The Investigation Of Organophosphorus Insecticide Resistance In Bombyx Mandarina And Fusion Expression Of The Resistance-related GST-Omega 1 Gene

Wild silkworm Bombyx mandarina (Lepidoptera: Bombycidae) has the same ancestor as the economic domestic silkworm Bombyx mori. It is generally thought that wild silkworm bears higher resistance to pesticides than that of domestic silkworm. There have been much more researches on the resistance of domestic silkworm to pesticides but only seldom researches on wild silkworm. Resistant strain of wild silkworm has not been reported yet.In the present study, organophosphate resistant strain of wild silkworm was selected to research its resistance to organophosphorus pesticides. Wild silkworms of different instars were collected from the field and selected by feeding with dichlorvos of various concentrations for several generations. The results showed that wild silkworm obtained much more resistance under the selection pressure of the pesticide. The resistance level of wild silkworm to dichlorvos increased by 2-fold and pupation rate also increased accordingly through selection for 3 generations. The preliminarily selected resistant strain of wild silkworm could be used as a good material for the cloning of resistance-related genes, the control of Lepidopteral pests, and the breeding of resistant strain of domestic silkworm.Glutathione S-transferases (GSTs) are a class of primary and secondary metabolic enzymes involved in the detoxification of organophosphate, organo-chlorine, and alleochemicals.A 771bp GST-Omega1 gene (encoded 256 aa) was cloned by RT-PCR technology from the midgut of wild silkworm. Analysis by conserved domains online tool at NCBI website indicated that the deduced amino acid sequence comprises the omega family conserved sequences such as 1 Cys residue and 8 GSH binding site. The GST-Omega1 gene was expressed using Bac-to-Bac baculovirus expression system. SDS-PAGE and Western blotting results showed that a specific band consistent with the expected size of fusion protein was detected around 33 kD. The recombinant protein was purified by His·Bind Resin. Km and Vmax for the CDNB as the substrate were 2.81μmol/L,and 2.70μmol /(mg·min)respectively.