Exploration And Functional Analysis Of Olfactory Related Genes In The Lucerne Plant Bug, Adelphocoris Lineolatus (Goeze)

In the insect kingdom, the relationships between individuals and the environment are often modulated by chemical communication. The olfaction system is essential for insects'survival and reproduction. As a highly specific and sensitive chemical sensor, it can 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. Elucidating the mechanism of insect olfactory perception can further facilitate the design and implementation of novel intervention strategies against these pests. The lucerne plant bug, Adelphocoris lineolatus (Goeze) (Hemiptera:Miridae), is one of the most destructive pests of Bt cotton in China and cause significant yield losses every year. Evidences showed that mirid can sense particular plant volatiles and sex pheromones to locate suitable host plant and mate partners. Olfactory related proteins have been demonstrated to be required for olfaction perception in insects. In A. lineolatus, they might represent new interesting targets for the control of their population. In this paper, two high-quality cDNA libraries were constructed from the antennae of A. lineolatus. Several olfactory related genes were discovered by particular bioinformatics analysis. The functional roles of related genes in the olfaction perception of A. lineolatus are investigated. The primary results are as follows:1.The antennae and antennal sensillae of A. lineolatus were observed by scanning electron microscopy. The results showed that the antennae of of A. lineolatus was in linear shape and made up of scape, pedicel and flagellum. There are four distinct antennal sensillae, including sensilla trichodea, sensilla chaeticum, sensilla basiconica and Bohm bristles.2.By using SMART method, two high-quality cDNA libraries were constructed from the male and female antennae of A. lineolatus. Several olfactory related genes are discovered by particular bioinformatics analysis, including 14 odorant binding prorein (OBP) genes,8 chemosensory protein (CSP) genes,2 odorant receptor (Or) genes and 8 putative odorant degrading enzyme (ODE) genes.3.The expression profiles of the 14 OBPs in different adult tissues were measured by real-time qPCR. These 14 OBPs were divided into four classes based on their expression intensity in antennae,heads, legs or other tissues, respectively. ClassⅠincluded nine OBP genes (Alin-OBP1,Alin-OBP2, Alin-OBP3,Alin-OBP4,Alin-OBP5,Alin-OBP6,Alin-OBP8,Alin-OBP12 and Alin-OBP13),which are dominantly expressed in the antennae tissues. ClassⅡincluded one OBP gene (Alin-OBP11), which was mainly expressed in the legs and heads. ClassⅢincluded one OBP gene (Alin-OBP14), which was predominantly expressed in the heads. ClassⅣincluded three OBP genes (Alin-OBP7, Alin-OBP9 and Alin-OBP10), which seemed ubiquitously expressed in male antennae, female antennae, heads and legs.4. A novel OBP (AlinOBP1)in the A. lineolatus was cloned, expressed and purification in laboratory. The binding properties of AlinOBP1 with 114 odorants of different chemical classes were measured by using a fluorescence probe 1-NPN with the fluorescence competitive binding method. The results revealed that AlinOBPl exhibits high binding abilities with two major putative pheromones, Ethyl butyrate and Trans-2-Hexenyl butyrate. In addition, it was observed that six volatiles released from cotton, Octanal, Nonanal, Decanal,2-Ethyl-l-hexanol, (3-caryophyllene andβ-ionone can also significantly bind with AlinOBPl.The binding experiments showed that the AlinOBPl can both bind with its putative pheromones and chemicals released from cotton, indicating there may exist a more multiple-function of AlinOBPl in the perireceptor events of olfaction. The predicted 3 D structure of AlinOBP1 was established with the BmorPBPl as the structural template. The AlinOBPl 3D modle 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. However, some hydrophilic residues (Asp19, Thr22, Asn23,Glu79 and Lys102) are also presented in the binding pocket, these hydrophilic residues are likely the initial recognition sites of ligands.5.A novel OBP (AlinOBP3) in the A. lineolatus was cloned, expressed and purification in laboratory. The binding properties of AlinOBP3 with nine cotton volatiles and five sex pheromone analogs were measured by fluorescence competitive binding assays with the fluorescence probe 1-NPN. The results revealed that of nine cotton volatiles, Myrcene,β-Ocimene andα-Phellandrene can bind with AlinOBP3.α-Phellandrene especially bound to AlinOBP3 with a high binding affinity, with a dissociation constant of 56.68μmol/L. Of the five sex pheromone analogs, Hexyl butyrate had the strongest binding affinity with AlinOBP3, with a dissociation constant as 59.53μmol/L. Butyl butyrate, Trans-2-Hexenyl butyrate and Ethyl butyrate had medium binding affinities with AlinOBP3,with dissociation constants of 227.39μmol/L,108.77μmol/L,143.47μmol/L, respectively. The results suggest that AlinOBP3 might be a pheromone binding protein (PBP) with a dual-function for the perception of sex pheromones and plant volatiles.