甘薯茎线虫乙酰胆碱酯酶基因Dd-ace-2全长cDNA的克隆和序列分析  

Molecular Cloning and Characterization of an Acetylcholinesterase Gene Dd-ace-2 from Sweet Potato Stem Nematode Ditylenchus destructor

作　　者：丁中[1] 彭德良[1] 黄文坤[1] 贺文婷[1] 高必达[2] 

机构地区：[1]中国农业科学院植物保护研究所植物病虫害生物学国家重点实验室,北京100094 [2]湖南农业大学生物安全科学技术学院,长沙410128

出　　处：《生物工程学报》2008年第2期239-244,共6页Chinese Journal of Biotechnology

基　　金：国家科技支撑计划课题(No．2006BAD08A08);~~植物病虫害生物学国家重点实验室开放基金项目(No．2006PD3)~~

摘　　要：利用RT-PCR、RACE技术克隆了甘薯茎线虫(Ditylenchus destructor)乙酰胆碱酯酶基因(Dd-ace-2)cDNA (GenBank登录号EF583058),用DNAMAN5.0、MEGA3.0进行了序列分析。克隆的Dd-ace-2基因cDNA全长2425 bp,包含一个2205 bp的开放阅读框,编码734个氨基酸。Dd-ace-2基因推导的氨基酸序列与南方根结线虫(Meloidogyne incognita)、秀丽小杆线虫(Caenorhabditis elegans)和动物寄生线虫胎生网尾线虫(Dictyocaulus viviparous)ace-2的氨基酸序列同源性分别达48.0%、42.7%和42.1%。在推导的734个氨基酸残基的前体蛋白中,前面的701个氨基酸残基是成熟的乙酰胆碱酯酶序列,其预测的分子量为79240.38 D。在一级结构中,形成催化活性中心的3个氨基酸残基(Ser291,Glu442和His574)、胆碱结合位点Trp(177),以及在亚基内形成二硫键的6个半胱氨酸完全保守;在电鳐乙酰胆碱酯酶分子的催化功能域中存在14个保守的芳香族氨基酸残基,其中10个在甘薯茎线虫乙酰胆碱酯酶中完全保守。与其它线虫和物种乙酰胆碱酯酶的聚类分析显示,甘薯茎线虫的乙酰胆碱酯酶与其它线虫乙酰胆碱酯酶ACE-2同属一个支系。A cDNA, named Dd-ace-2, encoding an acetylcholinesterase （ACHE, EC3.1.1.7）, was isolated from sweet-potato-stem nematode, Ditylenchus destructor. The nucleotide and amino acid sequences among different nematode species were compared and analyzed with DNAMAN5.0, MEGA3.0 softwares. The results showed that the complete nucleotide sequence of Dd-ace-2 gene of Ditylenchus destructor contains 2425 base pairs from which deduced 734 amino acids （GenBank accession No. EF583058）. The homology rates of amino acid sequences of Dd-ace-2 gene between Ditylenchus destructor and Meloidogyne incognita, Caenorhabditis elegans, Dictyocaulus viviparous were 48.0%, 42.7%, 42.1% respectively. The mature acetylcholinesterase sequences of Ditylenchus destructor may encode by the first 701 residues of deduced 734 amino acids.The conserved motifs involved in the catalytic triad, the choline binding site and 10 aromatic residues lining the catalytic gorge were present in the Dd-ace-2 deduced protein. Phylogenetic analysis based on AChEs of other nematodes and species showed that the deduced AChE formed the same cluster with ACE-2s.

关 键 词：甘薯茎线虫 乙酰胆碱酯酶 CDNA克隆 Dd-ace-2 

分 类 号：S435.31[农业科学—农业昆虫与害虫防治]