Abstract:

【Objective】The objective of this study is to clone the odorant binding protein 56h (OBP56h) gene from Drosophila suzukii, get the recombinant DsuzOBP56h protein, and characterize the binding profiles of DsuzOBP56h with some small molecular compounds.【Method】By means of specific primer, reverse transcription PCR (RT-PCR) was used to clone the full-length ORF of DsuzOBP56h. The sequences of insect OBPs with high similarity were downloaded from NCBI database for sequence alignment and analysis. Using NdeⅠand XhoⅠas restriction sites, OBP56h was ligated into pET-30a (+) prokaryotic expression vector, and transformed into Escherichia coli BL21 (DE3). IPTG was applied to induce the expression of recombinant DsuzOBP56h protein. The bacterial solution was collected and the protein was obtained through breaking cells by ultrasound, and then the protein was purified by the method of Ni-NTA resin. The purified protein was dialyzed by Tris-HCl and the concentration was determined by the method of BCA. The protein was diluted with 50 mmol·L ^-1 Tris-HCl (pH 7.4) to a final concentration of 2 μmol·L ^-1, and the ligand was diluted with chromatography-grade methanol to a final concentration of 1 mmol·L ^-1. The binding characterization of DsuzOBP56h with 18 small molecular compounds was investigated using bis-ANS as fluorescence probe. 【Result】 The full-length ORF of OBP56h in D. suzukii was amplified, which is 405 bp in total, including 19 amino acids of signal peptide in the N-terminal. It has 6 conserved cysteine sites, which is consistent with the typical characteristics of OBPs, and has the closest evolutionary relationship with D. melanogaster OBP56h. OBP56h was successfully inserted into pET-30a (+) and expressed at 1 mmol·L ^-1 IPTG and 28℃, then purified by the method of Ni-NTA resin. In the competitive fluorescence assay, the dissociation constant K_bis-ANS was 0.9568 μmol·L ^-1, indicating that bis-ANS is suitable to be a reporter of competitive fluorescence binding assay. Among 18 ligands, the binding affinity of bitter tastants berberine chloride and coumarin with DsuzOBP56h was strong, and the dissociation constant is 12.16 and 17.93 μmol·L ^-1, respectively. The dissociation constant of naringenin with DsuzOBP56h is 25.32 μmol·L ^-1. A volatile odorant β-cyclocitral, which is attractive to D. suzukii produced by strawberry leaves, can also bind to DsuzOBP56h, with the dissociation constant of 31.37 μmol·L ^-1.【Conclusion】The OBP56h in D. suzukii can bind with a variety of bitter tastants and volatile odors from plants, indicating that OBP56h may be involved in the gustatory and olfactory recognition of food in D. suzukii. The results can provide a theoretical basis for understanding the feeding behavior of D. suzukii, and provide a new idea for the ecological prevention and control of D. suzukii.

Key words: Drosophila suzukii, odorant-binding protein, prokaryotic expression, competitive binding