Preliminary Study On Function Of Two Odorant Binding Proteins In Adelphocoris Lineolatus

The lucerne plant bug, Adelphocoris lineolatus (Goeze) (Hemiptera:Miridae) is one of the most destructive pests of cotton in north China and cause significant yield losses every year. And also, lucerne plant bug can feed on a variety of plants. In the insect kingdom, the relationships between individuals and environment are often modulated by chemical communication. The olfaction system is essential for insects' survival and reproduction, it can sever as a highly specific and sensitive chemical sensor, detect, recognize and transfer chemical signals from the environment and trigger specific behavioral reactions, such as location host plant, mating with partners, avoidance of toxins and predators. In insects, perception of sex pheromones or plant volatiles is mediated by proteins located in the sensory hairs of the antennae, including odorant binding proteins (OBPs), chemosensory proteins (CSPs), olfactory receptors (ORs), odorant degrading enzymes (ODEs) and sensory neuron membrane proteins (SNMPs). Elucidating the mechanism of insect olfactory perception can further facilitate the design and implementation of novel intervention strategies against these pests. In this paper, the function of two odorant binding proteins (AlinOBP4 and the AlinOBP8) in Adelphocoris lineolatus was explored, and the main results are as follows:3D model of AlinOBP4 and AlinOBP8 were constructed by homology modeling methods. We found that the binding pocket of AlinOBP8 is bigger than that of AlinOBP4, and AlinOBP8 has more hydrophobic amino acids. We supposed that AlinOBP8 may have larger capacities and be able to bind with more odorant molecules than AlinOBP4. The 3D model demonstrated that the binding pocket is mainly organized by hydrophobic amino acids, which may be responsible for the hydrophobic interactions with the hydrocarbon part of the ligands. Nonetheless, some hydrophilic residues are presented in binding pocket, these hydrophilic residues are likely the initial recognition sites of ligands.The odorant binding protein AlinOBP8 was expressed in Escherichia coli, and the recombinant protein was harvested by affinity chromatography and gel filtration. The binding properties of AlinOBP8 with 85 volatile compounds were measured by using a fluorescence probe 1-NPN with the fluorescence competitive binding method. The results show that AlinOBP8 exhibits high binding abilities with green leaf volatiles, (E)-2-Hexenal with the binding constant 1.739 U mol/L, ethyl laurate laurinsaure-ethylester, (Z)-3-Hexen-l-ol, Farnesol and 2-butyl-l-octanol with the binding constant 0.462μmol/L,0.650μmol/L,0.665μmol/L and 0.784μmol/L, respectively. However, some sex pheromone analogues including Hexyl butyrate, trans-2-Hexenyl butanoate and the n-butyl butyrate have low binding affinities, and with the binding constant 3.164μmol/L,3.274μmol/L and 3.382μmol/L, respectively. Thus, we speculated that AlinOBP8 is a kind of general odorant binding protein (GOBP).Electroantennograms (EAG) were conducted to evaluate the electrophysiological responses of parasitoids antenna to 77 volatile compounds indoors. It was found that the EAG response value of aldehydes was the most intense, such as the trans-2-Hexenal,3,4-Dimethylbenzaldehyde, pentanal, octanal, etc. There are 20 compounds with large EAG response values, which are 6 aldehydes,5 eaters,3 ketones,3 alcohols and 3 hydrocarbon alkyls. Among them, there are 7 cotton volatiles and 5 sex pheromone analogs. There are eight kinds of volatiles can result in significantly different response between males and females, including 3,4-dimethyl-benzaldehyd, Caprylaldehyde, Benzaldehyde, Undecane,2-Hexanol, Octane, Dodecanal, Methyl salicylate.The microinjection approach was selected to silence the AlinOBP4 of the lucerne plant bugs by RNA interference. After injected siRNA in the thorax membranes of the bugs 48 hours, RT-PCR and the real-time fluorescence quantitative PCR asssy suggested that the transcript levels of the AlinOBP4 were reduced to less than half. And antennae of RNAi-treated goezes showed significantly lower electrophysiological responses to adelphocoris lineolatus sex pheromone analogs than that of non-treated goezes. These data suggest that AlinOBP4 is involved in the reception of some odorants attractants.Generally, the results mentioned above could help us finding potential chemical candidates for developing insect traps, and understanding the mechanism of the plant bug olfactory chemoreception.