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研究生:李培增
研究生(外文):Lee Pei-Tseng
論文名稱:十字花科蔬菜黑腐病菌Xanthomonascampestrispv.campestris致病基因orfF及其基因產物之定性
論文名稱(外文):Characterization of a virulent gene of Xanthomonas campestris pv. campestris, orfF, and its gene product
指導教授:陳建華陳建華引用關係
指導教授(外文):Jiann-Hwa Chen
學位類別:碩士
校院名稱:國立中興大學
系所名稱:分子生物學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:中文
論文頁數:112
中文關鍵詞:黃原菌黑腐病
外文關鍵詞:Xanthomonas campestris pv. campestrisblack root
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Xanthomonas campestris pv. campestris (Xcc) 能引起十字花科植物 (crucifers) 的黑腐病 (black rot)。本研究為探討其一致病品系Xcc11的orf F基因及其產物與病原性的關係。首先將一段攜帶有orf F基因的Xcc genomic DNA 片段於orf F基因中插入一段Kmr 基因,再利用一自殺質體將此orf F::Kmr片段送入Xcc11與另一致病品系Xcc17中進行DNA重組。先選取Kmr突變株,再以南方分析確定為orf F::Kmr突變株。以蕪菁幼苗進行病原性測試時,發現Xcc17的orf F::Kmr突變株致病力減弱,而Xcc11的orf F::Kmr突變株則完全喪失致病力。
利用大腸桿菌T7 promoter/ T7 RNA polymerase系統及35S Methionine,證明攜帶有orf F基因的Xcc11 genomic DNA 片段在大腸桿菌中可表現一與Orf F蛋白預測大小相同 (12.4 kDa) 的蛋白產物。進一步以蛋白質表現載體pET21b及親合性層析管柱獲得Orf F-His蛋白溶液。以HPLC分析此Orf F-His蛋白溶液,發現有兩種形式的Orf F-His蛋白存在,分子量皆為約13 kDa。將此Orf F-His蛋白溶液做為抗原,製備抗體,再以此抗體對Xcc11與Xcc17進行西方雜配分析,結果顯示兩品系Xcc菌的菌體蛋白及培養液蛋白中皆可偵測到雜配訊號。此結果顯示Orf F蛋白為一分泌蛋白。
由於Orf F蛋白在第28~30位置的氨基酸具有疑似nuclear localization signal (NLS) 的序列,因此將orf F 基因接到GUS報導基因並選殖入植物表現質體中,利用粒子傳遞系統 (biolistic system) 將質體送入洋蔥上表皮細胞並進行染色,發現Orf F-GUS融合蛋白會進入洋蔥細胞的細胞核中。但若將Orf F的NLS序列進行deletion或mutation後,進行相同的實驗,則發現deletion或mutation的Orf F-GUS融合蛋白就無法進入洋蔥細胞的細胞核中。利用yeast one-hybrid system發現Orf F蛋白在酵母菌中並不具有活化轉錄作用的能力。
Xanthomonas campestris pv. campestris (Xcc) is the causing agent of black rot of crucifies. The purpose of this study is to find out the role which orf F gene plays in pathogenicity. Insertion of a Kmr DNA fragment in the orf F gene was first made with an orf F-containing Xcc genomic DNA fragment, and the fragment was introduced into two strains of Xcc via a suicide vector. The orf F::Kmr recombinants were selected and confirmed by Southern hybridization. The orf F::Kmr mutants of one Xcc strain showed reduced pathogenicity toward turnip plants, whereas those of the other Xcc strain completely lost the pathogenicity.
By using the T7 RNA polymerase/T7 promoter system and 35S Methionine, it was found that the orf F-containing Xcc DNA fragment was capable of expressing a protein with expected size of Orf F. With the protein expression plasmid pET21b, an Orf F-His protein was generated in vivo and purified through affinity chromatography. However, further HPLC analysis indicated that the protein solution contained two forms of Orf F-His with same molecular weight.
The Orf F-His protein solution was used to prepare polyclonal antibody which was then to probe the total proteins of two Xcc strains and their cultural filtrates. Both strains demostrated hybridization signals with their cellular proteins and the cultrual filtrates, indicating that Orf F is a secretion protein.
The orf F gene was cloned into a plant expression vector with GUS reporter gene and delivered into onion epidermal cells via particle biolistic system. It was shown that the Orf F-GUS protein was localized in the nucleus of the onion dermal cells. The 28th to 30th residues of the predictive Orf F protein sequence are lysine residues and likely constitue of a nuclear localization signal. Deletion or mutation of the three residues abolished the nuclear localization capability of Orf F. Further yeast one-hybrid study indicated that Orf F did not have transcriptional activation activity in yeast.
縮寫字對照表 1中文摘要 3
英文摘要 5
壹、前言 7
貳、材料與培養條件 12
一、材料 12
二、培養條件 13
三、引子 14
參、試劑與方法 16
一、少量細菌質體DNA的抽取 16
二、勝任細胞 (competent cells) 之製備 17
三、轉形作用 18
四、DNA片段回收 19
五、聚合脢連鎖反應 (Polymerase Chain Reaction, PCR) 20
六、DNA黏接反應 21
七、十二烷基硫酸鈉聚丙烯醯氨板膠電泳法 SDS-PAGE 22
八、西方雜配分析 25
九、Xanthomonas campestris 染色體DNA的抽取 27
十、補平 (fill-in) 作用 28
十一、南方雜配分析 29
十二、-galactosidase活性測試 33
十三、基因槍轉殖法 35
十四、Histochemical染色 36
十五、DAPI staining 37
十六、蛋白質濃度測定 38
十七、三親交配 39
十八、pET system之蛋白質誘導表現及純化 40
十九、酵母菌 (S. cerevisiae) 之轉形 (electroporation) 42
二十、酵母菌 (S. cerevisiae) 全DNA抽取 44
二十一、X. campestris pv. campestris的病原性測試 44
二十二、X. campestris pv. campestris 的胞外蛋白之誘導 46
二十三、高壓液相色層分析法 (HPLC) 48
二十四、以35S-Methionine標定蛋白 49
肆、結果 51
一、Xcc17及Xcc11的orf F基因knock-out株的構築 51
二、Xcc17及Xcc11的orf F基因knock-out株的病原性測試 54
三、證明含有orf F基因之Xcc11 DNA片段在E. coli中可以在T7 55
promoter之啟動下表現Orf F蛋白
四、製備Orf F蛋白多價抗體 57
五、Xcc的病原性測試 61
六、Orf F蛋白NLS的定性分析 61
七、Orf F蛋白的活化轉錄作用的能力測試 65
伍、討論 67
圖表 74
參考文獻 102
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