关键词: 中华蜜蜂, 信息素结合蛋白, 原核表达, 分离纯化, 结合功能, 配基

Abstract: 【Aim】 To study the binding function of Acer-ASP1, a pheromone binding protein (PBP), in the Chinese honeybee (Apis cerana cerana) with pheromone and other plant volatiles. 【Methods】 In order to obtain the recombinant protein (Acer-ASP1), we successfully constructed the cloning and prokaryotic expression vector of Acer-ASP1, which was expressed in the optimized conditions. After the recombinant protein with biochemical activities was purified, the binding capability of Acer-ASP1 with pheromone and other odors was measured using competitive fluorescence assay where 1-NPN was applied as fluorescence probe. 【Results】 Seven of the 22 ligands tested showed stronger binding capability with Acer-ASP1 and were able to decrease the relative fluorescence intensity of 1-NPN by more than 50%. Among them, methyl 4-hydroxylbenzoate, a queen pheromone, showed the strongest capability to compete with 1-NPN, causing 99.31% reduction in the relative fluorescence intensity, and K_D=13.39 μmol/L; vanillyl alcohol, another queen pheromone, caused 95.5% decline in the relative fluorescence intensity, and K_D=98.44 μmol/L. Nevertheless, AcerASP1 did not bind with other kinds of pheromone at all except queen pheromone. In addition, five plant volatiles, i. e., methyl salicylate, phenylacetaldehyde, 3,4-dimethyl-benzaldehyde, 4-allylveratrole and β-ionone, showed capabilities to bind with ASP1 in various degrees. 【Conclusion】 The results indicate that Acer-ASP1 exhibits remarkable specificity with queen pheromone, and it can bind with some plant volatiles to some extent. This implies that Acer-ASP1 is probably a protein having complex physiological function, in which recognizing queen pheromone is deemed as its main function while recognizing plant volatiles its secondary function.

Key words: Apis cerana cerana, pheromone binding protein, prokaryotic expression, protein purification, binding function, ligand