Construction Of Transgenic Drosophila With Carboxylesterase Gene Of Aphis Gossypii And Study On Its Insecticide Resistance

With wildly using of insecticides, insects were forced to produce insecticides resistance to adaptatenvironmental changes and selecting pressure. Chemical insecticides were the effective action to insects,for example, organochlorines, organophosphates, carbamates and pyrethroids etc. For insect themechanisms of insecticides resistance can be basically classified metabolic resistance and target siteresistance. Over-expressed resistence proteins and changes of esterase involved in the metabolic resistance,for example, the quantitative changes and qualitative changes of carboxylesterases proteins and theamplification of P450monooxygenases and Glutathione S-transferases (GSTs) etc. Target site resistancemeans low sensitive target protein, which includes insensitive acetylcholinesterase, the GABA receptormutation and mutations in the voltage-gated sodium channel etc. Gene amplification or transcriptionalup-regulation and mutagenesis resulting in qualitative changes were the mechanisms of insectcarboxylesterases resistance.In our study, we used the technology of transgenic Drosophila, with classics Gal4/UAS system,successfully constructed three lines of Aphis gossypii carboxylesterases transgenic Drosophila, they arehomozygotes of Est-wild, Est-A151D and Est-W271L. The transgenic Drosophila lines cross with the Gal4lines and activate the expression of Aphis gossypii carboxylesterases in vivo. Through molecular assay ofthe transgenic Drosophila, the real time PCR results showed that nearly identical in the genome level andtranscriptome level of the three lines. Carboxylesterase activity were detected by starch gel electrophoresisand spectrophotometer, and the results showed the wild carboxylesterases activity were higher than themutation ones, and about2-3times enzymatic activity were quantified. Bioassays showed the mutagenesislines had higher chlorfenvinphos resistance, and no distinction among the transgenic Drosophila in thebioassays of malathion, monocrotophos, parathion and decamethrin, which illustrated that point mutation at151and271equivalent positions in carboxylesterases confered OP resistant to chlorfenvinphos and lostenzymatic activity simultaneously. Compare to the control W1118×Gal4, a surprising result was that all thetransgenic lines had higher insecticides resistance to malathion, parathion and decamethrin, and in the sameway the A151D mutagenesis ones accepted higher insecticides resistance to monocrotophos and chlorfenvinphos, all of which explained that the insects produced resistance through gene amplification,due to the allogenic gene join in the Drosophila genome. All our study results showed that the quantitativechanges will be main in the insect resistance evolution of insect carboxylesterases, and the qualitativechanges were only minority.