Identification And Functional Analysis Of GOBPs In The Geometrid, Ectropis Oblique Prout(Lepidoptera: Geometridae)

As one main Lepidopteran pest in tea garden, the larvae of tea geometrid damages mainly the tea leaves, which severely reduce the yield of tea products. Currently, the prevention of tea geometrid basically remains in chemical control. Spraying of pesticides can cause pesticides and environmental pollution, which could harm to human health. Therefore, the development of new method biological control is imminent. The function of insects olfactory plays an important role in feeding courtship and looking for spawning. Therefore, in this study, a series of studies were launched mainly around the general odorant binding protein in tea geometrid olfactory. Firstly, the espression profilings of Eobl GOBP1 and Eobl GOBP2 gene in tea geometrid were analyzed. Secendly, the recombinant Eobl GOBP2 was separated and purified by Escherichia coli gene expression system. The binding function of Eobl GOBP2 with several tea plant volatiles was studied in vitro here. The electroantennogram of E. oblique to tea plant volatiles was also tested. The interaction between imidacloprid and Eobl GOBP2 recombinant protein and the effect of binding function of Eobl GOBP2 with imidacloprid was studied. At last, the antennae structure of E. oblique was observed with scanning electron microscopy. The sub-cellular localization of Eobl GOBP2 on the antennae were detected by immune electron microscopy. The primary results as follows:1. The full-length c DNA sequences of Eobl GOBP1 and Eobl GOBP2 were obtained, their open reading frame is respectively 501 bp and 483 bp in length. The nuclear acid sequences analysis showed Eobl GOBP1 and Eobl GOBP2 encoding 166 and 160 amino acid residues, respectively. Both of their proteins have six conservative cysteines and contain a conservative domain that comprised by six α-helices. Their proteins have signal peptides of 17 amino acids,and 20 amino acids, respectively. Phylogentic tree showed that the kinship of Eobl GOBP1 and Eobl GOBP2 is far with GOBP1 and GOBP2 in other Lepidoptera insects respectively,but that the kinship of Eobl GOBP1 and Eobl GOBP2 is close to GOBP2 and GOBP1 respectively.2. The expression profiling of Eobl GOBP1 and Eobl GOBP2 in different tissues was measured by standard curve real-time PCR. The results revealed that Eobl GOBP1 was specifically expressed in the antennae, and Eobl GOBP2 was expressed in antennae, abdomen and wing at high abundance.3. The recombinant Eobl GOBP2 protein was obtained using constructed prokaryotic expression vector p ET32-Eobl GOBP2, and was purified by nickel agarose gel chromatography. New Zealand white rabbit was immunized with the recombinant Eobl GOBP2 protein to get the polyclonal antibodies.4. The binding properties of the recombinant purified Eobl GOBP2 with 19 tea volatiles was investigated by the fluorescence competition binding assays using 1-NPN as fluorescent reporter. The results showed that Eobl GOBP2 had strong binding capability with Dibutyl phthalate, Methyl salicylate, Benzaldehyde,(E)-2-hexenal,(E)-2-decenal, β-Ionone and Acetophenone. The following electrophysiological assay verified those ligands also elicited responses to the adults moth antennae. But the EAG value of the adults moth antennae with Dibutyl phthalate, which has the highest affinity with Eobl GOBP2, were the lowest. This is probably because the EAG test measures the antennal sensilla overall activity. It was inferred that Eobl GOBP2 bind with Dibutyl phthalate specificly.(E)-2-hexenal and Benzaldehyde both lead to strong electrophysiological responses in EAG trials.5. Multiple fluorescent spectra, UV absorption spectra, synchronous fluorescence spectra and circular dichroism(CD) spectra were combined to clarify the binding process of Eobl GOBP2 with imidacloprid. Basically, fluorescence intensity of Eobl GOBP2 could be considerably quenched by imidacloprid with an appropriate interaction distance(7 nm), indicating that a complex, which is more stable in lower temperature, was formed. The fact ΔH < 0, ΔS < 0, by thermodynamic analysis, indicated the vander Waals and hydrogen bond as main driving force. The result of molecular docking also show that there are four hydrogen bonds between imidacloprid and Eobl GOBP2. Moreover, synchronous fluorescence spectra and CD spectra analysis showed the change of partial hydrophilicity of Eobl GOBP2 and the decrease of α-helix after imidacloprid addition. In the case of the presence of imidacloprid, the binding constant of Eobl GOBP2 and(E)-2-hexenal decreased from 1.36×104 L·mol-1 to 1.13×104 L·mol-1. It indicated that imidacloprid weakened the binding ability of Eobl GOBP2 with(E)-2-hexenal.6. The antennae structure of E. oblique was observed with scanning electron microscopy. There are five kinds of receptors, respectively, Sensillum trichodeum, Sensillum chaeticum, Sensillum styloconicum, Sensillum squamiformium, Sensillum basiconicum. S. trichodeum is divided into two forms, AⅠand AⅡ. AⅠon the antennae of the male are far more than the female. S. chaeticum only exist on antennae of the female. The subcellular localization of Eobl GOBP2 on the chemosensilla in the antennae was labelled by colloidal gold labelling immune electron microscopy technique. The results displayed that Eobl GOBP2 was largely expressed in the AⅡS. trichodeum of the male. Eobl GOBP2 was alse expressed in AⅠS. trichodeum of the male.In conclusion, our studis laid an important foundation for the development and application of new biological control means based on the interference and suppression on olfactory function of tea geometrid.