Mechanism Of The Detoxification Metabolism Regulated By Transcription Factors AhR And ARNT In Nilaparvata Lugens

The brown planthopper,Nilaparvata lugens,is an important pest of rice,causing serious economic losses in China.At present,chemical control is the main measure for the control of N.lugens.Transcription factors,also known as trans-acting factors,could regulate the expression intensity of target genes,activate or suppress the transcription of related genes.Transcription factors are involved in regulating the expression of detoxification enzyme genes,which in turn affect the susceptibility of insect pest to insecticides.In this study,the brown planthopper is taken as the research object to explore the role of the transcription factors aryl hydrocarbon receptor(AhR)and aryl hydrocarbon receptor nuclear translocation(ARNT)in the detoxification metabolism of N.lugens.The main results are as follows:1.Based on the NCBI database,the open reading frame of NlAhR/NlARNT was amplified by PCR.NlAhR and NlARNT were found to be 2580 bp and 2109 bp in length encoding 841 and 703 amino acid residues,respectively.And both NlAhR and NlARNT had typical b HLH-PAS domains.Phylogenetic analysis showed that NlAhR has higher homology with its counterpart from Thrips palmi.NlARNT has higher homology with its counterpart from Neodiprion lecontei and Cephus cinctus.2.The expression of NlAhR and NlARNT in different developmental stages and different tissues of N.lugens was detected by q RT-PCR.The results showed that NlAhR and NlARNT were expressed in all developmental stages of N.lugens,and both highly expressed in the egg stage.NlAhR and NlARNT were expressed in all tissues mearsured in N.lugens.NlAhR was highly expressed in the head and cuticle,and NlARNT was highly expressed in the head and midgut.The expression of NlAhR and NlARNT was significantly increased in N.lugens with imidacloprid,etofenprox and isoprocarb treatment,respectively.NlAhR was up-regulated by 31%-99%,and NlARNT was upregulated by 47%-58%.But the expression of NlAhR and NlARNT had no significant difference with chlorpyrifos treatment.3.Knocking down NlAhR or NlARNT by RNAi,the susceptibilty of N.lugens to imidacloprid,etofenprox and isoprocarb increased significantly,but there was no significant difference to chlorpyrifos.After silencing NlAhR or NlARNT,the relative activities of detoxification enzymes P450,GST and Car E decreased significantly.It was further found that 1 of P450 genes,2 of GST genes and 2 of Car E genes were significant down-regulated with NlAhR knocked down.After silencing NlARNT,5 of P450 genes,4 of GST genes and 1 of Car E genes were significantly down-regulated.In addition,CYP301A1,Nl GSTt1 and NlCarE7 were significant decreased after injecting with ds NlAhR or ds NlARNT,among which the expression of NlCarE7 was significantly reduced by 85% and 93%,respectively.4.In order to further confirm the regulatory effect of NlAhR/NlARNT on NlCarE7,based on RNAi and dual luciferase reporter gene assay,the effect of NlCarE7 on the susceptibility of N.lugens and the effect of NlAhR/NlARNT on the promoter activity of NlCarE7 were tested.It was found that the susceptibility of N.lugens to etofenprox and isoprocarb was significantly increased after silencing NlCarE7,but no significant difference in susceptibility to imidacloprid.The NlCarE7 promoter region was cloned by PCR,and the transcription factor binding sites were analyzed.The results showed that there was a binding site of NlAhR/NlARNT in the NlCarE7 promoter region.NlAhR/NlARNT can significantly induce NlCarE7 promoter activity by dual luciferase reporter gene assay.Our results indicated that NlAhR/NlARNT could mediate the detoxification metabolism of ethofenprox and isoprocarb in N.lugens by regulating the expression of NlCarE7.