CaWRKY22在辣椒應(yīng)答青枯菌侵染中的作用及其機(jī)制分析
發(fā)布時(shí)間：2024-04-26 21:10
辣椒無論在中國(guó)還是全世界均是一種重要的蔬菜和工業(yè)原料作物,其生產(chǎn)效益高。然而,辣椒也是土傳病原菌多的典型茄科植物,病害的發(fā)生常常影響其產(chǎn)量和品質(zhì)最終導(dǎo)致其效益下降。培育和推廣應(yīng)用抗病的辣椒品種是解決其生產(chǎn)中病害問題的最經(jīng)濟(jì)有效對(duì)策,而闡明抗病分子機(jī)制則是開展辣椒有效遺傳改良的重要基礎(chǔ)。鑒于植物抗病在很大程度上受到轉(zhuǎn)錄水平的調(diào)節(jié),而WRKY轉(zhuǎn)錄因子在植物抗病中起重要調(diào)節(jié)作用,開展WRKY在辣椒等茄科植物抗病中的作用及機(jī)制研究是闡明抗病機(jī)制的重要途徑。鑒于此,本研究開展了一個(gè)lle WRKY轉(zhuǎn)錄因子成員在辣椒抗青枯病中的作用,并分析了其作用機(jī)制,主要研究結(jié)果如下:1、通過全基因組WRKY基因啟動(dòng)子上順式作用元件的掃描分析,發(fā)現(xiàn)一個(gè)WRKY家族成員啟動(dòng)子中含有應(yīng)答病原菌的順式作用元件,其推導(dǎo)氨基酸序列中含有保守的WRKY結(jié)構(gòu)域,在其C端含有(C-X5-C-X23-H-X1-H)鋅指基序,且發(fā)現(xiàn)該基因的推導(dǎo)氨基酸序列在野生種潘那利番茄、馬鈴薯、美花煙草和二倍體棉花等植物的所有WRKY家族成員中,分別與SpWRKY22、StWRKY22、NsWRKY22和GrWRKY22擁有最高的同源性,分別為...
【文章頁(yè)數(shù)】：90 頁(yè)
【學(xué)位級(jí)別】：博士
【文章目錄】：
Abbreviations
摘要
Abstract
1.Introduction
1.1.Plant immune responses to pathogens
1.2.Transcriptional reprogramming and phytohormones for plant defence
1.3.WRKY Transcription factors
1.4.Pepper and bacterial wilt disease
2.Review of Literature
2.1 Functional significance of WRKY TFs
2.2 WRKY TFs regulate plant innate immunity
2.3 WRKY TFs may positively or negatively regulate plant basal defense
2.4 Phytohormones and WRKY TFs
3.Material and Methods
3.1 Plant materials and growth conditions
3.2 Plant experimental materials cultivation
3.3 Culture and Infection of Agrobacterium
3.4 Inoculation of R.solanacearum
3.5 Vectors Construction
3.5.1 Gene amplification reactions
3.5.2 PCR programs
3.6 Isolation and refinement of target gene DNA fragment from Agarose Gel
3.6.1 Ligation process to ligate target gene with entry vectors by BP reaction mixture
3.6.2 Ligation with destination vectors by LR reaction mixture
3.7 Vectors transformation into E.coli cells
3.8 Plasmid extraction from E.coli
3.9 Transformation into Agrobacterium
3.10 Checking of Subcellular location
3.11 VIGS (Virus Induced Gene Silencing) of CaWRKY22 in pepper
3.11.1 Preparation of Infiltration Buffer
3.11.2 Procedure of VIGS virus induced gene silencing
3.11.3 Transient over-expression of CaWRKY22 in pepper leaves
3.12 Histochemical staining
3.12.1 Staining.by using Trypan.Blue
3.12.2 Samples preparation for Trypan blue staining
3.12.3 Destaining of trypan blue staining samples
3.12.4 DAB Staining (3,3'-Diaminobenzidine.)
3.12.5 Destaining of DAB (3,3'-Diaminobenzidine) stained samples
3.13 Extraction of RNA and cDNA synthesis
3.13.1 collection of samples for RNA extraction
3.13.2 Procedure of RNA Extraction
3.14 Synthesis of cDNA
3.14.1 Formulation of PCR mix
3.14.2 PCR procedure
3.15 (QRT-PCR) Quantitative-real-time-PCR
3.15.1 QRT-PCR Real time-PCR program
3.16 ChIP analysis-Chromatin immuno-precipitation
3.17 Extraction of Protein
3.17.1 Solution-1(12%)
3.17.2 Procedure
4.Results
4.1 Cloning and sequencing of CaWRKY22 cDNA
4.2 The expression of CaWRKY22 was transcriptionally modulated by RSI
4.3 CaWRKY22 localized to the nuclei
4.4 The silencing of CaWRKY22 reduced the resistance of pepper to Ralstonia solanacearum inoculationRSI
4.5 Transient over-expression of CaWRKY22 caused HR, cell death, and accumulation of H2O2 in theleaves of pepper plants
4.6 CaWRKY22 binds to the W-box and activates transcription of different marker genes in a W-boxdependent manner
4.7 The transcriptional modulation of marker genes by transient over-expression and virus inducedsilencing of CaWRKY22
4.8 The inter-relationship between CaWRKY22 and CaWRKY40
4.9 The inter-relationship between CaWRKY22 and other WRKYs including CaWRKY40, CaWRKY27,CaWRKY40 and CaWRKY58
Discussion
Conclusion
References
Appendices
Formulations of Solutions used
Chromatin Immuno-precipitation (ChIP)
Western Blotting Reagents
Laboratory Instruments used
Chemicals used in different experiments
Bioinformatics websites used
Supplementary Table 1
Supplementaiy Table 2
Supplementary Table3
Plasmid maps
本文編號(hào)：3964880
【文章頁(yè)數(shù)】：90 頁(yè)
【學(xué)位級(jí)別】：博士
【文章目錄】：
Abbreviations
摘要
Abstract
1.Introduction
1.1.Plant immune responses to pathogens
1.2.Transcriptional reprogramming and phytohormones for plant defence
1.3.WRKY Transcription factors
1.4.Pepper and bacterial wilt disease
2.Review of Literature
2.1 Functional significance of WRKY TFs
2.2 WRKY TFs regulate plant innate immunity
2.3 WRKY TFs may positively or negatively regulate plant basal defense
2.4 Phytohormones and WRKY TFs
3.Material and Methods
3.1 Plant materials and growth conditions
3.2 Plant experimental materials cultivation
3.3 Culture and Infection of Agrobacterium
3.4 Inoculation of R.solanacearum
3.5 Vectors Construction
3.5.1 Gene amplification reactions
3.5.2 PCR programs
3.6 Isolation and refinement of target gene DNA fragment from Agarose Gel
3.6.1 Ligation process to ligate target gene with entry vectors by BP reaction mixture
3.6.2 Ligation with destination vectors by LR reaction mixture
3.7 Vectors transformation into E.coli cells
3.8 Plasmid extraction from E.coli
3.9 Transformation into Agrobacterium
3.10 Checking of Subcellular location
3.11 VIGS (Virus Induced Gene Silencing) of CaWRKY22 in pepper
3.11.1 Preparation of Infiltration Buffer
3.11.2 Procedure of VIGS virus induced gene silencing
3.11.3 Transient over-expression of CaWRKY22 in pepper leaves
3.12 Histochemical staining
3.12.1 Staining.by using Trypan.Blue
3.12.2 Samples preparation for Trypan blue staining
3.12.3 Destaining of trypan blue staining samples
3.12.4 DAB Staining (3,3'-Diaminobenzidine.)
3.12.5 Destaining of DAB (3,3'-Diaminobenzidine) stained samples
3.13 Extraction of RNA and cDNA synthesis
3.13.1 collection of samples for RNA extraction
3.13.2 Procedure of RNA Extraction
3.14 Synthesis of cDNA
3.14.1 Formulation of PCR mix
3.14.2 PCR procedure
3.15 (QRT-PCR) Quantitative-real-time-PCR
3.15.1 QRT-PCR Real time-PCR program
3.16 ChIP analysis-Chromatin immuno-precipitation
3.17 Extraction of Protein
3.17.1 Solution-1(12%)
3.17.2 Procedure
4.Results
4.1 Cloning and sequencing of CaWRKY22 cDNA
4.2 The expression of CaWRKY22 was transcriptionally modulated by RSI
4.3 CaWRKY22 localized to the nuclei
4.4 The silencing of CaWRKY22 reduced the resistance of pepper to Ralstonia solanacearum inoculationRSI
4.5 Transient over-expression of CaWRKY22 caused HR, cell death, and accumulation of H2O2 in theleaves of pepper plants
4.6 CaWRKY22 binds to the W-box and activates transcription of different marker genes in a W-boxdependent manner
4.7 The transcriptional modulation of marker genes by transient over-expression and virus inducedsilencing of CaWRKY22
4.8 The inter-relationship between CaWRKY22 and CaWRKY40
4.9 The inter-relationship between CaWRKY22 and other WRKYs including CaWRKY40, CaWRKY27,CaWRKY40 and CaWRKY58
Discussion
Conclusion
References
Appendices
Formulations of Solutions used
Chromatin Immuno-precipitation (ChIP)
Western Blotting Reagents
Laboratory Instruments used
Chemicals used in different experiments
Bioinformatics websites used
Supplementary Table 1
Supplementaiy Table 2
Supplementary Table3
Plasmid maps
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