Proteomic Identification of Redox Sensitive Proteins and Fun
发布时间：2021-06-26 15:59
在环境胁迫下氧化还原代谢的失调导致细胞内活性氧（ROS）含量的增加,可能导致响应蛋白的结构和分子功能发生翻译后修饰（PTM）。氧化还原PTM是细胞信号传导和调控的重要途径,在作物的多种胁迫反应中,有多种蛋白质组学方法可以对PTM进行定量。盐胁迫通过触发多种活性氧含量的增加,从而在植物蛋白质组中产生动态变化,最终导致细胞内的氧化修饰,可以通过多种蛋白质组学方法对其进行定量。我们旨在通过分析甘蓝型油菜的短期盐胁迫,来鉴定大量的可逆半胱氨酸修饰生成的氧化还原蛋白质组学数据。为此,我们采用了iodoTMT方法分析了在200m M盐胁迫下持续四个小时的甘蓝型油菜幼苗的氧化还原蛋白质组。这种方法涉及iodoTMT标记半胱氨酸残基,HPLC分级分离,亲和富集和LC-MS/MS分析以及数据处理分析。使用这种方法,我们鉴定了1017种蛋白质中的2010种肽,其中909种蛋白质中的1809个位点发生氧化修饰。这些氧化修饰的蛋白质参与各种代谢,分子和细胞过程的调节。参与光合作用和氮代谢的蛋白质表达降低,而介导半胱氨酸和蛋氨酸代谢,精氨酸的生物合成,碳固定和蛋白质加工调控的蛋白质表达增加。我们定位了蛋白质中发...
【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】：116 页
【学位级别】：博士
【文章目录】：
摘要
ABSTRACT
LIST OF ABBREVIATIONS
CHAPTER Ⅰ.PROTEOMIC IDENTIFICATION OF REDOX SENSITIVE PROTEINS IN BRASSICA NAPUS L.BASED ON IODOTMT LABELLING OF CYSTEINE RESIDUES
1.1 BACKGROUND
1.1.1 Reactive oxygen species(ROS)
1.1.2 Signaling and regulatory role of ROS in plants
1.1.3 Redox homeostasis in plants
1.1.4 Redox mediated control of growth during abiotic Stress
1.1.5 ROS generation and signaling under salt stress
1.1.6 Effect of redox signaling on lipid composition
1.1.7 Cysteine modification by ROS
1.1.8 Proteomic approaches for identification of redox modified proteins
1.1.9 Shortcomings of previous redox proteomic approaches
1.1.10 Iodoacetyl tandem mass tags(Iodo TMT)method for redox proteomics
1.2 AIMS AND OBJECTIVES OF STUDY
1.3 MATERIALS AND METHODS
1.3.1 Plant material and stress conditions
1.3.2 Sample preparation
1.3.3 Iodo-TMT labeling
1.3.4 HPLC fractionation and affinity enrichment
1.3.5 LC-MS/MS analysis
1.3.6 Database search
1.3.7 Bioinformatics analysis
1.3.8 Enzyme activity assay
1.4 RESULTS
1.4.1 The iodo TMT method for redox proteome analysis
1.4.2 Quantitative analysis of redox proteomic data
1.4.3 Enrichment of modified proteins
1.4.4 Functional classification of modified proteins
1.4.5 Motif analysis and subcellular localization
1.4.6 Enzyme activity assay
1.5 DISCUSSION
1.5.1 Genes involved in carbon metabolism
1.5.2 Genes involved in amino acid metabolism
1.5.3 Proteins involved in stress response and detoxification
1.5.4 Proteins involved in biosynthesis
1.5.5 Scope of TMT based method
1.5.6 Importance of enzyme activity assay
1.5.7 Fructose1-6,bisphosphate
1.5.8 Phosphoglycerate kinase
1.6 CONCLUSIONS
CHAPTER Ⅱ.FUNCTIONAL CHARACTERIZATION OF FUMARASE IN ARABIDOPSIS AND BRASSICA NAPUS
2.1 INTRODUCTION
2.1.1 Krebs cycle/TCA cycle
2.1.2 Structure and operation of TCA cycle
2.1.3 Role of TCA cycle in plant metabolism
2.1.4 Fumarase is a component of TCA cycle
2.1.5 Functional significance of fumarase in plants
2.2 AIMS AND OBJECTIVES
2.3 MATERIALS AND METHODS
2.3.1 Gene identification in B.napus
2.3.2 Differential expression under stress treatments
2.3.3 Validation of RNA-seq data by q PCR
2.3.4 Vector construction
2.3.5 Transformation of Arabidopsis
2.3.6 Transformation of B.napus
2.3.7 Protein extraction and activity assays
2.3.8 Plant material,growth condition and stress treatments
2.3.9 Quantitative real time PCR
2.3.10 Measurement of lipid peroxidation
2.3.11 Relative electrolyte leakage measurement
2.3.12 Determination of H2O2 content
2.3.13 Determination of antioxidants
2.3.14 Lipid extraction
2.3.15 Fatty acid composition and oil content
2.3.16 Analysis of photosynthetic parameters
2.3.17 Agronomical traits analysis
2.3.18 Protein expression and invitro activity assay
2.3.19 Statistical analysis
2.4 RESULTS
2.4.1 The expression profile of fumarase
2.4.2 Fumarase OE lines performance under salt stress in Arabidopsis
2.4.3 Fumarase OE impact on fatty acid composition under short term salt stress in B.napus
2.4.4 Fumarase OE performance under long term salt stress in B.napus
2.4.5 Fumarase OE resulted in enhanced photosynthesis
2.4.6 Fumarase OE affected lipid composition
2.4.7 Fumarase OE lines showed improved developmental phenotype under both control and salt stress condition
2.4.8 Fumarase OE lines had more seed yield
2.4.9 Impact of fumarase overexpression on fatty acid composition in B.napus
2.5 DISCUSSION
2.5.1 Fumarase is a redox regulated enzyme
2.5.2 Fumarase overexpression promotes photosynthesis
2.5.3 Fumarase affects lipid composition
2.5.4 Fumarase over expression leads to improved biomass and seed yield
2.5.5 Fumarase overexpression improves oil content and composition
2.5.6 Alteration in fatty acid composition impacts stress response
2.5.7 Fumarase overexpression leads to improved physiological state in plants
2.5.8 Fumarase has role in early flowering
2.6 CONCLUSIONS
REFERENCES
APPENDIX Ⅰ:TABLE AND FIGURES
APPENDIX Ⅱ.PUBLICATIONS
ACKNOWLEDGEMENTS
【参考文献】：
期刊论文
[1]PROTEOME ANALYSIS OF RICE ROOT PROTEINS REGULATED BY GIBBERELLIN[J]. SETSUKO KOMATSU,HIROSATO KONISHI. Genomics Proteomics & Bioinformatics. 2005(03)
本文编号：3251635
【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】：116 页
【学位级别】：博士
【文章目录】：
摘要
ABSTRACT
LIST OF ABBREVIATIONS
CHAPTER Ⅰ.PROTEOMIC IDENTIFICATION OF REDOX SENSITIVE PROTEINS IN BRASSICA NAPUS L.BASED ON IODOTMT LABELLING OF CYSTEINE RESIDUES
1.1 BACKGROUND
1.1.1 Reactive oxygen species(ROS)
1.1.2 Signaling and regulatory role of ROS in plants
1.1.3 Redox homeostasis in plants
1.1.4 Redox mediated control of growth during abiotic Stress
1.1.5 ROS generation and signaling under salt stress
1.1.6 Effect of redox signaling on lipid composition
1.1.7 Cysteine modification by ROS
1.1.8 Proteomic approaches for identification of redox modified proteins
1.1.9 Shortcomings of previous redox proteomic approaches
1.1.10 Iodoacetyl tandem mass tags(Iodo TMT)method for redox proteomics
1.2 AIMS AND OBJECTIVES OF STUDY
1.3 MATERIALS AND METHODS
1.3.1 Plant material and stress conditions
1.3.2 Sample preparation
1.3.3 Iodo-TMT labeling
1.3.4 HPLC fractionation and affinity enrichment
1.3.5 LC-MS/MS analysis
1.3.6 Database search
1.3.7 Bioinformatics analysis
1.3.8 Enzyme activity assay
1.4 RESULTS
1.4.1 The iodo TMT method for redox proteome analysis
1.4.2 Quantitative analysis of redox proteomic data
1.4.3 Enrichment of modified proteins
1.4.4 Functional classification of modified proteins
1.4.5 Motif analysis and subcellular localization
1.4.6 Enzyme activity assay
1.5 DISCUSSION
1.5.1 Genes involved in carbon metabolism
1.5.2 Genes involved in amino acid metabolism
1.5.3 Proteins involved in stress response and detoxification
1.5.4 Proteins involved in biosynthesis
1.5.5 Scope of TMT based method
1.5.6 Importance of enzyme activity assay
1.5.7 Fructose1-6,bisphosphate
1.5.8 Phosphoglycerate kinase
1.6 CONCLUSIONS
CHAPTER Ⅱ.FUNCTIONAL CHARACTERIZATION OF FUMARASE IN ARABIDOPSIS AND BRASSICA NAPUS
2.1 INTRODUCTION
2.1.1 Krebs cycle/TCA cycle
2.1.2 Structure and operation of TCA cycle
2.1.3 Role of TCA cycle in plant metabolism
2.1.4 Fumarase is a component of TCA cycle
2.1.5 Functional significance of fumarase in plants
2.2 AIMS AND OBJECTIVES
2.3 MATERIALS AND METHODS
2.3.1 Gene identification in B.napus
2.3.2 Differential expression under stress treatments
2.3.3 Validation of RNA-seq data by q PCR
2.3.4 Vector construction
2.3.5 Transformation of Arabidopsis
2.3.6 Transformation of B.napus
2.3.7 Protein extraction and activity assays
2.3.8 Plant material,growth condition and stress treatments
2.3.9 Quantitative real time PCR
2.3.10 Measurement of lipid peroxidation
2.3.11 Relative electrolyte leakage measurement
2.3.12 Determination of H2O2 content
2.3.13 Determination of antioxidants
2.3.14 Lipid extraction
2.3.15 Fatty acid composition and oil content
2.3.16 Analysis of photosynthetic parameters
2.3.17 Agronomical traits analysis
2.3.18 Protein expression and invitro activity assay
2.3.19 Statistical analysis
2.4 RESULTS
2.4.1 The expression profile of fumarase
2.4.2 Fumarase OE lines performance under salt stress in Arabidopsis
2.4.3 Fumarase OE impact on fatty acid composition under short term salt stress in B.napus
2.4.4 Fumarase OE performance under long term salt stress in B.napus
2.4.5 Fumarase OE resulted in enhanced photosynthesis
2.4.6 Fumarase OE affected lipid composition
2.4.7 Fumarase OE lines showed improved developmental phenotype under both control and salt stress condition
2.4.8 Fumarase OE lines had more seed yield
2.4.9 Impact of fumarase overexpression on fatty acid composition in B.napus
2.5 DISCUSSION
2.5.1 Fumarase is a redox regulated enzyme
2.5.2 Fumarase overexpression promotes photosynthesis
2.5.3 Fumarase affects lipid composition
2.5.4 Fumarase over expression leads to improved biomass and seed yield
2.5.5 Fumarase overexpression improves oil content and composition
2.5.6 Alteration in fatty acid composition impacts stress response
2.5.7 Fumarase overexpression leads to improved physiological state in plants
2.5.8 Fumarase has role in early flowering
2.6 CONCLUSIONS
REFERENCES
APPENDIX Ⅰ:TABLE AND FIGURES
APPENDIX Ⅱ.PUBLICATIONS
ACKNOWLEDGEMENTS
【参考文献】：
期刊论文
[1]PROTEOME ANALYSIS OF RICE ROOT PROTEINS REGULATED BY GIBBERELLIN[J]. SETSUKO KOMATSU,HIROSATO KONISHI. Genomics Proteomics & Bioinformatics. 2005(03)
本文编号：3251635
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