梨(Pyrus bretschneideri)是受世界各地人們喜愛的重要水果,主要生長在溫帶地區(qū)。梨的果實多種多樣,大小、形狀、質地和風味都各有特點。依賴于有性生殖的成花坐果既是梨生產的主要目標,也是其研究的重要內容。被子植物的有性生殖過程需要雌雄生殖細胞相互協調配合,在雙受精過程中,花粉管和雌蕊之間交流頻繁。親和的花粉附著在花柱的柱頭上后,花粉萌發(fā)形成花粉管并向下生長,此過程中花粉管受到雌蕊細胞外基質中引導調控,進入胚珠。許多信號分子,如MLO(Mildew Locus O)也參與了這一過程。MLO是一種植物特異的具有七跨膜結構的蛋白,最初研究發(fā)現MLO影響白粉病的易感性,后來發(fā)現其廣泛參與生物/非生物脅迫響應,包括花粉與雌蕊的相互作用。已有研究表明,MLO基因家族存在于多種植物中。擬南芥MLO家族有15個成員,玉米至少有9個成員,而水稻中有12個成員。然而,至今沒有關于梨中MLO家族分析的報道。因此,為了揭示梨MLO家族的基因特征,本研究從梨數據庫中檢索了梨MLO成員,克隆對應基因,并對其在根、莖、葉、雌蕊、花粉和果實中的表達進行檢測。對梨中的MLO基因進行全基因組鑒定和分析,獲得...

【文章頁數】：108 頁

【學位級別】：博士

【文章目錄】：
ABSTRACT
摘要
Abbreviations
CHAPTER ONE:INTRODUCTION
    1.1. Double Fertilization
    1.2. MLO family
        1.2.1 Structure of MLO genes
        1.2.2 Function of MLO genes in pollen-pistil interaction
    1.3 Defensins-like protein
        1.3.1 The structure of defensin-like genes
        1.3.2 The function of DEFLs during reproduction
        1.3.3 The role of defensin in plant growth and development regulation
        1.3.4 The Main role of defensins-like genes in plant Immunity
        1.3.5 Other functions of defensin-like Genes
        1.3.6 Defensin-like genes under selection pressure
    1.4. The role of calcium in pollen-pistil
    1.5. Antisense oligodeoxynucleotide (ODN) technology
CHAPTER TWO: CHARACTERISATION OF MLO GENES IN PEAR
    2.1 Analysis of MLO gene family sequences in pear
        2.1.1 Materials and methods
            2.1.1.1 Sequence data search and domain detection
            2.1.1.2 Phylogenetic tree construction
            2.1.1.3 Multiple-sequence alignment and motif analysis
            2.1.1.4 Gene structure and transmembrane analysis
            2.1.1.5 Amino acid composition of MLO proteins in Pear
        2.1.2 Results
            2.1.2.1 Identification of MLO gene family in pear
            2.1.2.2 Transmembrane structure of PbrMLO proteins
            2.1.2.3 Multiple-sequence alignment and motif analysis
            2.1.2.4 Amino acid composition of MLO proteins in Pear
            2.1.2.5 Motif analysis of MLO proteins in Pear
            2.1.2.6 MLO gene structure analysis in pear
            2.1.2.7 Phylogenetic analysis of MLO gene in pear
        2.1.3 Discussion
        2.1.4 Summary
    2.2 Gene Cloning, Expression and Subcellular localization of MLO in Pear
        2.2.1 Introduction
        2.2.2 Material and Methods
            2.2.2.1 Plant Materials and Growth Conditions
            2.2.2.2 cDNA isolation
            2.2.2.3 Semi-quantitative PCR analysis
            2.2.2.4 Expression of MLO genes in pear
            2.2.2.5 Gene Cloning of PbrMLO5, PbrMLO23
            2.2.2.6 Construction of PbrMLO-GFP fusion expression vector
            2.2.2.7 Subcellular location of PbrMLO5 and pbrMLO23
        2.2.3 Results
            2.2.3.1 Expressions patterns of PbrMLO genes in different tissues
            2.2.3.2 Subcellular localization of PbrMLO5 and PbrMLO23
        2.2.4 Discussion
        2.2.5 Summary
    2.3 Functional analysis of PbrMLO23
        2.3.1 Introduction
        2.3.2 Materials and Methods
            2.3.2.1 Pollen Germination and Pollen Tube Growth
            2.3.2.2 ODN selection and pollen tube treatment
        2.3.3 Results
        2.3.4 Discussion
        2.3.5 Summary
CHAPTER THREE: INTERACTION BETWEEN MLO AND DEFL
    3.1 Introduction
    3.2 Materials and Methods
        3.2.1 Screening interacting proteins of PbrMLO23
        3.2.2 Yeast two hybrids for testing protein-protein interacttion
    3.3 Results
    3.4 Discussion
CHAPTER FOUR: CHARACTERISATION OF DEFL GENES IN PEAR
    4.1 Analysis of DEFL Gene Family Sequences in Pear
        4.1.1 Materials and methods
            4.1.1.1 Sequence database search
            4.1.1.2 Gene structure Analysis
            4.1.1.3 Motif analysis
            4.1.1.4 Chromosome location
            4.1.1.5 Phylogenetic tree construction
        4.1.2 Results
            4.1.2.1 Identification of DEFLs gene family members in Pear
            4.1.2.2 Gene structure Analysis
            4.1.2.3 Motifs analyses in Pear DEFLs protein family
            4.1.2.4 Chromosome location
            4.1.2.5 Phylogenetic Analysis of DEFLs genes in Pear
        4.1.3 Discussion
        4.1.4 Summary
    4.2 Gene cloning and Expression of DEFL genes
        4.2.1 Introduction
        4.2.2 Material and Methods
            4.2.2.1 Plant Materials and Growth Conditions
            4.2.2.2 cDNA isolation
            4.2.2.3 Semi-quantitative PCR analysis
            4.2.2.4 Expression of DEFL genes in pear
            4.2.2.5 Gene cloning of PbrDEFL21, PbrDEFL23, PbrDEFL35, PbrDEFL36
        4.2.3 Results
        4.2.4 Discussion
        4.2.5 Summary
    4.3 Functional analysis of DEFL genes
        4.3.1 Introduction
        4.3.2 Materials and Methods
            4.3.2.1 Pollen germination and pollen tube growth
            4.3.2.2 ODN Selection and Pollen tube treatment
            4.3.2.3 [Ca²⁺]_cyt measurement in pollen tube
        4.3.3 Results
            4.3.3.1 Knockdown PbrDELFs cause pollen tube growth changes
            4.3.3.2 Knockdown PbrDEFL23 cause increased [Ca²⁺]_cyt in pollen tube
        4.3.4 Discussion
Conclusion
Innovation points
    The achievements of this study are as follows
References
Publications



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