跳到主要內容

臺灣博碩士論文加值系統

(54.172.135.8) 您好!臺灣時間:2022/01/18 16:10
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:李振東
論文名稱:以金屬離子親和性管柱層析及酵母菌二雜交法分析十字花科黑腐細菌XpsE、XpsF、XpsL、XpsM與XpsN蛋白之複合體關係
論文名稱(外文):Affinity-chromatography and yeast-two hybrid analysis of the complex formation among XpsE, -F, -L, -M and -N in xanthomonas campestris
指導教授:呂維茗
學位類別:碩士
校院名稱:國立中興大學
系所名稱:農業生物科技學研究所
學門:農業科學學門
學類:農業技術學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:65
中文關鍵詞:金屬離子親和性管柱層析酵母菌二雜交法十字花科黑腐細菌
外文關鍵詞:Xanthomonas campestrisAffinity-ChromatographyYeast-two hybrid
相關次數:
  • 被引用被引用:3
  • 點閱點閱:822
  • 評分評分:
  • 下載下載:147
  • 收藏至我的研究室書目清單書目收藏:0
中文摘要
十字花科黑腐細菌在農業上是很重要的致病菌,與其它致病性細菌相同,它的分泌機器之完整與致病能力息息相關,其分泌機制隸屬於第二型的蛋白分泌系統,此分泌系統的最大特色是與SEC蛋白群有依賴關係,亦即欲分泌至胞外的蛋白在細胞質內產生後,需先透過SEC蛋白群送至細菌的細胞周質區(periplasm),再透過第二型分泌途徑送至胞外。黑腐細菌第二型分泌系統相關的蛋白至少有11個,已知其中XpsE為一細胞質蛋白,具有ATP binding motif,可藉由XpsL附著於內胞膜上,一般相信其最主要的角色應為提供分泌機器動作所須的能量;XpsL、XpsM、XpsN三者均為內膜蛋白,以XpsM蛋白居中形成複合體,倘若缺乏其中任何一種蛋白,則另二種蛋白含量均劇降;XpsD為一外膜蛋白,以至少12個左右的單元形成一規律的結構,推測其即為分泌的孔道。至於XpsF則為一穿過膜三次的內胞膜蛋白,推測其有兩種可能的角色,一作為組合分泌機器蛋白群的平台,或者作為輔助蛋白摺疊的chaperone,然而目前均欠缺證據,本篇研究根據XpsF絕大部份的蛋白區塊均面向細胞質的事實,推測XpsF應與XpsL或XpsE蛋白具有交互作用關係。
先前本實驗室曾利用XpsE、XpsF、XpsL、XpsM及XpsN蛋白共同過量表現的菌株,以免疫共沉澱法證實這五個蛋白可形成複合體,惟複合體含量不高。本篇研究則針對蛋白表現並不高但具有分泌能力的菌株,使用金屬離子親和性管柱大量吸附XpsL-(His)6融合蛋白,期望可同時吸附住XpsF蛋白,然而發現仍僅能偵測到XpsL/M/N蛋白複合體。在測試各種xps單一基因缺失的菌株後,我們結論在此些菌株中其XpsL/M/N與XpsL/E等蛋白複合體含量,均不會因為分泌動作無法完成而明顯改變。最後本研究使用酵母菌二雜交系統,分析除去嵌膜區域的各種Xps蛋白區塊是否具有交互作用,並定量分析其作用力的強弱。
Abstact
Xanthomonas campestris is an important phytopathogen in which the integrity of secretion machinery is critical for its pathogenicity. The type II secretion pathway (also known as the general secretion pathway) that requires the Sec proteins is known to exist in Xanthomonas. The type II secretion pathway is constituted by a stepwise process: secreted proteins are firstly translocated by sec-dependent mechanism through the inner membrane, released into the periplasm, and transported across the outer membrane by the so-called Gsp proteins. The Gsp proteins in Xanthomonas campestris contain at least 11 members. XpsE, with the ATP-binding motif, is a cytoplasmic protein associated with the cytoplasmic membrane through interaction with XpsL. XpsL, -M and -N are cytoplasmic membrane proteins that assembly as ternary complex. The stabilities of these three proteins are mutually dependent. XpsD is an outer membrane which forms the secretion pore. XpsG probably assemble with XpsH, -I, -J and -K and form pilus-like structure in periplasm. In contract to the above, roles of XpsF, a cytoplasmic membrane protein with two main domains facing inward, remain unknown thus far. It was speculated that XpsF may act, as platform for apparatus assemble or alternatively, as chaperon for protein folding. According to the predicated membrane topology, it is proposed that XpsF may interact with XpsE or XpsL, the other two Xps members with major cytoplasmic domains.
Demonstrated by co-immunoprecipitation, XpsF was found to form complex with XpsE and XpsL/M/N, although only to a scarce amount under overexpressed condition. Using metal-affinity chromatography, we aimed to detect the complex formation among the above five proteins in strains with secretion ability. Furthermore, xps chromosomal mutants were examined to evaluate the possibility that the secretion complex may be locked and stabilized while the machinery is partially defective. However, only XpsL/M/N and XpsL/E complex were sucessfully detected by the above two approaches. Using the yeast two-hybrid system, we further dissect the interaction domains by removing the membrane-anchor region of XpsF, XpsL and XpsM.

中文摘要 1
英文摘要 2
圖目錄 4
縮寫表 5
前言 6
材料與方法 13
結果 21
討論 27
參考文獻 31
圖表 33
附錄 49
參考文獻
林主祈, 2000. ”十字花科黑腐病菌中內膜蛋白XpsF與XpsLMN及XpsE間交互作用之探討”.國立中興大學農業生物科技學研究所碩士論文.
鍾宜靜, 1999. ”十字花科黑腐病菌XpsL蛋白在第二型胞外蛋白分泌過程中的角色探討” .國立中興大學農業生物科技學研究所碩士論文.
田孝威, 1999. ”十字花科黑腐病菌XpsM蛋白在第二型胞外蛋白分泌過途徑中的角色探討” 國立中興大學生物化學研究所碩士論文.
Binet, R., S. Letoffe, et al. (1997). "Protein secretion by Gram-negative bacterial ABC exporters--a review." Gene 192(1): 7-11.
Grass, S. and J. W. St Geme, 3rd (2000). "Maturation and secretion of the non-typable Haemophilus influenzae HMW1 adhesin: roles of the N-terminal and C-terminal domains." Mol Microbiol 36(1): 55-67.
Hege, T. and U. Baumann (2001). "Protease C of Erwinia chrysanthemi: the crystal structure and role of amino acids Y228 and E189." J Mol Biol 314(2): 187-93.
Holland, B. (1998). "Autotransporters: protein contortionists whose carboxyl termini translocate their own amino-terminal domains." Trends Microbiol 6(10): 388-9.
Hu, N. T., M. N. Hung, et al. (1992). "Molecular cloning, characterization and nucleotide sequence of the gene for secreted alpha-amylase from Xanthomonas campestris pv. campestris." J Gen Microbiol 138(Pt 8): 1647-55.
Hu, N. T., W. M. Leu, et al. (2002). "XpsG, the major pseudopilin in Xanthomonas campestris pv. campestris, forms pilus-like structure between cytoplasmic and outermembrane." Biochem J 4.
Jacob-Dubuisson, F., C. Locht, et al. (2001). "Two-partner secretion in Gram-negative bacteria: a thrifty, specific pathway for large virulence proteins." Mol Microbiol 40(2): 306-13.
Lee, H. M., S. W. Tyan, et al. (2001). "Involvement of the XpsN protein in formation of the XpsL-xpsM complex in Xanthomonas campestris pv. campestris type II secretion apparatus." J Bacteriol 183(2): 528-35.
Lee, H. M., K. C. Wang, et al. (2000). "Association of the cytoplasmic membrane protein XpsN with the outer membrane protein XpsD in the type II protein secretion apparatus of Xanthomonas campestris pv. campestris." J Bacteriol 182(6): 1549-57.
Lee, V. T., S. K. Mazmanian, et al. (2001). "A program of Yersinia enterocolitica type III secretion reactions is activated by specific signals." J Bacteriol 183(17): 4970-8.
Py, B., L. Loiseau, et al. (1999). "Assembly of the type II secretion machinery of Erwinia chrysanthemi: direct interaction and associated conformational change between OutE, the putative ATP-binding component and the membrane protein OutL." J Mol Biol 289(3): 659-70.
Py, B., L. Loiseau, et al. (2001). "An inner membrane platform in the type II secretion machinery of Gram-negative bacteria." EMBO Rep 2(3): 244-8.
Sagulenko, V., E. Sagulenko, et al. (2001). "VirB7 lipoprotein is exocellular and associates with the Agrobacterium tumefaciens T pilus." J Bacteriol 183(12): 3642-51.
Sandkvist, M. (2001). "Type II secretion and pathogenesis." Infect Immun 69(6): 3523-35.
Sasaki, J., T. Kita, et al. (1999). "Antibacterial activity of garlic powder against Escherichia coli O-157." J Nutr Sci Vitaminol (Tokyo) 45(6): 785-90.
Stathopoulos, C., D. L. Provence, et al. (1999). "Characterization of the avian pathogenic Escherichia coli hemagglutinin Tsh, a member of the immunoglobulin A protease-type family of autotransporters." Infect Immun 67(2): 772-81.
Voulhoux, R., G. Ball, et al. (2001). "Involvement of the twin-arginine translocation system in protein secretion via the type II pathway." Embo J 20(23): 6735-41.
Yen, M. R., C. R. Peabody, et al. (2002). "Protein-translocating outer membrane porins of Gram-negative bacteria." Biochim Biophys Acta 1562(1-2): 6-31.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top