葉片衰老是影響水稻生長發(fā)育和產(chǎn)量形成的重要因素,是水稻改良的重要目標。葉片衰老對植物的生長、發(fā)育、適應、存活和繁殖均具有重要意義。本文報道了一個新的水稻早衰突變體es5(early leaf senescence 5),它由甲基磺酸乙酯(EMS)處理的粳稻品種嘉禾212衍生而來。我們對其進行了表型特征和農(nóng)藝性狀的測定,生理生化特性、細胞死亡發(fā)生和ROS積累的檢測,ES5基因的遺傳定位、功能互補和脂質代謝產(chǎn)物譜分析。通過定位克隆方法,成功克隆了編碼磷脂酰絲氨酸合成酶的ES5基因。研究結果表明,ES5的突變引起水稻磷脂酰絲氨酸的積累,并導致水稻的早衰。研究結果如下:1.es5突變體在4葉期后表現(xiàn)葉片黃化。活性氧(ROS)和丙二醛(MDA)的高積累、葉綠體的解體、葉綠素含量和光合速率的降低證實了這一表型變化。受早衰影響,es5突變體的株高、單株有效穗、結實率及千粒重較野生型均顯著降低。此外,衰老相關基因(SAGs)和葉綠素降解相關基因在es5中上調(diào)表達,光合作用相關基因下調(diào)表達。2.ES5的候選區(qū)域被精細定位在第5染色體長臂37.57 kb的物理區(qū)間內(nèi)。該區(qū)間包含6個開放閱讀框(ORFs)。...

【文章頁數(shù)】：85 頁

【學位級別】：博士

【文章目錄】：
摘要
ABSTRACT
LIST OF ABBREVIATIONS
CHAPTER 1 INTRODUCTION
    1.1 GENERAL INTRODUCTION
    1.2 REVIEW OF LITERATURE
        1.2.1 Senescence
        1.2.2 Symptoms of leaf senescence
            1.2.2.1 Structural changes at the cellular level
            1.2.2.2 Biochemical changes
            1.2.2.3 Changes in photosynthetic apparatus
            1.2.2.4 Generation of reactive oxygen species
        1.2.3 The functional and regulatory shifts during leaf senescence
        1.2.4 Impact of senescence in agriculture
        1.2.5 Phospholipids
        1.2.6 Phosphatidylserine(PS)and its role in cell death signaling
        1.2.7 PS biosynthesis in plants
CHAPTER 2 IDENTIFICATION AND CHARACTERIZATION OF ES5 IN RICE
    2.1 INTRODUCTION
    2.2 MATERIALS AND METHODS
        2.2.1 Plant materials and growth condition
        2.2.2 Agronomic data recorded
        2.2.3 Measurement of photosynthetic pigments
        2.2.4 Measurement of net photosynthetic rate
        2.2.5 Transmission electron microscopy(TEM)
        2.2.6 Histochemical analysis
        2.2.7 Measurement of physio-biochemical parameters
        2.2.8 RNA isolation
        2.2.9 Synthesis of c DNA
        2.2.10 Quantitative real-time PCR(q RT-PCR)
        2.2.11 Statistical analysis
    2.3 RESULTS
        2.3.1 Phenotypic Characterization
        2.3.2 Comparison of agronomic traits
        2.3.3 Physiological characterization
        2.3.4 Ultra-structure of chloroplast
        2.3.5 Histochemical analysis
        2.3.6 Accumulation of MDA and ROS scavenging enzymes
        2.3.7 Reduction in total soluble protein content
    2.4 DISCUSSIONS
    2.5 CONCLUSION
CHAPTER 3 POSITIONAL CLONING OF ES5 IN RICE
    3.1 INTRODUCTION
    3.2 MATERIALS AND METHODS
        3.2.1 Plant materials and growth conditions
        3.2.2 DNA extraction for genotyping
        3.2.3 RNA isolation and complementary c DNA synthesis
        3.2.4 Polymerase chain reaction(PCR)
        3.2.5 Quantitative real-time PCR(q RT-PCR)
        3.2.6 Fine mapping of ESS
        3.2.7 Cloning of candidate gene and vector construction for functional complementation
            3.2.7.1 PCR amplification of genomic fragment of LOC_Os05g48060
            3.2.7.2 Preparation of p CAMBIA1300 plasmid
            3.2.7.3 In-fusion cloning procedure
        3.2.8 Amplification of CDS and vector construction for overexpression
            3.2.8.1 Synthesis of c DNA
            3.2.8.2 Amplification of target CDS
            3.2.8.3 Preparation of p CAMBIA1305-GFP plasmid
            3.2.8.4 In-fusion cloning procedure
        3.2.9 Protein sequence alignment and protein domain identification
        3.2.10 GUS assay
        3.2.11 Statistical analysis
    3.3 RESULTS
        3.3.1 Fine mapping of ESS
        3.3.2 Structure of ESS
        3.3.3 Complementation of ESS
        3.3.4 Protein structure prediction and analysis of homologous proteins
        3.3.5 Expression pattern of ESS
    3.4 DISCUSSIONS
    3.5 CONCLUSIONS
CHAPTER 4 FUNCTIONAL ANALYSIS OF ES5 IN RICE
    4.1 INTRODUCTION
    4.2 MATERIALS AND METHODS
        4.2.1 Plant materials
        4.2.2 Measurement of PSS
        4.2.3 Lipid metabolites extraction
        4.2.4 LC-MS/MS analysis
        4.2.5 Data processing and lipid metabolites annotation
    4.3 RESULTS
        4.3.1 es5 displayed increased PSS level
        4.3.2 es5 showed altered phospholipid contents
        4.3.3 Expression pattern of other homologs of ESS
        4.3.4 Metabolite profiling for lipid characterization in wild-type and es5 plants
            4.3.4.1 Repeated relevance assessment of the sample groups
            4.3.4.2 Orthogonal partial least squares discriminant analysis(OPLS-DA)
            4.3.4.3 Identification of differentially expressed metabolites(DEMs)
            4.3.4.4 KEGG pathway analysis of differentially expressed metabolites
    4.4 DISCUSSIONS
    4.5 CONCLUSION
CHAPTER 5 MAJOR FINDINGS AND FUTURE PROSPECTIVES
    5.1 MAJOR FINDINGS
    5.2 FUTURE PROSPECTIVES
REFERENCES
APPENDIX
ACKNOWLEDGEMENT
AUTHOR'S RESUME



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