Mechanism Analysis Of Interaction Between Olfactory Associated Proteins In Holotrichia Oblita(Falderman)

Underground pests are a harmful class group in agricultural industry in China, and their coverthabitats and strong adaptability result in difficulties in the prevention and control of pests. The scarabbeetle, Holotrichia oblita Faldermann (Coleoptera: Scarabaeidae), belongs to such a class and hascaused serious economic damage to crops, fruit trees and forest trees in China. The chemical methods ofcontroling underground pests could lead to many potential threats. Therefor, an environmentallyfriendly method for controlling of H. oblita is needed. The sophisticated insect olfactory system candetect and discriminate between different kinds of odorants, which are volatile small organic moleculesin the environment. This characteristic property plays a crucial role in insect behaviors, such as hostseeking, mating, ovipositing, as well as escape behaviors. Therefore, the secret that arousing a series ofinsect behavior reactions by perceiving and distinguishing amounts and kinds of odorants will beunveiled by investigating olfactory receptive mechanism, which contributes to exploit the newbiological agent to mess up communications among pests. This kind of new conception will providenew way for biological control, and gain our ends to protect crops from damage by pests. Ourconclusions are listed as follow.(1) According to registered OBP3and OBP4sequences from H. oblita in GenBank, we designedspecific primers and obtained ORF sequences of OBP3and OBP4from H. oblita. These two sequenceswere cloned into expression vector pET-30a respectively, which provided molecular basis for proteinfunctional analysis.(2) The crystal structure of Anopholes gambiae OBP20(AgamOBP20) was chosen as a templatefor both HoblOBP3and HoblOBP4to predict three-dimensional structures. The three-dimensionalmodel of HoblOBP3and HoblOBP4presented a large binding pocket, and the C-termini extended intothe binding pocket, which was similar to AgamOBP1. Six olfactory associated proteins of H. oblita,HoblOBP3, HoblOBP4, HoblCSP1, HoblCSP2, HoblOBP1and HoblOBP2, were expressed in E.coliand purified using Ni ion affinity chromatography. We selected42potential organic compounds on thebasis of competitive binding assays with proteins of HoblOBP3and HoblOBP4, which includedcompounds from volatile green plants and putative sex pheromones of some beetle species. The resultsshowed that HoblOBP4exhibited a broader spectrum of activity and well bound aliphatic compoundsconsisting of6carbon atoms, and putative sex pheromone compounds of some beetle species, such asL-isoleucine methyl ester. However, HoblOBP3only appeared high affinity to α-ionone and-ionone.In addition, the differences of position, variety and amount of functional groups, length difference ofcarbon chain, ring-shaped or ring-open structure of carbon chain, and optical isomerism in compoundswere the main impact factors that affected binding affinity. The interrelations and formation of dimerbetween olfactory associated proteins were discussed using competitive binding assays. We speculatedthat HoblOBP2could form dimer with HoblOBP1and HoblOBP4respectively.(3) We used the technique of colloidal gold post-embedding immunocytochemistry to detect colocalization of binary OBPs at the subcellsular level. The presented results demonstrated that thebinary mixtures of HoblOBP1/HoblOBP2and HoblOBP2/HoblOBP4were both coexpressed in sensillaplacodea and sensilla basiconica. This conclusion supported one assumption that OBPs could formdimer in order to perceive plant odors and sex pheromones, which provided evidence for thehydrophobic tunnel hypothesis.(4) The construction kits of DUALsystem Biotech were used to construct the normalized cDNAlibrary from H. oblita antenna in order to screen by yeast two-hybrid assay. Then, the completed librarywas cloned into vector pPR3-N expressing in yeast, which put up the platform for screening by yeasttwo-hybrid assay. Subsequently, the quality of the normalized cDNA library was tested. The resultsshowed that the number of independent transformants in the original library was1.1×106, and theaverage insert size was greater than1.2kb, which was up to the standard of the high-quality andhigh-spreadability cDNA library.(5) DUALhunter starter kits of DUALsystem Biotech were used to construct the bait vector ofHoblOBP1and HoblOBP2respectively. Then, the H. oblita antenna normalized cDNA library wasscreened. The screened results showed that six interactors screened with HoblOBP1bait vector andeight interactors screened by HoblOBP2bait vector were identified using PCR technique and analyzedusing Blast in GenBank. In the end, by assaying for detection of-galactosidase activity, the strongestpositive interactor, receptor for activated protein kinase C-like, was identified by HoblOBP1bait, andthe proclotting enzyme was viewed as the strongest positive interactor identified by HoblOBP2bait.Therefore, we speculated that the two positive interactors might work in the process of olfactoryrecognition.