Expression And Ligand-binding Analysis In Vitro Of Two Odorant Binding Protein Genes In Sesamia Inferens (Walker)

Insects,especially moths,have evolved a highly sophisticated and sensitive olfactory system to sense varieties of odorants from theexternal environment.By using this system,insects are capable of completing various important physiological and behavioral activities,such as seeking mates,locating oviposition sites and avoiding predators.The antenna is the principal olfactory organ,and particularly moth antenna,which houses tens of thousands of olfactory sensilla,is more sophisticated and sensitive to odorants.In the process of chemosensory reception,several proteins are involved in olfaction like odorant binding proteins(OBPs).The OBP exists in the olfactory sensilla lymph with a high concentration and is thought to bind and transport the lipophilic odorant molecules across theaqueous lymph to the specific odorant receptors(ORs).After that,the odorant molecules furtheractivate olfactory receptor neurons.Studies on OBPs and their functions from important agricultural pests not only assist in understanding insect olfactory mechanisms,but also provide potential molecular target candidates based on the olfaction.Sesamia inferens(Lepidoptera:Noctuidae)is a serious rice pest.Previously,large chemosensory genes in this species were identified and their expression profiles were investigated by RT-PCR and qPCR.To understand chemosensory system of this species,in this study we selected two antennal-high OBP genes and investigated their binding properties to different plant odorants by fluorescence competitive binding assays.The main results are as follows:1.Prokaryotic expression and ligand-binding analysis of S.inferens OBP15Based on the previous studies on relative expression levels of SinfOBP15 in different tissues of both sexes,SinfOBP15 was highly expressed in female and male antennae,suggesting that it plays roles in olfaction.In this study,we first used a bacterial system to express this protein with a final concentration of 1 mM IPTG.After inducing an additional 6 h,the bacterial extracts from different steps were further analyzed by SDS-PAGE.Results showed that SinfOBP15 was expressed mainly in inclusion bodies and thus denaturation and renaturation were performed using urea.The recombinant protein with His-tag was purified by a nickel affinity column and then the His-tag was removed by digestion with enterokinase.Finally,an expected band of about 14 KD was observed.Using 1-NPN as the fluorescent probe,binding properties of SinfOBP15 to 35 rice volatiles were studied by the fluorescence competitive binding assays.As a result,all tested odorants did not show obvious strong binding to SinfOBP15(Ki>50 ?M),possibly indicating that the best ligand(s)for SinfOBP15 hasbeen not found.Therefore,further bindingstudies of SinfOBP15 to many more odorants need to be conducted in future.2.Prokaryotic expression and ligand-binding analysis of S.inferens ABPxAntennal binding protein(ABP)is a sub-family of lepidopteran OBPs but so far related functions have been rarely done.From S.inferens transcriptome,a candidate OBP gene was identified and named as ABPx based on the phylogenetic tree and NCBI Blast result.Previously,studies showed that SinfABPx was highly expressed in antennae of both sexes.In this study,to explore the functional roles of SinfABPx in odorant perception we used the prokaryotic expression system,affinity chromatographic technique and fluorescence competitive binding assay to study its potential olfactory roles.Firstly,a purified target protein of 14 K D was obtained.Next,using 1-NPN as the fluorescent probe binding affinities of SinfABPx to 35 odorants with different structures were measured by the fluorescence competitive binding assays.Results showed that ?-ionone was the best ligand(Ki = 35.58 ?M)while other odorants proved to be poor ligands.This suggests that SinfABPx plays a role in the reception of ?-ionone.