CaWRKY22在辣椒应答青枯菌侵染中的作用及其机制分析

发布时间：2024-04-26 21:10

　　辣椒无论在中国还是全世界均是一种重要的蔬菜和工业原料作物,其生产效益高。然而,辣椒也是土传病原菌多的典型茄科植物,病害的发生常常影响其产量和品质最终导致其效益下降。培育和推广应用抗病的辣椒品种是解决其生产中病害问题的最经济有效对策,而阐明抗病分子机制则是开展辣椒有效遗传改良的重要基础。鉴于植物抗病在很大程度上受到转录水平的调节,而WRKY转录因子在植物抗病中起重要调节作用,开展WRKY在辣椒等茄科植物抗病中的作用及机制研究是阐明抗病机制的重要途径。鉴于此,本研究开展了一个lle WRKY转录因子成员在辣椒抗青枯病中的作用,并分析了其作用机制,主要研究结果如下:1、通过全基因组WRKY基因启动子上顺式作用元件的扫描分析,发现一个WRKY家族成员启动子中含有应答病原菌的顺式作用元件,其推导氨基酸序列中含有保守的WRKY结构域,在其C端含有(C-X5-C-X23-H-X1-H)锌指基序,且发现该基因的推导氨基酸序列在野生种潘那利番茄、马铃薯、美花烟草和二倍体棉花等植物的所有WRKY家族成员中,分别与SpWRKY22、StWRKY22、NsWRKY22和GrWRKY22拥有最高的同源性,分别为...

【文章页数】：90 页

【学位级别】：博士

【文章目录】：
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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