Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (7): 1311-1321.doi: 10.3864/j.issn.0578-1752.2023.07.009

• PLANT PROTECTION • Previous Articles     Next Articles

Interaction Mechanisms Between Bactrocera minax Odorant-Binding Protein BminOBP6 and Its Ligands

YANG Ling1(), TIAN XiaoLi2, GUI LianYou1, WANG FuLian1, ZHANG GuoHui1()   

  1. 1 College of Agriculture, Yangtze University, Jingzhou 434025, Hubei
    2 College of Life Science, Yangtze University, Jingzhou 434025, Hubei
  • Received:2022-12-10 Accepted:2023-01-17 Online:2023-04-01 Published:2023-04-03

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Abstract:

【Objective】 To explore the recognition mechanisms of BminOBP6 and its ligands, a C terminus-truncated BminOBP6 (TOBP6) was constructed in this study. Subsequently, the binding affinities of TOBP6 and the wild type protein BminOBP6 to 1-undecanol and (+)-limonene under different pH conditions were determined.【Method】 3D structure of BminOBP6 was built by homology modeling through the online software SWISS MODEL. Based on the established BminOBP6 3D model, the C terminus sequence after sixth α-helix of BminOBP6 was determined. This C terminus sequence is the sequence that will be removed from BminOBP6 in the subsequent study. Specific primers were designed to amplify the encoding sequence of BminOBP6 C terminus- truncated mutant TOBP6. Then the recombinant expression vector pET-32a/TOBP6 was constructed. Recombinant expression vectors pET-32a/TOBP6 and pET32a/BminOBP6 that has been made before in our lab were transformed into Escherichia coli cells BL21 (DE3) to express the TOBP6 and BminOBP6 recombinant proteins, respectively. Fluorescence competitive binding assays were conducted to determine the binding affinities of TOBP6 and BminOBP6 to 1-undecanol and (+)-limonene under different pH (7.4 and 5.0) conditions.【Result】 Amino acid sequence alignments revealed that BminOBP6 shared 62.6% identity with Culex quinquefasciatus CquiOBP1, and thus the 3D structure of CquiOBP1 was used as the template in the homology modeling of BminOBP6. The 3D structure of BminOBP6 indicated that the C terminus sequence after α6 of BminOBP6 is a sequence of 8 amino acid residues (D117-P124). Based on this result, the encoding sequence of TOBP6 was cloned and its recombinant expression vector pET-32a/TOBP6 was built. Recombinant expression vectors pET-32a/TOBP6 and pET32a/BminOBP6 were then transformed into E. coli cells BL21 (DE3) to express the TOBP6 and BminOBP6 recombinant proteins, respectively. The fluorescence competitive binding results showed that the binding affinity of BminOBP6 to 1-undecanol and (+)-limonene was quite strong at pH 7.4, the Ki values were 6.89 and 9.50 μmol·L-1, respectively. However, when the pH decreased to 5.0, BminOBP6 completely lost its binding capacity to 1-undecanol and exhibited an obvious decrease in binding to (+)-limonene (the Ki value increased from 9.50 μmol·L-1 to 31.26 μmol·L-1). The binding affinity of TOBP6 to 1-undecanol and (+)-limonene was completely abolished regardless of pH conditions.【Conclusion】 Changes in pH have a significant effect on the ligand binding and release of BminOBP6, and the C terminus of BminOBP6 is crucial for BminOBP6 to bind its ligands.

Key words: Bactrocera minax, odorant-binding protein (OBP), homology modeling, C terminus, prokaryotic expression, fluorescence competitive binding

Fig. 1

3D structure of BminOBP6 through homology modeling"

Fig. 2

cDNA sequence of BminOBP6 and deduced amino acid sequence he start codon is boxed; Signal peptide part is underlined; Conservative cysteine sites are circled; “*” stands for the stop codon; The green background represents cDNA of TOBP6; “→” and “←” mark positions of the upstream and downstream primers of TOBP6, respectively"

Fig. 3

SDS-PAGE analysis of expression and purification products of pET-32a/BminOBP6 and pET-32a/TOBP6"

Fig. 4

Fluorescence competitive binding property of BminOBP6 and TOBP6"

Table 1

Binding affinities of BminOBP6 and TOBP6 with odorant ligands"

BminOBP6 TOBP6
1-十一醇 1-Undecanol (+)-柠檬烯 (+)-Limonene 1-十一醇 1-Undecanol (+)-柠檬烯 (+)-Limonene
pH 7.4 IC50 (μmol·L-1) 8.13±0.24 11.20±0.53 - -
Ki (μmol·L-1) 6.89±0.20 9.50±0.45 - -
pH 5.0 IC50 (μmol·L-1) - 33.39±1.13 - -
Ki (μmol·L-1) - 31.26±1.06 - -
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