Cloning And Characterization Of NYD-OP7, A Novel Deltamethrin Resistance Associated Gene From Culex Pipiens Pallens

One mosquito opsin gene, NYD-OP7, has been cloned from Culexpipiens pallens. Within the sequence, an open reading frame (ORF) of1116 bp was found to encode a putative 371 amino acids protein whichexhibits high identity with opsins from Aedes and Anopheles mosquitoes.Transcript expression of NYD-OP7 was determined by real-timePCR in all life stages of deltamethrin-susceptible and -resistant strains ofthe Culex mosquito. The results demonstrated that this gene is expressedat all developmental stages, and it is expressed predominantly at thepupae and adult stages. Meanwhile, in pupae and adults, NYD-OP7 isoverexpressed in deltamethrin-resistant strain than in -susceptible strain.Importantly, stable expression of NYD-OP7 in the mosquito C6/36 cellscan confer moderate deltamethrin resistance.Our study provides the first direct evidence that increased expressionof an opsin gene may play some role in the development of deltamethrinresistance in Cx. pipiens pallens. Opsin 3 (OPN3), also called encephalopsin or panopsin, is a recentlydiscovered extraretinal photoreceptor, and its function is largely unknownuntil now.In this study, based on the RT-PCR results, we showed that OPN3was moderately expressed in the thymus, testis, liver, pancreas andplacenta and weakly in the prostate gland and kidney. Meanwhile, theexpression of OPN3 was significantly higher in the paclitaxel -resistantline than in the drug naive parental A549 cell line. Then we confirmed theoverexpression by real-time PCR analysis.Using cDNA transfection and RNA interference, we successfullyestablished stable transfectants with upregulation and downregulation ofOPN3 expression, respectively. In vitro drug sensitivity assaydemonstrated that overexpression of OPN3 sensitized A549 cells topaclitaxel whereas downregulation of OPN3 conferred A549_PTX cellresistance to paclitaxel.We also investigated cell localization of OPN3 by fusing it to anenhanced green fluorescent protein (EGFP). Green fluorescence signalwas found clearly in the nuclei, body and membrane of cells transfected with OPN3-EGFR In contrast, EGFP (vector control) was distributedmore or less evenly throughout the whole cell.Our data for the first time suggests that OPN3 gene may play somerole in the development of paclitaxel resistance in human lung cancercells.