Identification And Functional Analysis Of Odorant Binding Protein Genes In Sesamia Inferens (Walker)

During the long time of evolution, insects evolve highly developed olfactory system based mainly on the antennae. With this olfactory system, insects are able to recognize specific odorant information in the complex environments, and accordingly to respond with specific physiological or behavioral events, such as finding mates, food sources, habitat and oviposition sites. Lepidoptera insects are of complete metamorphosis, and adult moths of noctuidae are nocturnal, therefore they need more sensitive olfactory system to guide the nocturnal behaviors. Odorant binding proteins (OBPs) belong to a class of water-soluble small proteins which exist in insect antennal sensillum lumen with high concentrations. They are proposed to bind and transport the hydrophiac external oderants entered into the sensillum lumen to the olfactory receptors (OR) situated on the membrane of the receptive neuron. This is thought as the first biochemical step of olfaction. The pink rice borer, Sesamia infer ens (Lepidoptera:Noctuidae), has gradually emerged to be one of the most important rice pests in China since2000. Olfaction based behavioral manipulation technique has been successfully used in controlling many pests, however, the olfactory mechanism of insects are not well understood. Cloning olfactory genes and understanding their functions will undoubtedly help to develop more efficient techniques of behavioral manipulation. In this study, three pheromone binding protein (PBP) genes and two general odorant binding protein (GOBP) genes from S. inferens were obtained by transcriptome data analysis and molecular cloning, their tissue expression patterns were determined, and finally the physiological functions were preliminarily explored by fluorescence competitive binding experiments. The main results are as follows:1Gene cloning and sequence analysis of SinfOBPsBy degenerate PCR, transcriptomic analysis and RACE technology, full-length cDNAs of three PBPs and two GOBPs from S. inferens was identified, which were named as SinfPBPl (JF927621)> SinfPBP2(JN984058), SinfPBP3(JF927622), SinfGOBPl (KC887506) and SinfGOBP2(KF150178), respectively. Sequence analysis showed that all OBPs bear the characteristics of typical OBPs, including six conserved cysteines. The amino acid sequences of SinfPBPl, SinfPBP2, SinfPBP3, SinfGOBPl and SinfGOBP2had highest similarities of94%,88%,85%,91%,87%, respectively, with reported OBPs of other Noctudae insects. Phylogenetic trees showed that the five OBPs belong to the group PBP and GOBP, respectively. Distance between OBP genes in the tree well agreed with phylogenetic relations of these insects.2Expression patterns of SinfOBPs in different adult tissues and in antennae of different daysTissue and sex expression pattern of a gene may provide functional clues. The expression levels of SinfPBPl, SinfPBP2, SinfPBPS, SinfGOBP1and SinfGOBP2in different tissues of the adults were examined by RT-PCR. The results showed that the five OBPs expressed mainly in the antennae, but nearly none in other tissues. The qPCR measurements further conducted to investigate the sex difference and the temporal dynamics of the gene expression. The results indicated that five OBPs all reached the peak expression in the4th-day after the moth emergence. However, three PBPs displayed very different sex biased expression profiles, with SinfPBP1highly male biased, SinfPBP2moderately male biased, while SinfPBP3slightly female biased. Three PBP displayed a male/female expression ratios of11:1,6:1and1:1.6in the4-day old moths. Two SinfGOBPs displayed similar expression levels between males and females.3in vitro expression and purification of SinfPBPsIn order to study the function of PBPs in the chemical communication system, three SinfPBP genes were in vitro expressed using a prokaryotic expression system. The PBPs were built into the pGEX-4T-1(PBP1and PBP2) and pET-30a (+)(PBP3) expression vector, respectively, and then expressed in the Escherichia coli with induction by IPTG. SDS-PAGE analysis of pGEX/SinfPBP1, pGEX/SinfPBP2and pET/SinfPBP3cell culture showed obvious band of expected size of about42kDa,42kDa and22kDa, respectively; PBP1and PBP2were soluble, while PBP3was expressed as inclusion bodies. Recombinant proteins were purified by affinity chromatography, and the GST-tag or His-tag was removed by enzyme digestion.4Ligand-binding affinity analyses of SinfPBPsThe binding affinities of three SinfPBPs with30odorant compounds including three female sex pheromones were tested by fluorescence competitive binding experiments. The results showed that SinfPBP1exhibited high binding affinities to all3sex pheromone components (Ki=0.72-1.60); SinfPBP2showed high binding affinities to the alcohol and aldehyde components (Ki=0.78-1.71), but no binding to the ester component; while SinfPBP3showed very low binding affinity to all3sex pheromone components. Three SinfPBPs generally showed very low binding affinity to plant volatiles. Taken together, our results suggest that SinfPBPl plays major role, while SinfPBP3plays least role (if any) in reception of the female sex pheromones in S. inferens; SinfPBP2functions as a recognizer of alcohol and aldehyde components in the sex pheromone blend of S. inferens.