关键词: 暗黑鳃金龟, 气味结合蛋白, 组织表达, 蛋白纯化, 荧光竞争结合实验

Abstract: 【Aim】 The dark black chafer, Holotrichia parallela, locates the mate and host plant accurately and rapidly by binding of the odorant binding proteins (OBPs) with sex pheromones and plant volatiles. This study aims to clone the open reading frame (ORF) of odorant binding protein 15a (HparOBP15a) gene from H. parallela, as well as to gain insight into the characteristics of its encoding protein, its expression patterns, and characterize the binding profiles of OBP15a with a number of plant volatiles, and will provide a theoretical basis for a better understanding of the chemosensory mechanism of H. parallela.【Methods】 Based on transcriptome sequencing of the adult antenna of H. parallela, reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify the ORF of HparOBP15a. The mRNA levels of HparOBP15a in different tissues of adults were assayed by real-time quantitative PCR (qRT-PCR). Binding affinities of HparOBP15a towards 58 compounds, which are probably associated with the chemosensation of  H. parallela, were measured by fluorescent competitive binding assay. 【Results】 HparOBP15a has a full-length cDNA of 534 bp, encoding 147 amino acids, and its corresponding sequence was submitted to GenBank under the accession number AK1834747. The qRT-PCR assay indicated that  HparOBP15a mRNA was specifically expressed in the antennae of female adults. Among the 58 chemicals tested, HparOBP15a exhibited a good affinity to 46 kinds of ligands. Notably, it was found that HparOBP15a had the strongest binding affinity to dodecane and 1-dodecanol, with the dissociation constant of 8.5 and 11.3 μmol/L, respectively. HparOBP15a had a certain binding affinity toward the sex pheromone components methyl L-isoleucinate and (-)-linalool, with the dissciation constant of 21.0 and 18.5 μmol/L, respectively. 【Conclusion】 HparOBP15a protein shows a broad odorant-binding spectrum and has a strong binding affinity toward dodecane, a volatile from host elm leaves. This protein thus may play an important role in locating host of H. parallela.

Key words: Holotrichia parallela, odorant binding protein, tissue expression, protein purification, fluorescent competitive binding assay