Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/24048
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dc.contributor翁淑芬zh_TW
dc.contributor曾義雄zh_TW
dc.contributor.advisor林瑞文zh_TW
dc.contributor.advisorJuey-Wen Linen_US
dc.contributor.author陳弘偉zh_TW
dc.contributor.authorChen, Hong-Weien_US
dc.contributor.other中興大學zh_TW
dc.date2011zh_TW
dc.date.accessioned2014-06-06T07:21:52Z-
dc.date.available2014-06-06T07:21:52Z-
dc.identifierU0005-1708201017280000zh_TW
dc.identifier.citation蕭懿民 (2003) 十字花科黑腐病菌蛋白酶基因 prt1 與纖維酶基因 engA 之轉錄調控研究. 國立中興大學分子生物研究所博士論文. 陳智華 (2004) 十字花科黑腐病菌 Clp 蛋白之自我調控及對啟動子調控之研究. 國立中興大學分子生物研究所研究生論文進度期中報告研究計劃書. 李孟娟 (2003) Xanthomonas campestris pv. campestris 第四型纖毛合成所需 pilSRBA1A2CD 基因串之研究. 張曉娟 (2004) Stenotrophomonas maltophilia 噬菌體 SMA5 及 SMT13 之分離與分析. Asim K. J., and Purnendu.G. (1998). Xanthan biosynthesis in continuous culture: citric acid as an energy source. J F Bi 28, 961-970. Barber, C.E., Tang, J.L., Feng, J.X., Pan, M.Q., Wilson, T.J., Slater, H., Dow, J.M., Williams, P., and Daniels, M.J. (1997). A novel regulatory system required for pathogenicity of Xanthomonas campestris is mediated by a small diffusible signal molecule. Mol Microbiol 24, 555-566. Becker, A., Katzen, F., Puhler, A., and Ielpi, L. (1998). Xanthan gum biosynthesis and application: a biochemical/genetic perspective. Appl Microbiol Biotechnol 50, 145-152. Chan, J.W., and Goodwin, P.H. (1999). The molecular genetics of virulence of Xanthomonas campestris. Biotechnol Adv 17, 489-508. Chou, F.L., Chou, H.C., Lin, Y.S., Yang, B.Y., Lin, N.T., Weng, S.F., and Tseng, Y.H. (1997). The Xanthomonas campestris gumD gene required for synthesis of xanthan gum is involved in normal pigmentation and virulence in causing black rot. Biochem Biophys Res Commun 233, 265-269. Daniels, M.J., Barber, C.E., Turner, P.C., Sawczyc, M.K., Byrde, R.J., and Fielding, A.H. (1984). Cloning of genes involved in pathogenicity of Xanthomonas campestris pv. campestris using the broad host range cosmid pLAFR1. EMBO J 3, 3323-3328. de Crecy-Lagard, V., Glaser, P., Lejeune, P., Sismeiro, O., Barber, C.E., Daniels, M.J., and Danchin, A. (1990). A Xanthomonas campestris pv. campestris protein similar to catabolite activation factor is involved in regulation of phytopathogenicity. J Bacteriol 172, 5877-5883. Dow, J.M., Daniels, M.J., Dums, F., Turner, P.C., and Gough, C. (1989). Genetic and biochemical analysis of protein export from Xanthomonas campestris. J Cell Sci Suppl 11, 59-72. Dow, J.M., Feng, J.X., Barber, C.E., Tang, J.L., and Daniels, M.J. (2000). Novel genes involved in the regulation of pathogenicity factor production within the rpf gene cluster of Xanthomonas campestris. Microbiology 146 ( Pt 4), 885-891. Harding, N.E., Cleary, J.M., Cabanas, D.K., Rosen, I.G., and Kang, K.S. (1987). Genetic and physical analyses of a cluster of genes essential for xanthan gum biosynthesis in Xanthomonas campestris. J Bacteriol 169, 2854-2861. He, Y.W., Boon, C., Zhou, L., and Zhang, L.H. (2009). Co-regulation of Xanthomonas campestris virulence by quorum sensing and a novel two-component regulatory system RavS/RavR. Mol Microbiol 71, 1464-1476. He, Y.W., Ng, A.Y., Xu, M., Lin, K., Wang, L.H., Dong, Y.H., and Zhang, L.H. (2007). Xanthomonas campestris cell-cell communication involves a putative nucleotide receptor protein Clp and a hierarchical signalling network. Mol Microbiol 64, 281-292. He, Y.W., Xu, M., Lin, K., Ng, Y.J., Wen, C.M., Wang, L.H., Liu, Z.D., Zhang, H.B., Dong, Y.H., Dow, J.M., et al. (2006). Genome scale analysis of diffusible signal factor regulon in Xanthomonas campestris pv. campestris: identification of novel cell-cell communication-dependent genes and functions. Mol Microbiol 59, 610-622. He, Y.W., and Zhang, L.H. (2008). Quorum sensing and virulence regulation in Xanthomonas campestris. FEMS Microbiol Rev 32, 842-857. Horinouchi, S. (2007). Mining and polishing of the treasure trove in the bacterial genus streptomyces. Biosci Biotechnol Biochem 71, 283-299. Katzen, F., Ferreiro, D.U., Oddo, C.G., Ielmini, M.V., Becker, A., Puhler, A., and Ielpi, L. (1998). Xanthomonas campestris pv. campestris gum mutants: effects on xanthan biosynthesis and plant virulence. J Bacteriol 180, 1607-1617. Nishida, H., Ohnishi, Y., Beppu, T., and Horinouchi, S. (2007). Evolution of gamma-butyrolactone synthases and receptors in Streptomyces. Environ Microbiol 9, 1986-1994. Poplawsky, A.R., and Chun, W. (1997). pigB determines a diffusible factor needed for extracellular polysaccharide slime and xanthomonadin production in Xanthomonas campestris pv. campestris. J Bacteriol 179, 439-444. Poplawsky, A.R., and Chun, W. (1998a) Xanthomonas campestris pv. campestris requires a functional pigB for epiphytic survival and host infection. Mol Plant Microbe Interact 11, 466-475. Poplawsky, A.R., and Chun, W. (1998b). Synthesis of extracellular polysaccharide, extracellular enzymes, and xanthomonadin in Xanthomonas campestris: Evidence for the involvement of two intercellular regulatory signals. MPMI 11, 68-70. Poplawsky, A.R., Urban, S.C., and Chun, W. (2000). Biological role of xanthomonadin pigments in Xanthomonas campestris pv. campestris. Appl Environ Microbiol 66, 5123-5127. Poplawsky, A.R., Walters, D.M., Rouviere, P.E., and Chun, W. (2005). A gene for a dioxygenase-like protein determines the production of the DF signal in Xanthomonas campestris pv. campestris. Mol Plant Pathol 6, 653-657. Sambrook, J., Fritsch, E. F and Maniatis (1989). Molecular cloning: a laboratory manual, 2nd. Cold Spring Habor Press, Cold Spring Harbor, N. Y. Tang, J.L., Liu, Y.N., Barber, C.E., Dow, J.M., Wootton, J.C., and Daniels, M.J. (1991). Genetic and molecular analysis of a cluster of rpf genes involved in positive regulation of synthesis of extracellular enzymes and polysaccharide in Xanthomonas campestris pathovar campestris. Mol Gen Genet 226, 409-417. Thorne, L., Tansey, L., and Pollock, T.J. (1987). Clustering of mutations blocking synthesis of xanthan gum by Xanthomonas campestris. J Bacteriol 169, 3593-3600. Timothy, P.D. (1999) Autoregulator-dependent control of extracellular polysaccharide production in phytopathogenic bacteria. EJPP 105, 417-430.. Tseng, Y.H., Choy, K.T., Hung, C.H., Lin, N.T., Liu, J.Y., Lou, C.H., Yang, B.Y., Wen, F.S., Weng, S.F., and Wu, J.R. (1999). Chromosome map of Xanthomonas campestris pv. campestris 17 with locations of genes involved in xanthan gum synthesis and yellow pigmentation. J Bacteriol 181, 117-125. Wang, L.H., He, Y., Gao, Y., Wu, J.E., Dong, Y.H., He, C., Wang, S.X., Weng, L.X., Xu, J.L., and Tay, L., (2004). A bacterial cell-cell communication signal with cross-kingdom structural analogues. Mol Microbiol 51, 903-912. Williams, L.E., and Phillips, D.A. (1980). Effect of irradiance on development of apparent nitrogen fixation and photosynthesis in soybean. Plant Physiol 66, 968-972. Wilson, T.J., Bertrand, N., Tang, J.L., Feng, J.X., Pan, M.Q., Barber, C.E., Dow, J.M., and Daniels, M.J. (1998). The rpfA gene of Xanthomonas campestris pathovar campestris, which is involved in the regulation of pathogenicity factor production, encodes an aconitase. Mol Microbiol 28, 961-970.zh_TW
dc.identifier.urihttp://hdl.handle.net/11455/24048-
dc.description.abstractXanthomonas 屬於革蘭氏陰性嗜氧植物病原桿菌,菌落外觀為黃色黏濕狀。研究指出X. campestris pv. campestris (Xcc) B24 有七個轉錄單位與黃色素(xanthomonadin) 之表現有關,其中pigB 被認為是影響表現最重要的轉錄單位。pigB 包含兩個基因,分別主導 reductase/halogenase 以及 pteridine-dependent dioxygenase-like protein (簡稱 DogA)。已知 DogA 蛋白的功能之一為參與 Xcc 訊息因子 diffusible factor (DF) 之合成。為瞭解 Xc17 DF 的功能,其調控基因的表現模式,以及其與訊息調控因子 DSF 調控路徑之相關性,進行以下分析。首先構築 dogA 突變株 Xc17dogA::Gm,並觀察其特性。和野生株相比,dogA 突變株的胞外酵素 protease,α-amylase,與 cellulase 之分泌沒有明顯改變,運動性 (motility) 稍微增加。然而,附著性 (adhesion),xanthomonadin,胞外黏多醣 (exopolysaccharide, EPS) 以及致病性都明顯降低。Xc17 所分泌之 DF 則可互補這些突變株的外表型為野生型。實驗中取 Xc17,突變株 Xc17dogA::Gm,RM17F (rpfF 突變株),以及 TC817 (clp 突變株)之培養上清液,過濾去菌後,再分別添加於 DF 缺失的突變株 Xc17dogA::Gm 以及 DSF 缺失的突變株 RM17F 之液體培養基中,測試xanthomonadin,EPS,以及受到 DSF 正向調控之 engA 啟動子活性,以了解比較不同菌株上清液中訊息因子 DF 與 DSF 之含量。實驗結果顯示 DF 缺失的菌株中,其 DSF 產量或活性上升,而 DSF 缺失的突變株,其上清液中亦含有 DF ,但含量低於野生株。Clp 突變株的上清液中,DF 及 DSF 含量皆低於野生株。為了瞭解受到 DF 影響表現的蛋白,利用二維電泳分析 Xc17 以及 Xc17dogA::Gm 之全蛋白,發現六個可能受 DF 影響之蛋白質點,其中兩個蛋白質點的預測功能與 TCA cycle 有關。此外,Streptomyces A-factor 的化學結構與 DF 相似,推測 Xcc DF 在細胞中與對應的 receptor 結合後,可能啟動一個 (或數個) 重要之轉錄調節因子,此因子再調控其下游基因之表現,這些基因的產物包含 TCA cycle,xanthomonadin 合成以及 EPS 合成過程中的一些酵素等。因此,當 DF 缺失時 xanthomonadin 與 EPS 合成降低。zh_TW
dc.description.abstractXanthomonas are Gram-negative aerobic pathogenic bacteria with yellow mucoid. X. campestris pv.campestris (Xcc) B24 has seven transcription units related to the formation of cell pigment (xanthomonadin). Mutation in one of the transcription units, pigB, concerned reduction of xanthomonadin in bactaria.Region contains two genes, encoding reductase/halogenase and pteridine-dependent dioxygenase like protein (named DogA), respectively. It has been known that DogA protein is involved in the synthesis of Xcc signal factor diffusible factor (DF).The aim of this study is to understand the formation of DF in Xc17, and the relationship between DF and DSF regulated pathways.Firstly, the dogA mutant, Xc17dogA::Gm, was constructed. Compared to that of Xc17, extracellular enzyme levels of protease, α-amylase, and cellulase in Xc17dogA::Gm had not significant differecens.However, lower levels of xanthomonadin, EPS, and pathogenicity. DF were found in the mutant strain. In addition DF secreted by Xc17 can restore the mutations to the wild type.Supernatants of Xc17, Xc17dogA::Gm, RM17F (rpfF mutant strain), and TC817 (clp mutnat) were collected, membrane filtered, and added to the culture of DF deficient strain Xc17dogA::Gm and DSF deficient strain RM17F. Compared the DF and DSF levels among wild type and mutant strain xanthomonadin, EPS, and engA promoter activity. Results showed that DSF increased in the DF deletion strain, production or activity, the supernatant of DSF deficient strain, also contained DF with lower levels than that of the wild type strain, the supernatant of clp mutant strain, contained DF and DSF with lower levels than that of the wild type strain. To understand proteins affected by DF, two-dimensional gel electrophoresis was used to analyze proteins of Xc17 and Xc17dogA::Gm.Six proteins may be affected by DF, two of them are emzymes involved in the TCA cycle.In addition Streptomyces A-factor is similar to DF in chemical structure, indicating that Xcc DF may be recognized by a corresponding receptor, and exercise it's regulatory function on. The transcription factor then controls the transcription of enzymes that related in TCA cycle, xanthomonadin synthesis, and EPS synthesis. Therefor, loss of DF reduced the synthesis of xanthomonadin and EPS.en_US
dc.description.tableofcontents中文摘要 1 Abstract 2 前言 3 材料 7 一、菌種及質體 7 二、藥品 7 三、酵素 7 六、培養基 8 (一)液體培養基 8 (二)固體培養基 8 七、試劑與緩衝溶液 9 (一) 抽取質體 DNA 試劑 9 (二) DNA 電泳試劑 9 (三) SDS-聚丙烯醯胺凝膠試劑 9 (四) β-galactosidase 活性測試所需之試劑 10 (六) 二維電泳所需試劑 11 (七) 蛋白質銀染 (silver stain) 11 方法 12 一、細菌之培養與保存 12 二、小量質體製備 12 三、染色體 DNA 抽取 13 四、質體之構築 14 五、凝膠電泳分析 15 六、聚合酶連鎖反應 16 七、轉形作用 16 八、胞外多醣含量之測定 18 九、病原性 (pathogenicity) 測試 18 十、胞外酵素分泌能力測試 19 十一、西方墨點法分析(Western blot) 19 十二、黃色素(xanthomonadin)萃取分析 20 十三、二維電泳分析之蛋白質萃取 20 十四、蛋白質二維電泳分析 21 十五、蛋白質銀染 22 十六、啟動子之活性分析 22 結果與討論 24 I. dogA 基因之特性探討 24 一、dogA突變株之構築 24 二、生長曲線測試 25 三、色素缺失突變株 Xc17dogA::Gm 的 DF 互補測試分析 26 四、野生株與突變株 EPS 分泌之差異 27 五、測試 Xc17dogA::Gm突變株的胞外酵素分泌 28 六、致病性測試 29 七、附著性測試 29 八、motility 測試 30 II. 探討 DF 和DSF 兩訊息因子之相關性 31 一、比較Xc17、 Xc17dogA::Gm、 RM17F (rpfF 突變株) 以及 TC817 (clp 突變株) 的 DF 之含量 31 二、比較 Xc17、 Xc17dogA::Gm、 RM17F 以及 TC817 的 DSF 含量 32 三、DF 缺失突變株的 Clp 蛋白表現 33 III. 分析野生株及dogA突變株中具差異表現之蛋白 33 一、SDS-PAGE分析野生株及突變株 Xc17 dogA::Gm之蛋白表現 33 二、利用二維電泳分析野生株及突變株 Xc17dogA::Gm蛋白表現情形 34 三、LC/MS/MS分析 34 參考文獻 37 圖表 42zh_TW
dc.language.isoen_USzh_TW
dc.publisher生物化學研究所zh_TW
dc.relation.urihttp://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-1708201017280000en_US
dc.subject擴散因子zh_TW
dc.subjectDFen_US
dc.subject十字花科黑腐病zh_TW
dc.subjectXanthomonasen_US
dc.titleXanthomonas campestris diffusible factor 的調控功能之探討zh_TW
dc.titleInvestigation of the regulatory functions of diffusible factor in Xanthomonas campestrisen_US
dc.typeThesis and Dissertationzh_TW
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
item.languageiso639-1en_US-
item.openairetypeThesis and Dissertation-
item.grantfulltextnone-
item.fulltextno fulltext-
item.cerifentitytypePublications-
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