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Comprison Of Different Populations Sex Pheromone System And Functional Analysis Of Binding Protein OBP7 In Spodoptera Exigua

Posted on:2015-03-22Degree:MasterType:Thesis
Country:ChinaCandidate:J Y ZhuFull Text:PDF
GTID:2283330482470920Subject:Agricultural Entomology and Pest Control
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Spodoptera exigua (Huibner) is a worldwide important agricultural pest. It damages many crops and results in a great loss to the crop production. The control of this pest has depended on chemical insecticides for a long time, which results in not only pest resistance, but also killing of pest enemy and other non-target organisms, and thus alternative techniques are needed to be developed and employed. The sex pheromone of S. exigua, as a perspective technique, has been effectively used in the forecasting of the population, however, it has some shortcomings when used in the direct pest control, such as high cost, poor stability and inconvenience in application. Besides, efficiency of the sex pheromone lures is variable with the geographic regions, which is also an important factor to impede the wide use of this technique in the control of S. exigua. Therefore, it is important to investigate the differences of the sex pheromone communication systems between different filed S. exigua populations, for development and use of the population (region) based specific pheromone lures. In this paper, the titers and proportion of each sex pheromone component in the female pheromone glands, and the male EAG responses to the lures incorporated with binary blend of different proportions were investigated and compared between 4 field populations. To explore the mechanisms underlying the differences of the male response, the DNA sequences containing both exons and introns were analyzed and compared for pheromone binding protein (PBP) genes SexiPBPl and SexiPBP2. In addition, the function of an odorant binding protein (OBP) gene SexiOBP7 was explored with the recombinant SexiOBP7, using the fluorescence competitive binding assay. The main results were as follows.1. Differences in titers and proportions of sex pheromone components deteeted in pheromone glands among field populations of S. exiguaWith GC analysis, titers of sex pheromone components in the pheromone gland extracts from 4 field populations were investigated and analyzed. The results of investigations conducted in 2012 showed that all populations contained the same 4 pheromone components, which were Z9-14:OH (A)、Z9,E12-14:OH (B)、Z9-14:Ac (C) and Z9,E12-14:Ac (D). However, the titers and proportions of each component were different among populations. Proportion of Z9-14:OH in Shanghai population was significantly higher than those in Tianjin and Wuhan populations, and proportion of Z9,E12-14:Ac in Tianjing population was significantly higher than those in other 3 populations. The ratios between Z9-14:OH and Z9,E12-14:Ac was highest (60:40) in Shanghai population, and lowest (43:57) in Tianjing population. Similar results were achieved in assays of 2013. These results provided important basis for development of the population specific sex pheromone lures.2. Differences in EAG responses by male moths to binary pheromone blends of different ratios between two field S. exigua populationsThe electro-physiological responses of male moths from Taian and Shanghai populations were tested to the binary sex pheromone blends (Z9-14:OH and Z9,E12-14:Ac) of different ratios (4:6,5:5,6:4,7:3 and 9:1), by Electroantennogram (EAG) technology. The results showed that both populations could respond significantly to all the 5 tested ratios. In comparison, Taian population relatively prefer the blends of lower ratios of Z9-14:OH and Z9,E12-14:Ac, with the optimal ratio of 6:4; while the Shanghai population prefer the higher ratio blends, with the optimal ratio being 7:3. The differences at the behavioral level between the populations were to be measured by using the wind tunnel assays and the field trapping experiments.3. Differences in DNA sequences of SexiPBPl and SexiPBP2 between Taian and Shanghai populationsTo explore the mechanisms of difference in EAG response between populations, the DNA sequences of SexiPBP1 and SexiPBP2 were analyzed and compared. It was showed that the nucleic acids in both introns and exons displayed polymorphism. Two changes in exon of PBP1 resulted in amino acid substitution. A nucleic acid change of C123G occurred in both populations led to an amino acid substitution from asparagic acid toglutamic acid,while change of G373A in Taian population led to an amino acid substitution from Glycine to Serine, and change of C144G in Shanghai population led to an substitution from Valine to Alanine. The effect of these amino acid substitutions on the ligand binding needs to be further studied.4. in vitro expression and ligand-binding assay of SexiOBP7 from S. exiguaThe sexiOBP7 was expressed in E. coli by using pET-30a(+) expression vector. The resulted recombinant protein was purified by an affinity chromatography column with Ni Sepharose and enterokinase treatment. Using 1-NPN as the fluorescent probe, the functional study was further conducted with the recombinant SexigOBP7 by fluorescence competitive binding assay. Among the 35 tested odorant compounds, the major sex pheromone component Z9,E12-14:Ac and 3 host plant volatiles (acetophenone, farnesol and decyl acetate) showed the higher binding affinities, with the Ki values of 8.27,8.36, 9.46 and 11.15 μM, respectively. It suggests that sexiOBP7 might play roles in perception of these volatiles in adult S. exigua.
Keywords/Search Tags:Spodoptera exigua, sex pheromone communication system, field population, polymorphism, electroantennogram, ligand binding affinity
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