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cDNA cloning, prokaryotic expression, and ligand binding characterization of the odorant binding proteins CpunOBP3 and CpunOBP4 of the yellow peach moth, Conogethes punctiferalis (Lepidoptera: Crambidae)
Pages: 327-336
Year: Issue:  3
Journal: Acta Entomologica Sinica

Keyword:  Conogethes punctiferalisodorant binding proteingene cloningprokaryotic expressionplant volatilesfluorescence competitive binding assay;
Abstract: 【Aim】 This study aims to determine the physiological function of odorant binding proteins(OBPs) in the chemoreception of the yellow peach moth, Conogethes punctiferalis, so as to provide a theoretical basis for selecting OBPs that may be used as targets in C. punctiferalis biological control. 【Methods】 Two OBP genes(CpunOBP3 and CpunOBP4) were cloned from antennae of C. punctiferalis by PCR based on our previous antennal transcriptome data. The nucleotide and amino acid sequences of the two OBPs were analyzed with bioinformatics software. The recombinant expression vectors pET-30 a/CpunOBP3 and pET-30 a/CpunOBP4 were constructed. and the recombinant proteins CpunOBP3 and CpunOBP4 were obtained by prokaryotic expression and purification. The binding activity of the recombinant CpunOBP3 and CpunOBP4 to 24 ligands was analyzed by fluorescence competitive binding assay.【Results】 The open reading frame of CpunOBP3 gene(GenBank accession no.: GEDO010000010.1) is 387 bp in length, encoding a protein of 128 amino acids with the predicted molecular weight of 14.72 kD. The open reading frame of CpunOBP4 gene(GenBank accession no.: GEDO010000011.1) is 438 bp in length, encoding a protein of 145 amino acids. The predicted molecular weight of CpunOBP4 without signal peptide is 12.82 kD. CpunOBP3 and CpunOBP4 share the typical structural features of OBPs, including six conservative cysteine residues. Both the recombinant CpunOBP3 and CpunOBP4 were mainly expressed in the inclusion. Fluorescence competitive binding assay indicated that the recombinant CpunOBP3 showed the binding ability to seven plant volatiles tested, with the strongest binding ability to 3-carene(K_i=10.33 μmol/L), but not to two sex pheromones tested. The recombinant CpunOBP4 exhibited the binding ability not only to the two sex pheromones tested(cis-10-hexadecenal with the K_i value of 14.65 μmol/L and hexadecanoyl with the K_i value of 7.83 μmol/L), but also to eight plant volatiles tested, with the strongest binding ability to ethyl butyrate(K_i=4.32 μmol/L). 【Conclusion】 Based on these results, we inferred that CpunOBP3 plays an important role in the host location and shift of C. punctiferalis, while CpunOBP4 possesses dual functions in recognizing sex pheromones and plant volatiles. The results provide a theoretic basis for controlling the occurrence and damage of C. punctiferalis via disturbing its olfactory reception.
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