Cloning And Function Analysis Of Four P450 Genes Of Spodoptera Litura

Plants and insects have interacted on Earth for more than 400 million years.Plants produce toxic plant secondary metabolites to reduce the invasion of herbivorous insects,and insects have correspondingly evolved various detoxification metabolic mechanisms such as cytochrome P450 monooxygenase.Spodoptera litura,as a gluttonous and omnivorous pest,severely harms vegetable crops,food crops and economic crops in China.It can be immune to the defense system of various host plants,and the role of P450 detoxification genes in this process needs further research and clarification.In this study,Spodoptera litura was used as the research object,and based on the transcriptome data of four host plants?cotton,corn,cabbage,soybean?fed by Spodoptera litura in the laboratory,P450 genes with high expression and differential expression were selected.Using MEGA,Graphpad prism,DNAMAN,Prime,SPSS and other tools,through gene cloning,PCR,real-time fluorescence quantitative PCR,RNAi and other technologies.The structural characteristics,expression profiles,detoxification and metabolic functions of the plants secondary metabolites are clarified,including the structural characteristics,expression profiles of the four P450 genes?CYP6AB12,CYP321B1,CYP6AB60,CYP321A19?and the changes of related metabolites and metabolic pathways after CYP321B1 silencing.The results are as follows:1.Cloning and homology analysis of 4 P450 genes of Spodoptera litura.The target genes found in the transcriptome were cloned and sequenced to verify the results of the transcriptome data.The results showed that the length of the four P450 gene ORFs was approximately 1400 bp.Homology analysis of 4 amino acid sequences of P450 gene was carried out.It is closely related to Spodoptera exigua CYP321A16?ASO98018.1?,Spodoptera frugiperda CYP321B1?AGO62009.1?,Spodoptera exigua CYP6AB12?ASO97997.1?and Bombyx mori CYP6AB4?ABK27873.1?,amino acid homology was91.35%,88.89%,82.07%and 57.39%,respectively.Find the unique amino acid motif of P450 gene in the amino acid sequence:C-helix:WXXR,K-helix:EXXRXXP,PXXFXPXXF,Heme-binding:FXXGXXXCXG.2.Analysis of P450 gene expression profile of Spodoptera litura.Using RT-q PCR technology,target genes of 8 life stages?eggs,1-6 instar larvae and pupae?and 6 tissues?midgut,hemolymph,body wall,fat body,testis and ovary?of Spodoptera litura were tested.The results showed that the four P450 gene expression profiles were similar,and the relative expression levels were higher in the midgut and fat body tissue of the 4^thinstar larvae of Spodoptera litura.Among them,the relative expression of CYP6AB12 in the midgut is 53 times that of hemolymph tissue?control group?.The relative expression of CYP321B1 in the midgut and fat body is 6 times and 2 times that of hemolymph tissue,respectively.The relative expression of CYP6AB60 in the midgut and fat body is 97 times and 33 times that of the testis tissue?control group?.The relative expression of CYP321A19 in the midgut and fat body is 9 times and 27 times that of the testis tissue?control group?,respectively.3.P450 gene function verification.RNA interference was performed by ds RNA injection and ds RNA mixed nanomaterials smearing the body wall.Taking ds GFP as a control,after treatment,they were fed with artificial feed supplemented with plant secondarymetabolites?ds CYP6AB12-gossypol,ds CYP321B1-tannin,ds CYP6AB60-coumarin,soy isoflavones and ds CYP321A19-quercetin?.Samples of midgut and fat body were taken at 24,48 and 72 h after treatment,and the relative expression of P450 gene was detected by RT-q PCR.The larvae were weighed every 24hours after treatment.Statistical analysis of net weight gain and daily weight gain of larvae.The results showed that the relative expression of the four P450 genes in the midgut and fat body decreased significantly at 72 h compared with the control group.Among them,the relative expression of CYP6AB12 decreased to 9.2%and 29.6%.Similarly,the relative expression of CYP321B1 decreased to 19.3%and 12.5%.The relative expression of soybean isoflavone-treated CYP6AB60 decreased to 13.3%and27.3%,the relative expression of coumarin-treated CYP6AB60 decreased to 34.6%and57.7%.The relative expression of CYP321A19 decreased to 16.1%and 33.8%,respectively.Weight gain slowed significantly in the treatment group.It shows that the four P450 genes have a certain detoxification metabolism effect on the corresponding plant secondary metabolism.4.Metabolome analysis of differential metabolites and metabolic pathways.After ds CYP321B1 was injected for 24 h,the midgut of the 4^thinstar larvae of Spodoptera litura was dissected for extensive targeted metabolomic detection.Results found 185metabolites,combined with variable importance in projection?VIP?and p-value or fold change of variable analysis to further screen for differential metabolites,identified a total of 47 differential metabolites substances:including 13 kinds of amino acids and their metabolites,6 kinds of organic acids and their derivatives,5 kinds of benzene and its derivatives,5 kinds of lipids,5 kinds of nucleotides and their metabolites and the remaining 13 different metabolites.31 of 47 differential metabolites were up-regulated and 16 were down-regulated.Moreover,different metabolites belong to 56 metabolic pathways,among which,two metabolites in glutathione metabolism are down-regulated,while reduced glutathione in ABC transporter is up-regulated,and 2'-deoxyuridine is down-regulated.It was suggested that the silencing of CYP321B1 not only affected the detoxification metabolic process of tannin,but also affected the related detoxification metabolic pathway.In this study,four P450 genes of Spodoptera litura were cloned and analyzed,and the spatiotemporal expression patterns of the genes and their detoxification and metabolic functions in the corresponding host plants were clarified.The research results are helpful to deepen the role of P450 gene in the host adaptability of insects,lay the foundation for the new method of controlling Spodoptera litura in the future.