Preliminary Studies On The Functions Of Deltamethrin Resistance-associated NYD-Ch And NYD-Tr Of Culex Pipiens Pallens

[ Objective ] To research further the relationship between the genes NYD-Ch, NYD-Tr and insecticide resistance.[Method] The coding regions of NYD-Ch and NYD-Tr, which are highly expressed in resistance strain of Culex pipiens pallens, were amplified by PCR from the cDNA library constructed in resistance strain of the mosquito. After identification of T/A clone and sequencing, NYD-Ch and NYD-Tr were subcloned into the eukaryotic expression vector pIE1-3, and so the recombinant expression vectors pIE1-3/NYD-Ch and pIE1-3/NYD-Tr of insect cell were constructed. Identifications of restriction digestion and PCR were carried out before the above recombinant vectors were transfected respectively and co-transfected into Aedes albopictus C6/36 cell with pIE1-neo plasmid. Stable-transfected strains were established by screening culture with G418, and then the transcription and expression of the NYD-Ch and NYD-Tr were identified by RT-PCR and Western blotting. The viability of the transfected cells vs untransfected ones are detected by MTT and flow cytometry.[Results] It was confirmed that the expression vectors of insect cell were successfully constructed and that the stable-transfected cell lines were established. The expressions of the proteins of interest were identified by Western blotting. The following results are shown by MTT: (1) With the group transfected with pIE1-3/NYD-Ch merely, there was no significant difference compared with the control (p>0.05). (2) After exposed to 120μM deltmethrin for 120h, the viability of the group transfected with single pIE1-3/NYD-Tr increased by 38.20% (p<0.05) and 9.66% (p>0.05) respectively, compared with the cells that was transfected empty vector and untransfected. (3) In the group co-transfected with the both genes,expression of NYD-Ch and NYD-Tr increased the viability of deltmethrin-treated C6/36 cell, increased by 67.37 and 38.83% in the viability of the cells treated with 120μM, respectively (p<0.01 and p<0.001), increased by 48.83% and 24.26% in the viability of the cells treated with 160μM (p<0.05 and p<0.05), compared with the cells that were transfected with empty vector and untransfected.[Conclusion] It is demonstrated by MTT that NYD-Ch and NYD-Tr play an important role in being against the deltamethrin toxicity and thus provide cells certain protection, which suggests that there is a close relationship between the genes of NYD-Ch and NYD-Tr and the deltamethrin resistance.