梨MLO和DEFL调控花粉管生长的功能分析

发布时间：2023-04-22 04:38

　　梨(Pyrus bretschneideri)是受世界各地人们喜爱的重要水果,主要生长在温带地区。梨的果实多种多样,大小、形状、质地和风味都各有特点。依赖于有性生殖的成花坐果既是梨生产的主要目标,也是其研究的重要内容。被子植物的有性生殖过程需要雌雄生殖细胞相互协调配合,在双受精过程中,花粉管和雌蕊之间交流频繁。亲和的花粉附着在花柱的柱头上后,花粉萌发形成花粉管并向下生长,此过程中花粉管受到雌蕊细胞外基质中引导调控,进入胚珠。许多信号分子,如MLO(Mildew Locus O)也参与了这一过程。MLO是一种植物特异的具有七跨膜结构的蛋白,最初研究发现MLO影响白粉病的易感性,后来发现其广泛参与生物/非生物胁迫响应,包括花粉与雌蕊的相互作用。已有研究表明,MLO基因家族存在于多种植物中。拟南芥MLO家族有15个成员,玉米至少有9个成员,而水稻中有12个成员。然而,至今没有关于梨中MLO家族分析的报道。因此,为了揭示梨MLO家族的基因特征,本研究从梨数据库中检索了梨MLO成员,克隆对应基因,并对其在根、茎、叶、雌蕊、花粉和果实中的表达进行检测。对梨中的MLO基因进行全基因组鉴定和分析,获得...

【文章页数】：108 页

【学位级别】：博士

【文章目录】：
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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