水稻脂质合成基因的鉴定，特征研究与突变分析

发布时间：2020-12-19 17:43

　　脂质是所有植物细胞的重要成分,占其干重的5-10%。植物实现许多不同功能时常需要脂质,包括膜的生成、作为信号分子以及作为碳和能量的储存形式。水稻（Otyza sativa L.）是一种淀粉作物,谷物中脂质含量很低,糙米,麸皮和精米中的含量分别约为2.3%,18.3%和0.8%,其含量还取决于水稻品种,谷粒的成熟程度和生长条件。然而,谷物中脂质含量虽然很低但对谷物风味和贮藏性质有着重要影响,特别是精米中与淀粉形成复合物的磷脂。类似地,各种其他脂质和脂酶具有复杂的关系,从而维持细胞的自我调节并赋予稻谷以及其他器官各种功能特性。我们通过In silico分析鉴定了涉及脂质生物合成的495个基因,特别是那些涉及膜结构及其储存和降解的基因。我们根据这些基因的结构和功能,将其大致分为9个不同的过程或类别。包括脂肪酸延长,糖脂,甘油糖脂,鞘脂,固醇脂质,角质层蜡,磷脂酶和脂肪酶鉴定到的基因数分别是68、17、62、43、33、47、68、89和88个,有的基因涉及多种途径。对这些基因的通路特异性功能进行了计算分析,并与它们在拟南芥中的直系同源物进行比较,研究了它们的组织特异性表达。基于在不同发育时期... 

【文章来源】：浙江大学浙江省 211工程院校 985工程院校 教育部直属院校

【文章页数】：167 页

【学位级别】：博士

【文章目录】：
ACKNOWLEDGEMENTS
ABBREVIATIONS
ABSTRACT
摘要
CHAPTER 1 General Introduction
    1.1 Introduction to rice
    1.2 Significance of Oryza sativa L
    1.3 Rice and lipid: the quest for quality
    1.4 Reverse genetics approach for functional analysis
    1.5 Objectives of the study
    1.6 Overview of the whole study
CHAPTER 2 Review of Literatures
    2.1 Importance of lipids
        2.1.1 Classification of lipids
            2.1.1.1 Fatty acids
            2.1.1.2 Glycerolipids
            2.1.1.3 Glycerophospholipids
            2.1.1.4 Sphingolipids
            2.1.1.5 Sterol lipids
            2.1.1.6 Prenol lipids
            2.1.1.7 Saccharolipids
            2.1.1.8 Polyketides
        2.1.2 Biosynthesis of lipids
        2.1.3 Principal lipids
            2.1.3.1 Galactolipids
            2.1.3.2 Phospholipids
            2.1.3.3 Triacylglycerol
        2.1.4 Degradation of lipids
        2.1.5 Lipids storage in plants: In relation to rice
        2.1.6 Lipids accumulation during rice grain development
    2.2 Gene engineering using CRISPR/Cas9 system
        2.2.1 Defense system pathway in CRISPR/Cas system
        2.2.2 Nuclease activity of the Cas9
CHAPTER 3 Mining and Editing of Genes Involved in Lipid Biosynthesis in Rice
    3.1 Introduction
    3.2 Material and Methods
        3.2.1 Gene identification and selection
        3.2.2 Vector Construction for gene editing
        3.2.3 Agrobacterium mediated transformation
        3.2.4 Identification of mutants
        3.2.5 Growth and selection of mutants
    3.3 Results
        3.3.1 495 genes identified for various lipid biosynthesis
        3.3.2 Gene editing vectors constructed for 10 genes
        3.3.3 Efficiency of Agrobacterium mediated transformation
        3.3.4 Mutant lines of seven genes generated
    3.4 Discussion
CHAPTER 4 Loss of Function of OsDGD2β Results in Male Sterility in Rice
    4.1 Introduction
    4.2 Materials and Methods
        4.2.1 In silico analysis
        4.2.2 Mutant generation, identification and growth
        4.2.3 Characterization of mutant phenotype
        4.2.4 Measurement of chlorophyll and photosynthetic parameters
        4.2.5 Lipid composition analysis in leaf and anther
        4.2.6 Gene expression analysis
        4.2.7 Protein subcellular localization of OsDGD2β
    4.3 Results
        4.3.1 DGDG synthase in rice is encoded by five genes
        4.3.2 OsDGD2β is expressed highly in anther
        4.3.3 OsDGD2β is localized in chloroplast
        4.3.4 Two osdgd2β mutant lines were generated
        4.3.5 Changes in DGDG and total fatty acids content
        4.3.6 osdgd2β mutants are male sterile
        4.3.7 Effects of OsDGD2β mutation on transcription of other genes
    4.4 Discussion
        4.4.1 OsDGD2β is the sole highly expressed DGDG synthase gene in anther
        4.4.2 Mutation of OsDGD2β generates male sterile rice
        4.4.3 Future prospects: Hybrid rice breeding using osdgd2β
    4.5 Conclusion
CHAPTER 5 Mutation in OsMGD2 Alters Grain Quality and Affects Rice Productivity
    5.1 Introduction
    5.2 Materials and Methods
        5.2.1 In silico analysis
        5.2.2 Mutant generation, identification, growth and agronomic study
        5.2.3 Measurement of chlorophyll and photosynthetic parameters
        5.2.4 Fatty acid composition in milled rice
        5.2.5 RVA analysis
        5.2.6 Gene expression analysis
        5.2.7 Protein subcellular localization of OsMGD2
    5.3 Results
        5.3.1 MGDG synthase in rice is encoded by three genes
        5.3.2 Tissue specific expression of OsMGD2
        5.3.3 OsMGD2 protein is localized in the chloroplast
        5.3.4 Generation of two osmgd2 mutant lines
        5.3.5 Linoleic acid content decreased in the milled rice in mutants
        5.3.6 Alteration in viscosity of milled rice
        5.3.7 osmgd2 mutants had lowered photosynthetic parameters
        5.3.8 osmgd2 mutants had lower harvest yield
    5.4 Discussion
        5.4.1 OsMGD2 is expressed in anther and endosperm
        5.4.2 Involvement of OsMGD2 in photosynthesis
        5.4.3 Mutation of OsMGD2 affects seed quality and agronomic performance
    5.5 Conclusion
CHAPTER 6 OVERALL CONCLUSION AND FUTURE PERSPECTIVES
REFERENCES
APPENDIX
    Ⅰ Supplementary protocols
    Ⅱ Preparation of reagents
    Ⅲ List of primers used in the study
    Ⅳ List of Genes involved in lipid biosynthesis in rice


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期刊论文
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