Molecular cloning, expression and molecular modeling of chemosensory protein from Spodoptera litura and its binding properties with Rhodojaponin III

Abstract

Insects stimulate specific behaviors by the correct recognition of the chemicals in the external environment. Rhodojaponin III is a botanical grayanoid diterpenid oviposition deterrent isolated from Rhododendron molle. In this study we aimed to determine whether the CSPs involved in the recognition of Rhodojaponin III. A full-length cDNA encoding chemosensory protein was isolated from the antennae of Spodoptera litura Fabricius (CSPSlit, GenBank Accession No. DQ007458). The full-length cDNA of NlFoxA is 1789 bp and has an open reading frame (ORF) of 473 bp, encoding a protein of 126 amino acids, Northern blot analysis revealed that CSPSlit mRNA was mainly expressed in the antennae, legs, wings and female abdomens. A three-dimensional model of CSPSlit was constructed using homology modeling method, and its reliability was evaluated. The active site of CSPSlit was calculated using CDOCKER program indicated that the Tyr24, Ile45, Leu49, Thr64, Leu68, Trp79 and Leu82 were responsible ligand-binding active site on identifying Rhodojaponin III in the CSPSlit. The recombinant CSPSlit protein was expressed in Escherichia coli and purified using single-step Ni-NTA affinity chromatography. Fluorescence emission spectra revealed that the CSPSlit protein had significant affinity to rhodojaponin III. These results mean that CSPSlit is critical for insects identify the Rhodojaponin III.

Figure 1. Nucleotide and deduced amino sequences of CSPSlit.

The predicted signal peptide is underlined. The asterisk marks the translation-termination codon.

Figure 2. Phylogenetic analysis of CSP amino acid sequences.

Bootstrap support values based on 1000 replicates are indicated.

Figure 3. Northern blot analysis of RNA coding for CSPSlit in different tissues.

(a): The recovery and integrity of each RNA were assessed from the 18S rRNA pattern; (b): 1, antennas; 2, de-antennated heads; 3, foreleg; 4, mesopedes; 5, metapedes; 6, thoraces; 7, wings; 8, abdomens. (A): female; (B): male.

Figure 4. 3D structure of the CSPSlit. Six α-helixes were marked A–F.

Two disulfide bridges are represented by yellow stick.

Figure 5. The evaluation of the CSPSlit 3D structure by verify score (A) and Ramachardran plot (B).

Square: proline residues; triangle: glycine residues; circle: all other residues. blue and purple: favorable regions; all else: unfavorable regions.

Figure 6. Potential energy (A) and root-mean-square deviation (A) with respect to simulation time for 1000 ps free MD simulation on the CSPSlit model.

Figure 7. The complex (A) and detailed binding mode (B) of CSPSlit with rhodojaponin III.

The residues within 6 Å from ligand are shown.

Figure 8. Potential energy (A) and root-mean-square deviation (B) with respect to simulation time for 1000 ps molecular dynamics simulation on the CSPSlit- rhodojaponin III complex model.

Figure 9. Fluorescence spectra obtained titrating a 0.5 µM solution of CSPSlit with increasing amounts of 50 µM rhodojaponin III in methanol to final concentrations of 0, 50, 100, 150, 300 and 600 µM (a–f).

The intensity of this peak was used to measure the amount of rhodojaponin III bound to the protein.