Cloning, Sequence Analysis, Construction Of Expression Plasmids And Functional Identification Of Deltamethrin-resistance Associated Glycogen Branching Enzyme Gene (NYD-GBE) From Culex Pipiens Pallens

[Objective] To acquire the full length cDNA of glycogen branching enzyme(GBE) from the deltamethrin-resistant strain and identify the expression levels in resistant and susceptible strains of Cx. pipiens pallens. Construct prokaryotic and insect expression plasmids of NYD-GBE and further research the relationship between NYD-GBE and insecticide resistance.[ Methods ] Through RACE(Rapid amplifyication of cDNA ends) method , the full length GBE cDNA was acquired. The expression levels of NYD-GBE in the resistant and the susceptible strains were identified by Real time PCR. The open reading frame (ORF) sequence of NYD-GBE was cloned by RT-PCR, sequenced in an automatic DNA sequencer (ABI-100) and analyzed with bioinformatic softwares. The prokaryotic and insect expression plasmids of NYD-GBE were constructed by gene re-construction methods. The recombinant insect expression vectors were transfected into Aedes albopictus C6/36 cells. Stable-transfected strains were established by screening culture with blastin, and then the transcription and expression of the NYD-GBEwere identified by RT-PCR. The viability of the transfected cells and untransfected ones were detected by ~3H—TdR.[Results] A sequence of 2337 base pairs including an open reading frame of 2070 base pairs was gotten and named NYD-GBE (GenBank/NCBI DQ102393, 2005). NYD-GBE was acquired and expressed 19.67 fold in the resistant strain than in the susceptible strain by Real time PCR. The encoded protein had 689 amino acids with 82% and 72% homology of Anopheles gambiae(An. Gambiae), Melanogaster pseudoobscura (M. pseudoobscura) respectively, and the theoretic molecular weight and PI were 79811.25 Ku...