东方粘虫化学感受蛋白的功能分析
发布时间：2021-05-13 10:01
嗅觉在昆虫寄主定位、求偶等行为中起着关键性作用。目前已经明确,包括化学感受蛋白（CSPs）在内的多种蛋白参与昆虫嗅觉感受的过程中,但CSPs在昆虫体内具体的生理功能还不是很清楚。粘虫Mythimna separata是多种作物上重要的迁飞性害虫,在世界各地分布广泛。为了明确CSPs在粘虫中的生理功能,本研究对粘虫几个CSPs基因的表达模式、与配基的结合特性、分子对接等进行了研究,同时,还RNAi技术探讨了几种CSPs的功能。主要结果如下:化学感受蛋白基因表达谱克隆得到了 6个CSP基因（MsepCSP5,MsepCSP8,MsepCSP11,MsepCSP14,MsepCSP15和MsepCSP16）。通过RT-qPCR对MsepCSPs基因的表达模式进行分析,结果显示,MsepCSPs基因在粘虫各个发育阶段均有表达,在所检测的各个组织中也广泛分布。所有MsepCSPs基因在幼虫中均的相对表达量均高于在蛹期的表达。对于所有测试的MsepCSPs基因,雌性和雄成虫在不同发育节点显示出不同的表达模式:MsepCSP5,MsepCSP8和MsepCSP16基因在雌蛾羽化后第3d的表达量最高;...
【文章来源】：华中农业大学湖北省 211工程院校 教育部直属院校
【文章页数】：145 页
【学位级别】：博士
【文章目录】：
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
【参考文献】：
期刊论文
[1]2012年三代黏虫大发生原因初步分析[J]. 张云慧,张智,姜玉英,曾娟,高月波,程登发. 植物保护. 2012(05)
[2]粘虫雄蛾触角对其性信息素的电生理反应[J]. 汪新文,刘孟英,吴才宏. 昆虫学报. 1998(01)
本文编号：3183815
【文章来源】：华中农业大学湖北省 211工程院校 教育部直属院校
【文章页数】：145 页
【学位级别】：博士
【文章目录】：
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
【参考文献】：
期刊论文
[1]2012年三代黏虫大发生原因初步分析[J]. 张云慧,张智,姜玉英,曾娟,高月波,程登发. 植物保护. 2012(05)
[2]粘虫雄蛾触角对其性信息素的电生理反应[J]. 汪新文,刘孟英,吴才宏. 昆虫学报. 1998(01)
本文编号：3183815
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