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Cloning and Preliminary Studies on the Functions of EU073017 Gene from Culex Pipiens Pallens

Author: YangMiFang
Tutor: ZhuChangLiang
School: Nanjing Medical University
Course: Pathogen Biology
Keywords: Culex pipiens pallens deltamethrin resistance EU073017 gene real time PCR stable transfection cell viability
CLC: R383
Type: Master's thesis
Year: 2008
Downloads: 53
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Abstract


【Objective】To acquire EU073017 gene from Culex pipiens pallens and further research the relationship between EU073017 gene and insecticide resistance.【Methods】EU073017 gene was cloned from cDNA library of deltamethrin-resistant Cx.pipiens pallens,sequenced and analyzed with bioinformatic softwares.Transcriptions of EU073017 gene in the deltamethrin-susceptible strains and deltamethrin-resistant strains of Cx. pipiens pallens(fourth instar of larvae)was determined by Real-time PCR.The insect expression plasmids of EU073017 gene were constructed by gene reconstruction methods and transfected into Aedes albopictus C6/36 cells.Stable-transfected strains were established by screening culture with blasticidin.The viability of the transfected cells effected by various concentrations of deltamethrin were detected by Cell Counting Kit-8(CCK-8).【Results】EU073017gene were acquired with 398 bp including an CDS of 270 bp(GenBank no.EU073017,2007).Alignment of the deduced amino acid sequence with Anopheles funestus、An.gambiae and Ae. aegypti revealed sequence identities 86%、84%and 80%.EU073017 gene was overexpressed in deltamethrin-resistant strains than in deltamethrin-susceptible strains of Cx.pipiens pallens(fourth instar of larvae).Stable expression of EU073017 gene in C6/36 cells confer moderate deltamethrin resistantce.【Conclusion】EU073017 gene of Cx.pipiens pallens was cloned successfully and the gene is overexpressed at transcriptions level in the deltamethrin-resistant strains than in the deltamethrin-susceptible strains of Cx.pipiens pallens.EU073017 gene is related to deltamethrin resistance of the Culex mosquito.

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