朱砂叶螨乙酰胆碱酯酶基因的克隆与特征分析

Gene cloning and character analysis of acetylcholinesterase from Tetranychus cinnabarinus

乙酰胆碱酯酶(AChE)是有机磷和氨基甲酸酯类杀虫(螨)剂的主要作用靶标,为建立以朱砂叶螨(Tetranychus cinnabarinus)AChE为靶点的杀螨剂体外筛选体系,本研究拟克隆朱砂叶螨AChE基因,并分析其特征.采用同源基因克隆及RACE技术克隆基因,通过荧光定量PCR分析其表达特征.结果获得朱砂叶螨AChE的cDNA序列长为2 512 bp,其中开放阅读框(ORF)为2 064 bp,编码687个氨基酸,N端有信号肽,剪切位点在甲硫氨酸开始的第74-75个氨基酸残基之间,C端有糖酯锚定结构,GenBank登录号为AGI96546.1.序列分析显示朱砂叶螨与其他蜱螨目的AChE有高度的同源性,特别与同属叶螨属的二斑叶螨(Tetranychus urticae)AChE的同源性最高,但氨基酸序列比较分析显示两者酶的催化特性可能存在区别.朱砂叶螨不同发育期AChE的转录水平比较分析显示幼螨期的表达量最高,且从幼螨期开始表达量逐渐下降,成螨期最低.本研究表明朱砂叶螨与二斑叶螨的ace 1有较高的同源性,但酶特性存在差异,朱砂叶螨不同发育时期ace 1基因表达水平的不同可能与不同发育期螨的习性和代谢特性有关.

Acetylcholinesterase（AChE） is the major target for organophosphate and carbamate insecticides. However, ace gene of Tetranychus cinnabarinus has not been cloned. In order to construct an in vitro AChE inhibitor screening system, this study concentrated on cloning ace gene of T. cinnabarinus and analyzing its characteristics. Degenerate primers and RACE methods were used to clone ace 1 gene of T. cinnabarinus, and quantitative real-time PCR was used to measure the mRNA expression level of AChE. The results showed that the complete ace 1 cDNA sequence of an insecticide susceptible mite strain of T. cinnabarinus consisted of 2 512 bp with an open reading frame encoding an AChE precursor of 687 amino acid residues（GenBank accession number of AGI96546.1）. The ace 1 gene of T. cinnabarinus had the signal peptide at N-terminal, which was cleaved between the 74 th and 75 th residues from the first methionine. Furthermore, there was a glycolipid anchor in the C-terminus of ace 1 gene of T. cinnabarinus. The deduced amino acid sequence of T. cinnabarinus ace 1 gene was highly conserved with those of Acarina ace 1 genes, especially with T. urticae ace 1 genes, but there might be some differences in catalytic properties of T. cinnabarinus AChE and T. urticae AChE. Quantitative real-time PCR indicated that the highest mRNA expression level of AChE was in the larvae stage, and the AChE expression was slowed down after the larvae stage, with the lowest expression level in the adult stage. T. cinnabarinus ace 1 gene was shown in this study to be highly conserved with T. urticae ace 1 genes but with different catalytic properties. The difference in the AChE expression level betweendifferent developmental stages may be related to their habits and metabolism characteristics.