Cloning And Functional Identification Of Deltamethrin Resistance Associated Genes Of Cx. Pipiens Pallens And Primary Studies On Inhibiting Mosquito Resistance To Insecticide Using RNAi

Insect vectors seriously harm human health. According to the estimating, 2/3 infectious diseases are transmitted by insect vectors. Many known vector-borne diseases in the world, such as plague, macula typhoid, yellow fever and malaria etc. once brought on broad prevalence and arouse hundreds of thousands' human death. With progressively contacting of human being, many new and reemerging diseases transmitted by insect vectors are found. Although important advances continue to be made in the development of alternative control measures, insecticides will remain a vital part in the integrated control program of vector for the foreseeable future. Owing to largely and continuously using insecticides, it caused developing of resistance to insecticides. At present, resistance to insecticides has appeared in the major insect vectors from every genus included more than 504 species. Among them, 109 mosquitoes show resistance to one or more many insecticides. The resistance has developed to every chemical class of insecticide, including microbial drugs and insect growth regulators. It is worrying that insecticide resistance is expected to directly and profoundly affect the emergence and reemergence of some vector-borne diseases, and where resistance has not contributed to disease emergence, it is expected to threaten the diseases control. Therefore, it is very impending to studying of resistance mechanisms and proper resistance management.1. Molecular cloning and differential expression of a cytochrome P450 gene (CYP6F1) in the mosquito, Culex pipiens pattensTo clone a cytochrome P450 gene (CYP6F1) and determinedifferential expression at deltamethrin-resistant and susceptible strains of 4^th instar larvae, and all stages of resistant strains of Cx. pipiens pollens. CYP6F1 was isolated from deltamethrin-resistant 4th instar larvae of the mosquito using RT-PCR and RACE techniques, relative bioinformatics analysis was carried out using softwares, and the difference of expression was identified by real time PCR and RT-PCR in the deltamethrin-resistant and susceptible strains and all stages of the mosquito.The results showed that a new gene, CYP6F1 (GenBank/NCBI AY662654) and its allele, CYP6Flvl (GenBank/NCBI AY945841) with complete coding sequence in the cytochrome P450 family 6 were cloned and sequenced. Both the cDNA sequences of CYP6F1 and CYP6Flvl have an identical open reading frame of 1527 bp coding a putative protein of 508 amino acid residues. The deduced amino acid sequence of CYP6F1 and CYP6Flvl indicated that the encoded P450 has the conserved domains of a putative membrane-anchoring signal, putative reductase binding sites, a typical heme-binding site, an ETLR motif and substrate recognition sites. The CYP6F1 was expressed to a greater extent in the deltamethrin-resistant strain than in the susceptible strain. The expression levels of the CYP6F1 in the deltamethrin-resistant 1st, 2nd, 3 rd, 4th larvae and adult female mosquitoes differed, with the highest expression in the 4th larvae. It was suggested that CYP6F1 may be a deltamethrin-resistance associated gene of Cx pipiens pallens2. Primary functional expression of CYP6F1 from the deltamethrin-resistant mosquito, Cx. pipiens pallensIn order to identify the relationship between CYP6F1 and the resistance to deltamethrin in the mosquito, Cx. pipiens pallens. The entire coding region of CYP6F1 was amplified by PCR using the specific primers designed, and CYP6F1 cDNA was inserted into the expression vector pIB/V5-His-TOPO, and a recombinant vector, pIB/V 5-His/C YP6F1 was constructed. Then mosquito C6/36 cells was transfected with pIB/V5-His/CYP6F1, and the cells transfected with pIB/V5-His/CYP6F1 were screened with blasticidin and the stable expression cell lines were created. The cells were analyzed for transcriptand expression using RT-PCR and Western blot assays. After treating the cells transfected with pIB/V5-His/CYP6F1 and control cells with deltamethrin, the cell viability, growth curve, cell proliferation, cell cycle and morphologic changes were detect...