Biochemical Properties And CDNA Cloning Of Acetylcholinesterase In Liposcelis Bostrychophila Badonnel

Acetylcholinesterase (AChE) is the target of two major insecticide families, organophosphates (OPs) and carbamates. To elucidate the role of AChE in psocid resistance and the mechanism of AChE-mediated resistance, the biochemical characteristics and gene cloning of acetylcholinesterase (AChE) were investigated in Liposcelis bostrychophila Badonnel.The biochemical results showed that the contents of protein in resistant strains were significantly higher than that of the susceptible strain. The AChE activities in RDDVP (RF = 10.2105) and RPH3 (RF = 4.5083) were obviously reduced (P < 0.05). The comparison of the apparent Michaelis constant (K_m) showed that RDDVP strain increased by 1.33-fold, indicating that AChE might have changed in quality. However, AChE K_m of RPH3 strain has no obvious change, while its maximal velocity (F_max) decreased significantly (P < 0.05). Furthermore, the bimolecular reaction rate constant (K_i) values of the two resistant strains also declined significantly. It could be concluded that the target resistance existed in the two resistant strains of L. bostrychophila, and the resistance was correlated with the quality change of AChE. The structure modification leads to reduced affinity to substrate and insensitivity to insecticides.AChE insensitivity is a frequent resistance mechanism in insects. To elucidate the molecular mechanism of AChE-mediated resistance, two cDNA fragments of AChE gene in L bostrychophila were amplified with the degenerate primers from the conserved peptide sequences of AChEs in insect and acari species by reverse transcription-polymerase chain reaction (RT-PCR) method, 156 bp (GenBank accession number: AY762053) and 264 bp (GenBank accession number: AY762052). The cDNA fragment was inserted into pMD 18-T vector and cloned. The deduced amino acid sequence of AChE consisted of 134 amino acid residues, which shared numerous similarities with AChE of Aphis gossypii AChE2 (75%, AF502082), Schizaphis graminum (75%, AF321574), Leptinotarsa decemlineata (64%, L41180), Culex pipiens AChEl (63%, Q86GC8), Nephotettix cincticeps (62%, AF145235), Helicoverpa armigera (61%, AF369793), Anopheles gambiae AChEl (61%, AJ488492), Plutella xylostella (61%, AY061975), Aedes aegypti (59%, Q9TX11), C. tritaeniorhynchus (58%, AB122151), A. stephensi (58%, P56161), Drosophila melanogaster (56%, X05893), Torpedo califomica (55%, X03439), Bactrocera oleae (55%, AF452052), Musca domestica (55%, AF281161), Lucilia cuprina (55%, U88631), Myzus persicae (54%, AF287291), A. gossypii AChEl (53%, AF502081). The sequence analysis indicated there is high degree of amino acid sequence homology between L bostrychophila and other species. In addition, it includes several characteristic sequences of AChE. Hence, it could be concluded that the fragment encoded the AChE in L. bostrychophila.