大豆疫霉中CRN效应分子在本氏烟中的功能分析
发布时间：2024-02-22 20:26
大豆疫霉是一种半活体营养菌,能引起大豆根茎腐烂病。此微生物与真菌相似,能够快速克服寄主的防卫反应,涉及到多种机制。大豆疫霉的基因组为95Mb,能够编码上千个效应分子,它们进入寄主植物细胞促进病原菌的侵染与定殖。这些效应分子主要包括两类：分泌进入植物细胞间隙的质外体效应分子和转运进入植物细胞内的胞内效应分子,他们作用于不同的亚细胞结构。其中CRN是一类在植物病原卵菌中保守的胞内效应分子。大豆疫霉编码CRN效应分子的基因在侵染阶段高度上调表达,说明他们对致病性具有重要作用,但是我们对这类效应分子的作用机制还知之甚少。在本研究中,我们选择了3个效应分子(PsCRN70、PsCRN161和PsCRN115)通过在本氏烟中稳定表达研究了它们的功能。发现它们都定位在植物细胞核内,可以抑制由多种激发子诱导的细胞死亡,其中PsCRN70可以抑制植物的抗病反应促进病原菌的侵染,而另外两个效应分子能提高本氏烟的抗病和非生物逆境胁迫能力。主要结果和结论如下：1. PsCRN70能抑制本氏烟的抗病反应。分别利用瞬时和稳定表达技术,我们将PsCRN70在本氏烟中异源表达,发现它主要定位在植物细胞核中,可以抑制由...
【文章页数】：141 页
【学位级别】：博士
【文章目录】：
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
本文编号：3907217
【文章页数】：141 页
【学位级别】：博士
【文章目录】：
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
本文编号：3907217
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