Acta Entomologica Sinica ›› 2019, Vol. 62 ›› Issue (3): 275-283.doi: 10.16380/j.kcxb.2019.03.001

• RESEARCH PAPERS •     Next Articles

Gene expression profiling and ligand binding characterization of the odorant binding protein CforOBP8 in Cylas formicarius (Coleoptera: Brentidae)

JIA Xiao-Jian, GAO Bo, MA Juan, LI Xiu-Hua, CHEN Shu-Long, WANG Rong-Yan*   

  1. (Institute of Plant Protection, Hebei Academy of Agriculture and Forestry Sciences, Integrated Pest Management Centre of Hebei Province, Key Laboratory of Integrated Pest Management on Crops in Northern Region of North China, Ministry of Agriculture, Baoding, Hebei 071000, China)
  • Online:2019-03-20 Published:2019-03-14

Abstract: 【Aim】 This study aims to clarify the gene expression patterns of the odorant binding protein (OBP) OBP8 in Cylas formicarius and its ligand binding characteristics. 【Methods】 Based on the transcriptome data of C. formicarius adults, the odorant binding protein OBP8 gene was cloned from the antenna of C. formicarius adults and subjected to bioinformatical analysis. The expression patterns of this gene in adult antennae, mouthparts, wings and tarsi of C. formicarius were assayed by qPCR. Prokaryotic expression vector was constructed, and the recombinant protein was expressed and purified. The protein binding characteristics of the purified recombinant protein with 16 ligand compounds (one sex pheromone and 15 different plant volatiles) were assayed by fluorescence competitive binding assay. 【Results】 CforOBP8 (GenBank accession no.: MH549528) of C. formicarius was successfully cloned and sequenced. Its open reading frame is 414 bp in length, encoding a protein of 138 amino acids with the predicted molecular weight of 15.56 kD and the isoelectric point of 4.71. The N-terminal signal peptide of CforOBP8 consists of 21 amino acids. The mature protein possesses four conserved cysteines and can be classified into the Minus-C OBPs subfamily. The qPCR results showed that CforOBP8 was specifically expressed in the antenna of C. formicarius adults and slightly expressed in other tissues. SDS-PAGE analysis showed that the fusion protein was successfully expressed. The binding affinity assay of CforOBP8 to ligand compounds showed that CforOBP8 had the strongest binding affinity to sex pheromone (Z)-3-dodecen-1-yl(E)-2-butenoate with the dissociation constant (Ki) of 4.13 μmol/L, and a certain binding affinities to five plant volatiles including (Z)-3-hexen-1-ol acetate, limonene, nerol, nonanal and phenylacetaldehyde, with the Ki values of 7.54, 15.23, 16.31, 28.71 and 29.54 μmol/L, respectively. 【Conclusion】 CforOBP8 might be a pheromone binding protein of C. formicarius and play dual roles in the perception of sex pheromones and plant volatiles.

Key words: Cylas formicarius, odorant binding protein, sex pheromone, expression profiling, fluorescence competitive binding assay