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研究生:李佳蓉
研究生(外文):Chia-Jung Lee
論文名稱:火鶴花細菌性葉枯病病原菌Insertionsequence(IS)之分離與分析
論文名稱(外文):Isolation and characterization of insertion sequence (IS) from Xanthomonas campestris pv. dieffenbachiae, the casual agent of anthurium blight
指導教授:林長平林長平引用關係
指導教授(外文):Chan-Pin Lin
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:植物病理學研究所
學門:農業科學學門
學類:植物保護學類
論文種類:學術論文
論文出版年:2000
畢業學年度:88
語文別:中文
論文頁數:81
中文關鍵詞:轉位子火鶴花細菌性葉枯病火鶴花細菌性葉枯病病原菌
外文關鍵詞:insertion sequenceISanthurium blightXanthomonas campestris pv. dieffenbachiaeXCDISXCD1
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在本論文中,成功地使用pUCD800質體誘釣火鶴花細菌性葉枯病病原菌(Xanthomonas campestris pv. dieffenbachiae, 簡稱XCD)之轉位子(insertion sequence, IS),並且找到一新轉位子(ISXCD1)。實驗中使用電穿孔法(electroporation)將pUCD800質體導入XCD中,在蔗糖存在下,質體上所含sacBR基因所表現之levansucrase使導入質體之葉枯病菌出現族群數量遽減情形。篩選在蔗糖培養基上之葉枯病菌突變株,並對其pUCD800進行質體分析,經內鑑識酵素EcoRI及HindIII 酵解後圖譜分析觀察sacBR區域變化。實驗中總計篩選了186株突變株,其中70株(38.2﹪)為質體大小完全不變者,28株(15﹪)在sacBR區域有明顯插入1.2 kb片段,88株(46.77﹪)質體大小發生不可預期的重組現象。PCR測定及序列分析結果顯示此sacBR區域中之1.2 kb插入片段有21株為轉位子IS1051所引起之插入點不活化,另7株則為IS3 family之新轉位子。新轉位子(ISXCD1)(accession no. AF263433),全長為1,203 bp,包括兩端38 bp的不完全(imperfect)反向重複序列(inverted sequence, IR),其中包含有11 bp mismatchs,在突變株15-4及27-2中在sacBR基因上產生4 bp DR(direct repeat),胺基酸序列為2個部分重疊的orfA及orfB,在overlapping區域可發現IS3 family轉位酶常見的A7T motif,在orfA也可找到Helix-turn-helix motif(HTH motif)、orfB也可找到保守的DDE motif,其均為 IS3 family轉位酶之共通特性。由南方氏雜配法得知ISXCD1在XCD基因組中有9-11個雜配訊號,並且似乎也廣泛存在於G(-)及G(+)細菌中。IPCR(inverse polymerase chain reaction)的結果找到此轉位子在基因組中獨立的5個位置,比對轉位子之周圍序列並未發現有明顯插入點偏好性;而在H-2轉殖株中發現此ISXCD1正位於另一轉位子IS1051之中,並且在IS1051上造成4 bp之DR。依據轉位子之諸多特性,測試其應用於核酸探針之潛力;使用此轉位子內部序列引子對F570/ R1006(產物為436 bp大小)之聚合酵素連鎖反應對純化菌體DNA之偵測極限為1.5 pg,與前人研發的偵測用引子對Dif1/Dir1靈敏度(林,1999)效果相近。
A new insertion sequence was successful isolated from Xanthomonas campestris pv. dieffenbachiae(XCD)by applying the entrapment plasmid, pUCD800. The plasmid pUCD800 was introduced into XCD via electroporation. In the presence of sucrose, levansucrase produced by sacB of pUCD800 plasmid and caused XCD population decreased. Restriction enzyme analyses of these plasmids from 186 sucrose-resistant colonies revealed that the majority recovered that in the manner contained insertions in sacBR element of pUCD800(18.8﹪)or show no noticeable change in the structure of pUCD800(38.2﹪), and 46.77﹪ probably involved gross rearrangement of pUCD800. The new insertion sequence, ISXCD1, ( accession no. AF263433 ) is 1,203 bp in size and contains 38 bp inverted repeats with 11 bp mismatches at its termini. It generated 4-bp target site duplications in sacBR regions of pUCD800 harbored in 15-4 and 27-1 transformants, and carried two overlapping ORFs(orfA and orfB), with an A7T motif within the overlapping region. The deduced amino acid sequences of orfA and orfB contained a potential helix-turn-helix motif and a D, D(35)E domain of transposases, respectively. ISXCD1 is present about 9-11 copies in the XCD genome according to Southern hybridization. Southern hybridization analyses the distribution of the new IS is widely existed G(-)and G(+). Sequence analysis of 5 independent genomic copies of IPCR products indicated no preferred insertion site for the new insertion sequence. In H-2 transformant revealed that ISXCD1 exactly inserted into IS1051, and caused a 4 bp target site duplication. For the detection purpose, a PCR primer pair F570/ R1006 was designed based on the nucleotide sequence of the new IS. A minimum of 1.5 pg DNA of purified genomic DNA of XCD was sufficient to amplified a 436 bp PCR-product.
壹、 前言
貳、 前人研究
一、 火鶴花細菌性葉枯病病原菌之相關研究
二、 轉位子之相關研究
三、 轉位子在Xanthomonas屬細菌之相關報告
四、 pUCD800質體系統之相關研究
參、 材料與方法
一、 供試菌株來源、培養及保存
二、 細菌全DNA之抽取與濃度測定
(一) 以phenol/ CIAA步驟抽取細菌全DNA
(二) 以spin column步驟抽取細菌全DNA
(三) DNA濃度及純度測定
三、 pUCD800質體導入火鶴花細菌性葉枯病菌
(一) 以電穿孔法(electroporation)導入質體
(二) 轉形株之確認
四、 pUCD800自發性突變株的篩選及分析
(一) 自發性突變株的篩選
(二) 突變株質體大小之分析及分類
(三) 插入片段選殖
(四) 嵌入片段之定序
五、 火鶴花細菌性葉枯病病原菌新轉位子(ISXCD1)之特性分析
(一) 新轉位子在基因組上套數(copy number)之確定
(二) 新轉位子在Xanthomonas 屬及其它細菌中之分佈
(三) 轉位子插入點序列之分析
六、 新轉位子(ISXCD1)應用於開發核酸探針的潛力探討
肆、 結果
一、pUCD800質體導入火鶴花細菌性葉枯病菌
(一) 以電穿孔法(electroporation)導入質體
(二) 轉形株之確認
二、pUCD800自發性突變株的篩選及分析
(一) 自發性突變株的篩選
(二) 突變株質體大小之分析及分類
(三) 插入片段選殖
(四) 嵌入片段之定序
三、火鶴花細菌性葉枯病病原菌新轉位子(ISXCD1)之特性分析
(一) 新轉位子在基因組上套數(copy number)之確定
(二) 新轉位子在Xanthomonas 屬及其它細菌中之分佈
(三) 轉位子插入點序列之分析
四、新轉位子(ISXCD1)應用於開發核酸探針的潛力探討
伍、 討論
陸、 中文摘要
柒、 英文摘要
捌、 參考文獻
玖、 圖表
拾、 附圖
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