甘薯茎线虫乙酰胆碱酯酶基因Dd-ace-1全长cDNA的克隆和序列分析

Molecular cloning and characterization of a new acetylcholinesterase gene Dd-ace-1 from Ditylenchus destructor

利用反转录聚合酶链式反应,结合快速扩增cDNA末端(RACE)技术,克隆了甘薯茎线虫乙酰胆碱酯酶基因cDNA Dd-ace-1.该cDNA序列5′端存在反式剪接引导序列SL1,全长2196bp,包含1个1908bp的开放阅读框(GenBank登录号EF583059);在推导出的635个氨基酸残基的前体蛋白中,N端的前24个氨基酸残基为信号肽,随后的611个氨基酸残基是成熟的乙酰胆碱酯酶序列,其预测的相对分子质量为70144.12.在一级结构中,形成催化活性中心的3个氨基酸残基(Ser223、Glu360和His483)、胆碱结合位点Trp(106)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(Torpedo californica)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.该酶的氨基酸序列与南方根节线虫(Meloidogyne incognita)、胎生网尾线虫(Dictyocaulus viviparous)和秀丽小杆线虫(Caenorhabditis elegans)ACE-1的同源性为68.6%、67.2%和66.3%.与其他线虫乙酰胆碱酯酶的聚类分析显示,该乙酰胆碱酯酶(Dd-ACE-1)与其他线虫乙酰胆碱酯酶ACE-1同属一个支系.

A new gene, named Dd-ace-1, encoding an acetylcholinesterase （AChE , EC3.1.1.7） was cloned from the sweet potato stem nematode, Ditylenchus destructor. The full length cDNA, carrying the transspliced SL1 leader sequence, is 2 196 bp long with an open reading frame of 1 908 bp encoding 635 amino acid residues （ GenBank accession no. EF583059）. The complete amino acid sequence of AChE deduced from the cDNA consists of 24 residues for the putative signal peptide and 611 residues for the mature protein with a predicted molecular weight of 70 144.12. The conserved motifs involved in the catalytic triad, the choline binding sit and 10 aromatic residues lining the catalytic gorge were present in the Dd-ACE-1 deduced protein. The predicted protein shared with strong homology with Meloidogyne incognita ACE-1, Dictyocaulus viviparous ACE-1 and Caenorhabditis elegans ACE-1. Phylogenetic analysis based on other nematodes and species AChEs showed that the deduced AChE formed a cluster with ACE-1s.