嗅覺在昆蟲寄主定位、求偶等行為中起著關鍵性作用。目前已經(jīng)明確,包括化學感受蛋白（CSPs）在內(nèi)的多種蛋白參與昆蟲嗅覺感受的過程中,但CSPs在昆蟲體內(nèi)具體的生理功能還不是很清楚。粘蟲Mythimna separata是多種作物上重要的遷飛性害蟲,在世界各地分布廣泛。為了明確CSPs在粘蟲中的生理功能,本研究對粘蟲幾個CSPs基因的表達模式、與配基的結合特性、分子對接等進行了研究,同時,還RNAi技術探討了幾種CSPs的功能。主要結果如下:化學感受蛋白基因表達譜克隆得到了 6個CSP基因（MsepCSP5,MsepCSP8,MsepCSP11,MsepCSP14,MsepCSP15和MsepCSP16）。通過RT-qPCR對MsepCSPs基因的表達模式進行分析,結果顯示,MsepCSPs基因在粘蟲各個發(fā)育階段均有表達,在所檢測的各個組織中也廣泛分布。所有MsepCSPs基因在幼蟲中均的相對表達量均高于在蛹期的表達。對于所有測試的MsepCSPs基因,雌性和雄成蟲在不同發(fā)育節(jié)點顯示出不同的表達模式:MsepCSP5,MsepCSP8和MsepCSP16基因在雌蛾羽化后第3d的表達量最高;... 

【文章來源】：華中農(nóng)業(yè)大學湖北省 211工程院校 教育部直屬院校

【文章頁數(shù)】：145 頁

【學位級別】：博士

【文章目錄】：
Abstract
摘要
Chapter1:Background and Review Literature
    1.1 Introduction of Mythimna separata
    1.2 Insect chemoreception
    1.3 The physiological and morphological bases of chemoreception
    1.4 The molecular bases of chemoreception in insects
        1.4.1 Olfactory receptors(ORs)
        1.4.2 Ionotropic receptors(IRs)
        1.4.3 Odorant binding protein(OBP)
        1.4.4 Chemosensory protein(CSP)
    1.5 Functions of CSPs
        1.5.1 Detecting chemo-signals
        1.5.2 Pheromone glands:releasing semiochemicals
        1.5.3 Regeneration and development
        1.5.4 Nutrition
        1.5.5 Carriers of visual pigments
        1.5.6 Insecticide resistance
    1.6 Significance of chemoreception
    1.7 Insect olfaction and host plant selection
    1.8 RNA interference
        1.8.1 Introduction
        1.8.2 Interference by“dicing”and“slicing”
        1.8.3 Pathways of RNAi
        1.8.4 Cellular mechanism of RNAi
        1.8.5 Methods for delivery of RNAi
        1.8.6 Systemic RNAi
        1.8.7 RNAi as a tool for analysis of gene function in insects
    1.9 Objectives of the conducted studies
Chapter2:Expression profiling of chemosensory protein genes in oriental armyworm Mythimna separata
    2.1 Introduction
    2.2 Materials and Methods
        2.2.1 Insects and collection of tissues
        2.2.2 RNA extraction
        2.2.3 First strand cDNA synthesis
        2.2.4 Selection of genes for RT-qPCR
        2.2.5 Real-time quantitative PCR
    2.3 Results
        2.3.1 Characterization and sequence analysis of CSP genes of M.separata
        2.3.2 Tissue-specific expression of MsepCSP5
        2.3.3 Tissue-specific expression of MsepCSP8
        2.3.4 Tissue-specific expression of MsepCSP11
        2.3.5 Tissue-specific expression of MsepCSP14
        2.3.6 Tissue-specific expression of MsepCSP15
        2.3.7 Tissue-specific expression of MsepCSP16
    2.4 Discussion
    2.5 Conclusion
Chapter3:Functional analysis of MsepCSP5 a chemosensory protein from oriental armyworm Mythimna separata
    3.1 Introduction
    3.2 Material and Methods
        3.2.1 RNA extraction
        3.2.2 Reagents and kits
        3.2.3 Laboratory instruments
        3.2.4 First-strand cDNA synthesis
        3.2.5 Polymerase chain reaction(PCR)
        3.2.6 Gel extraction for DNA purification
        3.2.7 DNA ligation with pTOPO-T simple vector
        3.2.8 Transformation of plasmid DNA and sequence analysis
        3.2.9 Plasmid extraction
        3.2.10 Digestion with restriction enzymes
        3.2.11 Ligation of purified DNA
        3.2.12 Expression of recombinant protein
        3.2.13 Successful purification of recombinant protein
        3.2.14 Fluorescence binding assays
        3.2.15 Modeling of three-dimensional structures and molecular docking of ligands
        3.2.16 Y-tube olfactometer bioassay
        3.2.17 Double-stranded RNA synthesis
        3.2.18 Double-stranded RNA injection and gene expression analysis
    3.3 Results
        3.3.1 Cloning and sequence analysis of MsepCSP5
        3.3.2 Phylogenetic analysis
        3.3.3 Fluorescence binding assay
        3.3.4 Three-dimensional structure modeling and molecular docking
        3.3.5 Behavioral response of M.separata to the compounds with high binding affinities with MsepCSP
        3.3.6 RNAi-based silencing and behavioral responses of M.separata
    3.4 Discussion
    3.5 Conclusion
Chapter4:Functional analysis of MsepCSP8 a chemosensory protein from oriental armyworm Mythimna separata
    4.1 Introduction
    4.2 Material and Methods
        4.2.1 RNA extraction and cDNA synthesis
        4.2.2 Phylogenetic analysis
        4.2.3 Construction of recombinant-plasmid
        4.2.4 Expression and purification of recombinant-protein
        4.2.5 Fluorescence binding assay
        4.2.6 Modeling of three-dimensional structures and molecular docking of ligands
        4.2.7 Y-tube olfactometer bioassay
        4.2.8 Double-stranded RNA injection,gene expression analysis and post-RNAi bioassay
    4.3 Results
        4.3.1 Cloning and phylogenetic analysis of MsepCSP8
        4.3.2 Fluorescence binding assay
        4.3.3 Three-dimensional structure modeling and molecular docking
        4.3.4 Behavioral response of M.separata to the compounds with high binding affinities with MsepCSP
        4.3.5 Effectiveness of RNAi on MsepCSP8
    4.4 Discussion
    4.5 Conclusion
Chapter5:Functional analysis of MsepCSP14 a chemosensory protein from oriental armyworm Mythimna separata
    5.1 Introduction
    5.2 Material and Methods
        5.2.1 RNA extraction and cDNA synthesis
        5.2.2 MsepCSP14 sequence analysis
        5.2.3 Construction of recombinant-plasmid
        5.2.4 Expression and purification of recombinant-protein
        5.2.5 Fluorescence binding assay
        5.2.6 Modeling of three-dimensional structures and molecular docking of ligands
        5.2.7 Y-tube olfactometer bioassay
    5.3 Results
        5.3.1 Characterization and sequence analysis of MsepCSP14
        5.3.2 Phylogenetic analysis of MsepCSP14
        5.3.3 Recombinant protein expression and purification
        5.3.4 Fluorescence binding assay
        5.3.5 Three-dimensional structure modeling and molecular docking
        5.3.6 Behavioral response of M.separata to the compounds with high binding affinities with MsepCSP
    5.4 Discussion
    5.5 Conclusion
Chapter6:Summary and future perspectives
    6.1 Summary
    6.2 Novelty of work
    6.3 Future perspectives
References
Acknowledgements


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期刊論文
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