Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23594
標題: Small GTPase AtRab8蛋白質家族及可與其結合之AtRTNLB蛋白質於農桿菌感染過程之功能分析
Functional studies of small GTPase AtRab8 family and its interacting proteins AtRTNLB during Agrobacterium infections
作者: 張欣農
Chang, Hsing Nung
關鍵字: Arabidopsis
阿拉伯芥
Agrobacterium
RAB
RTNLB
農桿菌
RAB
RTNLB
出版社: 生命科學系所
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摘要: 摘要 Small GTPase (鳥糞嘌呤核苷三磷酸酶)為一廣泛存在於真核生物中的蛋白質家族,數量龐大,以單聚體的形式存在細胞中並作用,主要參與訊息傳遞及細胞內物質運輸等功能。Rab GTPase為small GTPases家族中最大的次家族,在細胞中後生合成後,會在其蛋白質C端進行轉譯修飾(post-translational modification),加上疏水性基團,幫助Rab鑲嵌於膜上,調控膜狀系統的運輸,囊泡的形成及輸送和胞吞作用(endocytosis)與胞釋作用(exocytosis)的進行等。許多病原菌可選擇性利用Rab蛋白質,藉此幫助病原菌躲避宿主防禦反應與佔據宿主細胞,已知阿拉伯芥的AtRab8B可與AtRTNLB1、 2、4及農桿菌(Agrobacterium tumefaciens)的致病蛋白質(virulence protein,Vir) VirB2結合。並且發現農桿菌感染植株時,植株內AtRab8B的表現量多寡會影響植株被農桿菌感染之效率;因此本研究進一步檢測阿拉伯芥AtRab8家族成員及可與其結合之AtRTNLB於農桿菌感染過程中扮演之角色。首先以酵母菌雙雜合(yeast two-hybrid)實驗得知AtRab8A和8D’也可與農桿菌VirB2蛋白質結合。並利用AtRab8A、8C及8D的T-DNA插入突變株,進行農桿菌感染分析實驗,結果顯示部分的rab8A與rab8C突變株腫瘤生成之效率比野生株稍微增加,而rab8D突變株中則與野生株無明顯差異,綜合上述結果推測AtRab8A極可能參與農桿菌感染過程。此外也以反轉錄聚合酶連鎖反應(RT-PCR)檢測AtRab8B基因靜默轉殖株(RNAi Rab8B)或大量表現轉殖株(O/E Rab8B)中的病原菌相關基因(pathogen-related genes 1-5,PR1-5)與植物防禦基因(plant defensin 1.2,PDF1.2)表現量有無改變。在AtRab8B 基因靜默轉植株中PDF1.2、PR1、PR2與PR5基因之mRNA累積量有顯著之提高,而在O/E Rab8B轉殖株中,PR1-5及PDF1.2之mRNA累積量整體皆有下降的趨勢,顯示AtRab8B在植物防禦途徑中可能扮演著重要的角色。也以酵母菌雙雜合實驗得知AtRab8A和8D’也可與植物的AtRTNLB2、4、8蛋白質結合,推測AtRab8A或8D’可能與AtRTNLB2、4、8及農桿菌的VirB2蛋白質形成複合體,進而參與農桿菌感染植物過程。另外,利用雙分子螢光互補(bimolecular fluorescence complementation,BiFC)實驗得知AtRab8A、8C、8D、8E、8D’ 與AtRTNLB1、2、4任兩種蛋白質皆可能阿拉伯芥原生質體中相互結合,在植物細胞形成一蛋白質複合體而共同參與植物細胞中重要功能。
Abstract The small GTPase family contains numerous protein members in eukaryotes and functions as a monomeric protein in plants cells and is involved in various signaling pathways and the regulation of vesicular trafficking. After the Rab protein is translated in cells, a hydrophobic group is linked to the C-terminal regions of the Rab protein by post-translational modifications, which helps the Rab protein integrate into membranes. The Rab proteins participate the regulation of membrane trafficking, the formation of vesicles transport, the endocytosis and exocytosis pathways. There are some bacterial pathogens utilize the Rab GTPase to avoid the host defense response and help bacteria to infect host cells. A previous study showed that the Arabidopsis AtRab8B protein interacts with the AtRTNLB1, 2, 4, and Agrobacterium VirB2 in yeast and in vitro. The Agrobacterium transformation efficiencies increase and decrease in the over-expression and RNAi AtRab8B transgenic plants, respectively. The possible roles of AtRab8 family and its interacting proteins AtRTNLB during Agrobacterium infections were characterized further in this study. In order to determine if other members of AtRab8 may participate Agrobacterium transformation process, we cloned AtRab8A, 8C-8E, and 8D' genes from Arabidopsis and performed yeast-two hybrid assays between AtRab8 proteins and several Vir proteins. Only AtRab8A and 8D' interacted with VirB2 in yeast. Additionally, we performed Agrobacterium transformation assays with Arabidopsis rab8A, rab8C and rab8D T-DNA insertion mutants. Some of the rab8A and rab8C mutants showed 25-35% increase in tumor formation efficiencies in comparison to wild-type plants. However, no significant difference of transformation efficiency between the rab8D mutant and wild-type plants were observed. These data suggest AtRab8A may be involved in the Agrobacterium-mediated plant transformation process. We also performed RT-PCR (reverse transcription polymerase chain reaction) to determine RNA accumulation levels of the pathogen-related gene 1-5 (PR1-5) and plant defesin gene 1.2 (PDF1.2) in AtRab8B RNAi and over-expression transgenic plants and wild-type plants. The AtRab8B RNAi plants accumulated more PDF1.2, PR1, PR2, and PR5 RNA, while the over-expression AtRab8B transgenic accumulated less PR1-5 and PDF1.2 than in wild-type plants. These data suggested that AtRab8B may participate the plant defense response. Yeast two-hybrid results also showed that AtRab8A and 8D' interacted with AtRTNLB2, 4, 8. Bimolecular fluorescence complementation (BiFC) test results demonstrated that AtRab8A ,8C-8E, 8D' and AtRTNLB1, 2, 4, may form a protein complex and may play a important role in plant cells.
URI: http://hdl.handle.net/11455/23594
其他識別: U0005-0302201216021700
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