葡萄休眠芽中低温驯化相关蛋白的鉴定和功能分析

发布时间：2023-02-15 20:34

　　葡萄是世界范围内广泛栽种的水果作物之一。中国作为葡萄的起源中心之一,拥有极其丰富的种质资源。温度是决定葡萄生长发育和地理分布的主要因素之一。在中国北方酿酒葡萄主产区,葡萄会遭受冬季极端低温的影响,需要埋土防寒以度过严冬。高抗寒性已成为这些地区葡萄品种筛选的主要目标。冷驯化是植物面对低温胁迫的一种适应策略,包括保护性蛋白质的积累等多个过程。与主栽的欧亚种葡萄(V.vinifera)相比,原产我国的山葡萄(V.amurensis)具有极强的耐寒性。解析山葡萄耐寒机制将为葡萄的抗性育种提供理论基础。本研究通过对山葡萄‘左山一号’和欧亚种‘京早晶’两种葡萄在冷驯化过程中的芽组织进行蛋白质组分析,以探讨两个葡萄品种在田间冷驯化的耐寒机制,并进一步选择低温应答蛋白Snakin-2进行功能分析。主要研究结果如下:1.葡萄休眠芽样品于2016年10月19号和12月2号在中国科学院植物研究所采集。利用差热分析发现,葡萄休眠芽的抗寒性随田间温度的逐步降低而增加,‘左山一号’的抗寒性明显高于‘京早晶’。因此拟通过样品之间的比较来揭示冷驯化下两个品种休眠芽在蛋白质组学水平的差异。2.通过比较蛋白质组学的方法,...

【文章页数】：113 页

【学位级别】：博士

【文章目录】：
摘要
abstract
List of Abbreviations
Chapter 1 Introduction and Literature Review
    1.1 Background
    1.2 Grapevine breeding history in China
    1.3 Acclimation to low temperature
    1.4 Plant Proteomics
        1.4.1 Overview of iTRAQ-based Proteomics Workflow
        1.4.2 Mass Spectrometry
        1.4.3 Proteomics studies in grapevine
    1.5 Candidate protein for cold acclimation
        1.5.1 Chemical structure of Snakin/GASA protein
    1.6 Research Aims
Chapter 2 iTRAQ-based proteomics uncovers the variation in budsof Vitis amurensis and Vitis vinifera during cold acclimation
    2.1 Introduction
    2.2 Materials and Methods
        2.2.1 Plant materials
        2.2.2 Protein extraction,in-solution digestion,and peptide extraction
        2.2.3 iTRAQ labeling and high-performance liquid chromatography(HPLC)fractionation
        2.2.4 Nano-LC-MS/MS analysis
        2.2.5 MS/MS data analysis and quality control
        2.2.6 Hierarchical clustering analysis and functional annotation of theproteins
        2.2.7 qRT-PCR analysis
        2.2.8 Statistical analysis
    2.3 Results
        2.3.1.Overview of the quantitative proteomics data
        2.3.2.Mass Spectrometry Quality Control
        2.3.3.PCA of DAPs during cold acclimation
        2.3.4.Analysis of the10 most upregulated DAPs in V.amurensis and V.vinifera during CA
        2.3.5.Functional categories of the overlapping CA-related DAPs in V.amurensis and V.vinifera
        2.3.6.Pathways specifically enriched in V.amurensis and V.vinifera duringCA
        2.3.7 The phenylpropanoid biosynthesis pathway is differently regulated inthe two grapevines during CA
        2.3.8 A qRT-PCR analysis revealed the correlation between transcriptionexpression and proteomic levels
    2.4 Discussion
        2.4.1 Carbohydrate and energy metabolism
        2.4.2 Stress response and defense-related proteins
        2.4.3 Protein biosynthesis and metabolism
        2.4.4 Phenylpropanoid biosynthesis pathway
    2.5 Correlation between transcriptional and proteomic expression
Chapter 3 Functional Analysis of the CA related protein Snakin-2
    3.1 Introduction
    3.2 Materials and methods
        3.2.1 Materials preparation
        3.2.2 RNA extraction and quantitative reverse transcription PCR(qRT-PCR)analysis
        3.2.3 Vector construction for gene transformation
        3.2.4 Transformation and generation transgenic callus from petiolesegments
        3.2.5 Molecular analysis of Vasnk-2 transgenic callus lines
        3.2.6 Subcellular localization of Snakin-2
    3.3 Results
        3.3.1 Quantitative expression of Snk/GASA2 after cold treatment
        3.3.2 Vector construction and gene transformation for overexpression in V.amurensis
        3.3.3.Molecular analysis of the transformed calli
    3.7 Discussion
Chapter 4 Conclusions and Future Prospects
    4.1 Conclusions
    4.2 Future prospects
References
Appendix
Acknowledgements
Student profile and publications



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