Prokaryotic expression and ligand binding characteristics of pheromone binding protein ASP1 in the Chinese honeybee (Apis cerana cerana)

【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. 39μmol/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.