| Spodoptera frugiperda（Smith）is a major invasive and omnivorous pest.Chemical control is an effective method to control S.frugiperda.However,the long-term irrational use of insecticides will inevitably lead to the generation of resistance to S.frugiperda,including chlorantraniliprole for the control of lepidopteran pests.The study on the molecular mechanism of resistance to chlorantraniliprole is an important basis for the resistance management of S.frugiperda.In this study,sublethal dose of chlorantraniliprole was used to treat S.frugiperda and transcriptome sequencing was performed to screen out differential expression of cytochrome P450 genes in response to chlorantraniliprole,and the roles of CYP9F1 and CYP6K2 genes in chlorantraniliprole resistance were verified by real-time fluorescence quantitative PCR（q RT-PCR）and RNA interference（RNAi）,and software was used to predict the mi RNA that may regulate the expression of CYP9F1 and CYP6K2 genes,and study the post-transcriptional regulation mechanism of the mi RNA on the expression of CYP9F1 and CYP6K2 genes,and clarify the molecular mechanism of the resistance to chlorantraniliprole in S.frugiperda.The main results are as follows:1.Screening of cytochrome P450 gene related to chlorantraniliprole resistance in S.frugiperda Leaf dipping method was used to determine the toxicity of chlorantraniliprole to the 2nd instarlarvae of S.frugiperda.The 2nd instar larvae were treated with LC10（10%lethal concentration）chlorantraniliprole for 24 h and transcriptome sequencing was performed.According to the sequencing data,cytochrome P450 genes responsive to chlorantraniliprole was screened out.The results showed that the LC50（median lethal concentration）and LC10（10%lethal concentration）of chlorantraniliprole against the2nd instar larvae of S.frugiperda were 2.087 mg/L and 0.931 mg/L,respectively.There were 109differentially expressed P450 genes affected by chlorantraniliprole,among which 83 P450 genes were up-regulated and 26 P450 genes were down-regulated.2.Effects of sublethal dose of chlorantraniliprole on CYP9F1 and CYP6K2 gene expression of S.frugiperdaThe 2nd instar larvae of S.frugiperda were treated with LC10（10%lethal concentration） chlorantraniliprole for 12 h,24 h,36 h,and 48 h,the expression levels of CYP9F1 and CYP6K2 genes were determined by q RT-PCR.The results showed that the expression levels of CYP9F1 and CYP6K2 genes were significantly up-regulated after 12 h,24 h,36 h,and 48 h of chlorantraniliprole treatment,indicating that the CYP9F1 and CYP6K2 genes may play important roles in chlorantraniliprole metabolism.3.Roles of CYP9F1 and CYP6K2 genes in chlorantraniliprole resistance of S.frugiperdaThe silencing efficiency of CYP9F1 and CYP6K2 and the sensitivity to chlorantraniliprole were determined by injecting ds RNA into the 2nd instar larvae of S.frugiperda.The results showed that silencing CYP9F1 and CYP6K2 genes significantly increased the sensitivity to chlorantraniliprole,suggesting that CYP9F1 and CYP6K2 genes play an important role in the detoxification metabolism of chlorantraniliprole resistance.4.Effects of chlorantraniliprole on mi RNA expression of S.frugiperdaBioinformatics software was used to predict the mi RNA that might regulate the expression of CYP9F1 and CYP6K2 genes,and q RT-PCR was used to determine the effects of chlorantraniliprole on the expression of mi RNA in S.frugiperda.The results showed that mi R-23a and mi R-190-5p may regulate the expression of CYP9F1 and CYP6K2 genes,respectively.The expressions of mi R-23a and mi R-190-5p were significantly down-regulated after chlorantraniliprole treatment,suggesting that CYP9F1 and CYP6K2 may have a negative regulatory relationship with mi R-23a and mi R-190-5p,respectively.5.Regulation of mi RNA on P450 gene expression in S.frugiperdaA series of experiments were conducted to verify the regulation of mi RNA on P450 gene expression by q RT-PCR and Dicer-1 gene silencing and double lucifase reporter gene detection and injection of mi RNA agomir and antagomir.the results showed that mi R-23a negatively regulates the CYP9F1 gene expression by binding to the 3’UTR target site of CYP9F1 gene,and mi R-190-5p negatively regulates the CYP6K2 gene expression by binding to the 3’UTR target site of CYP6K2 gene. |
