关键词: 梨小食心虫, 气味结合蛋白, 基因表达, 蛋白纯化, 荧光竞争结合实验

Abstract: 【Aim】 This study aims to clone the Minus-C odorant binding protein (OBP) gene of the oriental fruit moth, Grapholita molesta, and to measure its expression profiles in different adult tissues and the binding affinities of its recombinant protein with different ligands, so as to clarify its olfactory functions. 【Methods】 Based on the next generation sequencing of the female adult antenna of G. molesta, the complete coding sequence of a Minus-C OBP gene was cloned by using RT-PCR. The expression levels of this gene in different adult tissues (antenna, head with antennae removed, thorax, abdomen, leg and wing) of G. molesta were measured by qPCR. The recombinant protein was expressed by prokaryotic expression system and the purified protein was verified by SDS-PAGE and Western blot. The binding affinities of the recombinant protein with 35 ligands were analyzed using fluorescence competitive binding assay. 【Results】 A Minus-C OBP gene was successfully cloned from G. molesta and named GmolOBP14 (GenBank accession no. MF066361). The cDNA sequence of GmolOBP14 contains an ORF of 411 bp that encodes 136 amino acids including four conserved cysteine residues, and the encoded protein belongs to Minus-C OBPs subfamily. GmolOBP14 was expressed in different tissues of male and female adults, and had significantly higher expression levels in male wing and female antenna than in other tissues. The purified recombinant GmolOBP14 (rGmolOBP14) displayed binding abilities with 16 of the 35 tested ligands, and had higher binding affinities to pear ester and lauraldehyde with the dissociation constant Ki values of 6.92 and 12.74 μmol/L, respectively. rGmolOBP14 had medium binding abilities to decanal, tetradecanal, (E)-3-hexene-1-ol, benzyl alcohol and butyl hexanoate with the Ki values of 25.54, 20.61, 24.35, 23.44, and 23.33 μmol/L, respectively. rGmolOBP14 showed no binding activities to sex pheromones, suggesting that this protein is not involved in the perception and recognition of sex pheromones. 【Conclusion】 Based on the expression profiles of GmolOBP14 and the binding affinities of its recombinant protein to ligands, it is speculated that GmolOBP14 not only selectively binds and transports volatiles of the host plant but also participates in other physiological processes except for olfaction.

Key words: Grapholita molesta, odorantbinding protein, gene expression, protein purification, fluorescence competitive binding assay