Acta Entomologica Sinica ›› 2022, Vol. 65 ›› Issue (8): 977-985.doi: 10.16380/j.kcxb.2022.08.006

• RESEARCH PAPERS • Previous Articles     Next Articles

Gene cloning and ligand binding characterization of the odorant-binding protein HvarOBP2 in Hippodamia variegata (Coleoptera: Coccinellidae)

TANG Hao-Yu1,2, LIU Jing-Tao2, XIE Jiao-Xin2, YI Chao-Qun2,3, LIU Xiao-Xu2,3, ZHANG Yong-Jun2,*, SUN Yang1,*   

  1.  (1. Key Laboratory for Conservation and Use of Important Biological Resources of Anhui Province, Anhui Provincial Key Laboratory of Molecular Enzymology and Mechanism of Major Diseases, College of Life Sciences, Anhui Normal University, Wuhu, Anhui 241000, China; 2. State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China; 3. College of Plant Protection, Hebei Agricultural University, Baoding, Hebei 017000, China)
  • Online:2022-08-20 Published:2022-09-16

Abstract: 【Aim】 The objective of this study is to clone the odorant-binding protein (OBP) gene HvarOBP2 of Hippodamia variegata and to analyze the ligand binding characteristics of this protein. 【Methods】HvarOBP2 of H. variegata was screened from the antennal transcriptome of H. variegata by BLAST and other bioinformatics technologies and its full-length cDNA sequence was amplified by PCR. The recombinant HvarOBP2 was expressed by prokaryotic expression and purified by affinity chromatography, and its binding characteristics to 40 plant volatile compounds and 24 aphid-related volatile compounds were determined by fluorescence competitive binding assay. The molecular docking simulation between the recombinant HvarOBP2 and four ligands (β-ionone, dibutyl phthalate, octadecenoic acid and nerolidol) was performed using PyMOL1.9.0. 【Results】 HvarOBP(GenBank accession number: OK340816) has an open reading frame of 447 bp in length. Its encoded protein has a signal peptide consisting of 17 amino acids at the N-terminus, belonging to the classical OBPs subfamily with six conserved cysteine sites. The results of fluorescence competitive binding assay indicated that the recombinant HvarOBP2 had strong binding capabilities with plant volatile compounds octadecenoic acid, dibutyl phthalate, nerolidol, and β-ionone, with the dissociation constants (Ki values) of 1.68±0.04, 8.77±0.19, 15.93±0.33 and 10.79±0.24 μmol/L, respectively. Molecular docking of the recombinant HvarOBP2 with the above four ligands showed that Phe12 and Phe118 of HvarOBP2 are the key amino acid residues binding to multiple ligands. 【Conclusion】 The results suggest that HvarOBP2 may be involved in the recognition of plant volatiles and indirectly locating prey habitat, and these active binding compounds to HvarOBP2 play an important role in the process of searching and locating hosts for H. variegata.

Key words: Hippodamia variegata, odorant-binding protein, prokaryotic expression; fluorescence competitive binding, ligand binding characteristics