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DC 欄位 | 值 | 語言 |
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dc.contributor.author | 陳景康 | zh_TW |
dc.date.accessioned | 2021-07-01T08:13:53Z | - |
dc.date.available | 2021-07-01T08:13:53Z | - |
dc.date.issued | 1986 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75562 | - |
dc.description.abstract | 水稻白葉枯病病原菌之RNA聚合?在其溶解性噬菌體Xp10感染後,會遺失其σ次單位.為了探討σ次單位遺失之原因,乃純化出RNA聚合?核?(core enzyme),並以核?為抗原製備抗血清.以免疫沈澱法分析Xp10感染前後粗酵素萃取液中RNA聚合?組成,發現σ次單位會因Xp10的感染而降低對核?之吸附能力. 於純化過程中發現一個分子量為29,000的蛋白質ε,會與全?(holozyme)一起被純化出來.DEAE sephadex A 25管柱層析可將ε與全?分離.將ε與RNA聚合?重組後,發現可增進其轉錄活性,並可促進全?對Xp10 DNA的吸附能力.ε亦可於粗酵素萃取液中,以抗核?之抗血清沈澱出來.根據以上結果,認定ε為水稻白葉枯病病原菌RNA聚合?之次單位. 於實驗過程中亦發現核酸限制?XorII活性伴隨RNA聚合?出現.兩者間有無相關性,仍須進一步的研究. | zh_TW |
dc.description.abstract | The σ subunit of Xanthomonas campestris pv. oryzae RNA polymerase was loss during bacteriophage Xp10 infection. To study the mechanism of σ loss, RNA polymerase from uninfected cells was purified to homogeneity by DNase treatment, ultracentrifugation, ammonium sulfate fractionation, Heparin sepharose 4B, Bio Rex 70, Bio gel A 1.5 m and DEAE sephadex A 25 chromatography. Both holozyme and core enzyme from RNA polymerase could be obtained by these procedures. Core enzyme was used to prepare antiserum for further study. Immunoprecipitations of anti-core antiserum vs. infected and uninfected crude extracts were performed and compared by SDS-PAGE followed by silver stain. The amount of σ subunit coprecipitated with core enzyme of infected crude extract was far less than that of uninfected crude extract. Such result suggested that σ subunit exhibited less binding ability to core enzyme after Xp10 infection. A 29,000 daltons protein named ε was found to copurify with holozyme. It also coprecipitated with anti-core antiserum. ε separated from holozyme through DEAE sephadex A 25 chromatography. After reconstitution with holozyme in vitro, the increase of transcription activity on Xp10 DNA and poly d(A-T) was observed. ε alond did not bind to Xp10 DNA, however, after binding with holozyme, the increase of binding efficiency to Xp10 DNA was detected. It is concluded that ε is a subunit of X. campestris pv. oryzae RNA polymerase. A restriction enzyme activity similar to XorII was detected. The role of thus enzyme activity in RNA polymerase is still unknown, it needs further investigation. | en |
dc.description.provenance | Made available in DSpace on 2021-07-01T08:13:53Z (GMT). No. of bitstreams: 0 Previous issue date: 1986 | en |
dc.description.tableofcontents | 中文摘要 英文摘要 簡寫字對照表 壹.緒言……………………………………………………………………………………………………………………1 貳.材料與方法 一.材料 (一).生物材料………………………………………………………………………………………………………4 (二).藥品與酵素……………………………………………………………………………………………………4 (三).培養基…………………………………………………………………………………………………………5 (四).緩衝液…………………………………………………………………………………………………………6 二.方法 (一).細菌與噬菌體之培養…………………………………………………………………………………………8 (二).噬菌體Xp10 DNA的純化………………………………………………………………………………………9 (三).菌體之製備 …………………………………………………………………………………………………10 (四).RNA聚合?之萃取……………………………………………………………………………………………11 (五).RNA聚合?活性之測定………………………………………………………………………………………12 (六).蛋白質,KCl及核酸濃度之測定……………………………………………………………………………13 (七).管柱色層分析之製備………………………………………………………………………………………14 (八).凝膠電泳法,分析蛋白質…………………………………………………………………………………17 (九).洋菜膠電泳法,分析DNA……………………………………………………………………………………20 (十).S. aureus溶液之抽取………………………………………………………………………………………21 (十一).抗血清之製備……………………………………………………………………………………………21 (十二).抗血清效價之測定………………………………………………………………………………………22 (十三).免疫沈澱法…………………………………………………………………………………………………22 (十四).DNA片段尾端標幟法………………………………………………………………………………………23 (十五).能與蛋白質結合的DNA片段之測定………………………………………………………………………24 (十六).蛋白質對DNA吸附能力之測定……………………………………………………………………………25 (十七).照像,暗房技術及放射自動顯影術………………………………………………………………………25 參.結果 一.水稻白葉枯病病原菌RNA聚合?之純化 (一).全?部份………………………………………………………………………………………………………28 (二).核?部份………………………………………………………………………………………………………33 二.RNA聚合?次單位組成及分子量 (一).次單位組成……………………………………………………………………………………………………33 (二).次單位分子量…………………………………………………………………………………………………37 三.水稻白葉枯病病原菌與大腸桿菌RNA聚合?的比較 (一).抗原-抗血清反應………………………………………………………………………………………………37 (二).次單位組成及分子量…………………………………………………………………………………………37 四.蛋白質與RNA聚合?之關係 (一).構造上的探討--粗酵素萃取液中RNA聚合?次單位組成……………………………………………………40 (二).功能上的探討 1.對轉錄活性的影響………………………………………………………………………………………………43 2.對DNA吸附能力的影響…………………………………………………………………………………………43 3.對DNA片段選擇性吸附的影響…………………………………………………………………………………43 五.噬菌體Xp10感染後,寄主RNA聚合?組成之變化…………………………………………………………………43 六.RNA聚合?的變性及再活化…………………………………………………………………………………………47 七.核酸限制?XorII與RNA聚合?的關係………………………………………………………………………………47 肆.討論 一.水稻白葉枯病病原菌體內RNA聚合?的型式 ………………………………………………………………………54 二.RNA聚合?次單位組成 ………………………………………………………………………………………………54 三.次單位之功能…………………………………………………………………………………………………………55 四.噬菌體Xp10感染後,寄主RNA聚合?次單位組成之變化 …………………………………………………………56 五.RNA聚合?變性及再活化的條件 ……………………………………………………………………………………56 六.純化次單位的策略……………………………………………………………………………………………………57 七.酵素純化技術的改良…………………………………………………………………………………………………58 八.未來研究方向…………………………………………………………………………………………………………58 九.水稻白葉枯病病原菌RNA聚合?與核酸限制?XorII活性的關係…………………………………………………60 伍.參考文獻…………………………………………………………………………………………………………………61 | |
dc.language.iso | zh-TW | |
dc.date.schoolyear | 74-2 | |
dc.description.degree | 碩士 | |
dc.relation.page | 65 | |
dc.rights.note | 未授權 | |
dc.contributor.author-dept | 生命科學院 | zh_TW |
dc.contributor.author-dept | 植物科學研究所 | zh_TW |
顯示於系所單位: | 植物科學研究所 |
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