CabZIP53 and CaZNF830在辣椒应答青枯菌侵染中的作用及机制分析
发布时间：2021-04-06 05:08
辣椒（Capsicum annuum）是世界上最重要的蔬菜之一,疫霉和青枯菌引起的疫病和青枯病等疾病常常导致辣椒生产严重损失,在高温高湿条件下（HTHH）损失尤其严重。基于抗病遗传改良的辣椒抗病新品种选育是解决辣椒病害问题的根本有效的技术对策,而解析辣椒免疫的分子机制是其重要基础。由于植物抗病反应在很大程度上受转录水平的调控,各种转录因子在其中起重要作用。因此,剖析转录因子的功能及其作用机制是阐明植物抗病机制的重要途径和主要内容。然而,不同转录因子在辣椒抗病中的作用及其机制仍不清楚。在本研究中,对两个辣椒转录因子CabZIP53和CaZNF830的功能进行了功能鉴定,主要结果如下:1.一个推定的bZIP转录因子（CA04g07280）的启动子上含有多个应答病原菌的顺式元件,且通过ChIP分析发现它可能是CaWRKY40的靶基因,而我们的前期研究发现CaWRKY40在辣椒应答青枯菌侵染或高温高湿逆境的防御反应过程中均起正调节作用,暗示该基因可能在上述防御反应中起作用。通过克隆该基因的全长cDNA,发现其推导的氨基酸序列含有高度保守的bZIP结构域,且在番茄的bZIP家族所有成员中与Stb...
【文章来源】：福建农林大学福建省
【文章页数】：103 页
【学位级别】：博士
【文章目录】：
List of Abbreviations
摘要
Abstract
1.Introduction
2.Review of Literature
2.1.Immune functions of plant bZIPs
2.1.1.Sub-cellular localization of bZIPs and defense related proteins
2.1.2.Signaling molecules and bZIPs
2.1.3.Elicitors versus bZIPs
2.2.ZNFs are in involved in vegetative growth and development
2.2.1.Sub-Cellular localization of ZNFs
2.2.2.ZNFs are involved in plant defense responses to pathogens
3.Materials and Methods
3.1 Plant materials and growth conditions
3.2 Cultivation of plant experimental materials
3.3 Agrobacterium culture and infection
3.4 Ralstonia solanacearum inoculation
3.5 The construction of vectors
3.5.1 Reactions for Gene amplification
3.5.2 PCR-Machine program
3.6 Separation and Purification of target gene DNA （amplified product） from Agarose Gel
3.6.1 BP reaction for ligating target gene with entry vector
3.6.2 LR reaction mixture to ligate gene with destination vectors
3.7 Vector Transformation into E.coli competent cell
3.8 E.coli.Plasmid extraction
3.9 Agrobacterium Transformation
3.10 Sub-cellular compartmentalization
3.11 Virus Induced Gene Silence （VIGS） of CabZIP53in pepper plants
3.11.1 Infiltration solution preparation
3.11.2 Virus induced gene silence （VIGS） procedure
3.11.3 Transient over expression of CabZIP53 in pepper leaves
3.12 Histochemical staining
3.12.1 Trypan Blue Staining
3.12.2 Trypan blue staining samples preparation
3.12.3 Decolorization of trypan blue stainedleaves
3.12.4 DAB （3, 3'-Diaminobenzidine） Staining
3.12.5 Decolorization of DAB treated leaves
3.13 RNA Extraction and cDNA synthesis
3.13.1 Sample collection for RNA extraction
3.13.2 Procedure of RNA Extraction
3.14 cDNA synthesis
3.14.1 PCR mixture
3.14.2 PCR program
3.14.3 PCR program used was as follow
3.15 Quantitative real-time-PCR （qRT-PCR）
3.15.1 Real time RT-PCR program
3.16 Chromatin immune-precipitation （ChIP） assay
3.17 Fluorometric GUS enzymatic assay
3.18 Immunoblotting
3.19 Protein Extraction
3.19.1 Solution-I（12%）
3.19.2 Procedure
4.Results
4.1 The sequence analysis and cloning of CabZIP53
4.1.1 The expression of CabZIP53 was transcriptionally modulated by Ralstonia inoculation （RSI）and HTHH
4.1.2 CabZIP53 is targeted to the nuclei
4.1.3 Pepper Basal defense is compromised by the silencing of CabZIP53
4.1.4 The transient over-expression of CabZIP53 promotes HR response by modulating theexpression of immunity and thermo-tolerance related genes
4.1.5 Ca WRKY40 makes positive feedback loop with CabZIP53 for resistance against RSI and HTHH
4.1.6 The possible transcriptional regulation of CabZIP53 by CaWRKY6, CaWRKY40 andCabZIP63
4.1.7 The assay on the possible interaction between CabZIP53 to itself and to CabZIP63
4.2 Isolation and characterization of CaZNF830
4.2.1.The expression of CaZNF830was transcriptionally modulated by Ralstonia inoculation andHTHH
4.2.2.Cellular compartmentalization of CaZNF830
4.2.3.Silencing of CaZNF830 enhance bacterial growth, compromises plant defense and HR-mediated resistance to RSI and thermo-tolerance
4.2.4.Agrobacterium mediated transient over-expression of CaZNF830 induces hypersensitive celldeath response and defense responses
5.Discussion
5.1.Plant immunity against pathogens
5.1.1.CabZIP53 localizes to nucleus
5.1.2.CabZIP53 is induce by biotic or abiotic stresses
5.1.3.Transiently over-expressing CabZIP53 induce HR mimic cell death
5.1.4.CabZIP53 is targeted by Ca WRKY40
5.2.Nucleus based CaZNF830 regulate plant responses to stresses
5.2.1.Silencing of CaZNF830 impairs plant defense to stresses
5.2.2.Potential of CaZNF830 in crosstalk between plant response to biotic and abiotic stresses
Conclusion
References
Published papers and academic achievements during PhD program
Appendices
Solution formulations
ChIP
Western blotting
Plasmid maps
Acknowledgements
本文编号：3120823
【文章来源】：福建农林大学福建省
【文章页数】：103 页
【学位级别】：博士
【文章目录】：
List of Abbreviations
摘要
Abstract
1.Introduction
2.Review of Literature
2.1.Immune functions of plant bZIPs
2.1.1.Sub-cellular localization of bZIPs and defense related proteins
2.1.2.Signaling molecules and bZIPs
2.1.3.Elicitors versus bZIPs
2.2.ZNFs are in involved in vegetative growth and development
2.2.1.Sub-Cellular localization of ZNFs
2.2.2.ZNFs are involved in plant defense responses to pathogens
3.Materials and Methods
3.1 Plant materials and growth conditions
3.2 Cultivation of plant experimental materials
3.3 Agrobacterium culture and infection
3.4 Ralstonia solanacearum inoculation
3.5 The construction of vectors
3.5.1 Reactions for Gene amplification
3.5.2 PCR-Machine program
3.6 Separation and Purification of target gene DNA （amplified product） from Agarose Gel
3.6.1 BP reaction for ligating target gene with entry vector
3.6.2 LR reaction mixture to ligate gene with destination vectors
3.7 Vector Transformation into E.coli competent cell
3.8 E.coli.Plasmid extraction
3.9 Agrobacterium Transformation
3.10 Sub-cellular compartmentalization
3.11 Virus Induced Gene Silence （VIGS） of CabZIP53in pepper plants
3.11.1 Infiltration solution preparation
3.11.2 Virus induced gene silence （VIGS） procedure
3.11.3 Transient over expression of CabZIP53 in pepper leaves
3.12 Histochemical staining
3.12.1 Trypan Blue Staining
3.12.2 Trypan blue staining samples preparation
3.12.3 Decolorization of trypan blue stainedleaves
3.12.4 DAB （3, 3'-Diaminobenzidine） Staining
3.12.5 Decolorization of DAB treated leaves
3.13 RNA Extraction and cDNA synthesis
3.13.1 Sample collection for RNA extraction
3.13.2 Procedure of RNA Extraction
3.14 cDNA synthesis
3.14.1 PCR mixture
3.14.2 PCR program
3.14.3 PCR program used was as follow
3.15 Quantitative real-time-PCR （qRT-PCR）
3.15.1 Real time RT-PCR program
3.16 Chromatin immune-precipitation （ChIP） assay
3.17 Fluorometric GUS enzymatic assay
3.18 Immunoblotting
3.19 Protein Extraction
3.19.1 Solution-I（12%）
3.19.2 Procedure
4.Results
4.1 The sequence analysis and cloning of CabZIP53
4.1.1 The expression of CabZIP53 was transcriptionally modulated by Ralstonia inoculation （RSI）and HTHH
4.1.2 CabZIP53 is targeted to the nuclei
4.1.3 Pepper Basal defense is compromised by the silencing of CabZIP53
4.1.4 The transient over-expression of CabZIP53 promotes HR response by modulating theexpression of immunity and thermo-tolerance related genes
4.1.5 Ca WRKY40 makes positive feedback loop with CabZIP53 for resistance against RSI and HTHH
4.1.6 The possible transcriptional regulation of CabZIP53 by CaWRKY6, CaWRKY40 andCabZIP63
4.1.7 The assay on the possible interaction between CabZIP53 to itself and to CabZIP63
4.2 Isolation and characterization of CaZNF830
4.2.1.The expression of CaZNF830was transcriptionally modulated by Ralstonia inoculation andHTHH
4.2.2.Cellular compartmentalization of CaZNF830
4.2.3.Silencing of CaZNF830 enhance bacterial growth, compromises plant defense and HR-mediated resistance to RSI and thermo-tolerance
4.2.4.Agrobacterium mediated transient over-expression of CaZNF830 induces hypersensitive celldeath response and defense responses
5.Discussion
5.1.Plant immunity against pathogens
5.1.1.CabZIP53 localizes to nucleus
5.1.2.CabZIP53 is induce by biotic or abiotic stresses
5.1.3.Transiently over-expressing CabZIP53 induce HR mimic cell death
5.1.4.CabZIP53 is targeted by Ca WRKY40
5.2.Nucleus based CaZNF830 regulate plant responses to stresses
5.2.1.Silencing of CaZNF830 impairs plant defense to stresses
5.2.2.Potential of CaZNF830 in crosstalk between plant response to biotic and abiotic stresses
Conclusion
References
Published papers and academic achievements during PhD program
Appendices
Solution formulations
ChIP
Western blotting
Plasmid maps
Acknowledgements
本文编号：3120823
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