水稻OsFROs基因分子表征及其在逆境條件下的表達(dá)特性分析
發(fā)布時間：2023-04-22 11:12
鐵還原氧化酶(FRO)基因家族廣泛存在于植物中,在維持植物體內(nèi)金屬離子的穩(wěn)態(tài)、植物對金屬離子的耐受性以及其他復(fù)雜的信號網(wǎng)絡(luò)中扮演著重要角色,通過參與各類生物學(xué)過程來響應(yīng)一系列的非生物脅迫反應(yīng)。鐵氧化還原狀態(tài)的快速變化,可以在某種程度上刺激細(xì)胞功能的發(fā)揮,調(diào)節(jié)細(xì)胞的氧化還原平衡、電子傳遞過程以及其他新陳代謝過程。與此相反,鐵毒性卻具有高度的反應(yīng)活性,可以阻礙植物正常的生長和發(fā)育過程。早期研究表明,FROs在維持鐵離子(Fe)的穩(wěn)態(tài)、Fe的吸收和轉(zhuǎn)運機制中起重要作用,但其進(jìn)化歷史和核心功能位點仍不清楚,特別是在水稻中研究地更少。本研究初步分析了水稻FRO基因家族的系統(tǒng)發(fā)育關(guān)系,明確了水稻FRO基因的表達(dá)特征,然后利用遺傳轉(zhuǎn)化、形態(tài)學(xué)分析及生理測定等方法對水稻OsFRO1基因在脅迫響應(yīng)過程中的功能進(jìn)行了較深入的研究,取得的主要結(jié)果如下:1、為了探索FROs家族的系統(tǒng)發(fā)育關(guān)系,首先我們檢索了代表植物亞群的8個主要世系(即藍(lán)綠藻、紅藻、綠藻、苔蘚類、石松類、裸子植物、單子葉植物和真雙子葉植物)中的16個不同物種,從中鑒定到了50個植物類FROs基因和15個非植物體的類鐵還原氧化酶(FRO-lik...
【文章頁數(shù)】：129 頁
【學(xué)位級別】：博士
【文章目錄】：
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
本文編號：3797523
【文章頁數(shù)】：129 頁
【學(xué)位級別】：博士
【文章目錄】：
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
本文編號：3797523
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