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Structure Characterization And Functional Differentiation Of Pheromone Binding Proteins In Cotton Worm Helicoverpa Armigera(Hübner)

Posted on:2018-02-06Degree:DoctorType:Dissertation
Country:ChinaCandidate:K DongFull Text:PDF
GTID:1313330515478506Subject:Agricultural Entomology and Pest Control
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The cotton bollworm,Helicoverpa armigera,is one of the most serious agriculture pests worldwide.As manyother Lepidoptera species,the adults ofH.armigera also showed high sensitivity to conspecific pheromone components.However,the molecular mechanism of sex pheromone recognition in this pest is still need to be further investigated.Pheromone binding proteins(PBPs)are small water-soluble proteins which present in the lymph of olfactory sensilla with high concentration.Thses proteins are thought to be crucial in the process of pheromone recognition in insects.In order to explore the molecular mechanism and functional differentiation of PBPs in the pheromone detection of H.armigera,the morphology and ultrastructure of antennal in H.armigera moths were firstly investigated by scanning and transmission electron microscopy.Then the immunolocalization of HarmPBP1-3 proteins in chemosensilla on the antenna of male and female moths were characterizated by immunocytochemitry.At the same time,a pheromone binding protein(HarmPBP1)of H.armigera was expressed and the purified recombinant protein was obtained.The structures of the single protein and a complex with Z9-16:Ald were then determined.Based on the structure of HarmPBPl/Z9-16:Ald complex,We predict the key binding sites of HarmPBP1,and these predicted sites were verified by sitedirected mutagenesis and fluorescence competitive binding assay tests.Finally,the functional differentiation of three HarmPBP genes,HarmPBPl-3,was investigated by RNAi.The main results were as follow:1.The scanning and transmission electron microscopic examinations revealed that the gross morphology between male and female H.armigera moth antennae was threadlike.There were at least six types of olfactory sensilla in the antenna,including sensilla trichodea(str),sensilla chaetica(sch),sensilla basiconica(sba),sensilla auricillica(sar),sensilla coeloclnica(sco)and sensilla styloconica(sst).Sensilla trichodea are the most abundant sensillum type characterized on the antennae with thick cuticle and 1-3 dendrites in the sensillum lymph.Sensilla basiconica and sensilla auricillica are equipped with thin cuticular walls and multiple dendrites in the lymph.Sensilla chaetica own a terminal pore and thick wall,with dendrites in the inner lymph cavity.2.Sensilla trichodea in both sexes of H.armigera are labeled by all three anti-PBP antisera.In female antennae,only a few sensilla were abele compare with males.Other sensilla such as sensilla chaetica,sensilla basiconica and sensilla coeloconica were not labeled in both sexes.3.The crystal structure of single HarmPBP1 was composed by six a-helices,and the binding pocket was mainly formed by hydrophobic residues.All the structure showed high similarity with other typically Lepidoptera PBPs.Bsaed on the structure of HarmPBP1/Z9-16:Ald complex,we predict that Ser9,Phel2,Ser56 and Phe119 are the possibly key residues of HarmPBP1 binding with pheromone compounds.4.Five mutants of HarmPBP1,F12A,S56A,F119A,S9A and Q64A were obtained by site-directed mutagenesis.The results of fluorescence binding assays revealed that besides the random mutation,Q64A,other four mutants showed decreased binding affinities to both Z11-16:Ald and Z9-16:Ald.These results indicated that Ser9,Phe12,Ser56 and Phe119 are the key residues in the interaction between HarmPBP1 and pheromone compounds.5.Single and combinatorial RNAi were performed by injection of HarmPBP1-3 dsRNA.The results of qRT-PCR assay revealed that the expression levels of all three HarmPBP genes were reduced significantly after RNAi.The EAG assay showed that silence of single HarmPBP gene could not decrease the sensitivity of male antennae to any compounds.However,when HarmPBPl and HarmPBP2 were silenced together,the EAG response of male antennae to Z11-16:Ald showed a significant reduction.These results suggested that both HarmPBPl and HarmPBP2 could be associated with the recognition of Z11-16:Ald in H.armigera.Such conclution was enhanced by the results of wind-tunnel assays.
Keywords/Search Tags:cotton bollworm, pheromone binding protein, site-directed mutagenesis, fluorescence binding assays, RNAi
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