朱砂叶螨谷氨酸门控氯离子通道GluCl1基因的克隆与序列特征分析

朱砂叶螨是一种分布广泛、危害极大、难以防治和易产生抗性的农业害螨。为了研究朱砂叶螨杀螨剂神经靶标谷氨酸门控氯离子通道，进一步确定阿维菌素对朱砂叶螨的抗性机理，采用同源基因克隆以及RACE技术，克隆朱砂叶螨杀螨剂靶标谷氨酸门控氯离子通道(GluCl1)基因全长，分析其序列特征。结果表明，GluCl1基因序列全长为1856 bp，其中开放阅读框(ORF)为1338 bp，编码455个氨基酸，N端含25个氨基酸的信号肽，有4个跨膜结构域，GenBank登陆号为KC543353。同源比对分析表明，朱砂叶螨与其他蜱螨目GluCl基因有高度的同源性，特别与二斑叶螨的相似度最高。研究还发现朱砂叶螨敏感品系GluCl1第3个跨膜区的G314，与二斑叶螨敏感品系相应位点的氨基酸G323一致，而二斑叶螨抗性品系相应位点突变为D323。本研究进一步证实了G323突变为D323和二斑叶螨对阿维菌素产生抗性有关。本研究为今后建立以朱砂叶螨谷氨酸门控氯离子通道为靶点的选择性杀螨剂体外筛选体系奠定坚实基础。

Cloning and Characterization of Full-length Glutamate-gated Chloride Channel Gene from Tetranychus cinnabarinus

The aim of this study is to clone insecticide target glutamate-gated chloride channel (GluCl1) gene from Tetranychus cinnabarinus and analyze its sequence character, using a targeted degenerate PCR and RACE strategy. Full-length of GluCl1 gene was obtained. The cDNA sequence of 1856 bp was determined, which contains an open reading frame encoding an GluCl1 precursor of 455 amino acid residues, with a GenBank accession No. KC543353. The predicted signal peptide of T. cinnabarinus was 25 amino acid residues long at its N-terminal, and GluCl1 had four transmembrane domains. Alignment analysis showed that, the GluCl1 gene of T. cinnabarinus were homologous with those of others belonged to Acarina, especially with those of T. urticae. More interestingly, the amino acid site G314 in the 3rd transmembrane domain of GluCl1 of T. cinnabarinus sensitive population was identical to that of T. urticae sensitive population G323, but which was displaced by D323 in the GluCl1 gene of T. urticae insecticide-resistant population. This indicated that this mutation was related with the resistance of T. urticae to abamectin. This study laid the solid foundation for establishing an in vitro acaricide screening system targeted to glutamate-gated chloride channel of T. cinnabarinus in the future.