Identification Of P450 Genes Relevant To Lambda-Cyhalothrin Detoxification And Functional Analysis Of CYP6QE1 In Bradysia Odoriphaga

Bradysia odoriphaga is an important vegetable pest in China,feeding on more than 30plant species,especially Chinese chives.B.odoriphaga larvae cluster in the roots,bulbs,and even in immature stems of Chinese chives,making this pest hard to control.The application of insecticides is the main method of pest control in Chinese chives.However,insecticide use causes environmental pollution,may affect human health and may also lead to pesticide resistance.Cytochrome P450 monooxygenases（P450s）plays an important role in the metabolism of various endogenous and exogenous substances.Insecticide resistance in insects has been closely associated with overexpression of P450 genes.In this study,the differentially expressed P450s genes between lambda-cyhalothrin resistant and susceptible strains were investigated by RNA-seq integrally in B.odoriphaga,and CYP6QE1 gene was cloned and identified from B.odoriphaga.The expression profiles of CYP6QE1 in various tissues and at different developmental stages were analyzed by quantitative real time PCR（q RT-PCR）.The expression of CYP6QE1 was also studied after treatment with insecticides.RNAi mediated by double-stranded RNA（ds RNA）was used to study gene function.The main results are summarized as follows:1.Toxicity of lambda-cyhalothrin to susceptible and field populations in B.odoriphaga.The results showed that LC₅₀of lambda-cyhalothrin to susceptible populations and field populations was 79.509 mg/L and 473.01 mg/L,respectively.The resistance ratio of field populations was 5.949.2.RNA-Seq analysis of lambda-cyhalothrin resistant and susceptible populations in B.odoriphaga.The results showed that the lambda-cyhalothrin resistant population have 926 genes with up-regulated expression and 1,240 genes with down-regulated expression,compared with susceptible populations.The up-regulated genes include 17 P450s.Real-time PCR results showed that expression of 8 P450s,cluster19765,cluster23833,cluster27879,cluster5822,cluster43577,cluster33996,cluster26113,and cluster22792,were consistent with the transcriptome sequencing results,and the expression of cluster 23833 was highest,is247.19-fold.3.Cloning and sequence analysis of cytochrome P450 CYP6QE1 gene in B.odoriphaga.The full length of CYP6QE1 was cloned by RT-PCR and RACE technologies.The full length of CYP6QE1 was 1752 bp,encoding 511 amino acids.The isoelectric point and protein molecular weight were 8.96 and 58.314 k Da,respectively.Sequence alignment show that CYP6QE1 share 30%-50%identity with other known CYP6s and contains a series of typical conserved P450 motif,such as helix C WRNAR（residues 127-131）,helix K ETLR（residues371-374）and cytochrome P450 cysteine heme-iron ligand FGDGPRNCLG（residues448-457）.No signal peptide was found in this gene,and a transmembrane domain was formed between amino acids 5～27.4.Functional analysis of cytochrome P450 CYP6QE1 gene in B.odoriphagaThe results showed that relative expression levels of CYP6QE1 was the highest in the malpighian tube,followed by the midgut and fat body,and very low in the head.The expression of CYP6QE1 was significantly higher in larvae stage than that in other developmental stages,and was the highest in 3rd instar larvae,followed by 2nd instar.and rarely expressed in eggs,pupa and adults.The expression levels of CYP6QE1 were very low in in eggs,pupa and adults,and expression levels in male pupa and male adults were slightly higher than female pupa and female adults,but the difference was not significant.After exposure to LC₃₀lambda-cyhalothrin,the relative expression levels of CYP6QE1was increased significantly at 36h.After LC₅₀lambda-cyhalothrin treatment,the expression levels of CYP6QE1 was increased significantly at 36h and 48h.After treatment with LC₃₀chlorpyrifos and LC₃₀clothianidin,the expression levels of CYP6QE1 gene was significantly upregulated.CYP6QE1 gene was successfully silenced using artificial feeding in larvae.Compared to those of larvae fed on a diet containing ds GFP,the transcript levels of CYP6QE1 was decreased significantly at 48 h and 72?h after larvae fed on a diet containing ds CYP6QE1,which was 28.7%and 55.3%of the control.RNAi assays showed that the mortalities of ds CYP6QE1-treated larvae were significantly increased when exposure to lambda-cyhalothrin,chlorpyrifos and clothianidin.