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研究生:蕭懿民
研究生(外文):Yi-Min Hsiao
論文名稱:十字花科黑腐病菌蛋白酶基因prt1與纖維酶基因engA之轉錄調控研究
論文名稱(外文):Transcriptional Regulation of prt1 Gene and engA Gene in Xanthomonas campestris pv. campestris
指導教授:曾義雄曾義雄引用關係
指導教授(外文):Yi—Hsiung Tseng
學位類別:博士
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:2003
畢業學年度:91
語文別:中文
論文頁數:130
中文關鍵詞:十字花科黑腐病菌蛋白酶纖維酶轉錄調控
外文關鍵詞:Xanthomonas campestris pv. campestrisproteasecellulasetranscriptional regulation
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革蘭氏陰性的 Xanthomonas campestris pv. campestris 為十字花科蔬菜黑腐病 (black rot) 的植物病原細菌,造成農業上之嚴重損失。此菌所產生之大量胞外多醣 (exopolysaccharide) 稱為 xanthan gum,在工業上有許多用途,可作為懸浮劑、乳化劑、穩定劑及增稠劑等。本菌尚能分泌多種胞外酵素,如蛋白酶 (proteases)、果膠酶 (pectinases) 以及纖維酶 (endoglucanases,亦稱cellulases) 等。這些胞外酵素與胞外多醣為本菌之重要致病因子。上述致病因子的基因之調控,目前並不清楚。只知 clp 基因所主導之 Clp 蛋白 (cAMP receptor protein-like protein) 為一 global transcription factor,參與調控胞外酵素以及胞外多醣之產生與分泌。
為了獲得更多有關 X. campestris pv. campestris 致病因子基因調控之訊息,本研究選擇探討 X. campestris pv. campestris 之致病因子中主導主要胞外蛋白酶之基因 (prt1) 以及主導主要胞外纖維酶之基因 (engA) 之轉錄調控。
本研究之第一部份探討 X. campestris pv. campestris prt1 之上游區域。利用 5''-RACE (rapid amplification of cDNA ends) 技術來訂定 prt1 mRNA 之 5''—端,得知其位於轉譯起始點上游 84 nt 之核苷酸 ''''A'''' (為prt1之轉錄起始點),為 —10 序列下游 8 nt 處。另一方面,將含不同段落與長度的 prt1 上游片段 (Pprt1),選殖於泛寄主載體 pFY13-9 (為一 promoter-probing vector),並將所得 Pprt1-lacZ transcription fusion constructs 經電孔送入 X. campestris pv. campestris 野生株 (Xc17) 與 clp 基因缺失之突變株 (AU56E),針對啟動子活性加以分析。結果顯示,prt1 可在 Clp 存在下於 LB 或含 casein proteins 的培養基中被誘導,而且其表現在生長進入靜止期後仍持續增加。分析結果亦顯示 prt1 轉譯起始點上游 —392 至 —80 區域為 prt1 完全表現 (maximal expression) 所必須;而 —392 至 —207 區域則和 Clp-mediated regulation 與 casein proteins 之誘導有關。此外,prt1 啟動子在 heat stress 之情況下,並不受誘導;而利用 insertional mutation 構築的 prt1 突變株之生長速度與存活力與野生株並無差異。另一方面,本研究亦以 Clp 與 prt1 啟動子混合後,進行 gel retardation 試驗,證實二者並不互相結合,顯示 Clp 對於 prt1 之調控為藉由其他調控蛋白來達成,而該調控蛋白之表現則需要 Clp 的參與。
本研究之第二部份探討 X. campestris pv. campestris 主要胞外纖維酶基因 engA 之上游區域。利用 5''-RACE 技術所訂定之 engA mRNA 之 5''—端,位於 engA 轉譯起始密碼 ATG 上游 44 nt之核苷酸 ''''T'''' (轉錄起始點)。將含不同段落與長度的 engA 上游片段 (PengA),選殖於 pFY13-9,並將所得 PengA-lacZ transcription fusion constructs 經電孔分別送入 Xc17 與 AU56E,針對啟動子活性加以分析。分析結果得知,engA 轉譯起始點上游 —181 至 —23 區域之 159 bp 片段為其在 Clp 存在下完全表現所必須。此外,經由gel retardation 試驗得知 Clp 可和 engA 啟動子形成 DNA-蛋白複合物;而分析產生此 DNA-蛋白複合物所需之最短engA啟動子區域,顯示所需之最短片段為其轉譯起始點上游 —100 至 —75 之區域。此區域存在一不完全對稱 (imperfect symmetry) 之序列 TGTGGN6TCACA,位於 engA 轉譯起始點上游 —97 至 —76 處。進一步利用定點突變 (site-directed mutagenesis) 之方式,顯示該序列中所含預估之 Clp 結合所需之保留序列中之 GTG motif,不只為於活體外 (in vitro) 與 Clp 結合形成 DNA-蛋白複合物所必需,亦為在活體內 (in vivo) 之 engA 基因表現所必需。
由本研究之結果可知 Clp 為 prt1及 engA 基因表現之正調控因子 (positive regulator),但對二者的調控方式有所不同,對前者為間接參與,而對後者則為直接參與。
The Gram-negative plant pathogenic Xanthomonas campestris pv. campestris is the causative agent of black rot in crucifers, causing tremendous loss in agriculture. This bacterium produces great amounts of an exopolysaccharide, xanthan gum, which has a variety of applications in food, agriculture, cosmetics, and industry as a suspending, emulsifying, stabilizing, and viscosifying agent. In addition, it is capable of secreting several extracellular enzymes, including proteases, pectinases and endoglucanases (cellulases). These enzymes and xanthan have long been considered important virulence determinants. Gene regulation of these virulence factors is still unclear. It is only known that the global transcription factor Clp (cAMP receptor protein-like protein) encoded by clp gene regulates production and secretion of xanthan and these enzymes.
To explore regulation of the virulence genes in X. campestris pv. campestris, the aims of this study were to evaluate the transcriptional regulation of prt1 and engA genes encoding the major extracellular protease and endoglucanase, respectively.
In the first part of this study, the upstream region of prt1 was characterized. Using 5''-RACE (rapid amplification of cDNA ends) technique, nucleotide A at 84 nt upstream of the prt1 start codon (eight nt downstream of the —10 sequence) was mapped as the 5'' end of the prt1 mRNA, i.e. the transcription start site. Nested fragments of the prt1 upstream region were made to map the location of the promoter elements. The fragments were separately cloned into the broad host range promoter-probing vector pFY13-9 and the resultant Pprt1-lacZ transcriptional fusion constructs were introduced into the wild-type X. campestris pv. campestris 17 (Xc17) and clp mutant (AU56E) by electroporation. Results of reporter assays indicated that the expression is induced by LB medium or casein proteins in presence of Clp protein, with the expression levels continue to increase following cell growth until 30 hr after the cultures enter stationary phase. The region containing bp —392 to —80 relative to the prt1 translation initiation codon is required for maximal expression, in which bp —392 to —207 responds to the Clp-mediated regulation and the induction. In addition, the prt1 promoter shows no response to stressful conditions. The prt1 mutant constructed by insertional mutation showed no changes in growth rate or viability. Gel retardation assay showed no binding between Clp and prt1 promoter, indicating regulation by Clp to be exerted in an indirect manner, most likely via a regulatory protein(s) whose expression requires Clp.
In the second part of this study, the upstream region of engA was characterized. The 5''-end mapping of engA mRNA by 5''-RACE showed the transcription start site to be nucleotide T at 44 nt upstream of the engA ATG start codon. Reporter assays using PengA-lacZ transcriptional fusion constructs revealed that the 159-bp region containing bp —181 to —23 relative to the engA translation initiation site is sufficient to mediate transcription of engA in presence of Clp. Gel retardation assays indicated that Clp and engA promoter form a DNA-protein complex. Fine mapping defined the region encompassing bp —100 to —75 relative to the translation initiation codon, which has a sequence (TGTGGN6TCACA) between bp —97 and —76 with imperfect inverted repeats, to be necessary. Site-directed mutagenesis indicated that the GTG motif of the proposed Clp core consensus sequence is essential for both DNA-protein complex formation in vitro and engA gene expression in vivo.
This study thus provides evidence showing that Clp exerts positive control over expression of engA directly and that of prt1 indirectly in X. campestris pv. campestris.
目錄
壹. 中文摘要 1
貳. 英文摘要 3
參. 前言 5
肆. 材料 12
一. 菌種及質體 12
二. 一般藥品 12
三. 酵素 12
四. 培養基 13
1. 液體培養基 13
2. 固體培養基 13
五. 試劑與緩衝溶液 13
1. 抗生素濃度 13
2. 抽取染色體之緩衝溶液 13
3. 洋菜凝膠電泳試劑 13
4. SDS-聚丙烯醯胺凝膠試劑 14
5. 轉形所需之緩衝溶液 14
6. -galactosidase 活性測試所需之試劑 14
7. Clp透析所需之緩衝溶液 15
8. Xc17 與 AU56E cell lysate 製備所需之lysis buffer 15
9. Gel retardation 試驗所需之試劑 15
六. 引子 15
伍. 儀器 16
一. 吸光值測定 16
二. 離心機 16
三. 電孔儀 16
四. 聚合酶鏈鎖反應儀 16
五. 低溫迴轉式振盪培養箱 16
六. 雜交烘箱 16
七. 超音波細胞粉碎機 16
八. 紫外光照像系統 16
九. 電泳及轉漬設備 16
1. 洋菜凝膠電泳 16
2. 聚丙烯醯胺凝膠電泳 16
3. 轉漬 16
十. 其它 16
1. 水浴機 16
2. 酸鹼測定儀 16
3. 振盪器 16
4. 乾浴機 16
陸. 方法 17
一. 細菌之培養與保存 17
二. 蛋白酶活性測試 17
三. DNA 之製備 17
1. 小量質體 DNA 之抽取 17
2. 染色體 DNA 抽取法 18
四. 凝膠電泳分析 18
1. 洋菜凝膠之製備 18
2. SDS-聚丙烯醯胺凝膠之製備 18
3. 聚丙烯醯胺凝膠之製備 19
五. 聚合酶鏈鎖反應 19
六. Rapid amplification of cDNA ends (RACE) 20
1. 引子設計 20
2. 第一股互補 DNA 合成 20
3. 第一股互補 DNA 的純化 20
4. 互補 DNA 的tailing 反應 21
5. dC-tailed cDNA 的 PCR 反應與選殖 21
6. 巢式 PCR 增幅 21
七. 質體之選殖 21
1. PCR 增幅片段之選殖 21
2. 限制酶的切割分析 22
3. DNA 回收 22
4. DNA 的黏接 22
八. 轉形作用 22
1. E. coli 之轉形 22
2. X. campestris 之轉形 23
九. 啟動子之活性分析 24
1. 培養方式 24
2. -galactosidase 之活性測試 24
十. 熱休克處理 24
十一. 南方轉漬法 25
1. DNA 之轉漬與固定 25
2. 探針之製備 25
3. 雜配反應及偵測 26
十二. 生長曲線測定 26
十三. Xc17 total RNA 的純化 26
十四. Clp 之過度表現與純化 27
十五. Xc17 與 AU56E cell lysate 製備 28
十六. Gel retardation 28
1. DNA 模版之生物素標定 28
2. 結合反應 28
3. 電泳分析與轉漬 28
4. 偵測 29
柒. 結果與討論 30
第一部份:prt1 上游區域之選殖與轉錄調控之研究 30
一. 胞外蛋白酶活性之定性分析比較 30
二. prt1 轉錄起始點之訂定 31
1. 5-RACE 產物之製備流程 31
2. 巢式 PCR 產物之選殖與序列分析 31
三. prt1 上游片段之選殖 32
四. prt1 上游片段之啟動子活性分析 34
1. 含最長prt1 上游片段之transcription fusion construct 在Xc17 與AU56E、TC817、TC820 中之活性差異 34
2. 其餘含不同段落與長度之prt1 上游片段之transcription fusion constructs 在Xc17 與 AU56E 中之活性差異 35
3. 培養基中添加 skim milk,對於啟動子活性的影響 37
4. 熱休克處理對於啟動子活性的影響 38
五. Xc17 之 prt1 突變株之構築與特性分析 39
1. 構築質體 pME488 39
2. 同質互換之進行 40
3. 轉型株的南方轉漬法分析 40
4. 轉型株之特性分析 40
六. prt1 上游片段之 gel retardation 試驗 41
1. 結合反應所需 prt1 上游片段之選擇 42
2. Clp 之誘導表現與純化 42
3. Clp 與 prt1 上游片段之結合反應 43
4. Xc17 與 AU56E 之 cell lysate 與 prt1 上游片段之結合反應 43
七. prt1 之轉錄調控 44
第二部份:engA 上游區域之選殖與轉錄調控之研究 47
一. engA 轉錄起始點之訂定 47
1. 5-RACE 產物之製備流程 47
2. 巢式 PCR 產物之選殖與序列分析 47
二. engA 上游片段之選殖 49
三. engA 上游片段之啟動子活性分析 50
1. 不同之 transcription fusion constructs 在 Xc17 以及 AU56E 中之生長曲線分析 50
2. 不同之 transcription fusion constructs 在 Xc17 中之 -galactosidase 活性分析 50
3. 不同之 transcription fusion constructs 在 AU56E 中之 -galactosidase 活性分析 51
四. engA 上游片段之 gel retardation 試驗 52
1. 結合反應所需 engA 上游片段之選擇 52
2. 不同量之 Clp 對於 engA 上游片段之結合測試 52
3. Clp 對於不同段落與長度之 engA 上游片段之結合測試 54
五. engA 上游片段 DNA 序列之定點突變所造成的影響 55
1. engA 上游片段之 DNA 序列定點突變核苷酸之選擇 55
2. Clp-binding site(s) 位置 "5" 突變後,與Clp結合能力測試 57
3. Clp-binding site(s) 位置 "6" 突變後,與Clp結合能力測試 57
4. Clp-binding site(s) 位置 "7" 突變後,與Clp結合能力測試 58
5. 定點突變後之engA 上游片段對於轉錄活化之影響 58
六. engA 之轉錄調控 61
捌. 結論 65
玖. 參考文獻 67
拾. 表 77
拾壹. 圖 86
拾貳. 縮寫字對照表 128
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