Cloning And Primary Function Identification Of Ribosomal Protein L39 Gene From Culex Pipiens Pallens

Insect vectors seriously harm human health. According to theestimating, 2/3 infectious diseases are transmitted by insect vectors.Many known vector-borne diseases in the world, such as plague, maculatyphoid, yellow fever and malaria etc. once brought on broad prevalenceand arouse hundreds of thousands' human death. With progressivelycontacting of human being, many new and reemerging diseasestransmitted by insect vectors are found. Although important advancescontinue to be made in the development of alternative control measures,insecticides will remain a vital part in the integrated control program ofvector for the foreseeable future. Owing to largely and continuously usinginsecticides, it caused developing of resistance to insecticides. At present,resistance to insecticides has appeared in the major insect vectors fromevery genus included more than 504 species. Among them, 109mosquitoes show resistance to one or more many insecticides. Theresistance has developed to every chemical class of insecticide, includingmicrobial drugs and insect growth regulators. It is worrying thatinsecticide resistance is expected to directly and profoundly affect the emergence and reemergence of some vector-borne diseases, and whereresistance has not contributed to disease emergence, it is expected tothreaten the diseases control. Therefore, it is very impending to studyingof resistance mechanisms and proper resistance management.1. Molecular cloning and differential expression of a ribosomalprotein L39 gene (RPL39) in the mosquito, Culex pipiens pallensTo clone a ribosomal protein L39 gene (RPL39) and determinedifferential expression at deltamethrin-resistant and susceptible strains of4th instar larvae, and all stages of resistant strains of Cx. pipiens pallens,RPL39 was isolated from deltamethrin-resistant 4th instar larvae of themosquito using RT-PCR and RACE techniques, relative bioinformatiesanalysis was carried out using softwares, and the difference of expressionwas identified by real time PCR and RT-PCR in the deltamethrin-resistantand susceptible strains and all stages of the mosquito. The results showedthat a new gene, RPL39 (GenBank/NCBI DQ080075) with completecoding sequence was cloned and sequenced. The cDNA sequences ofRPL39 have an identical open reading frame of 156 bp coding a putativeprotein of 51 amino acid residues. Amino acid sequence alignment ofRPL39 by ClustalW software showed high conservation of RPL39 indifferent species. Phylogenetic relationships of RPL39 among Cx. pipienspallens and some other species showed RPL39 of Cx. pipiens pallens hasthe highest homology with Anopheles gambiae RPL39. SMART showedthere is no transmembrane domain in this protein, and a typical ribosomalprotein L39 domain is found. No signal peptide is predicted by SIGNALP3.0. Cellular localization analysis by PSORT indicated RPL39 likelyexisted in cytoplasm. The RPL39 was expressed to a greater extent in thedeltamethrin-resistant strain than in the susceptible strain. The expressionlevels of the RPL39 in the deltamethrin-resistant egg, 1st, 2nd, 3rd, 4th larvae, pupa and adult female mosquitoes differed, with the highestexpression in the adult female mosquito. It was suggested that RPL39may be a deltamethrin-resistance associated gene of Cx. pipiens pallens.2. Primary functional expression of RPL39 from the deltamethrin-resistant mosquito, Cx. pipiens pallensIn order to identify the relationship between RPL39 and theresistance to deltamethrin in the mosquito, Cx. pipiens pallens. The entirecoding region of RPL39 was amplified by PCR using the specific primersdesigned, and RPL39 cDNA was inserted into the expression vectorpIB/V5-His-TOPO, and a recombinant vector, pIB/V5-His/RPL39 wasconstructed. Then mosquito C6/36 cells was transfected with pIB/V5-His/RPL39, and the cells transfeeted with pIB/V5-His/RPL39 were screenedwith blasticidin and the stable expression cell lines were created. Thecells were analyzed for transcript and expression using RT-PCR andWestern blot assays. After treating the cells transfected with plB/V5-His/RPL39 and control cells with deltamethrin, the cell viability, growthcurve, cell proliferation, cell cycle and morphologie changes weredetected with ~3H-TdR etc..The results showed that the cell transfected with the RPL39 geneexpressed higher, the inhibitive effect (EC50) was higher than controlcells (p＜0.05), cell proliferation got much better and cell shape was bettercompared with the control cells after treating cells with deltamethrin. Itwas suggested that the cells transfected with RPL39 had an increasedresistance to deltamethrin as compared with the control cells and RPL39may be related to deltamethrin resistance in the mosquito, Cx. pipienspallens. 3. Primary downstream proteomic research of RPL39 from thedeltamethrin- resistant mosquito, Cx. pipiens pallensThe differential expression protein spots of cells transfected withRPL39 were identified by two-dimensional electrophoresis (2-DE) andpeptide mass fingerprinting (PMF) in order to discuss the insecticideresistance mechanism of RPL39 and the interaction with other proteins. Aseries of proteinsells were high expressed in cells transfected with RPL39,some proteins had high homology with enzyme protein of other species,e.g. prophenol oxidase, serine/threonine-protein kinase, serine protease,serine protein phosphatase, argininosuccinate lyase, tyrosinephospholipidase and multidrug resistance protein; some proteins had highhomology with transcriptive and translative regulate protein, e.g.ATP-binding cassette transporter subfamily B, transmembrane GTPase,DNA replication licensing factor, transcriptional regulator andretrotransposon gag protein; and some proteins had high homology withtransferrin, iron regulatory protein, selenophosphate synthetase andglycogen synthase. The insecticide resistance feature of cells transfectedwith RPL39 was predicted by increase detoxicating enzyme synergy,ensure the accurate transcription and translation, and enhance the viabilityof cells. The results provide a rationale for the insecticide resistancemechanism research of RPL39.