Screening And Verification Of Chilo Suppressalis P450 Enzymes Metabolizing Chlorantraniliprole

Chilo suppressalis(Walker)belongs to(Lepidoptera),family(Pyralidae),of Lepidoptera frequently occurs and harms in major rice regions of China,which has a serious impact on the yield and quality of rice.The main measure to control Chilo suppressalis in the field is the use of chemical insecticides.In 2008,a new bisamide insecticide,chlorantraniliprole,was registered in China,which has a good control effect on most Lepidoptera pests,so it has become the main insecticide to control Chilo suppressalis in various rice growing areas.After more than ten years of use,Chilo suppressalis has developed a high level of resistance to this fungicide in the rice regions of Jiangxi,Zhejiang and Hunan provinces in China.The resistance mechanism of Chilo suppressalis to chlorantraniliprole mainly includes the amino acid mutation of the target receptor ryanodine receptors and the enhancement of metabolic enzyme activity.In our previous study,we found that there was no positive correlation between the population of Chilo suppressalis with different levels of resistance to chlorantraniliprole in the field and the frequency of target ryanodine receptors mutation(G4910E).Therefore,the enhanced activity of metabolic enzymes in the field population of Chilo suppressalis resistant to chlorantraniliprole is an important mechanism.By comparing the resistant and susceptible strains,it has been found that several cytochrome P450 genes are overexpressed in the chlorophora-resistant Chilo suppressalis,and it is speculated that they may have the function of metabolizing insecticides.Due to the evolutionary plasticity of cytochrome P450 enzymes,that is,different P450 enzymes can be up-regulated in the same insect resistant population to the same insecticide;secondly,some potential P450 enzymes with metabolic insecticide activity are difficult to be recognized and understood by the above methods if they are not screened and up-regulated by chlorantraniliprole.In this study,four P450 genes of Chilo suppressalis that may metabolize chlorantraniliprole were screened from 28 Chilo suppressalis P450 genes by overexpression technique and bioassay of transgenic Drosophila.The phenomenon of overexpression of these four P450 genes was confirmed in different field resistant populations of Chilo suppressalis.Two of these P450 enzymes were successfully expressed in vitro by Bac-to-Bac insect baculovirus expression system.The main results are summarized as follows:1.Determination of sensitivity of Drosophila strain overexpressing Chilo suppressalis P450 genes to chlorantraniliproleUsing the Drosophila melanogaster transformed with P450 gene of Chilo suppressalis established in the previous laboratory,28 P450 genes of Chilo suppressalis were overexpressed in Drosophila melanogaster by GAL4/UAS system,and the sensitivity of Drosophila strains overexpressing P450 gene to chlorantraniliprole was determined.Compared with the control group da>9755,it was found that the sensitivity of four Drosophila strains overexpressing Cs CYP321F3,Cs CYP6AB45,Cs CYP4M38 and Cs CYP6CV4 genes to chlorantraniliprole was significantly decreased,and the sensitivity of Drosophila melanogaster overexpressing Cs CYP321F3 and Cs CYP6AB45 was significantly decreased.Therefore,the four P450 enzymes of Chilo suppressalis Cs CYP321F3,Cs CYP6AB45,Cs CYP4M38 and Cs CYP6CV4 may have the effect of detoxification and metabolism of chlorantraniliprole.2.Analysis of the expression of 4 P450 genes in different populations of Chilo suppressalis resistantion to chlorantraniliprole.Through field collection,three populations of Chilo suppressalis with resistance ratio of more than 100 times to chlorantraniliprole were obtained.The results of transcriptome sequencing showed that the expression of Cs CYP6AB45 was significantly up-regulated in Guangfu population(GF),Dayu population(DY)and Yujiang population(YJ);the expression of Cs CYP321F3 and Cs CYP4M38 was significantly up-regulated in GF and DY-resistant populations;while the expression of Cs CYP6CV4 was significantly up-regulated only in GF population.The results of the above transcriptome were verified by real-time fluorescence quantitative PCR.The results showed that the expression of P450 gene of Chilo suppressalis was basically consistent with that of the transcriptome,and the relative expression multiple was more than 4 times.Therefore,the over-expression of P450 enzymes in different resistant populations of Chilo suppressalis verified the rapid screening method of P450 enzymes in Chilo suppressalis established in this paper.3.In vitro expression of Cs CYP321F3 and Cs CYP6AB45 and determination of enzyme activity.In order to further verify the detoxification and metabolic activity of the screened P450 enzyme from Chilo suppressalis to chlorantraniliprole,we successfully expressed Cs CYP321F3 and Cs CYP6AB45 in Sf-9 cells and High five cells using Bac-to-Bac baculovirus expression system.The specific absorption peaks of the two recombinant P450 at 450nm were detected by CO difference spectroscopy,and the metabolic activities of recombinant Cs CYP321F3 and Cs CYP6AB45 on model substrate ECOD and PNA were determined.the results showed that compared with the control group of unloaded HTA,the activities of Cs CYP321F3 and Cs CYP6AB45 on PNA and ECOD were increased by 2.52 times and 3.56 times,respectively.The results showed that the two recombinant P450 enzymes had the ability of oxygen demethylation and oxygen deethylation,which laid a good foundation for further insecticide metabolism in vitro.To sum up,this study not only provided a rapid and comprehensive screening method for the study of pest resistance detoxification enzymes,but also laid a foundation for the follow-up function of Chilo suppressalis P450 metabolism of chlorantraniliprole.