E8,E 10-12∶OH对梨小食心虫性诱剂的增效作用及其与信息素结合蛋白和普通气味结合蛋白的结合机制

Synergistic effect of E8,E 10-12∶OH on the sex pheromone attractant to Grapholita molesta(Lepidoptera:Tortricidae)and its binding mechanisms with pheromone binding proteins and general odorant binding proteins

【目的】(反,反)-8,10-十二碳二烯-1-醇[(8 E,10E)-dodecadien-1-ol,E8,E 10-12∶OH]是苹果蠹蛾Cydia pomonella的主要性信息素成分,对梨小食心虫Grapholita molesta性诱剂具有引诱增效作用。本研究旨在测定E8,E 10-12∶OH对梨小食心虫性诱剂的增效能力,确定主要结合E8,E 10-12∶OH的信息素结合蛋白(pheromone binding proteins,PBPs)和普通气味结合蛋白(general odorant binding proteins,GOBPs)。【方法】利用触角电位(electroantennogram,EAG)仪测定梨小食心虫雄成虫对0.002,0.02,0.2,2,20,200和2000μg E8,E 10-12∶OH激发的EAG反应;利用田间诱集试验测定E8,E 10-12∶OH对梨小食心虫性诱剂的增效能力;利用荧光竞争结合实验测定梨小食心虫信息素结合蛋白GmolPBP1,GmolPBP2和GmolPBP3及普通气味结合蛋白GmolGOBP1,GmolGOBP2和GmolGOBP3与E8,E 10-12∶OH的抑制常数K i;利用分子动力学模拟的方法预测GmolGOBP2结合E8,E 10-12∶OH的关键氨基酸;利用定点突变和荧光竞争结合实验验证GmolGOBP2结合E8,E 10-12∶OH的关键氨基酸残基及弱相互作用力。【结果】梨小食心虫雄成虫对E8,E 10-12∶OH表现出EAG反应,雄成虫对2000μg E8,E 10-12∶OH的EAG反应值最高[(0.57±0.14)mV]。在梨小食心虫性诱剂中添加200μg E8,E 10-12∶OH时,增效效果最佳,增效倍数达2.46倍。GmolGOBP2结合E8,E 10-12∶OH的能力最强[K i=(1.92±0.05)μmol/L],是结合E8,E 10-12∶OH的主要OBP。分子动力学模拟表明GmolGOBP2的Phe18,Ile100,Glu104,Val117和Phe124结合E8,E 10-12∶OH的自由能最低,分别为-1.18,-1.33,-3.34,-1.19和-1.58 kj/kg,预测是GmolGOBP2结合E8,E 10-12∶OH的重要位点,将以上5个残基定点突变为丙氨酸Ala后,突变体蛋白E104A和F124A失去了结合E8,E 10-12∶OH的能力,表明Glu104和Phe124是GmolGOBP2结合E8,E 10-12∶OH的关键氨基酸。【结论】E8,E 10-12∶OH是开发梨小食心虫高效性诱剂的理想增效剂,GmolGOBP2在梨小食心虫识别种间性信息素E8,E 10-12∶OH的过程中发挥着重要作用,Glu104和Phe124是GmolGOBP2结合E8,E 10-12∶OH的关键氨基酸残基。

【Aim】(8 E,10E)-Dodecadien-1-ol(E8,E 10-12∶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,E 10-12∶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,E 10-12∶OH.【Methods】The electroantennogram(EAG)responses of male adults of G.molesta to 0.002,0.02,0.2,2,20,200 and 2000μg of E8,E 10-12∶OH were determined using EAG apparatus.The synergistic effect of E8,E 10-12∶OH on the sex pheromone attractant to G.molesta was determined via field trapping trials.The values of inhibition constant K i 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,E 10-12∶OH were measured through fluorescence competitive binding assay.The key amino acids of GmolGOBP2 involved in E8,E 10-12∶OH-binding were predicted by molecular dynamics simulation.The pivotal amino acid residues and weak interaction forces of GmolGOBP2 binding to E8,E 10-12∶OH were validated via site-directed mutagenesis and fluorescence competitive binding assay.【Results】Male adults of G.molesta exhibited EAG response towards E8,E 10-12∶OH,with the highest EAG response value of(0.57±0.14)mV to 2000μg E8,E 10-12∶OH.E8,E 10-12∶OH(200μg)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,E 10-12∶OH[K i=(1.92±0.05)μmol/L],and emerged as the main OBP binding to E8,E 10-12∶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,E 10-12∶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,E 10-12∶OH.Following site-directed mutagenesis of the above five residues to Ala,the GmolGOBP2 mutants E104A and F124A lost their binding affinities to E8,E 10-12∶OH,indicating that Glu104 and Phe124 are the key amino acids for GmolGOBP2 to bind to E8,E 10-12∶OH.【Conclusion】E8,E 10-12∶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,E 10-12∶OH,with Glu104 and Phe124 identified as the key amino acid residues for GmolGOBP2 to bind to E8,E 10-12∶OH.