CDNACloning And Expression Analysis Of CYP2and GST Gene Of Portunus Trituberculatus

Drug residues of aquatic products brought great harm to human healthand the environment. And the biotransformation of the drug is dependenton the drug metabolism enzymes, Drug-drug interactions which aregenerally associated with the induction and inhibition of drug metabolismenzymes activity may lead to the formation of aquacultural drugs residues or influence the drug effect. Cytochrome P450and glutathioneS-transferases are the important drug metabolism enzymes in the organism.However, only relatively few papers about the expression level andresearch on the effects of enzyme activity of aquacultural drugs on drugmetabolism enzymes in marine crustaceans. In this thesis, P.trituberculatuswas chosen as the experimental material, because they are one of theimportant economic crabs, which are famed in offshore waters. And studiedeffects of sulfadimidine （SM2）, sulfadiazine （SD） andsulfamonomethoxine（SMM）on CYP2and GST gene expression and theinfluence of enzyme activity. The study consists of three parts:The first part: The cDNA encoding CYP2was first cloned from P.trituberculatus using RT-PCR and Smart-Race. Analysis of the nucleotidesequence revealed that the cDNA clone had a full-length of1662bp andencoded a protein of492amino acids which had a predicted molecularweight of56.68kDa, with an estimated pI of6.384. It was predicted topossess the conserved K helik sequence (ExxR) and heme-binding motif(FxxGxxxCxG), suggesting that it belonged to the cytochrome CYP450subgroup. Comparison of amino acid sequences showed a similarity ofmore than75%between the CYP2in P. trituberculatus and Carcinusmaenas. Real-time PCR analysis revealed that CYP2was expressed in alltested tissues, including hepatopancreas, gills, muscle, hemolymph andeyestalk. The expression of CYP2in hepatopancreas increased afterintramuscular of the three kinds of sulphonamide on the different degree,where SD and SMM treated groups had a very significant decreasecompared to the SM2treated group, indicating that CYP2could be inducedby sulphonamide and might be involved in the drug-metabolic response ofP. trituberculatus.The second part: The cDNA encoding GST was first cloned fromP.trituberculatus using RT-PCR and Smart-Race. Analysis of the nucleotidesequence revealed that the cDNA clone had a full-length of1010bp andencoded a protein of216amino acids which had a predicted molecularweight of24.59kDa, with an estimated pI of5.294. It was predicted to possess the GST-N structure domain and GST-C structure domain,suggesting that it belonged to the GST subgroup. Comparison of aminoacid sequences showed a similarity of more than78%between the GST inP. trituberculatus and Eriocheir sinensis. Real-time PCR analysis revealedthat GST was expressed in all tested tissues, including hepatopancreas, gills,muscle, hemolymph and eyestalk. The expression of GST inhepatopancreas increased at the early stage and then reduce the late afterintramuscular of the three kinds of sulphonamide on the different degree,where SD and SMM treated groups had a very significant change comparedto the SM2treated group.The third part: We studied the effects of three kinds of sulphonamideon the activities of APND and GST in hepatopancreas of P.trituberculatus.The results on enzyme activity showed that compared to the control group,the activities of APND and GST in the S9mix were induced by the threekinds of sulphonamide on the different degree, where SD and SMM treatedgroups had a very significant decrease on the activities of APND and GSTcompared to the control group (P﹤0.01), while SM2treated group onlyincreased the activity of APND and GST significantly (P﹤0.05). Theseresults suggested that the three kinds of sulphonamide could widely inducethe activity of hepatopancreas CYP2and GST of P.trituberculatus.