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Identification Of Female Sex Pheromone,Cloning And Functional Analysis Of Binding Protein Genes In Orthaga Achatina (Butler)

Posted on:2013-09-14Degree:MasterType:Thesis
Country:ChinaCandidate:S J LiuFull Text:PDF
GTID:2283330467484937Subject:Agricultural Entomology and Pest Control
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The camphor tree, characterized by evergreen and fragrant leaves, is one of the most popularly planted landscape trees in China. Orthaga achatina (Butler) is an important pest of the camphor tree, and became more and more serious in recent years as the rapid increase in the plantage of the tree. Because of the hiding damage of the larvae in the nests, the traditional spraying with insecticides could not get satisfactory control effect, and caused serious pollutions in city area and scennary spots. Therefore, new green technologies for the management of O.achatina are badly needed. Insect sex pheromone has been successufully used in the management of many insects, and is hopfull to be used in control of O. achatina. However, there is no related study. In2009, our laboratory initiated a project on the identification and application of the sex pheromone in control of O. achatina. During the past years, we determined the mating behavior, sensillum types and their distribution on the antenna, and also identified a sex pheromone component. In this thesis, we conducted experiments to further identify remained components of the female sex pheromone, and to test the attractiveness of these pheromone components in both labortory and fields. In addition, in order to find key olfaction-related genes for potential use as new targets to design olfactory blockers in O.achatina, two odorant binding protein (OBP) genes were also cloned and functionally studied. The main results were as follows:1. The identification of O. achatina female sex pheromoneWith GC and GC-MS analysis, three compounds were chemically iedentified from the extracts of female sex pheromone gland of O. achatina, which were Z11-16:Ac, E11-16:OH and Z9-16:OH. To determine the position of the double bond in three compounds, the dimethyl disulfide (DMDS) reaction and GC-MS analysis were carried out. The results confirmed the positions of double bonds in Z11-16:Ac and E11-16:OH, but the double bond position in Z9-16:OH was not confirmed due to the low content in the extracts of the female gland.2. EAG and behavioral responses of O. achatina to sex pheromone and host plant odorantsThe three sex pheromone components and20plant odrants were tested for the electrophysilogical activity by electroantennogram (EAG) technology. The results showed that all of the three components caused obvious electroantennogram responses, and among these components Z11-16:Ac was highly effective than Z9-16:OH and E11-16:OH. All20plant volatiles were also capable to elicit EAG response in both male and female antennae. Among these plant volatiles, Z-3-hexenol butyrate, linalool, hexanol, hexyl acetate and camphor showed highest activigies for both males and females. Based on obove results, lures were designed and assessed in field experiments. The four lures numbered as3,6,7and N7all trapped a few moths when water-basin traps were used. However, the dry-collumn traps captured no moths of O. achatina.3. Molecular characterization of two OBP genes from O. achatinaThe degenerate primers were designed based on the pheromone binding protein (PBP), general odorant binding protein (GOBP) sequences of reported insect species, and RT-PCR and RACE experiments were conducted to clone the OBPs. As a result, a PBP and a GOBP were obtained from male antenna. These two genes were named OachPBP1and OachGOBP2, respectivly. The expression patterns of the two genes were further measured by qPCR method. Both OachPBPl and OachGOBP2were only expressed in female and male antenna. However, expression level of OachPBP1in male antenna were much (2.9times) higher than in female antenna, while OachGOBP2expressed at the same levels in male and female antenna.4. in vitro expression and ligand-binding assay of OachPBPl and OachGOBP2from O. achatinaThe two OBP genes were constructed into the pET-30a(+) expression vector, and the then expressed with the induction of IPTG in E. coli. Purification of the two proteins was performed by an affinity chromatography column with Ni Sepharose and enterokinase treatment. Using1-NPN as the fluorescent probe in the binding assay, three sex pheromones,10pheromone analogos, and19plant volatiles were tested for the binding ability. The results showed that OachPBPl bound with all13pheromones and analogos with high binding abilities, suggesting an important role of OachPBP1in the detection of sex pheromones in O.achatina. OachGOBP2, in addition to bound some plant volatiles, also bound highly with sex pheromoens, and thus OachGOBP2may play roles in the reception of both plant volatiles and sex pheromones. Very interestingly, farnesol and farnesene all showed high binding affinities with both OachPBPl and OachGOBP2, sugesting the physiological impotantance of the two plant volatiles in O.achatina.
Keywords/Search Tags:Orthaga achiatina, sex pheromone, pheromone binding protein, genoralodorant binding protein, fluorescence competitive binding assay
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