Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21393
標題: Analysis of the Xanthomonas campestris pv. campestris membrane proteins related to the infection of lytic phage L7
Xanthomonas campestris pv. campestris膜蛋白與fL7噬菌體感染有關的一些膜蛋白之分析
作者: 林珊瑩
Lin, Shen-Yen
關鍵字: membrane protein;膜蛋白;bacteriophage;Xanthomonas campestris pv. campestris;噬菌體;黃原菌
出版社: 分子生物學研究所
摘要: 
利用二維電泳分析,比較Xc11、Xc17及P20H等不同Xanthomonas分離株等電點4-7之膜蛋白,結果發現沒有明顯的差異。比較Xanthomonas不同生長時期膜蛋白表現的實驗中,也沒有發現表現量有差異的蛋白。另一方面,比較Xc17及ampG突變株(XKG) 的等電點4-7 及 6-11之膜蛋白,結果未發現有明顯的差異。可能AmpG在Xc17中表現量較低,利用此分析法無法偵測到。以噬菌體 L7感染 XcP20H感染時間30分鐘後,膜蛋白的量很少,推測可能是因為噬菌體進入菌體中產生溶菌作用的結果。
另外,比較phage resistant strain (B236) 及phage sensitive strain (P20H) 膜蛋白二維電泳圖,發現P20H在等電點5.0,分子量20 kDa處有一個膜蛋白,該蛋白在B236中的表現量明顯減少。將此蛋白經質譜儀定序結果發現為outer membrane protein P6 precursor (OmpP6)。依據Xanthomonas campestris pv campestris 33913 基因體核苷酸序列資料庫分析蛋白質之分子量為20.4 kDa, 等電點為5.5。針對ompP6基因進行突變,但未能獲得突變株因此先將互補質體 (pRKP6His) 送入菌中,再以電孔法送入欲進行double cross-over 之質體 (pOKP6Gm),亦無法得到突變株,推測此基因是菌體生長所必須利用西方墨點法證實了OmpP6為外膜蛋白,LPS量減少的菌株,其外膜上OmpP6 表現量減少。推測LPS影響OmpP6無法正確褶疊,因此外膜上的OmpP6量減少。FepA為外膜上的porin作為運送ferric enterobactin之通道。利用single cross-over基因重組策略破壞突變XcP20H的fepA基因後獲得突變株命名為PfepAM。其生長情形、對十字花科蔬菜致病力及胞外酵素之分泌之生理特性與野生株並無明顯差異。但以L7感染PfepAM後明顯觀察到其溶菌能力降低形程式菌體的也減少。推測此基因產物可能是L7感染 XcP20H 之接受器或接受器組成之一。

Comparing the membrane protein of different Xanthomonas isolates (Xc11, Xc17, and XcP20H) by two dimention electrophoresis (2D), the result show that Xanthomonas isolates membrane protein is not different. On the other hand, there is no difference between the Xc17 and XKG by membrane protein comparison. The AmpG of Xc17 expression level is too low to detect by two dimension electrophoresis . The experiment of comparison the membrane protein expression in different growth phase, the result reveals that membrane protein expression is not different. The most membrane protein isn't able to express after the L7 infect XcP20H 30 minutes, the result reveal that L7 will lyse P20H at 30 minutes.
From the 2D gels of phage (L7) resistance strain (B236) and phage sensitive strain (XcP20H), there is a membrane protein on B236 which expression level is less than XcP20H.The result of MALTI-TOF knows that this protein in an outer membrane protein P6 precursor (OmpP6), it's pI 5.5, MW 20.4kDa . Unfortunately, we can't get the ompP6 mutant by several ways, so the gene may be an essential gene for bacterial growth. From the Western blotting knows that the OmpP6 is an outer membrane protein and the OmpP6 folding is affected by lipopolysaccharide . Gram negative bacteria needs the FeⅢ transporter, the FepA is a outer membrane porin for ferric enterobactin. The PfepAM is a fepA gene mutant , the bacteriophage can't infect it. There is no difference the XcP20H and PfepAM of growth curve, virulence, and extracellular enzyme secretion. From the result of phage infection reveals that the FepA may be a receptor or component of receptor for L7.
URI: http://hdl.handle.net/11455/21393
Appears in Collections:分子生物學研究所

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