水稻OsFROs基因分子表征及其在逆境条件下的表达特性分析
发布时间：2023-04-22 11:12
铁还原氧化酶(FRO)基因家族广泛存在于植物中,在维持植物体内金属离子的稳态、植物对金属离子的耐受性以及其他复杂的信号网络中扮演着重要角色,通过参与各类生物学过程来响应一系列的非生物胁迫反应。铁氧化还原状态的快速变化,可以在某种程度上刺激细胞功能的发挥,调节细胞的氧化还原平衡、电子传递过程以及其他新陈代谢过程。与此相反,铁毒性却具有高度的反应活性,可以阻碍植物正常的生长和发育过程。早期研究表明,FROs在维持铁离子(Fe)的稳态、Fe的吸收和转运机制中起重要作用,但其进化历史和核心功能位点仍不清楚,特别是在水稻中研究地更少。本研究初步分析了水稻FRO基因家族的系统发育关系,明确了水稻FRO基因的表达特征,然后利用遗传转化、形态学分析及生理测定等方法对水稻OsFRO1基因在胁迫响应过程中的功能进行了较深入的研究,取得的主要结果如下:1、为了探索FROs家族的系统发育关系,首先我们检索了代表植物亚群的8个主要世系(即蓝绿藻、红藻、绿藻、苔藓类、石松类、裸子植物、单子叶植物和真双子叶植物)中的16个不同物种,从中鉴定到了50个植物类FROs基因和15个非植物体的类铁还原氧化酶(FRO-lik...
【文章页数】：129 页
【学位级别】：博士
【文章目录】：
摘要
ABSTRACT
Chapter 1 Review of literature
1.1 Introduction
1.2 Role of iron in plants
1.2.1 Iron deficiency, toxicity and Its impacts on rice
1.2.2 Mechanism of iron (Fe) uptake and transport in rice
1.3 Subcellular regulation of iron
1.3.1 Vacuoles; as largest pool of iron trafficking
1.3.2 Plasma membrane ferric reductase activity
1.3.3 Mitochondrial ferric reductase activity
1.3.4 Role of mitochondrial iron transporter (MIT) & nucleus in iron (Fe) transport
1.4 Loading of iron (Fe) into grain
1.5 Gene families related to iron (Fe) homeostasis
1.5.1 Nicotinamide Synthesis (NAS) Gene Family
1.5.2 Natural resistance-associated macrophage (NRAMP) gene family
1.5.3 Yellow stripe (YSL) gene family
1.5.4 Iron-regulated transporter (IRT) gene family
1.5.5 Ferric reduction oxidase (FRO) gene family
1.6 Rice FRO genes and their major functional domains
1.6.1 Subcellular localization and role of FROs in plant development
1.6.2 Transcriptomic changes of FRO genes during imbalance supply of iron
1.7 Approaches towards Iron (Fe) biofortification
1.7.1 Transgenic approach
1.7.2 Discovery of novel genes and their functional characterization
1.8 Aims and objectives of research work
Chapter 2 Expansion and Evolutionary Mechanism of Plant FRO Gene Family
2.1 Introduction
2.2 Methodology
2.2.1 Information of plant genomic databases
2.2.2 Identification of FRO family members
2.2.3 Phylogenetic analysis, chromosomal location and duplication
2.2.4 Gene structure, conserved domain and motif analysis
2.2.5 Analysis of promoter region
2.3 Results
2.3.1 Identification, classification, and annotation of FRO family members
2.3.2 Systematic evolutionary relationship of FRO genes
2.3.3 Structural diversity of FRO genes
2.3.4 Analysis of promoter region, conserved motifs and domains
2.3.5 Chromosomal location and gene duplication of FRO genes
2.4 Discussion
2.4.1. FRO genes expansion, duplication, and structural diversity
2.4.2. Evolution and functional diversity of conserved domains in plant FRO gene
2.5 Conclusion
Chapter 3 Expression Analysis of Rice FRO Genes in Response to Abiotic Stresses, Metalsand Hormone Applications
3.1 Introduction
3.1.1 Instruments
3.1.2 Reagents
3.2 Plant materials and growth conditions
3.2.1 Stress treatments and sample collection
3.2.2 Expression analysis
3.2.3 RNA extraction and c DNA synthesis
3.2.4 RNA extraction
3.2.5 cDNA Synthesis
3.2.6 qRT-PCR analysis
3.3 Antioxidant enzyme activities under metal stresses
3.4 Statistical analysis
3.5 Results
3.5.1 Tissue-specific expression profile of OsFRO1 and OsFRO7 genes
3.5.2 Inducible expression analysis of rice OsFRO1 and OsFRO7 gene under abioticand hormone applications
3.5.3 Inducible expression analysis of rice OsFRO1 and OsFRO7 genes againstdifferent heavy metals stresses
3.5.4 Determination of antioxidant enzyme activities
3.6 Discussion
3.6.1 Tissue-specific gene expression of rice FROs and response to metal stress
3.6.2 Inducible expression pattern of OsFRO1 and OsFRO7 genes against abioticstresses and hormones treatments
3.6.3 Changes in antioxidant activity under iron (Fe) and chromium (Cr) stress
3.7 Conclusion
Chapter 4 Functional Characterization of Os RO1 Gene and Stress Tolerance Mechanism
4.1 Introduction
4.2 Materials, Reagents and Instruments
4.2.1 Antisense RNA Vector Construction
4.2.2 Subcellular localization of OsFRO1
4.2.3 Histochemical beta-glucuronidase (GUS) analysis
4.2.4 Transfection of cloning vector into agrobacterium strain EHA-105
4.3 Genetic transformation of Os FRO1-RNAi into rice
4.3.1 Bacterial suspension and agrobacterium infection and co-cultivation
4.3.2 Regeneration and transgene conformation
4.4 Tissue Culture
4.4.1 Surface sterilization of seeds and Callus Induction
4.4.2 Selection, Regeneration and recovery of Transgenic Plants
4.5 Expression analysis of Os FRO1-RNAi
4.5.1 Tissue specific and inducible expression profile
4.5.2 Determination of antioxidant activity and Fe concentration in rice shoots
4.5.3 Determination of leaf pigments and root activity
4.6 Experimental data and statistical analysis
4.7 Results
4.7.1 Cloning of OsFRO1 gene and construction of expression vector
4.7.2 Agrobacterium mediated transformation
4.7.3 Agrobacterium growth and infection
4.7.4 Selection and transgene analysis
4.7.5 Subcellular localization of OsFRO1 in rice
4.7.6 Histochemical beta-glucuronidase (GUS) analysis
4.8 Analysis of expression profile of osfro1 in different tissues and young panicle
4.8.1 Phenotypic characterization of osfro1
4.8.2 Reduced tolerance of osfro1 silenced in rice roots
4.8.3 Knockdown effect of osfro1 in ROS network by modulation of enzymaticactivity
4.8.4 Knockdown osfro1 leads to Fe-sensitivity during germination and seedlinggrowth
4.8.5 Knockdown effect of osfro1 on abiotic stresses and hormones
4.8.6 Expression of osfro1 and chlorophyll content under Fe stress
4.9 Discussion
Chapter 5 General Summary and Future Perspectives
5.1 General summary
5.2 Future perspectives
References
Appendices
Appendix-1. List of symbols and abbreviations
Appendix-2. Media used for japonica rice tissue culture & transformation
Appendix-3. Media composition for bacterial growth
Appendix-4. Preparation of different reagents used in tissue culture media
Appendix-5. Plasmid extraction method
Appendix-6. Plant Tissue SOD, POD, CAT and MDA determination
Appendix-7. Primers used for vector construction and q RT-PCR analysis
Appendix-8. The domain structure of the FRO proteins
Appendix-9. Site of amino acids and motif location of FROs protein
Appendix-10. Schematic diagram of amino acid motifs of FRO, s proteins
Acknowledgements
CURRICULUM VITAE
本文编号：3797523
【文章页数】：129 页
【学位级别】：博士
【文章目录】：
摘要
ABSTRACT
Chapter 1 Review of literature
1.1 Introduction
1.2 Role of iron in plants
1.2.1 Iron deficiency, toxicity and Its impacts on rice
1.2.2 Mechanism of iron (Fe) uptake and transport in rice
1.3 Subcellular regulation of iron
1.3.1 Vacuoles; as largest pool of iron trafficking
1.3.2 Plasma membrane ferric reductase activity
1.3.3 Mitochondrial ferric reductase activity
1.3.4 Role of mitochondrial iron transporter (MIT) & nucleus in iron (Fe) transport
1.4 Loading of iron (Fe) into grain
1.5 Gene families related to iron (Fe) homeostasis
1.5.1 Nicotinamide Synthesis (NAS) Gene Family
1.5.2 Natural resistance-associated macrophage (NRAMP) gene family
1.5.3 Yellow stripe (YSL) gene family
1.5.4 Iron-regulated transporter (IRT) gene family
1.5.5 Ferric reduction oxidase (FRO) gene family
1.6 Rice FRO genes and their major functional domains
1.6.1 Subcellular localization and role of FROs in plant development
1.6.2 Transcriptomic changes of FRO genes during imbalance supply of iron
1.7 Approaches towards Iron (Fe) biofortification
1.7.1 Transgenic approach
1.7.2 Discovery of novel genes and their functional characterization
1.8 Aims and objectives of research work
Chapter 2 Expansion and Evolutionary Mechanism of Plant FRO Gene Family
2.1 Introduction
2.2 Methodology
2.2.1 Information of plant genomic databases
2.2.2 Identification of FRO family members
2.2.3 Phylogenetic analysis, chromosomal location and duplication
2.2.4 Gene structure, conserved domain and motif analysis
2.2.5 Analysis of promoter region
2.3 Results
2.3.1 Identification, classification, and annotation of FRO family members
2.3.2 Systematic evolutionary relationship of FRO genes
2.3.3 Structural diversity of FRO genes
2.3.4 Analysis of promoter region, conserved motifs and domains
2.3.5 Chromosomal location and gene duplication of FRO genes
2.4 Discussion
2.4.1. FRO genes expansion, duplication, and structural diversity
2.4.2. Evolution and functional diversity of conserved domains in plant FRO gene
2.5 Conclusion
Chapter 3 Expression Analysis of Rice FRO Genes in Response to Abiotic Stresses, Metalsand Hormone Applications
3.1 Introduction
3.1.1 Instruments
3.1.2 Reagents
3.2 Plant materials and growth conditions
3.2.1 Stress treatments and sample collection
3.2.2 Expression analysis
3.2.3 RNA extraction and c DNA synthesis
3.2.4 RNA extraction
3.2.5 cDNA Synthesis
3.2.6 qRT-PCR analysis
3.3 Antioxidant enzyme activities under metal stresses
3.4 Statistical analysis
3.5 Results
3.5.1 Tissue-specific expression profile of OsFRO1 and OsFRO7 genes
3.5.2 Inducible expression analysis of rice OsFRO1 and OsFRO7 gene under abioticand hormone applications
3.5.3 Inducible expression analysis of rice OsFRO1 and OsFRO7 genes againstdifferent heavy metals stresses
3.5.4 Determination of antioxidant enzyme activities
3.6 Discussion
3.6.1 Tissue-specific gene expression of rice FROs and response to metal stress
3.6.2 Inducible expression pattern of OsFRO1 and OsFRO7 genes against abioticstresses and hormones treatments
3.6.3 Changes in antioxidant activity under iron (Fe) and chromium (Cr) stress
3.7 Conclusion
Chapter 4 Functional Characterization of Os RO1 Gene and Stress Tolerance Mechanism
4.1 Introduction
4.2 Materials, Reagents and Instruments
4.2.1 Antisense RNA Vector Construction
4.2.2 Subcellular localization of OsFRO1
4.2.3 Histochemical beta-glucuronidase (GUS) analysis
4.2.4 Transfection of cloning vector into agrobacterium strain EHA-105
4.3 Genetic transformation of Os FRO1-RNAi into rice
4.3.1 Bacterial suspension and agrobacterium infection and co-cultivation
4.3.2 Regeneration and transgene conformation
4.4 Tissue Culture
4.4.1 Surface sterilization of seeds and Callus Induction
4.4.2 Selection, Regeneration and recovery of Transgenic Plants
4.5 Expression analysis of Os FRO1-RNAi
4.5.1 Tissue specific and inducible expression profile
4.5.2 Determination of antioxidant activity and Fe concentration in rice shoots
4.5.3 Determination of leaf pigments and root activity
4.6 Experimental data and statistical analysis
4.7 Results
4.7.1 Cloning of OsFRO1 gene and construction of expression vector
4.7.2 Agrobacterium mediated transformation
4.7.3 Agrobacterium growth and infection
4.7.4 Selection and transgene analysis
4.7.5 Subcellular localization of OsFRO1 in rice
4.7.6 Histochemical beta-glucuronidase (GUS) analysis
4.8 Analysis of expression profile of osfro1 in different tissues and young panicle
4.8.1 Phenotypic characterization of osfro1
4.8.2 Reduced tolerance of osfro1 silenced in rice roots
4.8.3 Knockdown effect of osfro1 in ROS network by modulation of enzymaticactivity
4.8.4 Knockdown osfro1 leads to Fe-sensitivity during germination and seedlinggrowth
4.8.5 Knockdown effect of osfro1 on abiotic stresses and hormones
4.8.6 Expression of osfro1 and chlorophyll content under Fe stress
4.9 Discussion
Chapter 5 General Summary and Future Perspectives
5.1 General summary
5.2 Future perspectives
References
Appendices
Appendix-1. List of symbols and abbreviations
Appendix-2. Media used for japonica rice tissue culture & transformation
Appendix-3. Media composition for bacterial growth
Appendix-4. Preparation of different reagents used in tissue culture media
Appendix-5. Plasmid extraction method
Appendix-6. Plant Tissue SOD, POD, CAT and MDA determination
Appendix-7. Primers used for vector construction and q RT-PCR analysis
Appendix-8. The domain structure of the FRO proteins
Appendix-9. Site of amino acids and motif location of FROs protein
Appendix-10. Schematic diagram of amino acid motifs of FRO, s proteins
Acknowledgements
CURRICULUM VITAE
本文编号：3797523
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