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Three Amino Acid Residues Bind Corn Odorants to Mcinobp1 in the Polyembryonic Endoparasitoid of Macrocentrus Cingulum Brischke

Tofael Ahmed
Bangladesh Sugarcane Research Institute, Ishurdi, Pabna, Bangladesh

Tian-tao Zhang
State Key Laboratory for the Biology of the Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China

Zhen-ying Wang
State Key Laboratory for the Biology of the Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China

Kang-lai He
State Key Laboratory for the Biology of the Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China

Shu-xiong Bai
State Key Laboratory for the Biology of the Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China

Abstract/ Executive Summary:

Odorant binding proteins (OBPs) play a central role in transporting odorant molecules from the sensillum lymph to olfactory receptors to initiate behavioral responses. In this study, the OBP of Macrocentrus cingulum McinOBP1 was expressed in Escherichia coli and purified by Ni ion affinity chromatography. Real-time PCR experiments indicate that the McinOBP1 is expressed mainly in adult antennae, with expression levels differing by sex. Ligand-binding experiments using N-phenyl-naphthylamine (1-NPN) as a fluorescent probe demonstrated that the McinOBP1 can bind green-leaf volatiles, including aldehydes and terpenoids, but also can bind aliphatic alcohols with good affinity, in the order trans-2-nonenal>cis - 3- hexen -1 -ol > trans-caryophelle, suggesting a role of McinOBP1 in general odorant chemoreception. We chose those three odorants for further homology modeling and ligand docking based on their binding affinity. The Val58, Leu62 and Glu130 are the key amino acids in the binding pockets that bind with these three odorants. The three mutants, Val58, Leu62 and Glu130, where the valine, leucine and glutamic residues were replaced by alanine, proline and alanine, respectively; showed reduced affinity to these odorants. This information suggests, Val58, Leu62 and Glu130 are involved in the binding of these compounds, possibly through the specific recognition of ligands that forms hydrogen bonds with the ligands, functional groups.

Keywords: r Agro-Scientific, Furnacalis, Plant Protection, Corn plants, Cloning, Macrocentrus cingulum

Location: In Bangladesh

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Diseases and insects, Insecticide

Objectives:

To determine the OBP of Macrocentrus cingulum McinOBP1, Escherichia coli and Ni ion affinity chromatography.

Methodology:

Insects: M. cingulum were collected from O. furnacalis larvae living on corn plants at the Langfang Experiment Station of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, China. A laboratory colony was established on host larvae of O. furnacalis that were reared on an artificial diet as described by Zhou et al. and maintained at 25uC with a photoperiod of 16 h:8 h, L:D. Adult parasitoid wasps were fed with 20% honey solution. RNA Extraction and cDNA Synthesis: Antennae, heads with maxillary palps (excluding antennae), legs, thoraxes and abdomen of female and male individuals were dissected 1–2 days after eclosion and immediately frozen in liquid nitrogen, then stored at –80uC until RNA extraction. Total RNA was extracted from the antennae or other tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s instructions. Before transcription, the RNAs were treat with DNase I (Invitrogen) to remove residual genomic DNA. cDNA was prepared from total RNA by reverse transcription, using the RT-PCR system (Promega) in accordance with the manufacturer's protocol. Cloning of McinOBP1-Wild Type in Expression Vectors: The pGEM plasmid containing the positive clones and the pET32a (+) were digested with NcoI and HindIII restriction enzymes for 2 hrs at 37uC and then the products were separated on agarose gel. The target fragments were purified from the gel and ligated into the digested pET32a (+) plasmid and the recombinant plasmids were transformed into TransT₁ competence cells and grown on LB solid medium with 10 ml ampicillin (10 mg/mL). Selected colonies were grown in LB liquid medium with ampicillin and then incubated in a shaker at 200 rpm, E. coli and transformed into BL21 (DE3)-competent cells. A single clone was identified and cultivated overnight in LB liquid medium including ampicillin on a shaker at 200 rpm in 37uC. The resulting plasmids were sequenced and shown to encode the mature proteins. Molecular Modeling and Ligands Docking: A three-dimensional model of McinOBP1 was generated using the I-TASSER Protein Structure and Function Predictions web server (http://zhanglab.ccmb.med.umich. edu/I-TASSER/) [40,41]. The sequence of McinOBP1 was compared to all known protein sequences that had a high sequence similarity with the target sequence on RCSB Protein Data Bank (PDB) (www.rcsb. org). The homology modeling of McinOBP1 was performed using build homology model of AaegOBP1. The best model was confirmed using the evaluation of PDF total energy, verify score and Ramachandran plot.

Source of Information: https://www.researchgate.net/publication/261408399; PLoS ONE · April 2014

Article Link (Online Journal): DOI: 10.1371/journal.pone.0093501 · Source: PubMed

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

These results suggest McinOBP1 is a general OBP that plays an important role in recognizing several plant volatiles. The three amino acids Val58, Leu62 and Glu130 appear to be involved in binding the aldehyde trans-2-nonenal as aldehyde and the terpenoid trans-caryophyllene. Seemingly these compounds interact with McinOBP1 with their hydrophobic chain inside the binding pocket and the functional group on the mount of the cavity. Future investigations with NMR tests or X-ray diffraction are needed to confirm these ligand-OBP interaction findings.

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