水稻脂質(zhì)合成基因的鑒定，特征研究與突變分析
發(fā)布時(shí)間：2020-12-19 17:43
脂質(zhì)是所有植物細(xì)胞的重要成分,占其干重的5-10%。植物實(shí)現(xiàn)許多不同功能時(shí)常需要脂質(zhì),包括膜的生成、作為信號(hào)分子以及作為碳和能量的儲(chǔ)存形式。水稻（Otyza sativa L.）是一種淀粉作物,谷物中脂質(zhì)含量很低,糙米,麩皮和精米中的含量分別約為2.3%,18.3%和0.8%,其含量還取決于水稻品種,谷粒的成熟程度和生長(zhǎng)條件。然而,谷物中脂質(zhì)含量雖然很低但對(duì)谷物風(fēng)味和貯藏性質(zhì)有著重要影響,特別是精米中與淀粉形成復(fù)合物的磷脂。類似地,各種其他脂質(zhì)和脂酶具有復(fù)雜的關(guān)系,從而維持細(xì)胞的自我調(diào)節(jié)并賦予稻谷以及其他器官各種功能特性。我們通過(guò)In silico分析鑒定了涉及脂質(zhì)生物合成的495個(gè)基因,特別是那些涉及膜結(jié)構(gòu)及其儲(chǔ)存和降解的基因。我們根據(jù)這些基因的結(jié)構(gòu)和功能,將其大致分為9個(gè)不同的過(guò)程或類別。包括脂肪酸延長(zhǎng),糖脂,甘油糖脂,鞘脂,固醇脂質(zhì),角質(zhì)層蠟,磷脂酶和脂肪酶鑒定到的基因數(shù)分別是68、17、62、43、33、47、68、89和88個(gè),有的基因涉及多種途徑。對(duì)這些基因的通路特異性功能進(jìn)行了計(jì)算分析,并與它們?cè)跀M南芥中的直系同源物進(jìn)行比較,研究了它們的組織特異性表達(dá)。基于在不同發(fā)育時(shí)期...
【文章來(lái)源】：浙江大學(xué)浙江省 211工程院校 985工程院校 教育部直屬院校
【文章頁(yè)數(shù)】：167 頁(yè)
【學(xué)位級(jí)別】：博士
【文章目錄】：
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
【參考文獻(xiàn)】：
期刊論文
[1]Characterization and Evaluation of OsLCT1 and OsN ramp5 Mutants Generated Through CRISPR/Cas9-Mediated Mutagenesis for Breeding Low Cd Rice[J]. LIU Songmei,JIANG Jie,LIU Yang,MENG Jun,XU Shouling,TAN Yuanyuan,LI Youfa,SHU Qingyao,HUANG Jianzhong. Rice Science. 2019(02)
[2]Development and Application of CRISPR/Cas System in Rice[J]. REN Jun,HU Xixun,WANG Kejian,WANG Chun. Rice Science. 2019(02)
[3]基于高分辨率熔解曲線技術(shù)的水稻伽瑪射線誘發(fā)突變的TILLIN體系（英文）[J]. Shan LI,Song-mei LIU,Hao-wei FU,Jian-zhong HUANG,Qing-yao SHU. Journal of Zhejiang University-Science B（Biomedicine & Biotechnology）. 2018(08)
[4]Rapid improvement of grain weight via highly efficient CRISPR/Cas9-mediated multiplex genome editing in rice[J]. Rongfang Xu,Yachun Yang,Ruiying Qin,Hao Li,Chunhong Qiu,Li Li,Pengcheng Wei,Jianbo Yang. Journal of Genetics and Genomics. 2016(08)
[5]Development of japonica Photo-Sensitive Genic Male Sterile Rice Lines by Editing Carbon Starved Anther Using CRISPR/Cas9[J]. Quanlin Li,Dabing Zhang,Mingjiao Chen,Wanqi Liang,Jiaojun Wei,Yiping Qi,Zheng Yuan. Journal of Genetics and Genomics. 2016(06)
[6]Phytohormones and their metabolic engineering for abiotic stress tolerance in crop plants[J]. Shabir H.Wani,Vinay Kumar,Varsha Shriram,Saroj Kumar Sah. The Crop Journal. 2016(03)
本文編號(hào)：2926312
【文章來(lái)源】：浙江大學(xué)浙江省 211工程院校 985工程院校 教育部直屬院校
【文章頁(yè)數(shù)】：167 頁(yè)
【學(xué)位級(jí)別】：博士
【文章目錄】：
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
【參考文獻(xiàn)】：
期刊論文
[1]Characterization and Evaluation of OsLCT1 and OsN ramp5 Mutants Generated Through CRISPR/Cas9-Mediated Mutagenesis for Breeding Low Cd Rice[J]. LIU Songmei,JIANG Jie,LIU Yang,MENG Jun,XU Shouling,TAN Yuanyuan,LI Youfa,SHU Qingyao,HUANG Jianzhong. Rice Science. 2019(02)
[2]Development and Application of CRISPR/Cas System in Rice[J]. REN Jun,HU Xixun,WANG Kejian,WANG Chun. Rice Science. 2019(02)
[3]基于高分辨率熔解曲線技術(shù)的水稻伽瑪射線誘發(fā)突變的TILLIN體系（英文）[J]. Shan LI,Song-mei LIU,Hao-wei FU,Jian-zhong HUANG,Qing-yao SHU. Journal of Zhejiang University-Science B（Biomedicine & Biotechnology）. 2018(08)
[4]Rapid improvement of grain weight via highly efficient CRISPR/Cas9-mediated multiplex genome editing in rice[J]. Rongfang Xu,Yachun Yang,Ruiying Qin,Hao Li,Chunhong Qiu,Li Li,Pengcheng Wei,Jianbo Yang. Journal of Genetics and Genomics. 2016(08)
[5]Development of japonica Photo-Sensitive Genic Male Sterile Rice Lines by Editing Carbon Starved Anther Using CRISPR/Cas9[J]. Quanlin Li,Dabing Zhang,Mingjiao Chen,Wanqi Liang,Jiaojun Wei,Yiping Qi,Zheng Yuan. Journal of Genetics and Genomics. 2016(06)
[6]Phytohormones and their metabolic engineering for abiotic stress tolerance in crop plants[J]. Shabir H.Wani,Vinay Kumar,Varsha Shriram,Saroj Kumar Sah. The Crop Journal. 2016(03)
本文編號(hào)：2926312
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