Cloning And Functional Characterization Of CYP9A Subfamily Genes Involved In Metabolism Of Lambda-cyhalothrin In Cydia Pomonella

The codling moth,Cydia pomonella,belongs to Lepidoptera,is a major agricultural invasive pest.The control of C.pomonella mainly relies on chemical insecticides,of which lambda-cyhalothrin is one of the most commonly used insecticide.The long-term unreasonable use made the C.pomonella had a medium-to-high level of resistance to lambda-cyhalothrin.Previous studies have shown that elevated enzyme activity of cytochrome P450 monooxygenases（P450s）is the reason for lambda-cyhalothrin resistance of C.pomonella,CYP9A is an important subfamily of P450 related to detoxification metabolism,CYP9A61 has been shown to be involved in the metabolism of lambda-cyhalothrin.Whether the other genes in the subfamily have the same function and the molecular mechanism of resistance formation are still unclear.In this paper,systematic identification of C.pomonella CYP9A genes by analyzing both the genome database and transcriptome.The spatiotemporal expression patterns and expression patterns after lambda-cyhalothrin exposed were analyzed.Moreover,we also analyzed the expression level between field strain and susceptible strain.The effects of three CYP9A genes silencing on the lambda-cyhalothrin sensitivity in C.pomonella larvae were detected by RNAi assay.Three CYP9A P450s were expressed in Sf9cells with the Bac-to-Bac baculovirus expression system,their ability to metabolize lambda-cyhalothrin were determined.Here are the main results:1.Identification and bioinformatics study of CYP9A genes of Cydia pomonellaBy screening and comparison of the genomic data and transcriptome data of the C.pomonella,we identified and characterized total of 4 CYP9A subfamily genes were identified,in addition to the previously reported CYP9A61,the other 3 were designated as CYP9A120,CYP9A121,and CYP9A122,respectively.The four CYP9A genes were located on the same chromosome（chr12）,and open reading frame（ORF）were ranged from 1593 bp to 1617 bp.The molecular weights of the proteins were similar,ranging from 61.06 k Da to 62.04 k Da,and the isoelectric points were 7.64 to 8.74.2.Expression Profiles of CYP9A genes of C.pomonellaThe real-time quantitative PCR（RT-qPCR）were carried to analyze the relative expression levels of CYP9A genes in different developmental stages,different tissues,larvae exposed to LD₁₀lambda-cyhalothrin and between field strain and susceptible strain.The results showed that CYP9A120,CYP9A121,and CYP9A122 were ubiquitously expressed at all developmental stages and mostly abundant in the larval stages,and the expression levels of CYP9A121 and CYP9A122 in the first instar larvae were higher than other developmental stages.The m RNA levels of CYP9A120,CYP9A121,and CYP9A122 in the midgut were higher than in the other tissues.LD₁₀lambda-cyhalothrin could induce the three CYP9A genes up-regulation of the expression levels,but showed different expression patterns at different times.The expression levels of CYP9A genes in lambda-cyhalothrin-resistant strain were significantly higher than in the susceptible strain.3.RNAi interference and insecticide susceptibility analysis on CYP9A genes of C.pomonellaAfter injecting double stranded RNA（dsRNA）,transcript levels and P450 activities of CYP9A120 and CYP9A121 were observed greatly reduced,while the silencing efficiency of CYP9A122 was lower and there was no significant change in P450 enzyme activity.The larvae were significantly more sensitive to LD₁₀and LD₅₀lambda-cyhalothrin after silencing CYP9A120 and CYP9A121.4.Expression of recombinant P450s protein and metabolism of lambda-cyhalothrin analysis of CYP9A genes of C.pomonellaCYP9A120,CYP9A121,CYP9A122 and CPR were expressed in Sf9 cells with the Bac-to-Bac baculovirus expression system.The results showed that the three CYP9A recombinant proteins were all active P450s and had catalytic activity to the two model substrates BOMR and BFC.CYP9A120,CYP9A121,and CYP9A122 can all metabolize lambda-cyhalothrin and generate hydroxylated metabolites,but they have regioselective catalysis for lambda-cyhalothrin:CYP9A121 mainly catalyzes lambda-cyhalothrin to4’-OH-metabolites,CYP9A122 mainly generated 2’-OH-metabolites;CYP9A120 has low metabolic activity to lambda-cyhalothrin,but can catalyze it to 2’-OH-and 4’-OH-metabolites.