Identification Of Odorant Binding Protein Genes In Apolygus Lucorum (Hemiptera: Miridae) And The Binding Specificity Analysis Of Odorant Binding Protein AlucOBP7

Odorant binding proteins (OBPs) are widely involved in olfactory perception and play a key role intransporting hydrophobic odorants across the sensillum lymph to the olfactory receptors (ORs). Thegreen plant bug,Apolygus lucorum(Meyer-Dür)(Hemiptera: Miridae), is oneof the mostimportantsucking mouthparts pests of Bt cotton in China and cause significant yield losses everyyear.To help us elucidate the mechanism of insect olfaction perception,a cDNA library was constructedfrom theantennae of A. lucorum.Twelve new odorant-binding related genes were discovered bybioinformaticsanalysis, and to lay the theoretical basis for controlling of green plant bugs and otherimportant target pests from interfering the insect olfaction perception, we tested the binding propertiesof AlucOBP7in the olfaction perception in the work. The primary results were as follows:1. A high-quality cDNA library was constructed from the antennae of A. lucorum using SMARTmethod, which analysed by particularbioinformatics, with a view todiscover the olfactory relatedgenes which were antennae-specificexpressed.12novel putative OBP genes were identified fromthe antennal cDNA library, and the expression profiles of the12OBPs indifferent tissues (antennae,heads, thoraxes, abdomens, legs and wings) were analyzed byreverse transcription PCR andreal-time quantitative PCR.The results revealed that AlucOBP2, AlucOBP7, AlucOBP8, AlucOBP11and AlucOBP12are highly expressed in antennae. At the same time there are other tissue-specificexpression OBPs.AlucOBP1, AlucOBP5andAlucOBP6were highly expressed in the heads, whereasAlucOBP3was specifically expressed only in the legs.In addition, AlucOBP4, AlucOBP9andAlucOBP10were almost expressed inall chemosensory tissues (antennae, heads, wings and legs).2. The novel AlucOBP7in theA. lucorumwas cloned, expressed and purification and the bindingproperties of AlucOBP7with10cotton volatiles and6sex pheromone analogs weremeasured byfluorescence competitive binding assays. Theresults revealed that AlucOBP7had the bindingaffinity with some cotton volatiles and also with some sex pheromone analogs. The results revealedthatAlucOBP7might have somesimilar function with a pheromone binding protein (PBP)and haddual-function for the perception of both sex pheromones and plant volatiles.3. We used immunohistochemicaltechnique to position and research AlucOBP7in the female andmale antennae of A. lucorum, and we found that AlucOBP7was present both in the sensillatrichodea (ST) and the sensilla basiconica (SB). The results suggested that AlucOBP7was not aodorant binding proteinwhich binding only with sex pheromone, and AlucOBP7might havedual-function to detectboth sex pheromones and plant volatiles.4. We used electroantennographic (EAG)to verify the responses of A.lucorum to66standardspecimens indoor.From theresults we found that female and maleA.lucorum had significantresponse to many standard samples, especially to the ester compound, and only two kinds of the sexpheromone analoguescaused a significant EAG responsedifference between female andmaleA.lucorum. The results suggested that ester compounds had an important role in regulating thebehavior of green plant bugs and had a potential application prospect.At the same time, we focus on the EAG valueof two kinds ofcotton volatiles and four kinds ofplant bugs sex pheromone analoguescombined with AlucOBP7, most of them can cause strong EAG response, the sequencedecreasingly was ethylbutyrate, butylbutyrate,4-oxo-(E)-2-hexenal, hexyl hexanoate,2-hexanoneand methylsalicylate.