在環(huán)境脅迫下氧化還原代謝的失調(diào)導致細胞內(nèi)活性氧（ROS）含量的增加,可能導致響應蛋白的結(jié)構(gòu)和分子功能發(fā)生翻譯后修飾（PTM）。氧化還原PTM是細胞信號傳導和調(diào)控的重要途徑,在作物的多種脅迫反應中,有多種蛋白質(zhì)組學方法可以對PTM進行定量。鹽脅迫通過觸發(fā)多種活性氧含量的增加,從而在植物蛋白質(zhì)組中產(chǎn)生動態(tài)變化,最終導致細胞內(nèi)的氧化修飾,可以通過多種蛋白質(zhì)組學方法對其進行定量。我們旨在通過分析甘藍型油菜的短期鹽脅迫,來鑒定大量的可逆半胱氨酸修飾生成的氧化還原蛋白質(zhì)組學數(shù)據(jù)。為此,我們采用了iodoTMT方法分析了在200m M鹽脅迫下持續(xù)四個小時的甘藍型油菜幼苗的氧化還原蛋白質(zhì)組。這種方法涉及iodoTMT標記半胱氨酸殘基,HPLC分級分離,親和富集和LC-MS/MS分析以及數(shù)據(jù)處理分析。使用這種方法,我們鑒定了1017種蛋白質(zhì)中的2010種肽,其中909種蛋白質(zhì)中的1809個位點發(fā)生氧化修飾。這些氧化修飾的蛋白質(zhì)參與各種代謝,分子和細胞過程的調(diào)節(jié)。參與光合作用和氮代謝的蛋白質(zhì)表達降低,而介導半胱氨酸和蛋氨酸代謝,精氨酸的生物合成,碳固定和蛋白質(zhì)加工調(diào)控的蛋白質(zhì)表達增加。我們定位了蛋白質(zhì)中發(fā)... 

【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)】：116 頁

【學位級別】：博士

【文章目錄】：
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


【參考文獻】：
期刊論文
[1]PROTEOME ANALYSIS OF RICE ROOT PROTEINS REGULATED BY GIBBERELLIN[J]. SETSUKO KOMATSU,HIROSATO KONISHI.  Genomics Proteomics & Bioinformatics. 2005(03)



本文編號：3251635