| The tobacco cutworm,Spodoptera litura Fabricius(Lepidoptera: Noctuidae),is an omnivorous insect which caused damage to 300 kinds of plants.Due to the unscientific uses of chemical insecticides,S.litura has developed different levels resistance to multiple insecticides such as cyantraniliprole,cypermethrin and abamectin.Metabolic resistance is one of the important resistance mechanisms in insects containing three phases.Cytochrome P450 monooxygenase(P450)and carboxylesterase(Car E)are the dominant genes in the phase I of detoxification in insects.Different signaling pathways in insects are activated by xenobiotics pressure to mediate transcription factors,which control the transcripts of downstream candidate genes.In this study,RNA-seq and q PCR were used to select upregulated P450 and Car E genes which response to xenobiotics exposure.These selected detoxification genes were functionally analyzed by using transgenic Drosophila and RNAi.Then,Y1 H,RNAi and EMSA were used to explore the transcriptional regulation mechanisms of key P450 s.Here are the results:RNA-seq results indicated that the terms of drug metabolism-cytochrome P450 and metabolism of xenobiotics by cytochrome P450 were included in the top 20 KEGG pathways in the three tissues under the cyantraniliprole,gossypol and nicotine treatments,suggesting that P450 s participate in detoxifying these three xenobiotics.RNA-seq results showed that 81 P450 s were differentially expressed in the S.litura’s fatbody,midgut and Malpighian tubule after cyantraniliprole,nicotine and gossypol exposure.A total of 31 P450 s were upregulated in the cyantraniliprole and nicotine treatments,and 32 P450 s were upregulated after gossypol exposure.Among them,the CYP3 and CYP4 genes accounted for the major numbers of these genes.In addition,the results of q PCR indicated the transcripts of CYP340 A and CYP340 L in the fatbody and the expression levels of CYP332A1,CYP6AB12,CYP6AB58,CYP6AB59 and CYP6AN4 in the Malpighian tubule were significantly upregulated after cyantraniliprole exposure.The expression of CYP6AB58 and CYP6AB59 was significantly increased in the Malpighian tubule after exposure to gossypol.The relative expression levels of CYP9A75 a and CYP9A75 b were simultaneously upregulated in the Malpighian tubule after cyantraniliprole,nicotine and gossypol exposure.The results of transgenic Drosophila bioassays by using topical application showed there were 2.30-,3.37-,1.71-,2.48-and 3.68-fold tolerance to cyantraniliprole in the overexpressed-CYP6AB59,CYP340 A,CYP6AB12,CYP9A75 a and CYP9A75 b lines,respectively,compared to the corresponding UAS-P450 s lines.The results of transgenic Drosophila bioassays by using feeding method indicated there were 2.58-,1.72-,1.51-,2.58-,2.35-,1.95-and 2.65-fold in the overexpressed CYP332A1,CYP6AB12,CYP6AB59,CYP6AN4,CYP340 A,CYP9A75a and CYP9A75 b lines,respectively,compared to the corresponding UAS-P450 s lines.In addition,the egglaying amounts of Act5 C > UAS-CYP9A75a/b lines were significantly higher than those in the UAS-CYP9A75a/b lines after nicotine exposure.The egg-laying amounts of Act5 C > UAS-CYP6AB58 and Act5 C > UAS-CYP6AB59 lines were significantly higher than those in the UAS-CYP6AB58 and UAS-CYP6AB59 lines after exposure to gossypol,respectively.According to the results of transgenic Drosophila bioassays,RNAi was used to verified the function of candidate P450 s in S.litura.The results indicated that the suppression of CYP332A1,CYP6AB12,CYP6AB59,CYP6AN4,CYP340 A,CYP9A75a and CYP9A75 b significantly increased the mortalities of S.litura to cyantraniliprole,respectively,compared to the control.Based on the abovementioned results,the transcriptional regulation of key P450 s,CYP9A75a and CYP9A75 b genes were explored.The potential TF-binding sites in the5’ flanking region of CYP9A75 a and CYP9A75 b were predicted by the online software package TRANSFAC 7.0,and the results showed that 7 TFs and 8 TFs were predicted in the 5’ flanking region of CYP9A75 a and CYP9A75 b,respectively.The results of Y1 H assays revealed that only FTZ-F1β1 may interact with the CYP9A75 b 5’ flanking region;however,no predicted TFs showed interactions with the CYP9A75 a 5’ flanking region.The RNAi results indicated that the knockdown of FTZ-F1β1 decreased the expression of CYP9A75 b to 70%.In addition,the EMSA and Y1 H assays were used to confirm the binding of FTZ-F1β1 to candidate DNA motifs in the CYP9A75 b 5’ flanking region.Y1 H assay showed that the promoter bait and FTZ-F1β1 prey grew normally on the selected medium,while the transformants containing the mutation of the FTZ-F1β1binding site(-430/-441)failed to grow normally under the same conditions.The results of EMSA demonstrated band shifting when recombinant GST-FTZ-F1β1 protein was incubated with the labeled probes of CYP9A75 b.p(-430/-441),and no shifted band was found when mutated probes were used.RNAi was used to determine whether p38,STAT5 B and Akt regulate the transcriptional activity of FTZ-F1β1.RNAi results showed the protein levels and phosphorylation of p38(p-p38: 48.91%;p38: 52.56%),STAT5B(p-STAT5B: 41.44%;STAT5B: 35.09%)and Akt(p-Akt: 59.57%;Akt: 50.69%)were decreased,respectively,compared to the control.Silencing of p38,STAT5 B and Akt significantly decreased the expression level of FTZ-F1β1(P < 0.05),resulting in the downregulation of CYP9A75 b.By feeding inhibitors,the phosphorylation and protein levels of p38 were downregulated to 55.02% and 83.89%,respectively,compared to the control;the phosphorylation and protein levels of STAT5 B were decreased to 20.36% and 2.03%,respectively,compared to the control;the phosphorylation and protein levels of Akt were downregulated to 46.12% and 83.26%,respectively,compared to the control.The expression levels of FTZ-F1β1 and CYP9A75 b were significantly downregulated with the expression decreases of p38,STAT5 B and Akt,respectively,compared to the control.RNA-seq and q PCR results indicated an inducible Car E gene of S.litura,COE030,was specifically expressed in the midgut,and its expression was significantly upregulated after cyantraniliprole and nicotine exposure.The results of transgenic Drosophila bioassays showed that there were 4.91-and 2.12-fold tolerance to cyantraniliprole and nicotine in the Esg > UAS-COE030 lines,respectively,compared to those in the UAS-COE030 line.The egg-laying amounts of Esg > UAS-COE030 line was significantly higher than that in the UAS-COE030 line.RNAi results revealed that the successful knockdown of COE030 in S.litura significantly enhanced S.litura’s sensitivity to cyantraniliprole.In addition,the results of HPLC demonstrated that the COE030 recombinant protein can metabolize cyantraniliprole in vitro.Taken together,P450 and Car E genes of S.litura which are related to the tolerance to cyantraniliprole,nicotine and gossypol were screened out via RNA-seq and q PCR.Combined with kinds of molecular biology,physiological and biochemical methods,these candidate genes were confirmed to be involved in the detoxification metabolism of xenobiotics.In addition,MAPK p38,JAK/STAT and PI3K/Akt signaling pathways were found to mediate the expression of FTZ-F1β1,regulating the changes of CYP9A75 b expression,which led CYP9A75 b played important roles in S.litura’s crosstolerance to insecticides and plant secondary metabolites.This study provides useful insights for transcriptional regulation of detoxification-related genes,which are mediated by different signaling pathways and cis-acting elements,and supports beneficial exploration and evidence for the insects resistance mechanism. |
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