Identification and Characterization of Flavonoid Modificatio
发布时间：2022-10-27 21:24
柑橘类水果是全世界广泛栽培和食用的水果,其口味宜人并且富含维生素,纤维,矿物质,类黄酮以及其他营养物质。同时柑橘属含有不同类别的次生代谢物。甜橙是最常食用和栽培的柑橘之一,拥有丰富的次生代谢物。然而,部分次生代谢物的调控机制仍不清晰。我们前期的转录组数据显示,一个CYPOCHROME P450基因和一个UDP-GLUCOSYL转位酶基因和代谢物积累以及响应不同非生物胁迫（强光和干旱）是正相关关系。因此,我们克隆了一个CYTOCHROME P450 75B1（Cs CYP75B1）基因和一个UDP-GLUCOSYL转移酶（Cs UGT78D3）基因,在拟南芥中超表达这两个基因,以探索他们在胁迫条件下的功能。结果表明,超表达Cs CYP75B1后,能明显诱导许多类黄酮类生物合成基因的表达上调2-12倍,所有转基因系中黄酮类化合物和酚类化合物的总含量较高。此外,所有转基因系植株的抗氧化活性和抗氧化能力均高于野生型和空载。当转基因植株受到干旱胁迫时,其根长显著增加,具有抗旱能力。经过14天的干旱胁迫,所有转基因系均表现出较强的耐旱性,积累较高的抗氧化类黄酮、花青素和酚类化合物,具有较高的总抗氧...
【文章页数】：148 页
【学位级别】：博士
【文章目录】：
ABSTRACT
摘要
List of Abbreviations
Chapter1 Literature Review
1.1 Origin and History of Citrus
1.2 Citrus production,challenges and possible solutions
1.3 Nutritional Importance of Citrus
1.4 Metabolites of Citrus
1.5 Flavonoid types in citrus
1.6 Biosynthesis of flavonoids in plants
1.7 Biology and function of flavonoid
1.8 Flavonoids role in stress acclimation
1.9 Genes in flavonoid and their regulation under stress
1.10 Cytochrome P450s and UDP-glycosyl-transferases genes role in plant metabolism
1.11 Significance and impact of Proposed Research
1.12 Objectives
1.13 The detailed road map
Chapter2 Functional analysis of a Citrus CYTOCHROME P450 gene
2.1 Introduction
2.2 Material and Methods
2.2.1 Plant type and growing condition
2.2.2 Vector preparation for transformation
2.2.3 Drought conditions and experimental details
2.2.3.1 Experimental design and treatments details
2.2.4 DNA extraction and PCR analysis
2.2.5 RNA isolation and Quantitative PCR
2.2.6 Chlorophyll?a?and?b?content
2.2.7 Extraction procedure of total flavonoid and total phenolics contents
2.2.7.1 Extraction
2.2.7.2 Estimation of total flavonoid content(TFC)
2.2.7.3 Total phenolics contents(TPC)
2.2.8 Total Anthocyanin contents(TAC)
2.2.9 Antioxidant enzymatic activity
2.2.10 Malondialdehyde and Electrolytic leakage
2.2.11 Hydrogen peroxide(H_2O_2)
2.2.12 Reactive oxygen species and superoxide radical?s determination
2.2.13 Antioxidant activity and capacity(DPPH free radical scavenging assay)
2.2.14 Trypan blue and Nitro-blue tetrazolium staining
2.2.15 Statistical analysis
2.3 Results
2.3.1 Selection of metabolic genes
2.3.2 Gene expression of Cs CYP75B1 and Cs UGT78D3 under abiotic stress
2.3.3 Gene expression results of selected genes under abiotic stress
2.3.4 Phylogenetic analysis of Cs CYP75B1 gene
2.3.4.1 Root elongation in Cs CYP75B1 transgenic lines
2.3.5 Up-regulation of flavonoid genes with high contents of flavonoids in transgenic Arabidopsis lines
2.3.6 Flavonoids accumulation in transgenic lines under drought stress
2.3.7 Antioxidant enzymatic activity under drought stress
2.3.8 Lower reactive oxygen species in all transgenic lines when exposed to drought stress
2.4 Discussion
Chapter3 Characterization and functional analysis of a citrus UDP-GLUCOSYL TRANSFERASE gene
3.1 Introduction
3.2 Material and Methods
3.2.1 Plant materials
3.2.2 Vector preparation and Agrobacterium mediated transformation
3.2.3 Growing and light stress conditions
3.2.3.1 Experimental design and treatment detail
3.2.4 DNA extraction and PCR analysis
3.2.5 RNA extraction and quantitative real time PCR analysis
3.2.6 Vanillin Assay for proanthocyanidins determination
3.2.7 Determination of proanthocyanidins contents(PAC)
3.2.8 Determination of chlorophyll content
3.2.9 Determination of total contents of phenolics and flavonoids
3.2.9.1 Extraction
3.2.9.2 Determination of total flavonoid content(TFC)
3.2.9.3 Total phenolics contents(TPC)
3.2.10 Total Anthocyanin contents(TAC)
3.2.11 Antioxidant capacity and activity(DPPH free radical scavenging assay)
3.2.12 Hydrogen peroxide(H_2O_2)
3.2.13 Superoxide radical?s determination
3.2.14 Statistical analysis
3.3 Results
3.3.1 Selection and correlation of UGTs gene with metabolites
3.3.2 Arabidopsis lines overexpressing Cs UGT78D3 gene
3.3.3 Transgenic lines accumulates proanthocyanidins in seed coats
3.3.4 Higher level of flavonoids and anthocyanins in all transgenic lines
3.3.5 Metabolic response of transgenic Arabidopsis under light stress
3.3.6 High light stress effects on biochemical attributes in transgenic lines
3.4 Discussion
Chapter4 Conclusion and future prospects
4.1 Conclusion
4.2 Innovations
4.3 Perspective for further research
REFERENCES
APPENDICES
List of Tables
List of Figures
AUTHOR INTRODUCTION
List of Publications
ACKNOWLEDGEMENTS
本文编号：3697241
【文章页数】：148 页
【学位级别】：博士
【文章目录】：
ABSTRACT
摘要
List of Abbreviations
Chapter1 Literature Review
1.1 Origin and History of Citrus
1.2 Citrus production,challenges and possible solutions
1.3 Nutritional Importance of Citrus
1.4 Metabolites of Citrus
1.5 Flavonoid types in citrus
1.6 Biosynthesis of flavonoids in plants
1.7 Biology and function of flavonoid
1.8 Flavonoids role in stress acclimation
1.9 Genes in flavonoid and their regulation under stress
1.10 Cytochrome P450s and UDP-glycosyl-transferases genes role in plant metabolism
1.11 Significance and impact of Proposed Research
1.12 Objectives
1.13 The detailed road map
Chapter2 Functional analysis of a Citrus CYTOCHROME P450 gene
2.1 Introduction
2.2 Material and Methods
2.2.1 Plant type and growing condition
2.2.2 Vector preparation for transformation
2.2.3 Drought conditions and experimental details
2.2.3.1 Experimental design and treatments details
2.2.4 DNA extraction and PCR analysis
2.2.5 RNA isolation and Quantitative PCR
2.2.6 Chlorophyll?a?and?b?content
2.2.7 Extraction procedure of total flavonoid and total phenolics contents
2.2.7.1 Extraction
2.2.7.2 Estimation of total flavonoid content(TFC)
2.2.7.3 Total phenolics contents(TPC)
2.2.8 Total Anthocyanin contents(TAC)
2.2.9 Antioxidant enzymatic activity
2.2.10 Malondialdehyde and Electrolytic leakage
2.2.11 Hydrogen peroxide(H_2O_2)
2.2.12 Reactive oxygen species and superoxide radical?s determination
2.2.13 Antioxidant activity and capacity(DPPH free radical scavenging assay)
2.2.14 Trypan blue and Nitro-blue tetrazolium staining
2.2.15 Statistical analysis
2.3 Results
2.3.1 Selection of metabolic genes
2.3.2 Gene expression of Cs CYP75B1 and Cs UGT78D3 under abiotic stress
2.3.3 Gene expression results of selected genes under abiotic stress
2.3.4 Phylogenetic analysis of Cs CYP75B1 gene
2.3.4.1 Root elongation in Cs CYP75B1 transgenic lines
2.3.5 Up-regulation of flavonoid genes with high contents of flavonoids in transgenic Arabidopsis lines
2.3.6 Flavonoids accumulation in transgenic lines under drought stress
2.3.7 Antioxidant enzymatic activity under drought stress
2.3.8 Lower reactive oxygen species in all transgenic lines when exposed to drought stress
2.4 Discussion
Chapter3 Characterization and functional analysis of a citrus UDP-GLUCOSYL TRANSFERASE gene
3.1 Introduction
3.2 Material and Methods
3.2.1 Plant materials
3.2.2 Vector preparation and Agrobacterium mediated transformation
3.2.3 Growing and light stress conditions
3.2.3.1 Experimental design and treatment detail
3.2.4 DNA extraction and PCR analysis
3.2.5 RNA extraction and quantitative real time PCR analysis
3.2.6 Vanillin Assay for proanthocyanidins determination
3.2.7 Determination of proanthocyanidins contents(PAC)
3.2.8 Determination of chlorophyll content
3.2.9 Determination of total contents of phenolics and flavonoids
3.2.9.1 Extraction
3.2.9.2 Determination of total flavonoid content(TFC)
3.2.9.3 Total phenolics contents(TPC)
3.2.10 Total Anthocyanin contents(TAC)
3.2.11 Antioxidant capacity and activity(DPPH free radical scavenging assay)
3.2.12 Hydrogen peroxide(H_2O_2)
3.2.13 Superoxide radical?s determination
3.2.14 Statistical analysis
3.3 Results
3.3.1 Selection and correlation of UGTs gene with metabolites
3.3.2 Arabidopsis lines overexpressing Cs UGT78D3 gene
3.3.3 Transgenic lines accumulates proanthocyanidins in seed coats
3.3.4 Higher level of flavonoids and anthocyanins in all transgenic lines
3.3.5 Metabolic response of transgenic Arabidopsis under light stress
3.3.6 High light stress effects on biochemical attributes in transgenic lines
3.4 Discussion
Chapter4 Conclusion and future prospects
4.1 Conclusion
4.2 Innovations
4.3 Perspective for further research
REFERENCES
APPENDICES
List of Tables
List of Figures
AUTHOR INTRODUCTION
List of Publications
ACKNOWLEDGEMENTS
本文编号：3697241
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