Cloning And Functional Characterization Of Homologous Odorant Binding Protein 11 Of Two Mirids

The last century, highly toxic pesticides have been used to against cotton bollwon Helicoverpa armigera Hubner, which has caused serious harm to cotton. With the use of pesticides, the resistance of cotton bollworm has increased gradually, until the cotton bollwon broke. Bt cotton has effective controlled cotton bollwon, which reduced the use of pesticides, leading to the population of secondary pests, mirid, are increasing rapidly, being the major pests. Because of the characteristics of miscellaneous eating habits, breeding fast, strong flight ability, enemy control weak, they became the dominant pests quickly, causing the huge losses. Chemical identification systems play an important role in the life of insects. Insect olfactory occupies an important place in survival and reproduction. Due to the characteristics of highly specificity and sensitivity, it can identify the chemical information in the environment and provide the basis of feeding, mating, avoiding the predators and selecting oviposition sites. The researches suggested that many kinds of proteins take part in olfactory activity, for example odorant binding proteins(OBPs), odorant receptors(ORs), chemosensory proteins(CSPs), sensory neuronmembrane proteins(SNMPs) and so on. Odorant binding proteins are studied extensively. In researches, OBPs were found in a large number of olfactory function, taste function rarely. With the constantly expanding and deepening, taste functions of OBPs have been found. In this study, taking an orthologous gene in two kinds of bugs as object, the taste functions of OBPs in Hemipera were explored.The main research contents are as follows:1. The sensilla identification of taste organ, styletScanning electron microscope results showed that it was distributed many sensors to the front of stylet, such as long sensilla chaetica, short sensilla chaetica and sensilla basiconica. The surfaces were smooth without holes. Sensilla basiconica was uniporous at the top.2. Cloning, binding specificity and immunolocalization of orthologous odorant binding protein, OBP11Orthologous genes were cloned in two species of mirid by specific primers. The identity of amino acid sequence was as high as 97.33%. At the m RNA and protein levels, it was confirmed that Afas OBP11(Adelphocoris fasciaticollis OBP11) and Asut OBP11(Adelphocoris suturalis OBP11) were more highly expressed in stylet than other tissues by the q RT-PCR and Western blot. Afas OBP11 and Asut OBP11 recombinant proteins were expressed and purified in prokaryotic expression system. Fluorescence competitive binding assay results dicated that two proteins had strong binding ability to the plant secondary metabolites(Ki<10μM), but also to some volatile chemicals. In details, Afas OBP11 can bind to two general cotton volitiles, one putative sex pheromone, one terpenoid and six plant secondary metabolites, while Asut OBP11 can bind to two general cotton volitiles, one putative sex pheromone, three terpenoids and seven plant secondary metabolites. Immunolocalization results demonstrated that the gold granules were strongly labeled in sensilla basiconica and tissue lymph cavity, but not neurons.3. RNAi of Asut OBP11 gene from Adelphocoris suturalisAfter 24 h injected Asut OBP11-ds RNA, the expression of Asut OBP11 gene was silenced to 80%. There was no significant difference in the EPG reaction.