Cloning of insecticide resistant genes based on conserved peptide sequences

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Overlapping cDNA sequences encoding acetylcholinesterase (AChE,EC 1.1.1.7) were amplified from an insecticide-susceptible strain of green rice leafhopper Nephotettix cincticeps by RT-PCR with degenerated primers based on conserved protein seqences of cholinesterases and by 3'RACE.The partial amino acid sequence of AChE deduced from the cDNA consisted of 507 residues involing the C-terminal for the precursor protein and it was equivalent to four fifth of a presumed size. The incomplete precrsor protein seqence had 80% and 65-86% of amino acid residues identical to those of Leptinotarsa decemlineata and three dipterpus species (Drosophila melanogaster, Anopheles stephensi, and Aedes aegypti)on an alignment window excluding hyper variable regions, respectively, and produced a remarkably similar hydropacy profile when compared to those of four insect species except for a highly hydrophilic region including 20-30 amino acid resides in the C-terminal half of the three dipterans. The three residues (SER,Glu and His) that putatively form the catalytic triad and Cys that form intra-subunit disulfide bonds were completely conserved when compared among AChEs from a broad range of animal species to date. All these features well established that the amplified cDNA encodes AChE in green rice leafhopper. A degenerated primer set used for the present study also worked for amplifying a cDNA of the housefly AChe which sequence is unknown, and so we suggest possibilities that the same primer set is applicable to the other insect AChE cDNAs.