title: 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.
authors: School of Basic Medicine and Biological Sciences, Soochow University, Suzhou, 215123, Jiangsu, People's Republic of China.
authors: Ju-mei
authors: JM
authors: Wang
authors: Bin-bin
authors: BB
authors: Wang
authors: Yi
authors: Y
authors: Xie
authors: Shan-shan
authors: SS
authors: Sun
authors: Zhi-ya
authors: ZY
authors: Gu
authors: Lie
authors: L
authors: Ma
authors: Fan-chi
authors: FC
authors: Li
authors: Yi-fan
authors: YF
authors: Zhao
authors: Bin
authors: B
authors: Yang
authors: Wei-de
authors: WD
authors: Shen
authors: Bing
authors: B
authors: Li
mesh_terms: D000110:Acetylcholinesterase / Q000737:chemistry / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000201:enzymology*; D002800:Cholinesterase Inhibitors / Q000737:chemistry*; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics*; D007305:Insecticide Resistance; D007306:Insecticides / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009946:Organothiophosphorus Compounds / Q000737:chemistry; D010830:Physostigmine / Q000737:chemistry; D061987:Sf9 Cells; D018411:Spodoptera
chemical_list: D002800:Cholinesterase Inhibitors; D019476:Insect Proteins; D007306:Insecticides; D009946:Organothiophosphorus Compounds; C003135:phoxim; D010830:Physostigmine; D000110:Acetylcholinesterase