Identification And Function Analysis Of Olfactory Relativeprotein In Frankliniella Occidentalis

The insect olfaction system is particularly sensitive and important in locating food sources, courtship, mating and oviposition sites. Within the insect olfaction system, chemosensory proteins(CSPs), odorant binding proteins(OBPs), olfactory receptors(ORs) and odorant degrading enzymes(ODEs), sensory neuron membrane proteins(SNMPs) are essential members. The western flower thrips, Frankliniella occidentalis(Thysanoptera, Thripidae), also called alfalfa thrips, is an invasive species. In this study, we identified, cloned, expressed and purified a few olfactory-related proteins of F. occidentalis, surveyed its expression in various developmental stages and adult tissues, determined its binding affinities for volatile substances using a competitive-binding fluorescence assay and studied immune localization, in order to further study more olfactory-related proteins of F. occidentalis, and understand the molecular mechanisms of the olfactory system of F. occidentalis. 1. Observation on sensilla of Frankliniella occidentalis with scanning electron microscopeOn antennal, there main are sensilla trichodea, sensilla chaetica, sensilla basiconica, mamillary sensilla. On legs, there main are sensilla trichodea, sensilla chaetica, sensilla basiconica, sensilla ear washing buob-shaped, mamillary sensilla, sensilla campaniform, B?hm bristle. On compound eyes, there is one class sensilla chaetica. 2. Cloning of olfactory-related protein genes of F. occidentalisWe cloned and identified 4 olfactory-related protein genes full-length c DNA and 1 c DNA fragment from the Western flower thrips, Frankliniella occidentalis, named Focc CSP1, Focc CSP2, Focc OBP1, Focc OBP2, Focc OR1 and the Genbank number are KM527949, KM035415, KM527948, KM527950, KM527951, respectively. The full-length c DNA of Focc CSP1, Focc CSP2, Focc OBP1, Focc OR1 genes are 597 bp, 521 bp, 660 bp, 1645 bp, contain 288 bp, 387 bp, 450 bp and 1374 bp open-reading frame encoding putative proteins of 95, 128, 149, 457 amino acids with a molecular weight of 11.377, 14.51, 16.388, 51.19 k Da and an isoelectric point of 4.72, 5.41, 7.46, 5.08, respectively. The deduced amino acid sequence of Focc CSP2 and Focc OBP1 contain putative signal peptide of 19 and 22 amino acid residues at the N-terminus, respectively. The c DNA fragment of Focc OBP2 gene is 880 bp, contains a 591 bp open-reading frame encoding a putative protein of 196 amino acids with a molecular weight of 23.6565 k Da and an isoelectric point of 7.41. 3. Expression profiling of olfactory-related protein genes of F. occidentalisIn developmental stages, Focc CSP1 in 1-day-old adult females had the highest expression, followed by second-instar nymphs. Focc CSP2 in First-instar nymphs had the highest expression, followed by 1-day-old adult females. Focc OBP1 in second and first instar nymphs had the higher expression, followed by 1-day-old females and males. Focc OBP2 in second instar nymphs had the highest expression, followed by 1-day-old females and males. Focc OR1 in 1-day-old adult females had the highest expression, followed by second instar nymphs and 1-day-old males. These genes were predominantly expressed in antennae. The transcript levels of Focc CSP1, Focc CSP2 and Focc OR1 were the greatest in the antennae, followed by leg tissue. Focc OBP1 transcript levels were the highest expression in the antennae than in the abdomen, were the lower expression in the legs, head and thorax than in the abdomen. Focc OBP2 transcript levels were the greatest in the antennae, followed by leg tissue. 4. Expression and purification of olfactory-related recombinant protein of F. occidentalisWe constructed the recombinant expression plasmid p ET-30a(+)/Focc CSP1, p ET-30a(+)/ CSP2, p ET-30a(+)/ OR1, and induced expression and purified the target proteins. Finally, purified His-tagged recombinant proteins were eluted by cleavage with recombinant enterokinase. The recombinant proteins were dtermined with the method of BCA protein concentration determination and the concentrations of Focc CSP1 and Focc CSP2 are 1.455 mg/m L and 1.013 mg/m L, respectively. 5. Fluorescence-based Ligand Binding Assay of F. occidentalisFocc CSP2 binding affinities for 19 ligands were determined using 1-NPN as a fluorescent reporter and 19 ligands as competitors. With the exception of salicylaldehyde, the ligands effectively displaced the fluorescent 1-NPN probe from the complex and decreased the fluorescence intensity of Focc CSP2/1-NPN to 50% of the initial value. Focc CSP2 binds strongly to anisic aldehyde, with a KD of 10.50 μM and IC50 of 11.3944μmol/L. Focc CSP2 also exhibited high-affinity binding to geraniol with KD value of 15.35μM and IC50 of 15.3463μmol/L. Focc CSP2 showed medium-affinity binding to ethyl nicotinate, methyl isonicotinate, benzaldehyde, ethyl benzene, citronellol, citronellal, pelargonic aldehyde, linalool, eucalyptol, hexane and eugenol, having KD values between 32.49 and 78.95 μM and IC50 values between 35.2426μmol/L and 85.6408μmol/L. Focc CSP2 binds weakly to other ligands. 6. The immunolocalization of Focc CSP1 protein in adult tissues of F. occidentalisPolyclonal antisera were prepared by immunizing New Zealand white rabbits with purified Focc CSP1 protein, and reacted with this protein in slices and 10 nm colloidal gold-affinipure goat anti-rabbit Ig G. The immunolocalization of Focc CSP1 protein in adult tissues was clarified by transmission electron microscope, indicating the immnological specificity of polyclonal antisera, which can bind aimed protein spectively and point out the distribution of Focc CSP1 protein. The immunocytochemistry results indicated that the hemolymph of antennae, legs and head were strongly labeled by anti-Focc CSP1 antisera, which is coincide to the results of q PCR, suggesting that Focc CSP1 might play an important role in olfactory reception, chemosensory and development regulation of F. occidentalis.