(3.238.88.35) 您好!臺灣時間:2021/04/11 17:37
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:::

我願授權國圖
: 
twitterline
研究生:江淑慧
研究生(外文):Shu-hui Chiang
論文名稱:十字花科黑腐病菌基因體內兩個rpoN之專一性研究
論文名稱(外文):The Specificity Study of the Two rpoN GenesIn Xanthomonas campestris pv.campestris
指導教授:胡若梅曾義雄曾義雄引用關係
指導教授(外文):Hu, R. M.Tseng, Y. H.
學位類別:碩士
校院名稱:臺中健康暨管理學院
系所名稱:生物資訊研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:72
中文關鍵詞:十字花科黑腐病菌啟動子
外文關鍵詞:σ54RpoN
相關次數:
  • 被引用被引用:2
  • 點閱點閱:117
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:16
  • 收藏至我的研究室書目清單書目收藏:0
細菌當面對環境的轉變,會藉著RNA聚合酶和所含的不同σ因子及相關的調控蛋白來活化或關閉一群受其調控的基因。σ54所辨認基因啟動子的區域是在轉錄起始位-24/-12兩個區域的保守序列(5''-TGGC-N8- TGCA/T-3''),其辨認基因啟動子區域及轉錄的機制與主要σ因子─σ70不同。
十字花科黑腐病菌為一革蘭氏陰性菌,會感染十字花科植物造成黑腐病。此菌在農業上會造成嚴重的災害,但是所產生的胞外黏多醣在工業上卻是有多重用途,可在食品加工、化妝品、製造業上作為膠化劑、乳化劑、塑化劑、安定劑之用。十字花科黑腐病菌基因體中含有兩套rpoN基因― rpoN1基因和rpoN2基因,這兩個RpoN蛋白彼此之間的相同度低(41%),和其他菌不同的是彼此可能存在個別的專一性。σ54調節子在其他的菌種曾經被研究過,但是在十字花科黑腐病菌未曾被研究過。生理學的初步研究顯示兩個 rpoN 基因的功能是獨立和不可互換的。在這次研究中,首先利用前不久發表的文獻提供完整的十字花科黑腐病菌的基因體序列,來預測基因體中所有可能受RpoN調控的基因啟動子。再以分子生物技術釐清上述搜尋到的基因啟動子是否受XCC中RpoN1或 RpoN2調控。本研究的結果發現受RpoN1調控的啟動子有glnA (麩醯胺合成酶基因)、nasA(硝酸塩轉運蛋白基因)、pilA1(纖毛蛋白基因)及prpB(丙酮烯醇磷酸轉位酶基因)四個基因的啟動子,受RpoN2調控的啟動子有flhF(鞭毛生合成基因)、flgG(鞭毛基底的桿狀物遠端結構蛋白基因)、flgB(鞭毛基底的桿狀物近端結構蛋白基因)、fliE(鞭毛MS環桿狀交界蛋白基因)及fliQ(鞭毛運輸蛋白基因)五個基因的啟動子。根據以上實驗的結果,進一步比較RpoN1或RpoN2所辨認基因啟動子保守序列的異同,發現RpoN1所辨認基因啟動子保守序列為5''-TTGGC-AN7-TGCG/T-3'',而RpoN2所辨認基因啟動子保守序列為 5''-TTTGGC-N8-TGCA -3''(粗體加底線的文字代表兩者的差異)。由此推論RpoN1或RpoN2具有專一性的決定因子。
Abstract
Bacteria are able to activate or switch off specific sets of genes as they face changing environmental conditions. This can be achieved through the activities of RNA polymerases (RNAP) containing alternate sigma factors and their cognate regulatory proteins. The sigma factor σ54 recognizes a conserved sequence motif centered at -24 and -12 nucleotides from the transcriptional start site (5''-TGGC-N8- TGCA/T-3'') and confers RNAP properties different from those of the major house-keeping form of RNAP such asσ70.
Xanthomonas campestris pv. campestris (XCC) is the Gram-negative bacterium causing black rot in crucifers, resulting in tremendous loss in agriculture. It produces great amounts of an exopolysaccharide, xanthan gum, which is used as viscosifying, emulsifying, plasticizer and stabilizing agent in food, cosmetics, agriculture and industry. The fully sequenced XCC genome has two homologues of the rpoN genes, rpoN1 and rpoN2. The two deduced RpoN sequences share only 41% of identity and both show different levels of homology to the RpoN proteins from other bacteria. Theσ54-dependent regulon has previously been studied in several groups of bacteria, but not in XCC. Preliminary physiological studies have demonstrated that the two rpoN genes are functionally independent and not interchangeable. In this study, I carried out searches on the recently published complete genome sequence of XCC in order to predict the possibleσ54-dependent genes. Molecular biological methods were then used to confirm whether the predicted σ54-dependent genes are indeed controlled by RpoN1 or RpoN2. The results revealed that glnA (encoding glutamine synthetase), nasA (encoding nitrate transporter), pilA1 (encoding pilin), and prpB (encoding carboxyphosphonoenolpyruvate phosphonomutase) are RpoN1-dependent, whereas flhF (encoding flagellar biosynthetic protein), flgG (encoding the distal rod protein of the flagellum basal body), flgB (encoding the proximal rod protein of the basal body), fliE (encoding MS-ring rod junction protein), and fliQ (encoding export component of flagellar protein) are RpoN2-dependent. Based on the RpoN-binding sequence in the upstream regions of the above genes and their homologs, the consensus RpoN1- and RpoN2-binding sites complied are 5''-TTGGC-AN7-TGCG/T- 3'' and 5''-TTTGGC-N8-TGCA-3'', respectively. In other words, the two Xcc RpoN proteins each has its specific recognition sequences.
壹.縮寫字對照表 1
貳.中文摘要 3
叁.英文摘要 4
肆.前言 5
伍.材料
一. 菌種及質體 10
二. 一般藥品 10
三. 酵素 10
四. 培養基 10
五. 試劑與緩衝溶液 11
1 抗生素濃度 11
2 抽取染色體之緩衝溶液 11
3 抽取質體 DNA試劑 11
4.β-galactosidase 活性測試所需之試劑 11
六 引子 12
陸.方法
I. 生物資訊的方法 13
II. 分生技術的方法 13
一. 細菌之培養與保存 13
二. DNA 之製備 14
1. 小量質體 DNA 之抽取 14
2. 染色體 DNA 抽取法 14
三. 洋菜膠體電泳分析 15
四. 聚合酶鏈鎖反應 15
五. 質體之選殖 16
1. 限制酶的切割分析 16
2. DNA 的黏接 16
六. 轉形作用 16
1. Escherichia coli 之轉形 16
2. Xanthomonas camperstris pv. campestris之轉形 17
七. 點測試法 17
八. 生長曲線測定 18
九. 啟動子之活性分析 18
1. 培養方式 18
2. -galactosidase 之活性測試 18
柒.結果與討論
第一部份:各基因啟動子區域之分析 19
一、 生物資訊的方法 19
I. 預測可能的RpoN-dependent promoters 19
II. 預測啟動子的分數的値 19
二、 分子生物實驗驗證 20
Ⅰ.基因重組質體之構築 20
П.諸基因啟動子之表現情形 21
1. 質體 pFYflgB啟動子活性的表現結果 21
2. 質體 pFYflgG啟動子活性的表現結果 21
3. 質體 pFYflF啟動子活性的表現結果 22
4. 質體 pFYnasA啟動子活性的表現結果 22
5. 質體 pFYglnA啟動子活性的表現結果 23
6.質體 pFYprpB啟動子活性的表現結果 23
7. 質體pFY13-9fliE之啟動子活性的表現結果 24
8. 質體pFY13-9fliQ之啟動子活性的表現結果 24
9.質體pFYfliL之啟動子活性的表現結果 25
10.質體 pFYpilA1之啟動子活性的表現結果 25
Ш 質體pFYglnA2之構築及啟動子活性的表現結果 26
Ⅳ 結論 27
第二部份:RpoN- binding site 專一性 28
I.分析RpoN1-dependent promoter sequence 28
II. 分析RpoN2-dependent promoter sequence 29
Ⅲ比較RpoN-dependent promoter sequence 29
Ⅳ.避免無意義的共有鹼基序列位置產生 29
第三部份:生物資訊預測啟動子分數與分子生物實驗所測啟動子活性關聯性 30
第四部份:生物資訊的預測性探討 30
第五部份:未來的挑戰 30
捌﹑結論 32
玖.表格 33
拾.圖 47
拾壹.參考文獻 70
Barrios, H., B. Valderrama, and E. Morett.(1999)Compilation and analysis of
σ54-dependent promoter sequences. Nucleic Acids Res. 27:4305-4313
Birnboim, H. C. and Doly, J. (1979) A rapid alkaline extraction procedure for
screening recombinant plasmid DNA. Nucleic Acids Res 7:1513-1523.
Blin, N. and Stafford, D. W. (1976) A general method for isolation of high molecular
weight DNA from eukaryotes. Nucleic Acids Res 3: 2303-2308.
Boyle-Vavra S, So M. and Seifert HS.(1993)Transcriptional control of gonococcal
pilE expression: involvement of an alternate sigma factor. Gene. 137:233-6.
Cohen, S. N., Chang, A. C. and Hsu, L. (1972) Nonchromosomal antibiotic
resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA.
Proc Natl Acad Sci U S A 69:2110-2114.
Chiurazzi M and Iaccarino M.(1990)Transcriptional analysis of the glnB-glnA
region of Rhizobium leguminosarum biovar viciae Mol Microbiol. 4:1727-35..
Choudhary M, Mackenzie C, Mouncey NJ and Kaplan S.(1999) RsGDB, the
Rhodobacter sphaeroides Genome Database. 27(1):61-2.
da Silva AC, Ferro JA, Reinach FC, Farah CS, Furlan LR, Quaggio RB,
Monteiro-Vitorello CB, Van Sluys MA, Almeida NF, Alves LM, do Amaral AM,
Bertolini MC, Camargo LE, Camarotte G, Cannavan F, Cardozo J, Chambergo
F, Ciapina LP, Cicarelli RM, Coutinho LL, Cursino-Santos JR, El-Dorry H,
Faria JB, Ferreira AJ, Ferreira RC, Ferro MI, Formighieri EF, Franco MC,
Greggio CC, Gruber A, Katsuyama AM, Kishi LT, Leite RP, Lemos EG, Lemos
MV, Locali EC, Machado MA, Madeira AM, Martinez-Rossi NM, Martins EC,
Meidanis J, Menck CF, Miyaki CY, Moon DH, Moreira LM, Novo MT, Okura
VK, Oliveira MC, Oliveira VR, Pereira HA, Rossi A, Sena JA, Silva C, de Souza
RF, Spinola LA, Takita MA, Tamura RE, Teixeira EC, Tezza RI, Trindade dos
Santos M, Truffi D, Tsai SM, White FF, Setubal JC, Kitajima JP.(2002)
Comparison of the genomes of two Xanthomonas pathogens with differing host
specificities. Nature 417:459-63.
Daniels, M. J., C. E. Barber, P. C. Turner, W. G. Cleary, and M. K. Sawczyc.
( 1984 ) Isolation of mutants of Xanthomonas campestris showing altered
pathogenicity. Microbiology 130:2447-2455.
Eisenstark, A. (1967) Bacteriophage technoques. In Methods in Viology, vol. 1, pp.
449-524. Edited by K. Maramorosch and H. Koprowski. New York: Academic Press.
Genschik, P., K. Drabikowski, and W. Filipowicz.(1998)Characterization of the
Escherichia coli RNA 3''-terminal phosphate cyclase and its σ54-regulated operon. J.
Biol. Chem. 273:25516-25526
Heuner K, Dietrich C, Skriwan C, Steinert M. and Hacker J.(2002)Influence of
the alternative σ28 factor on virulence and flagellum expression of Legionella
pneumophila. Infect Immun. 70(3):1604-8.
Horns T.and Bonas U.(1996) The rpoN gene of Xanthomonas campestris pv.
vesicatoria is not required for pathogenicity.
Ishimoto KS. and Lory S.(1989)Formation of pilin in Pseudomonas aeruginosa
requires the alternative sigma factor (RpoN) of RNA polymerase. Proc Natl Acad Sci
U S A. 86:1954-7.
Jin S, Ishimoto K. and Lory S.(1994)Nucleotide sequence of the rpoN gene and
characterization of two downstream open reading frames in Pseudomonas aeruginosa.
J Bacteriol. 176:1316-22.
Keseler, I. M., and Kaiser, D.(1997) σ54, a vital protein for Myxococcus xanthus.
Proc. Natl. Acad. Sci .USA 94:1979-1984.
Kohler T, Harayama S, Ramos JL. and Timmis KN.(1989) Involvement of
Pseudomonas putida RpoN sigma factor in regulation of various metabolic functions.
J Bacteriol. 171:4326-33.
Kullik, I., S. Fritsche, H. Knobel, J. Sanjuan, H. Hennecke, and H. M. Fischer.
(1991) Bradyrhizobium japonicum has two differentially regulated, functional
homologs of the σ54 gene (rpoN). J. Bacteriol. 173:1125-1138
Kustu, S., E. Santero, J. Keener, D. Popham, and D. Weiss. (1989)Expression of
σ54(ntrA) dependent genes is probably united by a common mechanism. Microbiol.
Rev. 53:367-376.
70
Lenz, O., A. Strack, A. tran-Betcke, and B. Friedrich. ( 1997 ) A
71
hydrogensensing system in transcriptional regulatin of hydrogenase gene expression
in Alcaligenes species. J. Bacteriol. 179:1655-1663
Miller, J. H. (1972) Experiments in molecular genetics. Cold Spring Habor
Laboratory, Cold Spring Harbor, N. Y.
Merrick, M., J. (1993)In a class of its own-the RNA polymerase sigma factor σ
N (σ54 ). Mol. Microbiol. 10:903-909
Michiels, J., M. Moris, B. Dombrecht, C. Verreth, and J. Vanderleyden. (1998)
Differential regulation of Rhizobium etli rpoN2 gene expression during symbiosis and
free-living growth. J. Bacteriol. 180:3620-3628
Michel-Reydellet N, Desnoues N, Elmerich C. and Kaminski PA.(1997)
Characterization of Azorhizobium caulinodans glnB and glnA genes: involvement of
the P(II) protein in symbiotic nitrogen fixation. J Bacteriol. 179:3580-7.
Mouz S, Coursange E. and Toussaint A.(2001)Ralstonia metallidurans CH34
RpoN sigma factor and the control of nitrogen metabolism and biphenyl utilization.
Microbiology. 147:1947-54
NCBI Bacteria Genomes, ftp://ftp.ncbi.nih.gov/genomes/Bacteria
Ninfa AJ, Mullin DA, Ramakrishnan G. and Newton A.(1989)Escherichia coli
sigma 54 RNA polymerase recognizes Caulobacter crescentus flbG and flaN flagellar
gene promoters in vitro. J Bacteriol. 171:383-91.
Reitzer L and Schneider BL.(2001)Metabolic context and possible physiological
themes of σ54-dependent genes in Escherichia coli. Microbiol Mol Biol Rev.
65:422-44
Reitzer L. J., Bueno R, Cheng WD, Abrams SA, Rothstein DM, Hunt TP, Tyler B,
and Magasanik B.(1987)Mutations that create new promoters suppress the σ54
dependence of glnA transcription in Escherichia coli. J Bacteriol. 169:4279-84.
PromScan, http://www.promscan.uklinux.net/software.html
Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: a
72
laboratory manual, 2nd. Cold Spring Habor Press, Cold Spring Harbor, N. Y.
Studholme D. J., and Dixon R.(2004)In silico analysis of the σ54-dependent
enhancer-binding proteins in Pirellula species strain 1. FEMS Microbiol Lett.
230:215-25.
Studholme, D. J., and M. Buck. (2000) The biology of enhancer-dependent
transcriptional regulation in Bacteria: insights from genome sequences. FEMS
Microbiol. Lett. 186:1-9
Totten PA, Lara JC, and Lory S.(1990)The rpoN gene product of Pseudomonas
aeruginosa is required for expression of diverse genes, including the flagellin gene. J
Bacteriol. 172:389-96.
Wang, T. W. and Tseng, Y. H. (1992) Electrotransformation of Xanthomonas
campestris by RF DNA of filamentous phage φLf. Lett Appl Microbiol 14:65-68.
Weiner, L., J. L. Brissette, and P. Model. (1991) Stress-induced expression of the
Escherichia coli phage shock protein operon is dependent on σ54 and modulated by
positive and negative feedback mechanisms. Genes Dev. 5:1912-1923
William, P. H. (1980) Black rot: a continuing treat to world crucifers. Plant Dis.
64:736-742.
Xu, H. and Hoover, T. R. (2001)Transcriptional regulation at a distance in bacteria.
Curr. Opin. Microbiol. 4:138—144.
Yang, B. Y. and Tseng, Y. H.(1988)Production of exopolysaccharide and levels of
protease and pectinase activity in pathogenic and non-pathogenic strains of
Xanthomonas campestris pv. campestris. Bot Bull Acad Sin 29:93-99.
Yang, M. H. (1997) Cloning and analysis of the promoter regions of filamentous
phage φLf. Master thesis, National Chung Hsing University.
Zhang, X., Chaney, M., Wigneshweraraj, S.R., Schumacher, J., Bordes, P.,
Cannon, W . and Buck, M. (2002) Mechanochemical ATPases and transcriptional
activation. Mol. Microbiol. 45:895—903
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
系統版面圖檔 系統版面圖檔