Abstract: 【Aim】 To study the function and binding characteristics with plant volatiles of the odorant-binding proteins in the oriental fruit moth, Grapholita molesta (Busck).【Methods】 The OBP cDNA from G. molesta was cloned by using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE), its tissue and developmental expression profiles were detected by RT-PCR and Real-time PCR, and the protein binding property was analyzed using fluorescence competitive binding assay with N-phenyl-1-naphthylamine (1-NPN) as the fluorescent probe. 【Results】A novel OBP cDNA from G. molesta was obtained, which was named as GmolOBP3 (GenBank accession no.: KF395363). GmolOBP3 contains a 492 bp open reading frame encoding a 163-amino-acid residue peptide. The predicted molecular weight and isoelectric point are 18.72 kDa and 4.93, respectively. The mature protein GmolOBP3 includes six typical conservative cysteine residues, which are the hallmark of insect OBPs. GmolOBP3 was expressed in the antennae and abdomen of female and male adults. In five days after eclosion, the expression level of GmolOBP3 in antennae of female moths increased with day-old after eclosion, but on the 5th day after eclosion, the expression quantity in antennae of male moths significantly reduced. In order to obtain the recombinant protein GmolOBP3, we constructed the prokaryotic expression vector of GmolOBP3, which was successfully expressed in the optimized condition. The recombinant protein was purified by anion exchange and Superose-12. The binding affinity of GmolOBP3 with 16 plant volatiles and 4 sex pheromone analogs indicated that GmolOBP3 could not bind with (E)-8-dodecenyl acetate and 1-dodecanol, while had weaker affinity with (Z)-8-dodecenyl acetate and (Z)-8-dodecenol with the association constants of 83.00 and 103.70 μmol/L, respectively. GmolOBP3 could also weakly bind with 16 plant volatiles with the strongest affinity to β-ionone and with a association constant of 49.36 μmol/L. 【Conclusion】 We so inferred that GmolOBP3 protein have characteristics of selective binding with various ligands.

Key words: Grapholita molesta, odorant binding proteins, cDNA cloning, tissue expression pattern, prokaryotic expression, fluorescence competitive binding assay