臺灣博碩士論文加值系統

本研究中首先構築包含部分熱休克調節基因hrcA DNA片段之質體pOKhrca，經轉殖送入十字花科黑腐病菌 (Xanthomonas campestris pv. campestris 17, Xc17) 後，篩選質體以 single crossover 嵌入染色體之hrcA突變株。分析培養溫度對菌體的效應時發現，於37℃時，突變株的生長速度略高於野生株。而hrcA突變株對十字花科蔬菜的致病力也與野生株相似。以北方轉漬法分析熱休克前後groESL的轉錄量，發現於正常生理溫度下，hrcA突變株的groESL轉錄量較野生株略多，但熱休克後，野生株與突變株groESL轉錄量均明顯上升。推測於Xc17中調控groESL表現的主要因子，可能為sigma factor s32，而非HrcA。以西方轉漬法分析結果顯示，於正常生長溫度下，野生株與突變株的GroEL之基礎表現就很高，於37℃熱休克之後增加的量並不顯著，於42℃熱休克二十分鐘後，野生株與突變株GroEL的表現有少量增加。以二維電泳分析正常生理溫度下之蛋白表現時，並沒有觀察到野生株與突變株有明顯差異表現之蛋白。但分析熱休克後之蛋白時，於分子量為20 kDa、pI為5.8的位置則有一明顯新增之蛋白，命名為Hsp20。將Hsp20經N端定序後，得到15個胺基酸序列，經比對後發現，此序列與Xylella fastidiosa之小分子熱休克蛋白有61 %的相似性。推測Hsp20為含有a-crystalline domain的小分子熱休克蛋白。利用Hsp20已知的N端序列，及小分子熱休克蛋白a-crystalline domain上較保守的區域，設計兩段引子，於Xc17的染色體上擴增出一段194 bp的DNA片段。定序並分析後，發現此序列所轉譯出的蛋白胺基酸序列與X. fastidiosa之小分子熱休克蛋白有86 %的相似度。以北方轉漬法分析，發現於37℃熱休克十五分鐘後，hsp20之RNA轉錄會大量表現，其轉錄產物之大小約為500 nt，但調控表現的機制則尚待探討。

Plasmid pOKhrca carrying a DNA fragment span within the heat shock regulatory gene hrcA was constructed. After electroporation of pOKhrca into Xanthomonas campestris pv. campestris 17, several Kmr transformants were obtained, and one of them was verified to be a Xc17 hrcA mutant by Southern blotting. The growth rate of the hrcA mutant was slightly faster than that of Xc17 at 37℃. Northern blot analysis suggested that the expression of groESL in Xc17was mainly regulated by sigma factor s32, not by HrcA. Western blot analysis showed high levels of GroEL in hrcA mutant and Xc17; after heat shock, only slight increase of GroEL was observed in both strains. 2-D gel electrophoresis results showed no obvious differences between protein profiles of Xc17 and hrcA mutant grown at 28℃. However, after heat shock at 37℃ for 20 min, a novel protein of 20 kDa, with a pI of 5.8 was observed on the 2-D map of the Xc17 or hrcA mutant, which was named Hsp20. The N-terminal 15 amino acids determined chemically share identity with the corresponding regions of small heat shock proteins from different organisms. According to the N terminal sequence of Hsp20 and the consensus sequence in the a-crystalline domain of the small heat shock proteins, two degenerated primers were designed. Using these primers, a 194 bp PCR fragment was amplified from Xc17 chromosome and sequenced. The amino acid sequence encoded by the 194 bp DNA fragment showed 86% identity to a low molecular weight heat shock protein of Xylella fastidiosa. The effect of heat shock response on the level of hsp20 expression was analyzed by Northern blot analysis. Results showed that the expression of hsp20 is greatly increased after heat shock at 37℃ for 15 min.

摘要…………………………………………………………………I
英文摘要……………………………………………………………II
前言…………………………………………………………………1
材料與方法…………………………………………………………8
一、少量質體DNA之萃取……………………………………8
二、中量質體DNA之萃取……………………………………8
三、染色體DNA之抽取………………………………………9
四、洋菜膠體 (agarose gel)電泳分析……………………………………10
五、DNA片段之回收……………………………………………………..10
六、DNA片段黏接反應 (ligation)……………………….10
七、勝任細胞 (competent cell)之製備…………………………………...10
八、轉形作用……………………………………………………………...11
九、電孔法………………………………………………………………...11
十、快速篩選法 (Rapid screen)…………………………………………..12
十一、DNA探針之製備……………………………………..……………12
十二、南方墨點雜交法 (Southern hybridization)……………………….13
十三、Xc17菌體內總量RNA之純化……………………………………13
十四、北方轉漬分析 (Northern blotting)…………………………………14
十五、SDS-PAGE之蛋白分析 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis)………………………………………...……….14
十六、西方墨點法分析 (Western blot)…………………………………..15
十七、二維電泳分析之蛋白萃取…………………………………………15
十八、蛋白質二維電泳分析………………………………………………16
十九、蛋白質銀染 (silver stain)…………………………………………..17
二十、蛋白質N端胺基酸定序之處理…………………………………..17
二十一、T-vector之製備………………………………………………….17
結果………………………………………………………………………………18
Xc17 hrcA突變株之構築………………………………………………….18
Xc17野生株及hrcA突變株之生理測試………………………………….19
北方轉漬分析……………………………………………………………...20
西方轉漬分析……………………………………………………………...20
二維電泳分析……………………………………………………………...21
熱休克蛋白Hsp20之N端定序及比對…………………………………..22
討論………………………………………………………………………………25
參考文獻…………………………………………………………………………29
圖表………………………………………………………………………………38
附錄………………………………………………………………………………57

吳承德 2000. 十字花科黑腐病菌hrcA基因之選殖定序與特性之研究。國立中興大學分子生物學研究所碩士論文。
許家智 2000. 十字花科黑腐病菌groESL基因之選殖及特性之研究。國立中興大學分子生物學研究所碩士論文。
黃蘭惠 1997. 十字花科黑腐菌rpoH基因之選殖及rpoD基因上游區域之探討。國立中興大學分子生物學研究所碩士論文。
戴盤銘. 1997. 十字花科蔬菜黑腐病菌dnaK及其上下游基因之選殖與定序. 國立中興大學分子生物學研究所碩士論文。
Aebersold, R.H., J. Leavitt, R.A. Saavedra, L.E. Hood, and S.B. Kent. 1987. Internal amino acid sequence analysis of proteins separated by one- or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose. Proc. Natl Acad. Sci. USA 84:6970-6974.
Babst, M., H. Hennecke ,and H.M. Fischer. 1996. Two different mechanisms are involved in the heat-shock regulation of chaperonin gene expression in Bradyrhizobium japonicum. Mol. Microbiol. 19:827-839.
Baird, P.N., L.M.C. Hall, and A.R.M. Coates. 1989 Cloning and sequence analysis of the 10 kDa antigen gene of Mycobacterium tuberculosis. J. Gen. Microbiol. 135:931-939.
Baldini, R.L., M. Avedissian, and S.L. Gomes. 1998. The CIRCE element and its putative repressor control cell cycle expression of the Caulobacter crescentus groESL operon. J. Bacteriol. 180:1632-1641.
Becker, A., F. Katzen, A. Puhler, and L. Ielpi. 1998. Xanthan gum biosynthesis and application: a biochemical/genetic perspective. Apple. Microbiol. Biotechnol. 50:145-152.
Braig, K., Z. Otwinowski, R. Hegde, D.C. Boisvert, A. Joachimiak, A.L. Horwich, and P.B. Sigler. 1994. The crystal structure of the bacterial chaperonin GroEL at 2.8 A. Nature 371:578-586.
Bucca, G., G. Ferina, A.M. Puglia, and C.P. Smith. 1995. The dnaK operon of Streptomyces coelicolor encodes a novel heat-shock protein which binds to the promoter region of the operon . Mol. Microbiol. 17:663-674.
Bukau, B. 1993. Regulation of the Escherichia coli heat-shock response. Mol. Microbiol. 9:671-680.
Chang, A.C.Y., and S.N. Cohen. 1979. Construction and characterization or amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J. Bacteriol. 134:1141-1156.
Cheng, M.Y., F.U. Hartl, J. Martin, R.A. Pollock, F. Kalousek, W. Neupert, E.M. Hallberg, R.L. Hallberg, and A.L. Horwich. 1989. Mitochondrial heat-shock protein Hsp60 is essential for assembly of proteins imported into yeast mitochondria. Nature 337:620-625
Craig, E.A., B.D. Gambill, and R.J. Nelson. 1993. Heat shock proteins:molecular chaperones of protein biogenesis. Microbiol. Rev. 57:402-414.
Craig, E.A., and C.A. Gross. 1991. Is hsp70 the cellular thermometer? TIBS 16:135-140
Derré, I., G. Rapoport, and T. Msadek. 1998. CtsR, a movel regulator of stress and heat shock response, controls clp and molecular chaperon gene expression in Gram-positive bacteria. Mol. Microbiol. 31:117-131.
Ehrnsperger, M., M. Gaestel, and J. Buchner. Structure and function of small heat-shock proteins. In: Molecular chaperones in the life cycle of proteins. edited by A. L. Find and Y. Goto. New York: Dekker, 1998, p.533-575.
Ellis, R.J. The molecular chaperone concept. 1990. Semin. Cell Biol. 1:1-9
Fink, A.L. Chaperone-mediated protein folding. 1999. Physiol. Rev. 79:425-449
Fink, A.L. The Hsp70 reaction cycle and its role in protein folding. In: Molecular Chaperones in the Life Cycle of Proteins, edited by A. L. Find and Y. Goto New York: Dekker, 1998, p 123-150.
Fischer, H.M., M. Babst, T. Kaspar, G. Acuña, F. Arigoni, and H. Hennecke. 1993. One member of a groESL-like chaperonin multigene family in Bradyrhizobium japonicum is co-regulated with symbiotic nitrogen fixation genes. EMBO J. 12:2901-2912
Georgopoulos, C., and W.J. Welch. 1993. Role of the major heat shock proteins as molecular chaperones. Annu. Rev. Cell Biol. 9:601-634.
Grossman, A.D., 1987. Sigma 32 synthesis can regulate the synthesis of heat shock proteins in Escherichia coli. Genes Dev. 1:179-184.
Hartl, F.U. 1996. Molecular chaperones in cellular protein folding. Nature 381:571-579
Hartl, F.U., R. Hlodan, and T. Langer. 1994. Molecular shaperones in protein folding:the art of avoiding sticky situations. Trends Biochem. Sci.. 19:20-25.
Hecker, M., W. Schumann, and U. Völker. 1996. Heat-shock and general stress response in Bacillus subtilis. Mol. Microbiol. 19:417-428.
Hendershot, L., J. Wei, J. Gaut, J. Melnick, S. Aviel, and Y. Argon. 1996. Inhibition of immunoglobulin folding and secretion by dominant negative BiP ATPase mutants. Proc. Natl. Acad. Sci. USA 93:5269-5274.
Herman, C., D. Thevenet, R. d’Ari, and P. Bouloc. 1995. Degradation of s32, the heat shock regulator in Escherichia coli, is governed by HflB. Proc Natl Acad Sci USA 92:3516-3520.
Hill R.B., J.M. Flanagan, and J.H. Prestegard. 1995. 1H and 15Nmagnetic resonance assignments, secondary structure, and tertiary fold of Escherichia coli DnaJ(1-78). Biochemistry 34:5587-5596.
Holt, J.G., N.R. Krieg, P.H.H. Sneath, J.T.Staley, and S.T. Willtams. 1994. Group 4 Gram-Negative aerobic/microaerophilic rods and cocli/subgroup 4A genus Xanthomonas, p100. In J. G. Holt et al. (ed.), Bergey’s manusl of determinative bacteriology-9. Williams & Wilkins press, Maryland USA.
Karzai, A.W., and R. Mcmacken. 1996. A bipartite signaling mechanism involved in DnaJ-mediated activation of the Escherichia coli DnaK protein. J. Biol. Chem. 271: 11236-11246.
Kenji, N., E.Z. Ron, H. Yanagi, and T. Yura. 1999. Differential and independent roles of a s32 homolog and HrcA repressor in the heat shock response of Agrobacterium tumefaciens. J. Bacteriol. 181:7509-7515.
Kennedy, J.F., and I.J. Bradshaw. 1984. Production, properties and application of xanthan. Prog Ind. Microbiol. 19:319-371.
Kilstrup, M., S. Jacobsen, K. Hammer, and F.K. Vogensen. 1997. Induction of heat shock proteins DnaK, GroEL, and GroES by salt stress in Lactococcus lactis. Appl. Environ. Microbiol. 63:1826-1937.
Koch, B., and M. Kilstrup, F.K. Vogensen, and K. Hammer. 1998. Induced levels of heat shock proteins in a dnaK mutant of Lactococcus lactis. J. Bacteriol. 180:3873-3881.
Laufen, T., U. Zuber, A. Buchberger, and B. Bukau. 1998. DnaJ proteins in:Molecular chaperones in the life cycle of proteins, edited by A. L. Fink and Y. Goto. New York: Dekker, p. 241-274.
Lee, G.J., A.M. Roseman, H.R. Saibil, and E. Vierling. 1997. A small heat shock protein stably binds heat-denatured model substrates and can maintain a substrate in a fokding-competent state. EMBO J. 16:659-671
Li, M., and S.-L. Wong. 1992. Cloning and characterization of the groESL operon from Bacillus subtilis. J. Bacteriol. 174:3981-3992.
Lindquist, S., and E.A. Craig. 1988. The heat-shock proteins. Annu. Rev. Genet. 22:631-677.
Marchuk, D., M. Drumm, A. Saulino, and F.S. Collins. 1991. Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR products. Nucleic Acids Res. 19:1154.
McCarty, J.S., A. Buchberger, J. Reinstein, and B. Bukar. 1995. The role of ATP in the functional cycle of the DnaK chaperone system. J. Mol. Biol. 249:126-137
Missiakas, D., and S. Raina. 1997. Protein misfolding in the cell envelope of Escherichia coli: new signaling pathways. Trends Biochem Sci 22:59-63.
Model, P., G. Jovanovic, and J. Dworkin. 1997. The Escherichis coli phage-shock-protein (psp) operon. Mol Microbiol 24:255-261.
Mogk, A., A. Völder, S. Engelmann, M. Hecker, W. Schumann, and U. Völker. 1998. Nonnative proteins induce expression of the Bacillus subtilis CIRCE regulon. J. Bacteriol. 180:2895-2900.
Mogk, A., G. Homuth, C. Scholz, L. Kim, F.X. Schmid, and W. Schumann. 1997. The GroE chaperonin machine is a major modulator of the CIRCE heat shock regulon of Bacillus subtilis. EMBO J. 16:4579-4590.
Münchbach, M., A. Nocker, and F. Narberhaus. 1999. Multiple small heat shock proteins in Rhizobia. J. Bacteriol. 181:83-90.
Narberhaus, F., and H. Bahl. 1992. Cloning, sequencing and molecular analysis of the groESL operon of Clostridium acetobutylicum. J. Bacteriol. 174:3282-3289.
Narberhaus, F., K. Giebeler, and H. Bahl 1992. Molecular characterization of the dnaK gene region of Clostridium acetobutylicum, including grpE, dnaJ, and a new heat shock gene. J. Bacteriol. 174:3290-3299
Neidhardt, F.C., and R.A. VanBogelen. 1987. The heat shock response. In Escherichia coli and Salmonella typhimurium:Cellular and Molecular Biology. Neidgardt, F.C., J.L. Ingraham, K.B. Low, B. Magasanik, M. Schaechter, and H.E. Umbarger (eds). Washington, DC:American Society for Microbiology Press, pp. 1334-1345.
O’Farrell, P.H. 1975. High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250:4007-4021.
Pennington, S.R., M.R. Wilkins, D.F. Hochstrasser, M.J. Dunn. 1997. Proteome analysis:From protein characterization to biological function. Trends in Cell Biology 7:168-173.
Qian, Y.Q., D. Patel, F.U. Hartl, and D.J. Mccoll. 1996. Nuclear magnetic resonance solution structure of the human Hsp40(HDJ-1) J-domain J. Mol. Biol. 260:224-235.
Roberts, R.C., C. Toochinda, M. Avedissian, R.L. Baldini, S.L. Gomes, and L. Shapiro. 1996. Identification of a Caulobacter crescentus operon encoding hrcA, involved in negatively regulating heat-inducible transcription, and the chaperone gene grpE. J. Bacteriol. 178:1829-1841
Rose, R.E. 1988. The nucleotide sequence of pACYC177. Nucleic Acids Res. 16:356
Rudiger, S., L. Germeroth, J. Schnerder-Mergener, and B. Bukau. 1997. Substrate specificity of the DnaK chaperone determined by screening cellulose-bound peptide libraries. EMBO J. 16:1501-1507.
Sambrook, J., E.F. Fritsch, and T. Maniatic. 1989. Molecular cloning:A Laboratory Manual. Cold Spring HarborLaboratory Press, Cold Spring Harbor, NY.
Sandford, P.A., and J. Baird. 1983. Industrial utilization of polysaccharides, p411-490. In G. O. Aspinall (ed.), The polyusaccharide, Vol. 2. Academic press, New York. N. Y. USA.
Schmidt, A., M. Schiesswohl, U. Volker, M. Hecker, and W. Schumann. 1992. Cloning, sequencing, mapping, and transcriptional analysis of the groESL operon from Bacillus subtilis. J. Bacteriol. 174:3993-3999.
Schröder, H., T. Langer, F.-U. Hartl, and B. Bukau. 1993. DnaK, DnaJ and GrpE form a cellular chaperone machinery capable of repairing heat-induced protein damage. EMBO J. 12:4137-4144.
Schulz, A., B. Tzschaschel, and W. Schumann. 1995. Isolation and analysis of mutants of the dnaK operon of Bacillus subtillis. Mol. Microbiol. 15:421-429.
Segal, G., and E.Z. Ron. 1996a. Heat shock activation of the groESL operon of Agrobacterium tumefaciens and the regulatory roles of the inverted repeat. J. Bacteriol 178:3634-3640.
Segal, G., and E.Z. Ron. 1996b. Regulation and organization of the groE and dnaK operons in Eubacteria. FEMS Microbiol. Lett. 13.8:1-10.
Servant, P., and P. Mazodier. 1996. Heat induction of hsp18 gene expression in Streptomyces albus G:Transcriptional and posttranscriptional regulation. J Bacteriol 178:7031-7036.
Spohn, G., and V. Scarlato. 1999. The autoregulatory HspR repressor protein governs chaperone gene transcription in Helicobacter pylori. Mol. Microbiol. 34:663-674.
Straus, D.B., and W.A. Walter. 1990. DnaK, DnaJ, and GrpE heat shock proteins negatively regulate heat shock gene expression by controlling the synthesis and stability of sigma 32. Genes Dev. 4:2202-2209.
Straus, D.B., W.A. Walter, and C.A. Gross. 1989. The activity of sigma 32 is reduced under conditions of excess heat shock protein production in Escherichia coli. Genes Dev. 3:2003-2010.
Szabo, A., R. Korszun, F.U. Hartl, and J. Flanagan. 1996. A zinc finger-like domain of the molecular chaperone DnaJ is involved in binding to denatured protein substrates. EMBO J. 15:408-417.
Szyperski, T., M. Pellecchia, D. Wall, C. Georgopoulos, and K. Wuthrich. 1994. NMR structure determination of the Escherichia coli DnaJ molecular chaperone: secondary structure and backbone fold of the N-terminal region (residues 2-108) containing the highly conserved J domain. Proc. Natl. Acad. Sci. USA 91:11343-11347.
Taylor, J., N.L. Anderson, A.E. Scandory, K.E. Villard, and N.G. Anderson. 1982. The TYCHO system for computer analysis of two-dimensional gel electrophoresis patterns. Clin. Chem. 28:861-866.
Tilly, K., J. Spence, C. Georgopoulos. 1989. Modulation of stability of the Escherichia coli heat shock regulatory factor sigma. J. Bacteriol. 171:1585-1589.
Tomoyoasu, T., J. Gamer, B. Bukau, M. Kanemori, H. Mori, A.J. Rutman, A.B. Oppenheim, T. Yura, K. Yamanaka, H. Niki, S. Hiraga, and T. Ogura. 1995. Escherichia coli FtsH is a membrane-bound, ATP-dependent protease which degrades the heat-shock transcription factor s32. EMBO J. 14:2551-2560
Vandekerckhove, J,. G. Bauw, M. Puype, J. Van Damme, and M. Van Montagu. 1985. Protein-blotting on polybrene-coated glass-fiber sheets. Eur. J. Biochem. 152:9-19.
Vauterin, L., J. Smings, K. Kersters, M. Gillis, T.W. Mew, M.N. Schroth, N.J. Palleroni, D.C. Hildebrand, D.E. Stead, E.L. Civerolo, A.C. Hayward, H. Maraite, R.E. Stall, A.K. Vidaver, and J.F. Brabury. 1990. Towards an improved taxonomy of Xanthomonas. Int. J. Syst. Bacteriol. 40:312-316.
Vieira, J., and J. Messing. 1991. New pUC-derived cloning vectors with different selectable markers and DNA replication origins. Gene 100:189-194.
Viitanen P.V., T.H. Lubben, J. Reed, P. Goloubinoff, D.P. O’keefe, and G.H. Lorimer. 1990. Chaperonin-facilitated refolding of rebulosebisphosphate csarboxylase and ATP hydrolysis by chaperonin 60 (GroEL) are K+ dependent. Biochemistry 29:5665-5671.
Wang, T.-W., and Y.-H. Tseng. 1992. Electro transformation of Xanthomonas campestris by RF DNA of filamentous phage fLf. Lett. Appl. Microbiol. 14:65-68
Wetzstein, M., U. Völker, J. Dedio, S. Löbau, U. Zuber, M. Schiesswohl, et al. 1992. Cloning, sequencing, and molecular analysisi of the dnaK locus from Bacillus subtillis. J. Bacteriol. 174:3300-3310
Wilkins, M.R., C. Pasquali, R.D. Appel, K. OU, O. Golaz, J.C. Sanchez, J.X. Yan, A.A. Gooley, G. Hughes, I. Humphrey-Smith, K.L. Williams, D.F. Hochstrasser. 1996. From proteins to proteomes:Large-scale protein identification by two-dimensional electrophoresis and amino acid analysis. BioTechnology 14:61-65.
Williams, P.H. 1980. Black rot:a continuing threat to world crucifers. Plant Dis. 64:736-742.
Wu, C.C., M.D. Sun, J.J. Hua, and K.C. Tzeng. 1990. Collection, authentication, identification and preservation of plant pathogenic bacteria in Taiwan (III). Food Industry research and Development Institute, Hsinchu, Taiwan, R.O.C. Research Report No. 634.
Xu, Z.H., A.L. Horwich, and P.B. Sigler. 1997. The crystal structure of the asymmetric GroEL-GroES-(ADP)7 chaperonin complex. Nature 388:741-750
Yang, B.Y., and Y.H. Tseng. 1988. Production of exopolysaccharide and levels of protease and pectinase activity in pathogenic and non-pathogenic strains of Xanthomonas campestris pv. campestris. Bot. Bull. Academia Sinica. 29:93-99.
Ye, R., and S.-L. Wong. 1994. Transcriptional regulation of the Bacillus subtilis glucitol dehydrogenase gene. J. Bbacteriol. 176:3314-3320.
Yuan, G., and S.-L. Wong. 1995. Isolation and characterization of Bacillus subtilis groE regulatory mutants: evidence for orf39 in the dnaK operon as a repressor gene in regulating the expression of both groE and dnaK. J. Bacteriol. 177:6462-6468.
Yura, T., H. Nagai, and H. Mori. 1993. Regulation of the heat-shock response in Bacteria. Annu Rev Microbiol 47:321-350
Zahn, R., S. perrett, and A.R. Fersht. 1996a. Conformational states bound by the molecular chaperones GroEL and SecB: a hidden unfolding (annealing) activity. J. Mol. Biol. 261:43-61.
Zahn, R., S. perrett, G. Stenberg, and A.R. Fersht. 1996b. Catalysis of amide proton exchange by the molecular chaperones GroEL and SecB. Science 271:642-645.
Zuber, U., and W. Schumann. 1994. CIRCE, a novel heat shock element involved in regulation of heat shock operon dnaK of Bacillus subtilis. J. Bacteriol. 176:1359-1363.