Abstract:

【Objective】The objective of this research is to explore the function of Anomala corpulenta odorant binding protein 11 (AcorOBP11) by studying the binding characteristics of AcorOBP11 with the host plant volatiles, and to lay the foundation for clarifying the olfactory molecular mechanism of AcorOBP11.【Method】Reverse transcription PCR (RT-PCR) was used to clone the full-length ORF ofAcorOBP11 by specific primers, and sequences with high similarity were downloaded for sequence comparison and analysis by BLAST. Prokaryotic expression of AcorOBP11 was conducted by Escherichia coliprotein expression system, the OBP11 was inserted into the expression vector pET28a, and the recombinant plasmid was transferred into E. coli competent cells BL21 (DE3). The target protein was purified by using recombinant enterokinase to remove the His-tag and purified again by using a nickel column. The purified protein was diluted with 50 mmol·L^-1 Tris-HCl (pH 7.4) to a dilution of 2 μmol·L^-1, and the odorant was diluted with chromatographic grade methanol to a final concentration of 1 mmol·L^-1, and 1-NPN was used as a fluorescent probe to determine the binding characteristics of AcorOBP11 to 37 host plant volatiles by fluorescence competitive binding assay. Modeller was used to obtain the three-dimensional structure of AcorOBP11 by using AgamOBP48 (PDB ID: 4ij7) as a template. Autodock semi-flexible docking was used to simulate the binding of AcorOBP11 to host plant volatiles.【Result】The full-length ORF ofAcorOBP11 was amplified, which is 639 bp in total, including 17 amino acids of signal peptide at the N-terminal. With eight conserved cysteine sites, AcorOBP11 belongs to the Plus-C OBP subfamily. The evolutionary tree results indicated that it had the closest evolutionarily relationship with HparOBP9. AcorOBP11 was successfully inserted into pET-28a and expressed at 0.25 mmol·L^-1 IPTG and induced expression at 37℃. The target protein was obtained by nickel column purification twice. The results of competitive binding experiments showed that AcorOBP11 has a good binding ability to 13 kinds of host plant volatiles, such as α-ionone, 3-methylbutanal, 2-ethylhexyl acrylate, bornyl acetate, cinene, trans-2-hexenal, 2-ethylhexanal, isovaleric acid. Among them, α-ionone has the best binding ability, and its competitive dissociation constant is 22.78 μmol·L^-1. The molecular docking results showed that AcorOBP11 has the lowest binding free energy with α-ionone, which is -24.74 kJ·mol^-1, and forms a hydrogen bond at Cys163, indicating that it has the strongest binding ability with α-ionone, which is consistent with the fluorescence competition binding result. 【Conclusion】AcorOBP11 can recognize a variety of host plant volatiles, and it is speculated that it plays an important role in locating host plants for A. corpulenta. The results will provide new insights forA. corpulenta ecological control.

Key words: Anomala corpulenta, odorant binding protein 11 (OBP11), prokaryotic expression, fluorescence competitive binding, homology modeling, molecular docking