Screening And Functional Analysis Of Differential Expression CYP450 Genes After P.okadai Treatment With Beta-cypermethrin

Objective: This article intends to screen for differential expression CYP450 genes in P.okadai with beta-cypermethrin treatment.The up-regulation Pocyp4d46 was identified by bioinformatic analysis results and gene expression profiling as a possible candidate gene for beta-cypermethrin insecticide detoxification reaserch in P.okadai,and the results of Pocyp4d46 experimental studies would lay the ethical foundation for further discussion of the insecticides resistance mechanism of P.okadai.Methods: 1.Screening of differentially expressed CYP450 genes in P.okadai under betacypermethrin stress: The up-regulated CYP450 genes were screened from the differential transcriptomes database and validated by PCR amplification,then the DNAMAN 6.0、MEGA 5.2 and TMHMM Server v.2.0 softwares were used to predict their bioinformatics.2.The patiotemporal expression profiles of differentially expressed CYP450 genes: The candidate gene was determined by the differential expression of CYP450 genes detected by RT-qPCR in the P.okadai of Fat body、Midgut and Malpighian tube,after the P.okadai females were treated with beta-cypermethrin at different action time of 0 h、0.5 h、1 h、2 h、3 h、4 h.3.Effect of Po CYP4D46 recombinant protein on viability of S2 cells with betacypermethrin treatment:(1)Eukaryotic expression of Pocyp4d46 in Drosophila melanogaster S2 cells: The recombinant p Fast Bac1-Pocyp4d46 and Bacmind were constructed by eukaryotic expression system successively,and the recombinant Bacmind was transfected into S2 cells,then the Po CYP4D46 recombinant protein was identified by SDS-PAGE,Immunofluorescence and Western blot.(2)The effect of Po CYP4D46 recombinant protein on the inhibition of S2 cells viability with beta-cypermethrin treatment by CCK8 assay,and the experiment was divided into blank group(S2),beta-cypermethrin group,Bacmind-Po CYP4D46 group and Bacmind-Po CYP4D46+betacypermethrin group.4.Effect of siRNA interference with Pocyp4d46 on beta-cypermethrin sensitivity of P.okadai.(1)The effect of siRNA interference on the expression level of Po CYP4D46 recombinant protein detected by Western blot and immunofluorescence,and the experiment was divided into Bacmind group,Bacmind+Pocyp4d46-NC group and Bacmind+Pocyp4d46-siRNA group.(2)Fluorescence quantitative PCR detection of the Pocyp4d46-siRNA interference effect on different tissues.(3)The detection of mortality changes in P.okadai which under beta-cypermethrin stress after Pocyp4d46-siRNA interference.Results: 1.The three up-regulated expression P.okadai CYP450 genes of cyp4d46,cyp49a1 and cyp28d2 were screened by PCR verification and bioinformatics analysis.2.The spatiotemporal expression profiles of CYP450 genes showed that the relative expression levels of cyp4d46,cyp49a1 and cyp28d2 in midgut,fat body and Malpighian tube were significantly increased with beta-cypermethrin treatment(P<0.05),and the cyp4d46 gene had the highest up-regulation in the midgut action 1h later(P<0.05).3.The Po CYP4D46 recombinant protein was identified by SDS-PAGE,Immunofluorescence and Western blot,which was about 47.5 kilodalton and expressed in the S2 cells lysis precipitate mainly,and distributed in the cytoplasm by Immunofluorescence detection.4.The CCK8 assay showed that the viability of S2 cells were both decreased significantly in the beta-cypermethrin group and Bacmind-Po CYP4D46+beta-cypermethrin group compared with blank group(P<0.01 or P<0.05),but the viability of cells in the BacmindPo CYP4D46+beta-cypermethrin group was significantly higher than the beta-cypermethrin group(P<0.01)and lower than the Bacmind-Po CYP4D46 group(P<0.01).5.The results of siRNA interference with Pocyp4d46 on beta-cypermethrin sensitivity of P.okadai showed that:(1)The Po CYP4D46 recombinant protein expression level was decreased significantly in the Bacmind+Pocyp4d46-siRNA group compared with the Bacmind group and Bacmind+Pocyp4d46-NC group(P<0.01)detected by Western blot;the Immunofluorescence detection showed that,the count of cells immunofluorescently labeled with Po CYP4D46 recombinant protein were significantly reduced for Pocyp4d46-siRNA interferenced.(2)The relative expression of Pocyp4d46 gene in the Fat body,Midgut and Malpighian tube of P.okadai were all significantly decreased after siRNA interference at different action time of 24 h 、 48 h and 72 h(P<0.05),and the interference effect of Pocyp4d46 relative expression in Midgut tissue at 24 h was the lowest(37.28%).(3)The mortality detection of P.okadai treated by beta-cypermethrin insecticide after siRNA interference: the mortality rate of Pocyp4d46-siRNA group for 15.0%(3h)and 27.5%(6h)were significantly increased compared with Pocyp4d46-NC group for 6.25%(3h)and15.0%(6h).(4)Effect of continuous beta-cypermethrin pesticide stress on survival curves after siRNA interference: the Pocyp4d46-siRNA group P.okadai were all died within 24 hours by beta-cypermethrin continued treatment with drug film method,while the Pocyp4d46-NC group P.okadai were all died within 36 hours,which was about 12 hours later.Conclusion:1.There are three differentially expressed CYP450 genes were screened in P.okadai under beta-cypermethrin stress such as cyp4d46、cyp49a1 and cyp28d2.2.The expression of Pocyp4d46 in the midgut,fat body and Malpighian tube may related to the pyrethroid insectivides detoxification of P.okadai.