Abstract
Chemosensory proteins (CSPs) belong to a family of small water-soluble proteins that can selectively bind and transport odorant molecules for olfactory communication in insects. To date, their definite physiological functions in olfaction remain controversial when compared with odorant binding proteins (OBPs). To investigate the functions of CSPs in the oriental fruit moth Grapholita molesta, we determined the tissue expression patterns and binding properties of the CSP, GmolCSP8. The key binding sites of GmolCSP8 with a representative ligand were evaluated using molecular flexible docking, site-directed mutagenesis and ligand-binding experiments. Multiple sequence alignment and phylogenetic analysis showed that GmolCSP8 possesses a typical conserved four cysteines motif and shares high sequence identity with some CSP members of other Lepidopteran insects. GmolCSP8 was predominantly expressed in the wings and antennae of both male and female adults and may be involve in contact chemoreception. Recombinant GmolCSP8 (rGmolCSP8) exhibited specific-binding affinities to small aliphatic alcohols (C4–12) and had the strongest binding affinity to 1-hexanol. The three-dimensional structure of GmolCSP8 was constructed using the structure of sgCSP4 as a template. Site-directed mutagenesis and ligand-binding experiments confirmed that Thr27 is the key binding site in GmolCSP8 for 1-hexanol binding, because this residue can form hydrogen bond with the oxygen atom of the hydroxyl group in 1-hexanol, and Leu30 may play an important role in binding to 1-hexanol. We found that pH significantly affected the binding affinities of rGmolCSP8 to ligand, revealing that ligand-binding and -release by this protein is related to a pH-dependent conformational transition. Based on these results, we infer that GmolCSP8 may participate in the recognition and transportation of 1-hexanol and other small aliphatic alcohols.
Highlights
The oriental fruit moth, Grapholita molesta Busck (Lepidoptera: Tortricidae), is a major pest species of fruit trees belonging to the family Rosacea, and has caused substantial losses in fruit yields throughout the world (Myers et al, 2007)
Three incorrect basic groups were identified in the Unigene of GmolCSP8, obtained by antennal transcriptome sequencing, and the verified sequence was deposited in GenBank under accession number KR003781
Using the NCBI BLASTx program, we found that GmolCSP8 shared relatively high sequence similarity with other insect Chemosensory proteins (CSPs), with 82% identity with Lobesia botrana LbotCSP8 (GenBank No AXF48705.1), and almost 60% identities with Ostrinia furnacalis OfurCSP6 (GenBank No BAV56810.1), Spodoptera litura SlitCSP12 (GenBank No ALJ30223.1), Cnaphalocrocis medinalis CmedCSP31, Dendrolimus punctatus DpunCSP10 (GenBank No ARO70314.1), Conogethes punctiferalis CpunCSP8 (GenBank No AHX37222.1), and Ectropis obliqua EoblCSP20 (GenBank No ALS03845.1)
Summary
The oriental fruit moth, Grapholita molesta Busck (Lepidoptera: Tortricidae), is a major pest species of fruit trees belonging to the family Rosacea, and has caused substantial losses in fruit yields throughout the world (Myers et al, 2007). The adult moths exhibit pronounced seasonal host-transfer behavior in adjacent areas of peach and pear, or peach and apple orchards (Rajapakse et al, 2006). Changes in the components of host-plant volatiles are the primary cause of host transfer for this pest (Najar-Rodriguez et al, 2013b). G. molesta is predominantly monitored and controlled by sex pheromone trapping of male moths. This species is able to undergo multiple mating (up to fours), and the females can present a higher mating rate in orchards controlled by sex attractants (de Morais et al, 2012). Female G. molesta are considered the main colonists and can cause a serious threat to pear and apple orchards in the vicinity of peach crops. Investigating the function of olfactionrelated genes will help to better understand the olfactory system of G. molesta
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