| Cytochrome P450,as a mixed-function oxidase in insects,plays a very vital role in insect life activities.In addition,CYP4G,a cytochrome P450 subfamily genes,is directly involved in the synthesis of insect cuticular hydrocarbons and plays a key role in insect development and response to stress.Glyphodes pyloalis is a major pest of mulberry,which has strong adaptability and causes serious losses to mulberry production in major sericulture producing areas in China every year,thus seriously restricting the development of sericulture industry.Due to the long-term unreasonable use of chemical pesticides,G.pyloalis have developed resistance to common pesticides in some regions of mulberry areas,resulting in failures of pest controling.Whether P450 is involved in the development of G.pyloalis resistance remains unclear.On the other hand,there are still many problems to be solved and clarified in the detailed process of CYP4G participation in insect cuticular hydrocarbon biosynthesis,and it has not been involved in the related research of G.pyloalis.Therefore,clarifying the mechanism of CYP4G in response to insecticide stress and regulation of cuticular hydrocarbon biosynthesis will provide an important vision for further research to reveal the function of CYP4G in G.pyloalis,and also provide an important idea for the development of efficient management for G.pyloalis.In this study,the P450 gene of G.pyloalis was identified based on RNA-seq to explore its mechanism in response to pesticide stress and regulation of cuticular hydrocarbon biosynthesis.The main results are as follows:1.57 cytochrome P450 genes were identified by building RNA-seq database and sequence analysis of the transcriptome of G.pyloalis.Sequence analysis showed that all P450 genes identified of G.pyloalis had five conserved domains of cytochrome P450 gene.Including Helix-C,Helix-I,Helix-K,PERF and Heme-binding regions.Phylogenetic tree analysis showed that cytochrome P450 identified belonged to four families of CYP mito,CYP2,CYP3 and CYP4 mito,and each family was composed of 8,8,14 and 27 genes,respectively.Six P450 gene were selected from the above four families for further study.The protein structure of these cytochrome P450s was obtained by homology modeling.RT-q PCR showed that CYP301A1 had the highest expression level in the 4thinstar larvae and the lowest expression level in the adult stage.The expression of CYP304F17 was significantly expressed in pupae stage than in larvae and adults stages.On the other hand,CYP6AW1 and CYP6CV1 had the highest expression level in the midgut,while CYP304F17had the highest expression in the hemolymph.The induction levels of them by different insecticides were further analyzed.The results showed that the expression level of CYP6AW1 was also significantly up-regulated after treatment with deltamethrin and lambda-cyhalothrin,suggesting that CYP6AW1 may be involved in the detoxification and metabolism in G.pyloalis against these two pyrethroid insecticides.The expression of CYP301A1 was significantly up-regulated after chlorfenapyr treatment,indicating that CYP301A1 may be involved in the detoxification of chlorfenapyr.2.Two CYP4G subfamily genes（CYP4G112 and CYP4G113）were identified in the transcriptome of G.pyloalis.Sequence alignment analysis showed that the amino acid sequences encoded by CYP4G112 and CYP4G113 had CYP4G subfamily-specific conserved domain.Phylogenetic analysis showed that CYP4G112 and CYP4G15 of Drosophila melanogaster clustered together,while CYP4G113 and CYP4G1 of Drosophila melanogaster clustered together.RT-q PCR analysis showed that the expression levels of CYP4G112 and CYP4G113 have highest expression in adult stage,and the lowest expression in 2-5 instar larvae.CYP4G112 had the highest expression in epidermis,while in different tissues,CYP4G113 had the highest expression in fat body.Under pesticide stress,the expression level of CYP4G113 in G.pyloalis was up-regulated by 13.7 folds after 24 h treatment with sublethal dose of chlorfenapyr,and the expression level of CYP4G112 in 3rdinstar larvae of G.pyloalis was up-regulated by 11.0 folds after 48 h treatment with sublethal dose of lambda-cyhalothrin.It is speculated that CYP4G family genes may be involved in the detoxification process of common insecticides.In addition,under desiccation stress,the expression levels of CYP4G112 and CYP4G113 increased significantly after 24 h and 48 h treatment in the environment with relative humidity of 10%,suggesting that CYP4G may have the function of responding to insecticides and desiccation stress.3.After silencing CYP4G112 expression by RNAi,G.pyloalis appeared lethal phenotype in pupal stage.At the same time,the cuticular hydrocarbon biosynthesis process of G.pyloalis was limited,and the triglyceride was significantly accumulated.RT-q PCR results showed that after RNA interference with CYP4G112 and CYP4G113,the expression levels of fatty acid synthesis pathway genes including fatty acid synthase FAS was up-regulated,while the expression levels of desaturase Desat,elongase of elongation of very long chain fatty acid ELO and fatty acyl reductase FAR decreased significantly.The gas chromatography technology was used to determine that the content of partial unsaturated fatty acids in the larvae of G.pyloalis increased after silencing CYP4G112.At the same time,after silencing CYP4G113,the water loss rate and weight of G.pyloalis increased significantly in desiccation environment.The above results showed that CYP4G affected the growth and development of G.pyloalis and its adaptation to desiccation conditions by regulating the biosynthesis of cuticular hydrocarbon.In summary,this study revealed that CYP4G and other P450 detoxifying enzyme genes of G.pyloalis responded to common insecticides stress by up-regulating expression.CYP4G plays an important role in cuticular hydrocarbon biosynthesis and regulate the expression of genes in cuticular hydrocarbon biosynthesis pathway and lead to lipid accumulation.In addition,CYP4G contributes to normal pupation and enhances the ability of stem borers to cope with drought stress.This study has important reference for revealing the function of cytochrome P450 in important physiological activities of G.pyloalis and exploring new targets for green management of G.pyloalis. |
