柑橘因其為人類提供豐富的健康物質和不同風味而深受消費者喜愛。風味是果實重要的感官品質之一,也是決定果品市場性的重要因素。柑橘果實的風味主要決定于果實汁胞中的糖酸水平及它們的比例,而果實中的糖酸水平取決于可溶性糖、有機酸在果實細胞液胞中貯藏能力,其中蔗糖和檸檬酸檸檬酸分別是果實積累的重要糖分和有機酸。已有研究表明,液胞膜上存在三種質子泵,液胞型H⁺-ATPase（V-ATPase）、質膜型H⁺-ATPase（P-type V-ATPase）和液胞型H⁺-pyrophosphatase（V-PPase）,它們通過提供電化學梯度和能量在促進物質進入液胞貯藏中起到非常重要的作用。有些研究認為I型V-PPase基因在適應逆境或在特定組織中能夠促進有機酸和花青素在液胞中積累中起到重要作用,不過它們是否在可溶性糖和檸檬酸（柑橘果實的主要風味物質）積累與液胞中起作用,目前還不清楚。本研究首先從柑橘基因組中鑒定了V-PPase基因,然后通過對不同品種和不同處理（干旱和ABA）情況下果實中的可溶性糖、檸檬酸含量和基因表達的共分析、以及轉基因... 

【文章來源】：華中農業(yè)大學湖北省 211工程院校 教育部直屬院校

【文章頁數】：138 頁

【學位級別】：博士

【文章目錄】：
摘要
ABSTRACT
CHAPTER1:INTRODUCTION
    1.1 RESEARCH RATIONALE
    1.2 RESEARCH ADVANCES IN SUGAR AND CITRIC ACID ACCUMULATION IN THECITRUS FRUIT
        1.2.1 Sugar Accumulation in the Citrus Fruit
        1.2.2 Citric Acid Accumulation in Citrus Fruit
        1.2.3 The Connection between Sugar and Organic Acid Metabolism
    1.3 RESEARCH ADVANCES IN VACUOLAR PROTON PUMPS
        1.3.1 The V-ATPase
+-ATPase">        1.3.2 The P-type H⁺-ATPase
        1.3.3 The V-PPase
    1.4 RESEARCH OBJECTIVES,CONTENT,AND SIGNIFICANCE
+-PYROPHOSPHATASEGENES">CHAPTER2:IDENTIFICATION,CLONING AND MOLECULARCHARACTERIZATION OF CITRUS VACUOLAR H⁺-PYROPHOSPHATASEGENES
    2.1 INTRODUCTION
    2.2 MATERIALS AND METHODS
        2.2.1 Plant Material
        2.2.2 RNA Extraction and c DNA Synthesis
        2.2.3 Gene Isolation and Sequencing
        2.2.4 Bioinformatics Analysis
        2.2.5 Full-length Sequence Cloning of Putative Genes
+-Pyrophosphatase Genes">        2.2.6 Subcellular Localization of Citrus Vacuolar H⁺-Pyrophosphatase Genes
        2.2.7 Gene Expression Analysis
        2.2.8 Statistical Analysis
    2.3 RESULTS
+-Pyrophosphatase Genes">        2.3.1 Identification of Citrus Vacuolar H⁺-Pyrophosphatase Genes
+-Pyrophosphatase Genes">        2.3.2 Subcellular Localization of Citrus Vacuolar H⁺-Pyrophosphatase Genes
+-PyrophosphataseGenes">        2.3.3 Spatial Expression Analysis of Citrus Type I Vacuolar H⁺-PyrophosphataseGenes
    2.4 DISCUSSION
    2.5 CONCLUSION
+-PYROPHOSPHATASE GENES EXPRESSION IN CITRUS FRUITS">CHAPTER3:CHARACTERIZATION OF CITRUS TYPE I VACUOLAR H⁺-PYROPHOSPHATASE GENES EXPRESSION IN CITRUS FRUITS
    3.1 INTRODUCTION
    3.2 MATERIALS AND METHODS
        3.2.1 Plant Material
        3.2.2 Determination of Soluble Sugars,and Organic Acids Content
        3.2.3 RNA Extraction and Gene Expression Analysis
        3.2.4 Statistical Analysis
    3.3 RESULTS
        3.3.1 Changes of Organic Acid and Soluble Sugar Contents in Citrus Fruitsduring Development
+-Pyrophosphatase Genes Expression Profiles During Fruit Development and Ripening of Three Citrus Cultivars">        3.3.2 Analysis of Organic Acid and Soluble Sugar Contents,and Type I VacuolarH⁺-Pyrophosphatase Genes Expression Profiles During Fruit Development and Ripening of Three Citrus Cultivars
+-Pyrophosphatase Geneswith Organic Acid and Sugar Profiles in Citrus Fruits">        3.3.3 Correlation Analysis of Citrus Type I Vacuolar H⁺-Pyrophosphatase Geneswith Organic Acid and Sugar Profiles in Citrus Fruits
    3.4 DISCUSSION
    3.5 CONCLUSION
+-PYROPHOSPHATASE GENES">CHAPTER4:EFFECT OF DROUGHT STRESS AND ABA TREATMENT ONSUGAR AND ORGANIC ACID CONTENTS AND EXPRESSION PROFILES OFCITRUS TYPE I VACUOLAR H⁺-PYROPHOSPHATASE GENES
    4.1 INTRODUCTION
    4.2 MATERIALS AND METHODS
        4.2.1 Plant Material
        4.2.2 Drought Application and Sampling
        4.2.3 ABA Treatment and Sampling
        4.2.4 RNA Extraction and Gene Expression Analysis
        4.2.5 Physical and Inclusion Indicators
        4.2.6 Determination of Soluble Sugars and Organic Acids Content
        4.2.7 Quantification of ABA Contents
        4.2.8 Statistical Analysis
    4.3 RESULTS
        4.3.1 Physical and Biochemical Changes in Citrus Fruits in Response to DroughtStress
        4.3.2 Physical and Biochemical Changes in Citrus Fruits in Response to ABAApplication
+-Pyrophosphatase Genes in Response to Drought Stress">        4.3.3 Changes in Soluble Sugars,Organic Acids,and Expression Profiles ofCitrus Type I Vacuolar H⁺-Pyrophosphatase Genes in Response to Drought Stress
+-Pyrophosphatase Genes in Response to ABAApplication">        4.3.4 Changes in Soluble Sugars,Organic Acids,and Expression Profiles ofCitrus Type I Vacuolar H⁺-Pyrophosphatase Genes in Response to ABAApplication
    4.4 DISCUSSION
    4.5 CONCLUSION
+-PYROPHOSPHATASE GENES">CHAPTER5:FUNCTIONAL CHARACTERIZATION OF CITRUS TYPE IVACUOLAR H⁺-PYROPHOSPHATASE GENES
    5.1 INTRODUCTION
    5.2 MATERIALS AND METHODS
        5.2.1 Plant Material
        5.2.2 Construction of Expression Vectors
        5.2.3 Transfection of Citrus Fruits and Leaves
        5.2.4 Application of V-PPase Inhibitors in Citrus Fruits and Leaves
        5.2.5 RNA Extraction and Gene Expression Analysis
        5.2.6 Preparation of Crude Membrane and Enzymatic Activity Assays
        5.2.7 Quantification of Pyrophosphate
        5.2.8 Determination of Sucrose and Citrate Contents
        5.2.9 Statistical Analysis
    5.3 RESULTS
+-Pyrophosphatase Genes in Both Citrus Fruits and Leaves">        5.3.1 Transient Overexpression of Citrus Type I Vacuolar H⁺-Pyrophosphatase Genes in Both Citrus Fruits and Leaves
        5.3.2 Application of V-PPase Inhibitors in Citrus Fruits and Leaves
+-Pyrophosphatase Genes Expression">        5.3.3 Transcript Response of Sucrose Transporter Genes to the Alteration ofCitrus Type I Vacuolar H⁺-Pyrophosphatase Genes Expression
    5.4 DISCUSSION
    5.5 CONCLUSION
CHAPTER6:ASSESSING THE ROLE OF TYPE I V-PPASE IN A SWEETORANGE（C.SINENSIS）BUD MUTANT‘HONG ANLIU’HAVING DIFFERENT ACCUMULATION PROFILES OF SOLUBLE SUGARS FROM ITS WILDPARENT’ANLIU’ORANGE
    6.1 INTRODUCTION
    6.2 MATERIALS AND METHODS
        6.2.1 Plant Materials
        6.2.2 Physical and Inclusion Indicators
        6.2.3 Measurement of Soluble Sugars Content
        6.2.4 Extraction of Total RNA and Quantitative Real-Time PCR（QRT-PCR）
        6.2.5 Statistical Analysis
    6.3 RESULTS
        6.3.1 Variations in Sugar contents during‘HAL’and‘AL’Fruit Developmentand Ripening
        6.3.2 Comparative Expression Analysis of Invertase Genes in Developing JS
        6.3.3 Comparative Expression Analysis of Sucrose Synthase Genes in DevelopingJS
        6.3.4 Comparative Expression Analysis of Sucrose Phosphate Synthase Genes inDeveloping JS
        6.3.5 Comparative Expression Analysis of Sugar Transporter and VacuolarPyrophosphatase Proton Pump-Related Genes in Developing JS
    6.4 DISCUSSION
    6.5 CONCLUSION
SUMMARY AND FUTURE PERSPECTIVES
REFERENCES
APPENDICES
LIST OF PUBLICATIONS
ACKNOWLEDGMENT


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期刊論文
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