Cloning of insecticide resistant genes based on conserved peptide sequences

基于保守肽序列的杀虫剂抗性基因克隆

• 批准号: 07660067
• 负责人: TOMITA Takashi
• 金额: $ 1.34万
• 依托单位: National Institute of Health
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07660067/
• 关键词: insecticide resistance; green rice leaf hopper; housefly; cDNA; mmolecular biology; acetylcholinesterase; チトクロム P450

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.

根据胆碱酯酶保守蛋白序列设计简并引物，采用RT-PCR和3 'RACE技术，从绿色灰叶蝉（Nephotettix cincticeps）敏感品系中扩增出乙酰胆碱酯酶（AChE，EC 1.1.1.7）的重叠cDNA序列，推导出AChE的部分氨基酸序列，由507个残基组成，涉及前体蛋白的C-末端，相当于推测大小的5分之4。不完整的前体蛋白序列与马铃薯叶甲和三种双翅目昆虫的氨基酸残基分别有80%和65-86%的相同（黑腹果蝇、斯氏按蚊和埃及伊蚊）分别在不包括高变区的比对窗口上进行比较，并且当与四种昆虫物种的那些相比时，产生了非常相似的亲水性分布，除了包括20- 30%的高度亲水性区域之外。30个氨基酸残基位于三个双翅目昆虫的C-末端一半。三个残基（SER，Glu和His），purone形成的催化三联体和半胱氨酸，形成亚基内二硫键是完全保守的AChE之间进行比较时，从广泛的动物物种。这些特征证明所扩增的cDNA编码绿色稻叶蝉AChE。本研究中使用的简并引物组也用于扩增家蝇乙酰胆碱酯酶的cDNA，其序列是未知的，所以我们建议的可能性，相同的引物组是适用于其他昆虫乙酰胆碱酯酶cDNA。