关键词: 中华蜜蜂, 气味结合蛋白, 蛋白纯化, 气味化合物, 荧光竞争结合实验

Abstract: 【Aim】 Odorant binding proteins (OBPs) play an important role in host localization, egg-laying site selection and other behaviors of insects. Clarifying the binding characteristics of AcerOBP14 with ligands helps to elucidate the molecular mechanism of olfactory recognition in Apis cerana cerana. 【Methods】 The expression levels of OBP14 in the antennae of the 20-day-old adult workers and drones of A. c. cerana and pollen foragers of A. c. cerana and Apis mellifera ligustica were detected by qRT-PCR. The prokaryotic expression vector pET28a/AcerOBP14 was constructed, and the recombinant AcerOBP14 protein was expressed and purified. The binding characteristics of AcerOBP14 with 37 odorant compounds were analyzed by fluorescence competition binding assay. 【Results】 The qRT-PCR analysis revealed that the expression level of OBP14 in the antennae of pollen foragers of A. c. cerana was significantly higher than those in the antennae of the 20-day-old adult workers and drones of A. c. cerana and pollen foragers of A. m. ligustica. Fluorescent competition binding assay results showed that AcerOBP14 had the binding ability with queen bee pheromone, alarm pheromone, Nasonov pheromone and a variety of plant volatiles, and showed the strongest binding ability to β-ocimene with the dissociation constant Ki value of 0.297 μmol/L. 【Conclusion】 AcerOBP14 has a broad ligand-binding spectrum, suggesting that it may be involved in a variety of physiological and behavioral responses of A. c. cerana, and play an important role in the pollen foraging behavior of A. c. cerana.

Key words: Apis cerana cerana, odorant binding protein, protein purification, odorant compound, fluorescence competitive binding assay