請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76143
標題: | 水稻白葉枯病病原菌的去氧核醣核酸聚合?的部份純化與特性分析 Partial Purification and Characterization of a DNA Polymerase in Xanthomonas campestris pv.Oryzae |
作者: | 紀文章 |
出版年 : | 1995 |
學位: | 碩士 |
摘要: | 多種DNA聚合?在原核生物已被詳細研究,但水稻白葉枯病病原菌的DNA聚合?尚無人研究;本論文乃嘗試於分離純化水稻白葉枯病病原菌的DNA聚合?,以瞭解噬菌體Xp12感染後DNA聚合?之變化;其純化步驟如下:細胞粉碎器打破細菌細胞,然後高速離心去除細胞殘骸;透析後,經過離子管柱層析DEAE-cellulose、Phospho-cellulose,再經過親和性管柱heparin-Sepharose、DNA-cellulose純化;此純化酵素步驟可增加比活性232.3倍;由酵素活性與電泳圖之蛋白質濃度對照,推測DNA聚合?為分子量42 kDa、66 kDa或此兩蛋白質複合體之蛋白質。 初步分析顯示:水稻白葉枯病病原菌的DNA聚合?需要四種去氧核?三磷酸為受質,以DNA為範本及二價離子如錳離子或鎂離子。DNA合成的最適當溫度及酸鹼值分別在37℃及pH 8.2,鎂離子與錳離子的最適當濃度分別20mM及0.4mM。鹽類:氯化鉀、氯化鈉、硫酸銨、氯化銨均可抑制DNA聚合?活性。至於範本的專一性,以經pancreatic-DNase處理過產生缺口之salmon sperm DNA為最佳,以超音波處理產生缺口之salmon sperm、calf-thymus DNA,與人工合成之poly(dA)-p(dT)12-18為次佳。完全無法利用雙股、單股DNA及RNA作為範本。水稻白葉枯病原菌DNA聚合?不受化學抑制劑aphidicolin、N-ethylmaleimide、Ara-CTP所抑制,但會稍受ddTTP抑制,此特性類似E. coli DNA polymerase I。 受噬菌體Xp12感染30分鐘之水稻白葉枯病病原菌,經離子管柱層析DEAE-cellulose發現在0.39M KC1溶離處,有另一DNA聚合?的活性出現,此可能為噬菌體Xp12之DNA聚合?。 Though many prokaryotic DNA polymerases have been well documented, the DNA polymerase of Xanthomonas spp. has not been reported. Thus we isolated and characterized this DNA polymerase and monitored its activity profile after infection by phage Xp12. The DNA polymerase was purified as follows: The cell extracts were harvested by French pressure homogenization followed by high-speed centrifugation. After dialysis the cell extracts were fractionated by DEAE-cellulose and Phospho-cellulose ion exchange chromatography, followed by heparin-Sepharose and DNA-cellulose affinity chromatography. The specific activity of DNA polymerase was enriched for 232.3 fold. Based on the results of activity assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weight of DNA polymerase was suspected to be either 42 kD, 66 kD or both. Preliminary characterizations were done. Utilizing divalent cations such as Mn2+ or Mg2+ as cofactors and DNA as a template, the X. oryzae DNA polymerase would polymerize the four dNTPs by incorporating them into the template in vitro. The optimal temperature and pH was measured to be 37℃ and pH 8.2 while the optimal concentration of magnesium and mangenese ion was 20 mM and 0.4 mM. Salts such as KCl, NaCl, (NH4)2SO4, and NH4Cl could inhibit the enzyme activity. In terms of template specificity, pancreatic DNase-treated salmon sperm DNA is better than sonicated salmon sperm DNA, sonicated calf-thymus DNA, or synthetic poly(dA)-poly(dT)12-18. This enzyme could not use double-stranded DNA, single-stranded DNA, or RNA as templates. Similar to E. coli, X oryzae DNA polymerase was found to be insensitive to chemical inhibitors, such as aphidicolin, N-ethylmaleimide, Ara-CTP, while moderately sensitive to ddTTP. If X oryzae was infected by phage Xp12 for 30 minutes, another DNA polymerase activity was found in the 0.39M KCl eluate of DEAE-Cellulose fractionation. This activity was plausibly the DNA polymerase from Xp12. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76143 |
全文授權: | 未授權 |
顯示於系所單位: | 植物科學研究所 |
文件中的檔案:
沒有與此文件相關的檔案。
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。