关键词: 橘小实蝇； 烟碱型乙酰胆碱受体； 基因克隆； 序列分析； 表达谱, 实时定量PCR

Abstract: 【Aim】 The oriental fruit fly, Bactrocera dorsalis, is an important worldwide quarantine pest that damages many fruits and vegetables and has developed resistance to many insecticide classes including neonicotinoids. In this study, based on the identification of the cDNA of nicotinic acetylcholine receptor α9 subunit gene in B. dorsalis, we analyzed its molecular characterization and polygenetic relationship, and detected its expression pattern in different developmental stages and adult tissues of B. dorsalis, which will lay the foundation for further understanding the potential function properties and the role of Bdα9 in insecticide resistance in B. dorsalis. 【Methods】 Based on the transcriptome data of B. dorsalis by next-generation sequencing, nicotinic acetylcholine receptor gene sequence was successfully screened, assembled and identified by homologous blast. The full-length cDNA sequence was cloned by using RT-PCR and RACE technology, and bioinformatics analysis software package was used to predict its general biological information. Quantitative real-time polymerase chain reaction was used to investigate the expression profiles of this gene in different developmental stages of the fly and the head, thorax and abdomen of adult B. dorsalis. 【Results】 The nicotinic acetylcholine receptor α9 subunit gene was cloned from B. dorsalis by specific primers designed based on the predicted gene sequence. The full-length cDNA contains 1 486 bp with 1 281 bp of open reading frame encoding 426 amino acids with a predicted molecular weight of 49.1 kD and an isoelectric point of 6.56. The gene was named Bdα9 with the GenBank accession number of JQ178254. The amino acid sequence homology and phylogenetic tree analysis indicated that Bdα9 possesses the typical characteristics of nAChR α subunit, clusters together with Agα9 and Dmβ3, and shares 22%-27% amino acid sequence identity with nAChR α9 subunits from other insects. The qPCR results showed that Bdα9 was expressed in all developmental stages of B. dorsalis. The relative expression level of Bdα9 in the adult was significantly higher than those in the egg, the 2nd instar larva, the 3rd instar larva and the pupa. The expression level of Bdα9 in adult head was the highest and significantly higher than those in adult thorax and abdomen. 【Conclusion】 The nicotinic acetylcholine receptor gene, Bdα9, was identified, and its expression profiles in different developmental stages and adult tissues of B. dorsalis were examined, with the highest expression level in adults and the adult head, respectively, implying that it may play a critical role in the central nervous system of adult.  

Key words: Bactrocera dorsalis; nicotinic acetylcholine receptor, gene cloning, sequence analysis, expression profile, qPCR