Study On Cloning, Mutation, Expression And Application Of Housefly (Musca Domestica) AChE Gene

Acetylcholinesterase (AChE, EC 3.1.1.7) catalyzes the hydrolysis of the neurotransmitter acetylcholine (ATCh) at cholinergic synapses to keep the normal transmission of nerve impulses. It is the target of the organophosphate and carbamate compounds. The intensive use of insecticide has led to the pollution of environment and many insects have developed resistance. So studying the interaction of the target enzyme and insecticide, screening out the insecticides through mutant protein has become a very important project. Housefly acetylcholinesterase was considered to be the most sensitive enzyme in detecting pesticides, and the crude enzyme was used to be the enzyme sources, but this method has many defects, such as the crude enzyme contains many other proteins which disturb the enzyme sensitivity. In this research, acetylcholinesterase gene was amplified from the sensitive and resistant housefly strains, the 1224 site was mutated. The acetylcholinesterase genes were expressed in baculovirus system and purified products were used in the detection of pesticides. The major results are summarized as following:1. We named the housefly susceptible and resistant strain as SS, RR respectively, and four mutationsV261L, G343A, F408Y, D421V were found in the AChE from resistant strain. SS and RR AChE genes were cloned and sequenced. Also a mutant AChE gene (V261L/G343A/D421V) in which the A in 1224 nt of RR was changed to T (tyrosine to phenylalanine) was constructed2. The genes were recombined into the baculovirus vector bacmid to construct the recombinants, Bacmid-SS, Bacmid-RR and Bacmid-V261L/G343A/D421V and expressed in Tn cells. SDS-PAGE and affinity chromatography showed that there is a specific band in supernatant, whose molecular weight is about 66 kDa. The IC50 of these three recombinant AChEs to organophosphare and carbamate insecticides showed the combination of V261L/G343A/D421V has similar sensitivity to the resistance strain but far exceed to the susceptible strain.3. AChE gene from susceptible housefly strain was expressed in the Tn cells on a large scale. The protein was purified by procainamide affinity chromatagraphy. The result of application of purified enzyme in detection of organophosphate and carbamate insecticides showed this enzyme was more sensitive to carbamates than to organophosphates, but the most sensitive was Dichlorvos which is an organophosphate insecticide.