Acta Entomologica Sinica ›› 2016, Vol. 59 ›› Issue (1): 25-32.doi: 10.16380/j.kcxb.2016.01.004

• RESEARCH PAPERS • Previous Articles     Next Articles

Molecular cloning, prokaryotic expression and lignand-binding characterization of a novel pheromone binding protein OBP10 in Apis cerana cerana (Hymenoptera: Apidae)

WU Fan, HUANG Jun-Jun, TAN Jing, TANG Ming-Zhu, LI Hong-Liang*   

  1. (Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, College of Life Sciences, China Jiliang University, Hangzhou 310018, China)
  • Online:2016-01-20 Published:2016-01-20

Abstract: 【Aim】 Odorant-binding proteins (OBPs) play an important role in olfactory system of Chinese bee, Apis cerana cerana. In this study, we aim to research the binding characteristics of pheromones and some plant volatiles with a novel pheromone-binding protein OBP10 of A. cerana cerana. 【Methods】 We successfully cloned the gene  AcerOBP10 from A. cerana cerana (GenBank accession no. KP717060.1) by RT-PCR, constructed its prokaryotic expression vector pET30-AcerOBP10, and conducted its expression in the optimized conditions in vitro. The recombinant protein with biochemical activities was purified by the method of Ni2+-agarose affinity chromatograph. Using 1-NPN as a fluorescence probe, the binding capability of AcerOBP10 with diverse bee pheromones and plant volatiles was measured by competitive fluorescence assay. 【Results】 By analyzing the alignment of the deduced homologous proteins of other insect species, we speculated that AcerOBP10 is a novel PBP of  A. cerana cerana. The results of competitive fluorescence assay showed that eight out of 14 ligands were able to decrease the relative fluorescence intensity of 1-NPN by more than 50%. Among them, methyl 4-hydroxylbenzoate (HOB), one of queen pheromone components, showed the strongest capability to compete with 1-NPN, causing 93.94% reduction in the relative fluorescence intensity with the KD value of 11.04 μmol/L. AcerOBP10 was also found to have the capability of binding with worker bee pheromones (geraniol and nerol) and alarm pheromones (heptanone and isoamyl acetate), suggesting that it plays a significant role in recognizing pheromones in the bee olfactory system. AcerOBP10 also had strong binding force with some plant volatiles, like β-ionone. 【Conclusion】 As a pheromone-binding protein, AcerOBP10 has a strong binding capability with diverse bee pheromones. Compared with another PBP (AcerASP1), AcerOBP10 seems to have more extensive pheromone binding profiles. The results provide a theoretical basis to explore the molecular mechanism of pheromone recognition and transduction in A. cerana cerana.

Key words: Apis cerana cerana, pheromone-binding protein, pheromone, fluorescent competition, binding characteristics