Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/97779
DC FieldValueLanguage
dc.contributor賴爾珉zh_TW
dc.contributorErh-Min Laien_US
dc.contributor.author施博原zh_TW
dc.contributor.authorPo-Yuan Shihen_US
dc.contributor.other生物科技學研究所zh_TW
dc.date2018zh_TW
dc.date.accessioned2019-03-22T06:05:12Z-
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dc.identifier.urihttp://hdl.handle.net/11455/97779-
dc.description.abstract農桿菌是一種會讓植物生成癌腫病的土壤病原菌,藉由第四型分泌系統把特定DNA片段轉殖到植物染色體上,進而促使植物細胞增生形成腫瘤,因此科學家利用此機制來進行基因轉殖。過去的研究發現,農桿菌感染的成效並不單取決於細菌本身,許多植物內部系統,例如免疫系統和賀爾蒙反應都會影響到轉殖的效率,然而關於植物因子如何調控農桿菌的轉殖效率仍有很多需要被研究的地方。因此本研究利用轉錄體學來找出會影響轉殖效率的植物因子,從野生型阿拉伯芥Col-0小苗被農桿菌短暫型轉殖後的基因表現中,我發現根部和莖部的反應基因主要參與在免疫反應、生長調控、次級代謝物代謝、接受外界環境刺激和基礎細胞內反應之上。進一步的研究發現,次級代謝物glucosinolates和camalexin可能在防禦機制上扮演著重要的角色,利用突變株和代謝體學分析,我發現indole glucosinolate的水解對於初期防禦農桿菌感染很重要,而camalexin的累積則會抑制後期的感染和腫瘤發育;另外aliphatic glucosinolate的水解產物則對短暫型的轉殖效率有多樣的影響。這些研究成果提供了一個新的思考方向,將來或許可以利用植物次級代謝物來控制癌腫病或是操控轉殖效率。除此之外,由於EFR已被證實是個上游的免疫受器,能辨識農桿菌的轉錄延伸因子(EF-Tu)來啟動免疫反應並抑制農桿菌轉殖,因此本研究也比較Col-0和efr-1突變株中的基因表現差異,期望找出能調控農桿菌轉殖效率的EFR下游基因。我初步發現許多根部和莖部基因的表現量在Col-0和efr-1中有顯著的差異,暗示著這些基因可能是受到EFR的調控。由於已知的資料顯示EFR主要表現在莖部而非根部,且多數篩選出的莖部基因也同時受到EF-Tu辨識序列elf26的調控,這些結果都支持EFR主要負責莖部對農桿菌的反應而非根部。因此我利用不同的策略,從莖部的資料中篩選潛在的EFR調控基因,利用小苗短暫型轉殖系統在突變株的實驗結果顯示,部分突變株確實會影響到農桿菌的轉殖效率,這些證據支持了上述的篩選結果能提供合適的研究名單,將來則可利用阿拉伯芥的原生質體轉殖系統來做更進一步的功能性分析,以探討EFR是如何抑制農桿菌感染的機制;另一方面,由於植物根部的反應主要並非受EFR調控,所以我們目前先挑選出在Col-0和efr-1皆高度表現的基因,來做未來的功能性探討。zh_TW
dc.description.abstractAgrobacterium tumefaciens is a plant pathogenic bacterium which can cause crown gall disease by transferring a piece of DNA from bacterial cells into plant genome via a Type IV secretion system (T4SS). Due to this unique inter-kingdom DNA transfer capability, A. tumefaciens has been widely used as a gene transfer tool to generate transgenic plants. Previous studies have shown that many plant factors such as immune system and hormone responses are important to determine transformation efficiency. However, the plant genes and mechanisms involved in the transformation process are not well understood. In this study, I employed Agrobacterium-mediated transient transformation system on Arabidopsis seedling and used transcriptome analysis of seedling shoots and roots to dissect plant genes responding to Agrobacterium infection. From the gene expression profiles in wild type Col-0 Arabidopsis thaliana seedlings, common and distinct differentially expressed genes involved in growth regulation, response to stimuli, secondary metabolism and cellular reactions were enriched in both shoots and roots. Further analysis on secondary metabolism ontology revealed that the plant secondary metabolites, glucosinolates (GS) and camalexin, may play important roles to modulate Agrobacterium infection and impact transformation efficiency. By combining metabolism and transformation assays using relevant Arabidopsis mutants, the results suggest their differential roles at different infection stages. Indole GSs can be rapidly hydrolyzed to inhibit Agrobacterium infection at the early stage, and then accumulation of camalexin can further inhibit later infection to reduce tumor formation. Moreover, the hydrolysis products of aliphatic GSs have various effects on the transient transformation efficiency. These findings provide a new insight into using plant secondary metabolites to protect plants from Agrobacterium infection or to manipulate transformation efficiency. In addition, I also compared the gene expression profiles between Col-0 and an immune receptor mutant, efr-1, which is defective in perceiving conserved N-terminal epitope (elf26) of the elongation factor Tu, a potent microbe-associated molecular pattern (MAMP) of Agrobacterium. EFR has been shown as the first layer of immune receptor to suppress Agrobacterium transient transformation. I found several differentially expressed shoot and root genes displaying dramatic difference of their expression levels between Col-0 and efr-1, suggesting their gene expression regulated by EFR. The EFR expression patterns in the available microarray data suggested EFR mainly expresses in shoots but not roots, and the comparison between the identified genes and the elf26-responsive genes indicated a half of identified shoot genes can be regulated by elf26. This data supported that EFR mainly contributes to shoot but not root responses to Agrobacterium infection. The seedling transient transformation assay, assessed by T-DNA-encoded beta-glucuronidase (GUS) activity, on the selected mutants suggested that some of EFR-dependent shoot genes may regulate transformation efficiency. To this end, Arabidopsis protoplast system has been used to dissect the roles of EFR-dependent genes in EFR-dependent immune signaling pathways which may regulate Agrobacterium-mediated transformation efficiency. In addition, the genes that are highly expressed in roots of both Col-0 and efr-1 could be used as candidate genes to study underlying mechanisms how roots respond to Agrobacterium infection.en_US
dc.description.tableofcontentsAcknowledgement i 中文摘要 ii Abstract iv Table of Contents vii List of Tables ix List of Figures x List of Appendices xii Chapter 1 General introduction 1 1.1 Agrobacterium virulence genes and their roles in T-DNA transfer 2 1.2 Plant factors involved in Agrobaceterium-mediated plant transformation 4 1.3 Agrobacterium–mediated transformation and Plant pattern-triggered immunity (PTI) 8 1.4 Agrobacterium-mediated transformation and Plant hormone system 13 1.5 Plant transcriptome analysis in response to Agrobacterium infection/transformation 15 Chapter 2 Transcriptome analysis of Arabidopsis seedlings to Agrobacterium infection reveals tissue-specific regulation and differential roles of glucosinolates and camalexin in Agrobacterium-mediated transformation efficiencies 19 Abstract 20 Introduction 22 Materials and Methods 25 Results 30 Discussion 43 Tables 51 Figures 64 Chapter 3 Identification of Arabidopsis genes involved in EFR-dependent pathways in response to Agrobacterium infection 79 Abstract 80 Introduction 82 Materials and Methods 85 Results 90 Discussion 98 Tables 106 Figures 122 Chapter 4 Conclusions and future prospects 136 References 142 Appendices 161 Resume 166zh_TW
dc.language.isoen_USzh_TW
dc.rights同意授權瀏覽/列印電子全文服務,2021-01-16起公開。zh_TW
dc.subject農桿菌zh_TW
dc.subject農桿菌媒介轉殖法zh_TW
dc.subjectglucosinolatezh_TW
dc.subjectcamalexinzh_TW
dc.subjectEFRzh_TW
dc.subject植物免疫zh_TW
dc.subject癌腫病zh_TW
dc.subject轉錄體學zh_TW
dc.subjectAgrobacterium tumefaciensen_US
dc.subjectAgrobacterium-mediated plant transformationen_US
dc.subjectglucosinolateen_US
dc.subjectcamalexinen_US
dc.subjectcrown gallen_US
dc.subjectEFRen_US
dc.subjectplant immuneen_US
dc.subjecttranscriptomeen_US
dc.title利用轉錄體學研究農桿菌媒介阿拉伯芥轉殖效率之基因 及次級代謝物zh_TW
dc.titleEmployment of transcriptome analysis for studies of Arabidopsis genes and secondary metabolites involved in Agrobacterium-mediated transformationen_US
dc.typethesis and dissertationen_US
dc.date.paperformatopenaccess2019-01-16zh_TW
dc.date.openaccess2021-01-16-
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
item.openairetypethesis and dissertation-
item.cerifentitytypePublications-
item.fulltextwith fulltext-
item.languageiso639-1en_US-
item.grantfulltextrestricted-
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