The Role Of Three Types Of Metabolic Enzymes In Culex Pipiens Pallens In Deltamethrin Resistance

Mosquitoes are a group of important vector for malaria,dengue fever,filariasis,and Japanese encephalitis,and pose a serious threat to human health.Widespread and inappropriate use of insecticides has caused mosquito resistance to insecticides and it has become an important obstacle to mosquito control.Metabolic resistance is the main mechanism of insecticide resistance.Cytochrome P450 monooxygenases(CYPs),carboxylesterases(CCEs)and glutathione-S-transferases(GSTs)are the three major detoxification enzymes involved in insecticide detoxification.At present,it was reported in many insects that many members of CYPs,CCEs,and GSTs are associated with insecticide resistance.But how do these resistance genes work together to coordinate the regulation?How to find the main effect gene?These are urgent problems to be solved.Expression Quantitative Trait Loci(eQTL)is a newly developed whole genome research method.The mRNA expression of each gene is used as a quantitative trait and QTL analysis are performed together with the genetic polymorphism data to analyze the regulatory relationships and synergies between genes,find upstream or downstream regulated genes,and finally establish regulation network,for clarifying the mechanism of gene regulation.In this study,we selected Culex pipiens pallens and deltamethrin as material to perform eQTL analysis to find the deltamethrin resistance-related genes of mosquitoes,establish regulatory networks of these genes,and search for major genes.There are 3 major results as below.1.There were 467 possible metabolic resistance related gene sequences,including 183 CYPs,244 CCEs,and 40 GSTs.After further gene sequence alignment,a total of 85 genes belonging to the families of detoxification enzymes were identified,of which 57 were CYPs,13 were CCEs,and 15 were GSTs.All differentially expressed CYPs,CCEs and GSTs were classified according to sequence similarity and homology analysis.57 CYPs,13 CCEs and 15 GSTs were further divided into 8 families,5 families and 5 families,respectively.In addition,each enzyme was given a full name.2.18 P450 genes,8 CCEs genes and 8 GSTs genes were selected and qRT-PCR was used to verify their expression patterns.Among them,12 CYPs,5 CCEs and 5 GSTs were consistent with RNA-seq results.3.We selected 85 differentially expressed metabolic enzyme unigenes and 42,196 SNP loci data for downstream analysis using eQTL.Used Cytoscape,we drew a regulatory network of diffrently expressed genes to find the major regulatory genes in the network.After regulatory network analysis,qRT-PCR verification,siRNA microinjection,and US CDC bottle bioassay,we found one major regulatory gene CYP9AL1 and a regulatory factor CCEJHE3.CYP9AL1 up-regulates the conserved hypothetical protein,CYP329B1 and mitochondrial ferrochelatase gene;TATA-binding protein-associated factor 172 may be the upstream regulatory factor of CCEJHE3.In addition,in the regulatory network,GSTD10 as a possible major regulatory factor has yet to be further studied.This study explored the interactions among mosquito-resistant genes,screened the major genes or regulatory factors related to insecticide resistance and discussed the molecular basis of mosquitoes resistance from levels of expression and regulation.The research results provide a scientific basis for the study of the regulation mechanism of mosquitos metabolic resistance as well as the detection and treatment of insecticide resistance.