关键词: 柑橘大实蝇, BminOBP25, 气味结合蛋白, 基因克隆, 组织表达分析, 荧光定量PCR

Abstract: 【Aim】 The odorant binding proteins (OBPs) of insects are closely related to olfactory recognition and play a key role in the successful delivery of fat-soluble odorant molecules in the lymph nodes of the antennal sensory receptors to the olfactory receptors. In order to understand the roles of OBPs in olfactory recognition and to lay foundation for studying the molecular mechanisms of olfactory transmission in Bactrocera minax, an OBP gene was cloned and its expression profiles were analyzed. 【Methods】 The full-length cDNA sequence of OBP gene was cloned from B. minax using RT-PCR and RACE techniques, and subjected to bioinformatics analysis. The recombinant expression vector pET28a(+)-BminOBP25 was constructed and transformed into Escherichia coli BL21 (DE3). The recombinant protein was identified by SDS-PAGE and Western blotting. The expression profiles of OBP gene in different tissues of B. minax adults were detected by quantitative real-time PCR (qPCR). 【Results】 An OBP gene was cloned from B. minax and named BminOBP25 (GenBank accession no.: MH181875). Its ORF is 447 bp in length encoding 148 amino acids with a predicted molecular weight of 17.5 kD. The encoded protein has typical six conserved cysteines and six α-helices. The recombinant expression vector pET28a(+)-BminOBP25 was constructed and the target protein was stably expressed in host bacteria in the form of 6×His tag fusion protein after IPTG induction. qPCR analysis showed that BminOBP25 mRNA was expressed in antennae, head (without antennae), thorax, abdomen, leg, wing and ovipositor of adults, with higher expression levels in antenna, head (without antennae), leg and ovipositor.【Conclusion】 BminOBP25 has high transcriptional activity in antennae, heads, legs and ovipositors of B. minax adults, suggesting that BminOBP25 may also have physiological functions in non-olfactory tissues, and especially may play important roles during the selection of insect feeding and spawning sites. Its functions need further study. In this study, the prokaryotic expression of BminOBP25 was achieved, laying a foundation for further study of its functions.

Key words: Bactrocera minax, BminOBP25, odorant binding protein (OBP), gene cloning, tissue expression profiling, qPCR