Functional Analysis And Regulatory Mechanisms Of Two Delta Class GSTs In Procambarus Clarkii

Procambarus clarkii is introduced into China,and is rapidly developed into an important economic freshwater shrimp.With the rise of rice and shrimp farming industry,the toxic effects of pesticide residues in water and soil on shrimp also become the focus of attention.The GST gene of P.clarkii is further studied and is of great significance to rice shrimp culture and human health food.Therefore,in this paper,the two Delta classes GST of P.clarkii was investigated.:1.Cloning and analysis of the Pc GSTD1 and Pc GSTD2 genesThe full length c DNA of two delta glutathione S-transferases(Pc GSTD1 and（Pc GSTD2）were cloned from P.clarkii using PCR and RACE PCR techniques.The5’UTR,ORF,3’UTR and total length of Pc GSTD1 c DNA are 66 bp,654 bp,486 bp and 1206 bp respectively,and the number of encoded amino acids is 218,the molecular weight of the protein is 24.17 k Da and the theoretical isoelectric point is 5.35.The5’UTR,ORF,3’UTR and total length of Pc GSTD2 are 75 bp,657 bp,384 bp and 1116bp respectively,and the number of encoded amino acids is 219,with the molecular weight of 24.39 k Da and the theoretical isoelectric point of 5.80.Protein structure analysis showed that the mature peptides of Pc GSTD1 and Pc GSTD2 consisted of two typical structural domains,GST＿N and GST＿C.Both Pc GSTD1 and Pc GSTD2showed the highest similarity to Homarus americanus with 72.02%and 76.71%,respectively.The phylogenetic tree showed that both Pc GSTD1 and Pc GSTD2belonged to the Delta class GST.2.Analysis of expression patterns of Pc GSTD1 and Pc GSTD2Pc GSTD1 and Pc GSTD2 were detected in various tissues by q PCR,with the highest expression in hepatopancreas,Pc GSTD1 was least expressed in stomach and Pc GSTD2 was least expressed in muscle.subcellular localization showed that both Pc GSTD1 and Pc GSTD2 were expressed in the cytoplasm as cytoplasmic proteins.Pc GSTD1 and Pc GSTD2 were up-regulated in both hepatopancreas and gills within24h of stimulation with different concentrations of imidacloprid.The results of enzyme activity assay showed that the GST activity in hepatopancreas and gill increased after the stimulation of different concentrations of imidacloprid.To verify whether Pc GSTD1 and Pc GSTD2 were regulated by Pckeap1b,Pc Nrf1 and Pc Maf K and participate in the detoxification process of P.clarkii,the m RNA expression of Pc GSTD1 and Pc GSTD2 was examined by q PCR after exogenous stimulation and interference knockdown.The expression of Pc GSTD1 and Pc GSTD2 was increased in the Pckeap1b and Pc Nrf1 interference groups and decreased in the Pc Maf K interference group compared with the control group by using ds RNA to effectively silence Pckeap1b,Pc Nrf1,and Pc Maf K genes,respectively.Prokaryotic expression of Pcb ZIP and Pc Maf K fusion proteins was obtained for GST Pull-down assay,and the results showed that they could bind in vitro.The results of gel mobility shift assay showed that Pc Maf K protein was able to bind to Pc GSTD2promoter in vitro.Dual luciferase reporter showed that the core region of Pc GSTD1promoter is at-440 bp～+54 bp and the core region of Pc GSTD2 promoter is at-1609bp～-1125 bp.These results indicate that both Pc GSTD1 and Pc GSTD2 were involved in the process of resistance to imidacloprid in the P.clarkii,and expression is influenced by Pckeap1b,Pc Nrf1 and Pc Maf K.3.Enzymatic properties and antioxidant functions of Pc GSTD1 and Pc GSTD2The fusion proteins Pc GSTD1-His and Pc GSTD2-His were obtained by induction of recombinant protein expression,and were detected by SDS-PAGE protein gel electrophoresis,which showed that recombinant proteins were present in both supernatant and precipitate.The optimum temperature and p H of Pc GSTD1 and Pc GSTD2 were 20℃and 30℃,p H8 and p H7,respectively.The results of the disk diffusion assay showed that BL21（DE3）of the expressing Pc GSTD1 and Pc GSTD2proteins were more resistant to H₂O₂ than the control.