,gill,and hepatopancreas.Both real-time PCR and western blotting showed that Pc-amy was mainly expressed in hepatopancreas.To investigate the effects of ecdysteroid injection on the expression levels of Pc-amy in hepatopancreas,real-time PCR and western blotting were performed.The results revealed that the expression of Pc-amy declined significantly after 48 hours.These results suggest that Pc-amy involved in the responses to ecdysteroid stimulation.