臺灣博碩士論文加值系統

十字花科黑腐病菌 Xanthomonas campestris pv. campestris 為革蘭氏陰性菌。 P20H之突變株TC820為由mini-Tn5致變在clp基因所得。 clp基因蛋白產物與cyclic AMP receptor protein （CRP） 相似，因此稱其為 Clp (CRP-like protein)。 在本研究中，主要萃取P20H與 TC820之總體蛋白，進行二維電泳分析。 在 P20H 與TC820之2D gels中，分別標記有201 spots和148 spots，彼此之間有125 個蛋白相符合。 將六個表現量有差異之蛋白進行質譜分析後，其中一個蛋白為GroEL，受Clp正調控，此蛋白是由與上游groES基因組成一操縱子（operon）的groEL 基因所轉譯出來。
為了了解groESL基因的轉錄是否會受Clp調控，所以我們分析以LacZ為報導基因之融合質體β-galactosidase 的表現量。 其中一個構築體（pWHP552）含有相對於groES基因轉譯起始點的 -32 到 -583 的啟動子片段，在此片段中有 Clp-binding site （-243 到 -264）、 CIRCE element（-51 到 -77）及 σ32 結合區域（-10/-35 , 分別在-118~-127 和 —142~-148之間）。 另一個構築體（pWHP150）含有一個大小為150 bp（-29 到 -178）但不含Clp結合區域的片段。 這結果指出，groESL 基因之啟動子活性在 clp 基因突變株TC820之活性比在 P20H 野生株中降低約 3倍。 藉由西方點墨法分析，使用GroES之專一性抗體，顯示GroES在clp突變株TC820表現量下降，證實Clp正調控groESL operon，此為認定stress-responsive groESL為 Clp-dependent 的首次報導。

Xanthomonas campestris pv. campestris is a Gram-negative bacterium causing black-rot disease in crucifers. TC820 was a mutant derived from strain P20H by mini-Tn5 transposition at clp gene, encoding a protein homologous to cyclic AMP receptor protein (CRP), Clp (CRP-like protein). In this study, crude extracts prepared from P20H and TC820 were subjected to two-dimensional gel electrophoresis. In P20H and TC820, 201 and 148 spots were labeled, respectively, with 125 of the spots matched. Six of the proteins showing differences in amounts were analyzed by mass spectrometry. One of them, upregulated by Clp, was identified as GroEL encoded by groEL, a gene forming an operon with the upstream groES gene.
To understand whether transcription of groESL gene is regulated by Clp, transcriptional fusion constructs using LacZ as the reporter were assayed for β-galactosidase levels. One construct (pWHP552) carried the region from —32 to —583 relative to groESL initiation codon, containing a putative Clp-binding site（-243 to -264）, a CIRCE element（-51 to -77）and a σ32 binding region（-10/-35 at -118~-127 and -142~-148, respectively）. The other (pWHP150) contained a 150 bp fragment（-29 to -178）without Clp-binding site. The results indicated that the activity of groESL promoter is about 3-fold lower in clp mutant TC820 than that in the parental P20H. Using antibodies specific to GroES for Western blotting revealed the amounts of GroES were found to be reduced in clp mutant TC820, confirming that Clp upregulates the expression of groESL operon. This is the first report identifying the stress-responsive groESL as Clp dependent.

中文摘要
英文摘要
縮寫字對照表 1
前言 3
材料與方法
I. 材料
一、 菌種及質體 。 9
二、 藥品、酵素。 9
三.、菌種培養基。 9
1. 液體培養基。 9
2. 固體培養基。 9
四、 引子。 10
五、 試劑與緩衝溶液。 10
1. 染色體DNA抽取之試劑。 10
2. 質體DNA抽取之試劑。 10
3. 一般電泳試劑。 10
六. 二維電泳分析系統。 11
七. 二維電泳藥品 11
II. 實驗方法
一、菌種保存。 11
二、質體 DNA 之抽取。 12
三、染色體 DNA 之抽取。 12
四、洋菜膠體電泳分析 (Agarose gel electrophoresis)。 12
五、DNA片段回收。 13
六、DNA 黏接 (Ligation)。 13
七、質體 DNA 之快速篩選法 (Rapid screen)。 13
八、轉形作用 (transfomation)。 14
九、聚合酶鏈鎖反應 (Polymerase chain reaction, PCR)。 15
十、點測試法 (Spot test)。? ? ? ? ? ?? ? ?? 15
十一、生長曲線測定 (growth curve)。 16
十二、b-galactosidase 之活性測試。 16
十三、 SDS-聚丙醯銨凝膠電泳(SDS-PAGE)。? ? ? ? ? 17
十四、西方點墨分析（Western blot）。 17
十五、二維電泳分析之蛋白質萃取。 18
十六、蛋白質二維電泳分析。 18
十七、蛋白質銀染 ( silver stain)。 19
十八、SYPRO Ruby stain。 19
結果
一、利用二維電泳分析 P20H 及 clp 基因突變株TC820蛋白表現 21
情形。
1. 生長曲線測試。 21
2. 二維電泳分析菌體內總體蛋白質表現情形。 21
二、利用ImageMasterTM 2D Elite software 分析2Dgels? ? ? ? 22
三、質譜分析。 ? ? ? ? ? ? ? ? 23
四、利用西方點墨法分析。 23
五、groESL基因啟動子在 P20H 及 clp 基因突變株之表現情形。 24
1. groESL基因啟動子之選殖與質體之構築。 24
2. 轉型株之篩選。 25
3. groESL基因啟動子之表現情形。 25
（1）在LB medium 之表現情形。 25
（2）在XOLN medium含2﹪Xylose為碳源之表現情形。 26
4. 熱休克對groESL 基因啟動子在P20H野生株及clp基因 26
突變株TC820活性表現情形。
討論 28
參考文獻 34
圖表 42

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