Synergistic effect of E8,E10-12∶OH on the sex pheromone attractant to Grapholita molesta(Lepidoptera:Tortricidae)and its binding mechanisms with pheromone binding proteins and general odorant binding proteins
[Aim](8E,10E)-Dodecadien-1-ol(E8,E10-12∶OH)is the main component of sex pheromone in Cydia pomonella,and has a synergistic effect on the sex pheromone attractant to Grapholita molesta.The objective of this study is to detect the synergistic effect of E8,E10-12∶OH on the sex pheromone attractant to G.molesta,and identify the pheromone binding proteins(PBPs)and general odorant binding proteins(GOBPs)that primarily bind to E8,E10-12∶OH.[Methods]The electroantennogram(EAG)responses of male adults of G.molesta to 0.002,0.02,0.2,2,20,200 and 2 000 μg of E8,E10-12∶OH were determined using EAG apparatus.The synergistic effect of E8,E10-12∶OH on the sex pheromone attractant to G.molesta was determined via field trapping trials.The values of inhibition constant Ki of the pheromone binding proteins GmolPBP1,GmolPBP2 and GmolPBP3,and the general odorant binding proteins GmolGOBP1,GmolGOBP2 and GmolGOBP3 of G.molesta binding to E8,E10-12∶OH were measured through fluorescence competitive binding assay.The key amino acids of GmolGOBP2 involved in E8,E10-12∶OH-binding were predicted by molecular dynamics simulation.The pivotal amino acid residues and weak interaction forces of GmolGOBP2 binding to E8,E10-12∶OH were validated via site-directed mutagenesis and fluorescence competitive binding assay.[Results]Male adults of G.molesta exhibited EAG response towards E8,E10-12∶OH,with the highest EAG response value of(0.57±0.14)mV to 2 000 μg E8,E10-12∶OH.E8,E10-12∶OH(200 μg)displayed a significant synergistic effect on the sex pheromone attractant to G.molesta,with the maximum synergistic multiplier of 2.46-fold.GmolGOBP2 had the strongest binding affinity to E8,E10-12∶OH[Ki=(1.92±0.05)μmol/L],and emerged as the main OBP binding to E8,E10-12∶OH.Molecular dynamics simulations showed that the amino acids Phe18,Ile100,Glu104,Val117 and Phe124 of GmolGOBP2 exhibited the lowest binding free energy when binding to E8,E10-12∶OH,being-1.18,-1.33,-3.34,-1.19 and-1.58 kj/kg,respectively.These amino acids were predicted as important residues for GmolGOBP2 to bind with E8,E10-12∶OH.Following site-directed mutagenesis of the above five residues to Ala,the GmolGOBP2 mutants E104A and F124A lost their binding affinities to E8,E10-12∶OH,indicating that Glu104 and Phe124 are the key amino acids for GmolGOBP2 to bind to E8,E10-12∶OH.[Conclusion]E8,E10-12∶OH emerges as an ideal synergist for developing efficient sex pheromone attractant for G.molesta.GmolGOBP2 plays an important role in perceiving interspecies pheromone E8,E10-12∶OH,with Glu104 and Phe124 identified as the key amino acid residues for GmolGOBP2 to bind to E8,E10-12∶OH.
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