柑橘類水果是全世界廣泛栽培和食用的水果,其口味宜人并且富含維生素,纖維,礦物質(zhì),類黃酮以及其他營養(yǎng)物質(zhì)。同時(shí)柑橘屬含有不同類別的次生代謝物。甜橙是最常食用和栽培的柑橘之一,擁有豐富的次生代謝物。然而,部分次生代謝物的調(diào)控機(jī)制仍不清晰。我們前期的轉(zhuǎn)錄組數(shù)據(jù)顯示,一個(gè)CYPOCHROME P450基因和一個(gè)UDP-GLUCOSYL轉(zhuǎn)位酶基因和代謝物積累以及響應(yīng)不同非生物脅迫（強(qiáng)光和干旱）是正相關(guān)關(guān)系。因此,我們克隆了一個(gè)CYTOCHROME P450 75B1（Cs CYP75B1）基因和一個(gè)UDP-GLUCOSYL轉(zhuǎn)移酶（Cs UGT78D3）基因,在擬南芥中超表達(dá)這兩個(gè)基因,以探索他們?cè)诿{迫條件下的功能。結(jié)果表明,超表達(dá)Cs CYP75B1后,能明顯誘導(dǎo)許多類黃酮類生物合成基因的表達(dá)上調(diào)2-12倍,所有轉(zhuǎn)基因系中黃酮類化合物和酚類化合物的總含量較高。此外,所有轉(zhuǎn)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載。當(dāng)轉(zhuǎn)基因植株受到干旱脅迫時(shí),其根長顯著增加,具有抗旱能力。經(jīng)過14天的干旱脅迫,所有轉(zhuǎn)基因系均表現(xiàn)出較強(qiáng)的耐旱性,積累較高的抗氧化類黃酮、花青素和酚類化合物,具有較高的總抗氧... 

【文章頁數(shù)】：148 頁

【學(xué)位級(jí)別】：博士

【文章目錄】：
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



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