Functional study on the mutations in the silkworm (Bombyx mori) acetylcholinesterase type 1 gene (ace1) and its recombinant proteins

Abstract

The acetylcholinesterase of Lepidoptera insects is encoded by two genes, ace1 and ace2. The expression of the ace1 gene is significantly higher than that of the ace2 gene, and mutations in ace1 are one of the major reasons for pesticide resistance in insects. In order to investigate the effects of the mutations in ace1’s characteristic sites on pesticide resistance, we generated mutations for three amino acids using site-directed mutagenesis, which were Ala(GCG)303Ser(TCG), Gly(GGA)329Ala(GCA) and Leu (TCT)554Ser(TTC). The Baculovirus expression system was used for the eukaryotic expression of the wild type ace1 (wace1) and the mutant ace1 (mace1). SDS-PAGE and Western blotting were used to detect the targeting proteins with expected sizeof about 76 kDa. The expression products were purified for the determination of AChE activity and the inhibitory effects of physostigmine and phoxim. We observed no significant differences in the overall activity of the wild type and mutant AChEs. However, with 10 min of physostigmine (10 μM) inhibition, the remaining activity of the wild type AChE was significantly lower than that of the mutant AChE. Ten min inhibition with 33.4 μM phoxim also resulted in significantly lower remaining activity of the wild type AChE than that of the mutant AChE. These results indicated that mutations for the three amino acids reduced the sensitivity of AChE to physostigmine and phoxim, which laid the foundation for future in vivo studies on AChE’s roles in pesticide resistance.

Monitoring of Target-Site Mutations Conferring Insecticide Resistance in Spodoptera frugiperda

“…Point mutations linked to organophosphate resistance have been described for Cydia pomonella (F399V), Chilo suppressalis (A314S) and P. xylostella (D131G, A201S, G227A and A441G) [56][57][58][59][60]. Moreover, heterologous expression of AChE mutants (A303S, G329A and L554S) from the silkworm (Bombyx mori) have supported the reduction in AChE sensitivity towards carbamate and organophosphate insecticides [61].…”

Guided Evolution of Recombinant Bombyx mori Acetylcholinesterase II by Homology Modeling to Change Pesticide Sensitivity

“…For proteins where the crystal structure is not available, the amino acid sequence information or the predicted 3D-structure obtained by molecular modeling could be used as starting point for a rational design of variants. Indeed, an A303S/G329A/L554S mutant of B. mori AChE with a similar enzyme activity but lower pesticide sensitivity than wild type AChE was created based on sequence information only, by Ju-Mei et al [ 15 ]. Tan et al [ 16 ] generated Y327D and I374D mutants of Musca domestica AChE after sequence alignment and 3D structure modeling, which lead to a 14–16-fold improvement of the sensitivity to particular pesticides.…”

Identification and characterization of ace1-type acetylcholinesterase in insecticide-resistant and -susceptible Propylaea japonica (Thunberg)

“…It is commonly believed that the reduced sensitivity of AChE due to ace1 mutation is a major factor relating to the resistance of insects to organophosphates (OPs) and carbamates (CBs) (Fournie & Mutéro, 1994; Revuelta et al , 2011; Wang et al , 2014). In general, the expression of ace1 gene in insects is significantly higher than that of ace2 (Huchard et al , 2006; Kim & Lee, 2013; Wang et al , 2014). It was believed that mutation(s) in ace1 alone induced the resistance in insects that have both of the genes (Huchard et al, 2006; Wang et al, 2014).…”

“…In general, the expression of ace1 gene in insects is significantly higher than that of ace2 (Huchard et al , 2006; Kim & Lee, 2013; Wang et al , 2014). It was believed that mutation(s) in ace1 alone induced the resistance in insects that have both of the genes (Huchard et al, 2006; Wang et al, 2014). In Propylaea japonica (thunberg), a high activity on AChE1 (coded by ace1 ) was shown, but none could be detected on AChE2 (coded by ace2 ) (Kim & Lee, 2013).…”