Cloning Of Arrestin Gene From Culex Pipiens Pallens And Its Preliminary Characterization In Deltamethrin Resistance

Culex pipiens pallens is an important home-dwelling disease vector in our country,having serious hazard to human health. Chemical control has been the main effectivemeasure to reduce populations of disease vectors since the1950s. However, continuousand excessive application of insecticides has resulted in rapid development of insecticideresistance in many mosquito species including Cx. pipiens pallens. To identify newresistance genes and to study their effects on the resistance has been considered as one ofthe most challenging issues in modern applied entomology. The previous studies in ourlaboratory discovered and proved that opsin gene was a gene associated with insecticideresistance; in further study on how opsin is involved in insecticide resistance mechanisms,we found that arrestin gene was expressed higher in the deltamethrin-resistant (DR)strain than in the deltamethrin-susceptible (DS) strain of Cx. pipiens pallens. Similarly,there ware other reports that arrestin was highly expressed in Cx. quinquefasciatusresistant to permethrin and in Drosophila melanogaster resistant to DDT.Based on these studies, we cloned the full length cDNAs of arrestin gene from Cx.pipiens pallens with PCR and RACE (rapid amplification of cDNA end). Then wesurveyed the mRNA levels of arrestin gene in the mosquito life cycle of DR and DSstrain of Cx. pipiens pallens by quantitative real-time PCR. In addition, we identified therelationship between arrestin and deltamenthrin(DM) resistance using strategies ofgenetic overexpression and RNAi of arrestin in mosquito cells. Cell viability wasanalyzed by CCK8after DM treated. We also analyzed the mRNA levels of cytochromeP4506A1(CYP6A1, GenBank ID:FJ423553) and opsin between the transfected cells andthe controls. In our study, the complete sequence of arrestin gene was cloned from Culex pipienspallens, an open reading frame (ORF) of1,155bp being found to encode a deduced384amino acid protein (GenBank ID: HQ833831,2011), and the putative amino acidsequence shareing99%,92%and71%identity with arrestin from Cx. quinquefasciatus,Anopheles gambiae and Drosophila melanogaster respectively. It was found that arrestingene was expressed throughout the life cycle in all life stages of DS and DR strains of Cx.pipiens pallens. Moreover, at egg, pupae, male and female adults’ stages, arrestin wassignificantly up-regulated in DR strain compared with DS strain(p<0.05).Over-expression of arrestin comparably increased cell viability and knockdown ofarrestin by siRNA decreased cell viability in mosquito cells under DM treatment.Meanwhile, the mRNA levels of CYP6A1and opsin were up-regulated in mosquito cellstransfected with arrestin and were down-regulated with knockdown of arrestin in cells,suggesting that the arrestin gene may be a deltamethrin resistance related gene and playsome role in the development of DM resistance in combination with opsin to regulatetranscriptional level of CYP6A1which finally leads to resistance.In this study, we presented the first evidence that arrestin is associated withinsecticide resistance in Cx. pipiens pallens, and preliminarily investigated the resisitancemechanism of arrestin. Our results provided a new clue for clarifying the molecularmechanisms of insecticide resistance and establishing the new resistance testing methods.It has an important theoretical value and potentially practical application.