大豆疫霉是一種半活體營(yíng)養(yǎng)菌,能引起大豆根莖腐爛病。此微生物與真菌相似,能夠快速克服寄主的防衛(wèi)反應(yīng),涉及到多種機(jī)制。大豆疫霉的基因組為95Mb,能夠編碼上千個(gè)效應(yīng)分子,它們進(jìn)入寄主植物細(xì)胞促進(jìn)病原菌的侵染與定殖。這些效應(yīng)分子主要包括兩類：分泌進(jìn)入植物細(xì)胞間隙的質(zhì)外體效應(yīng)分子和轉(zhuǎn)運(yùn)進(jìn)入植物細(xì)胞內(nèi)的胞內(nèi)效應(yīng)分子,他們作用于不同的亞細(xì)胞結(jié)構(gòu)。其中CRN是一類在植物病原卵菌中保守的胞內(nèi)效應(yīng)分子。大豆疫霉編碼CRN效應(yīng)分子的基因在侵染階段高度上調(diào)表達(dá),說(shuō)明他們對(duì)致病性具有重要作用,但是我們對(duì)這類效應(yīng)分子的作用機(jī)制還知之甚少。在本研究中,我們選擇了3個(gè)效應(yīng)分子(PsCRN70、PsCRN161和PsCRN115)通過(guò)在本氏煙中穩(wěn)定表達(dá)研究了它們的功能。發(fā)現(xiàn)它們都定位在植物細(xì)胞核內(nèi),可以抑制由多種激發(fā)子誘導(dǎo)的細(xì)胞死亡,其中PsCRN70可以抑制植物的抗病反應(yīng)促進(jìn)病原菌的侵染,而另外兩個(gè)效應(yīng)分子能提高本氏煙的抗病和非生物逆境脅迫能力。主要結(jié)果和結(jié)論如下：1. PsCRN70能抑制本氏煙的抗病反應(yīng)。分別利用瞬時(shí)和穩(wěn)定表達(dá)技術(shù),我們將PsCRN70在本氏煙中異源表達(dá),發(fā)現(xiàn)它主要定位在植物細(xì)胞核中,可以抑制由...

【文章頁(yè)數(shù)】：141 頁(yè)

【學(xué)位級(jí)別】：博士

【文章目錄】：
Dedication
ABBREVIATIONS
ABSTRACT
摘要
CHAPTER 1
    1. GENERAL INTRODUCTION
    2. OBJECTIVES OF THIS STUDY
CHAPTER 2 RESEARCH BACKGROUND
    1 PHYTOPHTHORA SOJAE
        1.1 Life cycle of Phytophthora sojae
        1.2 Phytophthora sojae effector proteins
    2. MOLECULAR PLANT-PATHOGEN INTERACTIONS
        2.1 PAMP Triggered Immunity
        2.2 Effector Triggered Immunity
    3. LOCALIZATION OF OOMYCETE EFFECTORS
    4. OOMYCETE EFFECTOR SUPPRESS PLANT IMMUNITY
CHAPTER 3 PHYTOPHTHORA SOJAE EFFECTOR CRN70 SUPPRESSES PLANT DEFENSE INNICOTIANA BENTHAMIANA
    Abstract
    1. Introduction
    2. Material and Methods
        2.1 Plant material, bacterial strain and growth condition
        2.2 Plasmid construction
        2.3 Generation of the PsCRN70-transgenic N. benthamiana
        2.4 Agrobacterium-mediated transient expression
        2.5 Phytophthora parasitica inoculation assay
        2.6 RNA extraction and quantitative RT-PCR
        2.7 Confocal microscopy
        2.8 Protein extraction and Western blot analyses
        2.9 DAB staining
    3. Results
        3.1 Generation of thePsCRN70-transgenic N. benthamiana
        3.2 Expression of the PsCRN70 in N. benthamiana enhance susceptibility to P. parasitica
        3.3 PsCRN70 suppresses cell death in N. benthamiana
        3.4 Expression of the PsCRN70 impairs the H₂O₂ accumulation in N. benthamiana
        3.5 Expressions of the PsCRN70 reduce the expressional levels of the plant defense-associated genes
    4. DISCUSSION
CHAPTER 4 OVEREXPRESSION OF PHYTOPHTHORA SOJAE EFFECTOR CRN161 IN NICOTIANABENTHAMIANA ENHANCES DISEASE RESISTANCE AND INDUCES TOLERANCE TOSALT AND DROUGHT STRESS
    Abstract
    1. INTRODUCTION
    2 MATERIAL AND METHODS
        2.1 Plant material, growth conditions, and treatments
        2.2 Construction of the transformation vector
        2.3 Generation of the PsCRN161-transgenic N. benthamiana
        2.4 DNA isolation from T1 leaves
        2.5 Total RNA Isolation
        2.6 Protein extraction and western blots
        2.7 Confocal microscopy
        2.8 Agrobacterium-mediated transient expression
        2.9 Phytophthora inoculation and resistance assay
        2.10 3,3-Diaminobenzidine (DAB) staining assays
        2.11 SYBR Green quantitative reverse transcription polymerase chain reaction (RT-PCR) assay
        2.12 Salt and drought stress analysis
    3. RESULTS
        3.1 Overexpression of the PsCRN161 gene in N. benthamiana
        3.2 Subcellular localization of PsCRN161
        3.3 Enhanced resistance of PsCRN161-transgenic plants to oomycetes Phytophthora infection
        3.4 Overexpression of PsCRN161 suppresses cell death in N. benthamiana
        3.5 Induction of expressional levels of the defence-related genes in PsCRN161-transgenic plants
        3.6 PsCRN161-transgenic plants enhanced H₂O₂ accumulation
        3.7 PsCRN161-transgenic plants increased the salt tolerance
        3.8 PsCRN161-transgenic plants increased the drought tolerance
        4. Discussion
CHAPTER 5 PHYTOPHTHORA SOJAE EFFECTOR CRN115 IMPROVES DROUGHT AND SALTTOLERANCE AND ENHANCED DEFENCE RESPONSES TO PATHOGEN INFECTION INTRANSGENIC NICOTIANA BENTHAMIANA
    Abstract
    1. Introduction
    2. Material and Methods
        2.1 Plant growth and treatments
        2.2 Generation of the PsCRNl 15-transgenic N. benthamiana
        2.3 Vector construction and genetic transformation
        2.4 DNA isolation from T1 leaves
        2.5     Total RNA Isolation
        2.6 Protein extraction and western blots
        2.7 Subcellular localization of the PsCRN115 protein
        2.8 Pathogen infection
        2.9 3,3-Diaminobenzidine (DAB) staining assays
        2.10 Quantitative reverse transcription polymerase chain reaction (RT-PCR) assay
        2.11 Analysis oftransgenic tobacco plants for abiotic stress tolerance
    3. Results
        3.1 Transformation and Molecular Analyses of PsCRN115 gene in N. benthamiana Plants
        3.2 PsCRN115 is localized to the nucleus
        3.3 Enhanced Phytophthora resistance in PsCRN115-transgenk plants
        3.4 Detection of reactive oxygen species accumulation in PsCRN115-transgenic plants
        3.5 Altered expression of defence-related genes in PsCRN115-transgenic plants
        3.6 PsCRN115-transgenic plants display increased salt tolerance
        3.7 PsCRN115-transgenic plants display increased drought tolerance
    4. Discussion
SUMMARY AND CONCLUSIONS
REFERENCES
APPENDIX
PUBLICATIONS
ACKNOWLEDGEMENTS



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