CabZIP53 and CaZNF830在辣椒應(yīng)答青枯菌侵染中的作用及機(jī)制分析
發(fā)布時(shí)間：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導(dǎo)致辣椒生產(chǎn)嚴(yán)重?fù)p失,在高溫高濕條件下（HTHH）損失尤其嚴(yán)重�；诳共∵z傳改良的辣椒抗病新品種選育是解決辣椒病害問(wèn)題的根本有效的技術(shù)對(duì)策,而解析辣椒免疫的分子機(jī)制是其重要基礎(chǔ)。由于植物抗病反應(yīng)在很大程度上受轉(zhuǎn)錄水平的調(diào)控,各種轉(zhuǎn)錄因子在其中起重要作用。因此,剖析轉(zhuǎn)錄因子的功能及其作用機(jī)制是闡明植物抗病機(jī)制的重要途徑和主要內(nèi)容。然而,不同轉(zhuǎn)錄因子在辣椒抗病中的作用及其機(jī)制仍不清楚。在本研究中,對(duì)兩個(gè)辣椒轉(zhuǎn)錄因子CabZIP53和CaZNF830的功能進(jìn)行了功能鑒定,主要結(jié)果如下:1.一個(gè)推定的bZIP轉(zhuǎn)錄因子（CA04g07280）的啟動(dòng)子上含有多個(gè)應(yīng)答病原菌的順式元件,且通過(guò)ChIP分析發(fā)現(xiàn)它可能是CaWRKY40的靶基因,而我們的前期研究發(fā)現(xiàn)CaWRKY40在辣椒應(yīng)答青枯菌侵染或高溫高濕逆境的防御反應(yīng)過(guò)程中均起正調(diào)節(jié)作用,暗示該基因可能在上述防御反應(yīng)中起作用。通過(guò)克隆該基因的全長(zhǎng)cDNA,發(fā)現(xiàn)其推導(dǎo)的氨基酸序列含有高度保守的bZIP結(jié)構(gòu)域,且在番茄的bZIP家族所有成員中與Stb...
【文章來(lái)源】：福建農(nóng)林大學(xué)福建省
【文章頁(yè)數(shù)】：103 頁(yè)
【學(xué)位級(jí)別】：博士
【文章目錄】：
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
本文編號(hào)：3120823
【文章來(lái)源】：福建農(nóng)林大學(xué)福建省
【文章頁(yè)數(shù)】：103 頁(yè)
【學(xué)位級(jí)別】：博士
【文章目錄】：
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
本文編號(hào)：3120823
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