柑橘液胞型焦磷酸质子泵基因鉴定及其在蔗糖和柠檬酸积累调控中的功能验证

发布时间：2021-02-23 13:07

　　柑橘因其为人类提供丰富的健康物质和不同风味而深受消费者喜爱。风味是果实重要的感官品质之一,也是决定果品市场性的重要因素。柑橘果实的风味主要决定于果实汁胞中的糖酸水平及它们的比例,而果实中的糖酸水平取决于可溶性糖、有机酸在果实细胞液胞中贮藏能力,其中蔗糖和柠檬酸柠檬酸分别是果实积累的重要糖分和有机酸。已有研究表明,液胞膜上存在三种质子泵,液胞型H⁺-ATPase（V-ATPase）、质膜型H⁺-ATPase（P-type V-ATPase）和液胞型H⁺-pyrophosphatase（V-PPase）,它们通过提供电化学梯度和能量在促进物质进入液胞贮藏中起到非常重要的作用。有些研究认为I型V-PPase基因在适应逆境或在特定组织中能够促进有机酸和花青素在液胞中积累中起到重要作用,不过它们是否在可溶性糖和柠檬酸（柑橘果实的主要风味物质）积累与液胞中起作用,目前还不清楚。本研究首先从柑橘基因组中鉴定了V-PPase基因,然后通过对不同品种和不同处理（干旱和ABA）情况下果实中的可溶性糖、柠檬酸含量和基因表达的共分析、以及转基因... 

【文章来源】：华中农业大学湖北省 211工程院校 教育部直属院校

【文章页数】：138 页

【学位级别】：博士

【文章目录】：
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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