臺灣博碩士論文加值系統

十字花科黑腐病菌 ( Xanthomonas campestris ) 屬於格蘭氏陰性菌，需仰賴第二型蛋白分泌系統將各種水解酵素分泌至胞外，藉此感染十字花科植物。第二型蛋白分泌機器由XpsD~XpsO等十二個蛋白所組成。XpsE是唯一的胞內蛋白，和內胞膜上的XpsL蛋白有交互作用關係；並且擁有四個nucletide binding motifs，具有微弱的ATPase活性。在與ATP結合情況下，會促進XpsE形成六聚體，進而與XpsL朝向胞內的N端區域 ( XpsLN ) 結合，隨後XpsLN則會促進XpsE蛋白水解ATP。XpsF為穿膜三次的內胞膜蛋白，由穿膜區將其切割成N端（XpsFN）及C端（XpsFC）兩個位於胞內的蛋白區塊，目前對其功能尚有待釐清。過去分析XpsF蛋白功能時，發現xpsF基因缺損會使菌株喪失分泌澱粉酵素的功能，顯示XpsF蛋白在此機器運作中扮演必要角色。先前利用yeast two hybrid system針對位於胞內的Xps蛋白區塊之間相互作用的分析，發現XpsFc分別與XpsFN、全長XpsE以及XpsLN皆有交互作用發生，暗示XpsE、XpsF以及XpsL三者之間有互動關係。為了進一步探討XpsF蛋白與XpsL或XpsE之間的相互作用，本文利用in vitro pull down 實驗進行分析，發現無論是否添加不同的nucleotides，XpsFN或XpsFC與全長XpsE蛋白或與XpsLN的交互作用皆極微弱，即使有MBP-XpsLN或XpsFC蛋白的存在也無顯著促進現象。其次，利用ADP-AlF4模擬ATP transition state偵測XpsE與XpsLN的結合情形以及XpsF在這當中可能扮演的角色，發現XpsE與ADP-AlF4結合不會影響它與XpsLN的交互作用，且XpsE與XpsLN的結合也沒有因為XpsFN或XpsFC的存在而改變。Gel filtration結果指出XpsE結合ADP-AlF4會促進六聚體的形成，因此推測當XpsE水解ATP進入transition state時，可能仍維持在六聚體的狀態 ，並依然結合在XpsL上，而XpsF在此分泌機制中所擔任的任務仍有待進一步探討。

Xanthomonas campestris pv. campestris is a Gram-negative bacterium that relies on type II secretion system for secreting hydrolytic enzymes in their infection of plant leaves. The X. campestris type II secretion system is composed of 12 Xps protein components, of which XpsE is the only cytoplasmic protein consisting of four conserved nucleotide binding motifs. It is known to interact with the inner membrane protein XpsL. Triggered by ATP-binding, XpsE oligomerizes and associates with the N terminal domain of XpsL. Such interaction appears to stimulate ATP hydrolysis by XpsE. XpsF is predicted to be a polytopic inner membrane protein with two large cytoplasmic domains divided by three transmembrane sequences. Its biological function in type II secretion system remains poorly understood. Previous yeast two hybrid analyses revealed that the C-terminal domain of XpsF (XpsFC) interacts with its own N-terminal domain (XpsFN), with the N-terminal domain of XpsL (XpsLN) and with XpsE, implicating interactive relationship among XpsE, XpsF and XpsL. To further characterize physical interaction of XpsF with XpsL and with XpsE, detailed in vitro pull down assays were performed in this study. The results indicated that the interactions between XpsFN or XpsFC and XpsLN or XpsE, with or without preincubating with different nucleotides, are all very weak. Neither could stimulation be observed in presence of MBP-XpsLN or Nus-XpsFch,. Interaction between XpsE and XpsLN were also analyzed in presence of ADP-AlF4, an analogue mimicking ATP transition state, or in presence of XpsFN or XpsFC. Pull down assay revealed that ADP-AlF4 has no effect on the interaction between XpsE and XpsLN. Neither does XpsFN or XpsFC. Gel filtration chromatography revealed that preincubation of XpsE with ADP-AlF4 stimulated its oligomerization, implying XpsE bound to ATP transition state remains oligomeric and associated with XpsLN. As to the role of XpsF in type II secretion system, further studies are required.

前言 ……………………………………………………………………1
材料方法…………………………………………………………………7
材料
一、菌種及質體 ........................................7
二、培養機、抗生素、試劑與緩衝液........................7
實驗方法
一、蛋白質的純化與萃取....................................7
1.XpsE-Strep蛋白之純化..................................7
2.XpsFNh與Nus-XpsFch蛋白之純化..........................7
3.MBP-XpsLN蛋白之純化...................................8
二、Pull down assay.......................................8
1.XpsFNh pull down assay.................................8
(1)蛋白定量..............................................8
(2)XpsFNh pull down assay................................9
2.Nus-XpsFch pull down assay............................9
3.MBP-XpsLN pull down assay.............................10
三、蛋白質SDS-PAGE（sodium dodecyl sulfate-polyacrylamide gel electrophoresis）電泳分析............................11
四、西方墨點法（Western blot）...........................11
五、分子篩管柱層析（Size exclusion chromatography）......12
結果.....................................................13
討論.....................................................20
參考文獻.................................................25
圖.......................................................31
附表.....................................................47
附錄.....................................................49

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