水稻早衰基因ES5的克隆及功能分析

发布时间：2023-03-29 04:04

　　叶片衰老是影响水稻生长发育和产量形成的重要因素,是水稻改良的重要目标。叶片衰老对植物的生长、发育、适应、存活和繁殖均具有重要意义。本文报道了一个新的水稻早衰突变体es5(early leaf senescence 5),它由甲基磺酸乙酯(EMS)处理的粳稻品种嘉禾212衍生而来。我们对其进行了表型特征和农艺性状的测定,生理生化特性、细胞死亡发生和ROS积累的检测,ES5基因的遗传定位、功能互补和脂质代谢产物谱分析。通过定位克隆方法,成功克隆了编码磷脂酰丝氨酸合成酶的ES5基因。研究结果表明,ES5的突变引起水稻磷脂酰丝氨酸的积累,并导致水稻的早衰。研究结果如下:1.es5突变体在4叶期后表现叶片黄化。活性氧(ROS)和丙二醛(MDA)的高积累、叶绿体的解体、叶绿素含量和光合速率的降低证实了这一表型变化。受早衰影响,es5突变体的株高、单株有效穗、结实率及千粒重较野生型均显著降低。此外,衰老相关基因(SAGs)和叶绿素降解相关基因在es5中上调表达,光合作用相关基因下调表达。2.ES5的候选区域被精细定位在第5染色体长臂37.57 kb的物理区间内。该区间包含6个开放阅读框(ORFs)。...

【文章页数】：85 页

【学位级别】：博士

【文章目录】：
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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