关键词: 光肩星天牛, 气味结合蛋白, 嗅觉, 基因表达, 荧光竞争结合实验

Abstract: 【Aim】 Cloning and identification of the odorant binding protein (OBP) genes and clarifying their expression features and ligand-binding characteristics with host-plant volatiles are helpful to address the molecular mechanisms of olfaction in the Asian longhorned beetle, Anoplophora glabripennis. 【Methods】 Based on the antenna transcriptome data of A. glabripennis female adult, the complete coding sequence of OBP12 was cloned using RT-PCR and bioinformatically analyzed. The expression levels of OBP12 in the antenna, head (without antennae), thorax, abdomen, leg and wing were assayed by realtime quantitative PCR (qRT-PCR). The recombinant OBP12 protein was prokaryotically expressed and then purified by Ni ion affinity chromatography. The binding affinities of the recombinant OBP12 with 39 ligands were assessed by using fluorescent competitive binding assay. 【Results】 AglaOBP12 of A. glabripennis was successfully cloned and sequenced (GenBank accession no.: KX890109). Its ORF is 414 bp in length, encoding 137 amino acids with the signal peptide of 18 amino acids at the N-terminal. The matured protein possessed six conserved cysteines and could be classified into the Classical OBP subfamily. qRT-PCR results showed that AglaOBP12 primarily expressed in the antenna and slightly expressed in other tissues. Among the 39 chemicals tested, the recombinant AglaOBP12 had only binding activities to 19 compounds, suggesting that AglaOBP12 has obvious selective binding characteristics to host plant volatiles. The recombinant AglaOBP12 showed higher binding affinities to dodecanol, tetradecanol, farnesol, dodecanal, cis-3-hexenyl acetate and β-caryophyllene, with the Ki values of 196, 0.96, 1.03, 0.82, 0.77 and 0.74 μmol/L, respectively. 【Conclusion】 The nucleotide and deduced amino acid sequences of AglaOBP12 were characterized in this study. AglaOBP12 shows particularly strong binding affinities to alcohols, aldehydes and terpenes with 12 carbon atoms in the main-chain. Based on the results of qRT-PCR and fluorescent competitive binding assay, we speculated that AglaOBP12 in the Asian longhorned beetle plays an important role in locating trophic host plants.

Key words: Anoplophora glabripennis, odorant binding protein, olfactory, gene expression, fluorescence competitive binding assay