Cloning of insecticide resistant genes based on conserved peptide sequences

根据乙酰胆碱酯酶的保守蛋白质序列和3‘RACE序列，采用简并的RT-PCR方法，从一个对杀虫剂敏感的稻纵卷叶蝉品系中扩增出编码乙酰胆碱酯酶(AChE，EC 1.1.1.7)的重叠序列，其氨基酸序列由507个氨基酸残基组成，与前体蛋白的C末端有关，相当于推测大小的五分之四。不完全前体蛋白序列在不包括高变异区的比对窗口上分别含有80%和65-86%的氨基酸残基，与Deptinotarsa decemlineata和三种双翅目昆虫的氨基酸序列相同，并产生了与四种昆虫非常相似的亲水性图谱，除了三种双翅目昆虫的C-末端一半有20-30个氨基酸的高亲水性区域外。三个残基(SER，Glu和His)可能形成催化三联体，Cys形成亚基内二硫键，当与来自广泛动物物种的ACh进行比较时，它们是完全保守的。所有这些特征都证实了扩增的AChE基因编码绿色稻叶蝉体内的AChE。本研究中使用的一组简并的引物也可以扩增出一个序列未知的家蝇AChE的cDNAs，因此我们认为同样的一组引物也适用于其他昆虫AChE的cDNAs。

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.