Gene Cloning, Expression Pattern And Functional Analysis Of Two Odorant Binding Protein Genes In Spodoptera Exigua

During the long time of evolution, insects evolve complicate and sensitive olfactory system. With this system, insects are able to perceive the thousands of chemical volatiles in the environments, and respond with specific physiological and behavioral adaption, such as feeding, mating and ovipositing. Odorant binding proteins (OBPs) belong to a class of water-soluble small proteins, which exist in insect antennal sensillum lumen with high concentrations. OBPs are proposed to bind and transport the hydrophilic external odorants to the olfactory receptors (ORs) situated on the dendrite membrane of the receptive neuron. Spodoptera exigua (Lepidoptera:Noctuidae) is a polyphagous and important agricultural pest. The studies on OBPs of S. exigua are not only helpful for better understanding of the olfactory mechanism, but also can provide potential target for development of olfaction based control techniques. In this study, by transcriptome data analysis and PCR experiments, we obtained 2 new OBP genes from S. exigua, investigated their tissue and temporal expression patterns, and explored their physiological functions by gene in vitro expression, fluorescence competitive binding assays and behavioral experiments. The main results are as follows.1 Gene cloning and sequence analysis of SexiOBP7 and SexiOBP13By analyzing the transcriptomic data, cDNA fragments of two OBP genes were obtained, and then the full length sequences were further characterized by RACE technology from S. exigua. One OBP gene was named as SexiOBP7 (GenBank accession No. JX962790.1) and the other one was named by other people as SexiOBP13 (KC817145). Sequence analysis showed that both OBPs have all characteristics of typical OBPs including six conserved cysteines. The amino acid sequences of SexiOBP7 had highest similarity of 87% with reported HarmOBP2, while SexiOBP13 had highest similarity of 80% with SlitOBP23. SexiOBP7 had an open reading frame (ORF) of 429 bp encoding 143 amino acids, with the 21 amino acids fragment at the hydrophobic N-terminus being the predicted signal peptide. The predicted molecular mass and isoelectric point of the mature protein were 16.34 kDa and 8.88, respectively. SexiOBP13 contains an ORF of 438 bp that encode 146 amino acids, with the 24 amino acid at the hydrophobic N-terminus being the signal peptide. The predicted molecular mass and isoelectric point of the mature SexiOBP13 were 15.41 kDa and 6.71, respectively.2 Tissue and temporal expression patterns of SexiOBP7 and SexiOBP13The expression pattern is an important way to explore the functions of a gene. The spatial and temporal expression patterns of SexiOBP7 and SexiOBP13 were examined by qPCR. The results showed that SexiOBP7 was very weakly expressed in larval stage, but highly and specifically expressed in the adult antennae. In contrast, SexiOBP13 was highly expressed in larval stage but did not in adult, and in larvae it was specifically expressed in the heads. As for the temporal dynamics, SexiOBP1 reached the peak expression in the three day old males, and showed the highest expressoin in the one day and four day females. SexiOBP13 displayed the highest expression in the heads of the third instar larvae, and the expression sharply decreased after then. The results suggest that SexiOBP7 play roles in adult olfaction, while SexiOBP13 in the larval olfaction in S. exigua.3 Prokaryotic expression and purification of SexiOBP7 and SexiOBP13To explore the function of SexiOBP7 and SexiOBP13, prokaryotic expression system was used to express these two SexiOBPs. The OBP sequences were first inserted into the pET-30a (+) expression vector, and then were expressed in Escherichia coli with induction by IPTG, and finally purified through nickel ion affinity chromatography. The purified recombinant proteins were further treated with enterokinase to remove the His-tags, and purified again through the affinity chromatography. SDS-PAGE analysis of pET/SexiOBP7 and pET/SexiOBP13 cell cultures showed the obvious band of expected size of about 22 and 21 kDa, respectively. Both proteins were expressed in the soluble forms.4 Ligand-binding affinity analyses of OBP7 and OBP13The binding affinities of two SexiOBPs with 37 odorant compounds including sex pheromone components and plant volatiles were tested by fluorescence competitive binding assays, with the 1-NPN as the fluorescence probe. The results showed that SexiOBP7 exhibited high binding affinities to nonyl acetate (Ki=22.81 μM), nerolidol (Ki=22.03 μM) and (3-ionone (Ki=9.49 μM); while SexiOBP13 showed high binding affinities to Z9,E12-14:Ac (Ki=3.82 μM), Z9,E12-14:OH (Ki=16.39 μM), farnesol (Ki=24.01 μM) and nerolidol (Ki=27.59 μM). Taken together, SexiOBP7 may play a role in adult olfaction of host plants volatiles such as β-ionone, and SexiOBP13 play roles in larval olfaction of to the sex pheromone component Z9,E12-14:Ac.5 Trend assay of larvae to sex pheromone Z9,E 12-14:AcAccording to the ligand binding assay results, S. exigua larvae were postulated to be able to perceive a female pheromone component To confirm this, a diet choice assay was conducted to measure the behavioral response of 3 day old larvae to Z9, E12-14:Ac. The results displayed that the number of larvae that was attracted to the diet plus Z9, E12-14: Ac was significantly (1.62 fold) higher than that to the control diet. This result indicates that larvae of S. exigua can indeed recognize and behaviorally respond to Z9, E12-14:Ac. Taken together, Z9,E12-14:Ac may serve as a food signal, and SexiOBP13 plays roles in the recognition of Z9, E12-14:Ac.