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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 main target of organophosphorus and carbamate insecticides (mites). In order to establish an in vitro screening system of acaricide against Tetranychus cinnabarinus AChE, Cloning the AChE gene of Tetranychus cinnabarinus and analyzing its characteristics.The gene was cloned by homologous gene cloning and RACE and its expression characteristics were analyzed by fluorescence quantitative PCR.Results The cDNA sequence of AChE was 2.212 bp, The ORF was 2 064 bp, encoding a protein of 687 amino acids with a signal peptide at the N-terminus. The cleavage site was between the 74th and 75th amino acid residues of methionine, , GenBank accession number AGI96546.1. Sequence analysis showed that there was high homology between AChE and other AChE, especially the homology with AChE of Tetranychus urticae, However, the comparative analysis of the amino acid sequences showed that the catalytic properties of the two enzymes may be different.Comparison of the transcriptional levels of AChE at different developmental stages shows that the expression level of AChE is the highest during the young mite stage, and gradually decreases from the young mite stage, The lowest period There was a high homology between Acetinae sp. And Tetranychus urticae ace 1, but there were differences in enzyme characteristics. The different expression levels of ace 1 gene in different developmental stages of Tetranychus cinnabarinus may be related to the habits and metabolic characteristics related.