Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/53773
標題: Functional Studies of the Agrobacterium Tumefaciens Tzs Protein and Plant Hormone, Cytokinin, on Agrobacterium-Mediated Plant Transformation Processes
功能分析農桿菌反玉米素合成蛋白質及植物細胞分裂素對土壤農桿菌感染植物過程之影響
作者: 黃皓瑄
賴爾??
關鍵字: 基礎研究
Agrobacterium tumefaciens
生物科學類
農桿菌
反玉米素合成蛋白質
Tzs
trans-zeatin
摘要: 土壤農桿菌感染自然界中許多重要經濟作物,使作物出現腫瘤病徵,作物因而生長遲緩,收成減少。這是由於菌體內Ti 質體(tumor-inducing plasmid)上一段轉移DNA (transfer-DNA, T-DNA)會被嵌入植物基因體中所致。為進一步了解農桿菌如何感染植物,我們利用蛋白質體學的方法,發現農桿菌中的反玉米素合成蛋白質(Tzs:trans-zeatin synthesizing protein)會被acetosyringone (AS,為一已知的感病基因誘導物)誘導表現。tzs 基因在農桿菌中可產生異戊烯轉移酶(iso-pentenyl transferase),此酵素負責催化細胞分裂素(cytokinin)的合成。為進一步瞭解tzs 基因,如何參與農桿菌感染植物過程。我們構築了tzs 基因的突變株,並利用此突變株在阿拉伯芥(Arabidopsis thaliana)及馬鈴薯上進行感染測試。結果顯示:tzs 突變株致病力較野生種低。同時突變株的短暫轉殖效率也較野生種低,我們推測Tzs 蛋白質可能參與農桿菌感染植物過程,且可能是在T-DNA 嵌入植物基因體的步驟之前。我們同時使用tzs 突變株在不同種植物上進行測試。初步結果顯示tzs 突變株感染不同種植物之能力各不相同。另外,在AS 處理下,tzs突變株生長較為遲緩或甚至死亡。因此,我們將進行一為期三年的研究,進一步瞭解Tzs 蛋白質調控及功能分析。首先進行基因及化學互補實驗(genetic and chemicalcomplementation tests)以測試tzs 突變株感染力降低及生長遲緩,是否因tzs 基因不表現和農桿菌中細胞分裂素量減少所致。我們也將在農桿菌野生種及數個致病(vir)基因的突變種中,檢測Tzs 蛋白質表現量,以進一步瞭解Tzs 蛋白質如何被調控。另外,我們將利用基因體學及蛋白質體學方式,來分析在tzs 突變株中其他基因的表現量,再進一步分離會受tzs 基因調控的細菌基因和蛋白質。我們也將利用基因體學及蛋白質體學方式,分析及分離會受細胞分裂素調控的細菌基因和蛋白質。此研究成果可幫助我們進一步瞭解Tzs 和細胞分裂素(cytokinin)在農桿菌感染植物過程所扮演角色。
Agrobacterium tumefaciens is a soil phytopathogen that genetically transforms host cells causingcrown gall tumors, an agronomically important disease that affects most dicotyledonous plants. A.tumefaciens transforms plant cells by transferring the T-DNA (transfer-DNA) of the Ti plasmid(tumor-inducing plasmid) to plant cells and integrating it into the nuclear genome. In order to elucidatefurther the molecular mechanism of the A. tumefaciens-mediated plant transformation process, we haveidentified several acetosyringone (AS)-induced A. tumefaciens proteins using two-dimensional gelelectrophoresis (2-DE). Several known AS-induced proteins including Tzs and two newly discoveredAS-induced proteins, Y4mc and HspL, were identified. The tzs (trans-zeatin synthesizing) gene in theNopaline strains of A. tumefaciens encodes a cytokinin biosynthetic prenyl transferase. To determinethe role(s) of tzs in A. tumefaciens virulence, tzs deletion and frame-shift mutants were generated andcharacterized. Quantitative tumor assays demonstrated that tzs deletion and frame-shift mutantsdecreased their ability to cause tumors on Arabidopsis roots and potato tubers. Additionally, tzsmutants reduce transient transformation efficiencies in Arabidopsis roots, suggesting that Tzs might beinvolved in step(s) prior to T-DNA integrations. Tumor assays of other host plants were also tested todetermine if Tzs may be a host range factor. The tzs mutants were able to enhance transformationefficiencies on green pepper and cowpea stems, reduce transformation efficiency on pai-tsai, loose leaflettuce, and carnation, but not on other plant species tested so far. Interestingly, the tzs deletion mutantsare impaired in cell viability and/or growth in AS-induced conditions. Here, we propose a three-yearresearch proposal to elucidate the AS-induced regulation analyses of Tzs and the potential role(s) of Tzsand cytokinin (trans-zeatin) in A. tumefaciens virulence and bacterial growth. We will perform geneticand chemical complementation tests of tzs mutants to determine if the absence of tzs and/or cytokininwill cause the decrease of virulence and bacterial growth in tzs mutants. We will examine the Tzsprotein expressions in the wild-type A. tumefaciens and various vir mutants under different conditions todetermine the regulation of Tzs in A. tumefaciens. Additionally, genomic and proteomic analyses of tzsmutants will be conducted to examine if other bacterial gene expressions will be affected by the absenceof tzs in A. tumefaciens. Similarly, genomic and proteomic analyses of Agrobacterium under thetreatments of cytokinins will be performed to determine if any bacterial gene expression will be affected.The results gained from this research proposal shall provide important information on the role(s) of Tzsand/or cytokinin in Agrobacterium-mediated plant transformation process and bacterial growth underinfection conditions.
URI: http://hdl.handle.net/11455/53773
其他識別: NSC97-2311-B005-002-MY3
Appears in Collections:生命科學系所

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