臺灣博碩士論文加值系統

摘要

Lf 為一能專一性感染十字花科植物黑腐病菌之線狀噬菌體。 利用跳躍子突變法破壞得到對 Lf 感染呈現抗性之 X. campestris strain P20H::miniTn5 (Tc)，經定位後發現其插入點為位於隸屬細菌雙組件系統中主導轉錄調節之 algR 基因，接續利用插入法致變構築algR 基因突變株與其相對應 sensor 的 algZ 基因突變株，其突變株之特性分析結果如下：(1) algR 突變株對 Lf 具有抗性，algZ 則否。 (2) algR 及 algZ基因破壞後，其菌落外觀、生長情形、胞外酵素分泌與野生株相較並無明顯差異。 (3) algR 突變株於半固態洋菜膠之運動能力較其他菌株緩慢，但將帶有 algR 基因之 pBBADalgR 質體送入 algR突變株後，則可恢復同野生株之 Lf sensitive 性狀與swarming motility。
經蛋白序列分析發現，AlgR 的 C 端具有專一性結合於[TA][AC][CA]GTTN[AG][TG] 序列之 LytTR DNA binding domain (FhRhH [RK] [SNQ]hhVN)，為了尋找可能受 algR 調控的基因，於是利用 RSAT consensus program 搜尋 X. campestris 基因體中之AlgR 結合序列，並挑選其中五個基因 (fabA、gltD、plcN、phoR、slp) 作啟動子活性分析。 結果顯示，AlgR 並非 fabA、gltD、plcN、phoR 基因表現所必須；slp 基因之啟動子活性於 algR 突變株皆遠低於野生株與 algZ 突變株，即代表 AlgR 以正向轉錄調控 slp 基因表現。 由於噬菌體早期的感染需要 type IV pilus 輔助，而 pilA1 基因主導生成 type IV pilus mayor pilin，經啟動子活性結果顯示，AlgR 以正向轉錄調控 pilA1 基因表現。

Abstract

Lf is a filamentous bacteriophage which specifically infects Xanthomonas campestris pv. campestris. A Lf resistant X. campestris strain was isolated by mini-Tn5 transposon insertional mutagenesis. The insertion site was mapped within algR, gene encoding for a transcriptional activator belonging to the microbial two component systems. Mutants of algR and algZ, the cognate sensor gene of algR, were reconstructed by insertional mutagenesis and the phenotypes of mutants were analyzed and summarized: (1) algR mutant is resistant to Lf infection and algZ mutant is still sensitive to Lf. (2) algR and algZ mutants showed no difference in colony morphology, growth rate and extracellular enzymes secretion. And (3) algR mutant, but not algZ mutant, moved slower than their parental strain on a semisolid plant. The phage sensitivity and mobility deficiency of the algR mutant were recovered after introducing an algR gene containing plasmid, pBBADalgR.
A DNA-binding domain LytTR (FhRhH[RK][SNQ]hhVN) which binds specifically to [TA][AC][CA]GTTN[AG][TG] sequence, was identified in the c-terminal of AlgR. To identify putative AlgR-regulated gene, RSAT consensus program was used to search in X. campestris genome for AlgR-binding consensus sequence. Five genes (fabA, gltD, plcN, phoR and slp) were selected for promoter activity assay. The results indicated that expression of fabA, gltD, plcN and phoR genes were not dependent on AlgR or AlgZ. slp gene promoter activity mutant was significantly lower in an algR mutant than in an algZ mutant or in their parental strain Xc17. This suggests that slp gene is positively regulated by algR. The early step of Lf infection depends on type IV pilus. Gene pilA1 encodes for the mayor pilin of type IV pilus. The promoter assay demonstrated that the expression of pilA1 gene is positively regulated by AlgR.

目錄

縮寫字對照表 I
胺基酸縮寫字對照表 V
菌種縮寫字對照表 VI
中文摘要 VIII
英文摘要 IX

壹、 前言 1

貳、 實驗材料 5

一、基因體序列 5
二、菌種、噬菌體及質體 5
三、引子 5
四、儀器設備 5
五、藥品 6
六、酵素 6
七、Marker 6
八、抗生素使用濃度 7
九、培養基 7
十、試劑與緩衝液 7

參、 實驗方法 10

一、細菌之培養 10
二、菌種之保存 10
三、噬菌體之增殖與保存 10
四、噬菌體濃度測定 (titer) 11
五、溶菌斑測試 (spot test) 12
六、生長曲線測定 12
七、細菌染色體 DNA 之製備 13
八、小量質體 DNA 之製備 13
九、聚合酶連鎖反應 (polymerase chain reaction, PCR) 14
十、質體 DNA 之限制酶切割 15
十一、DNA片段回收 15
十二、DNA接合反應 16
十三、洋菜膠體電泳分析 16
十四、勝任細胞 (competent cells) 之製備 16
十五、細菌之轉形作用(transformation) 17
十六、運動性測試 (Swaring motility) 19
十七、胞外酵素分泌測試 19
十八、蛋白質之誘導表現 20
十九、蛋白質樣本之製備 20
二十、SDS-PAGE之製備 21
二十一、蛋白質之 SDS-膠體電泳分析 21
二十二、蛋白質 Coomassie blue 染色 22
二十三、凝膠封片 22
二十四、西方墨點法 (Western blot) 22
二十五、轉印膜呈色 23
二十六、半乳糖苷酶 (-galactosidase) 酵素活性分析 24
二十七、啟動子區域預測 25
二十八、生物統計分析 25

肆、 結果與討論 26

第一部份：algR 基因之探討

一、X. campestris 之algR 基因發現 26
二、基因序列特性分析 26
三、X. campestris algR 及 algZ 基因突變株之構築 27
四、algR 及 algZ 基因突變後對X. campestris所造成之性狀影響 28
五、algR 基因互補試驗 32
六、互補株與轉殖株之特性分析 34
七、AlgR 蛋白於不同表現載體系統之蛋白表現 36

第二部分：AlgR 轉錄調控之探討

一、尋找 X. campestris中可能受AlgR所調控之標的基因 39
二、標的基因上游啟動子區域之選殖與β-galactosidase 活性分析 40
三、algR突變株之PilA1表現量 54

第三部分：X. campestris 雙組件訊號傳遞系統之探討 56

伍、 表 63

一、本試驗中所使用之菌種與噬菌體 63
二、本試驗中所使用與構築之質體 66
三、本試驗中所使用之引子與選殖區域 68
四、基因序列特性分析 69
五、菌株性狀分析 70
六、Pseudomonas之 AlgR binding site 71
七、生物資訊方法建構之alignment matrix 72
八、每個核苷酸出現機率相對應之積分 73
九、受AlgR調控之可能標的基因與其DNA結合相關位置 74
十、X. campestris 可能受AlgR調控之標的基因啟動子區域預測 75
十一、X. campestris AlgR蛋白與其他菌種比對結果 76
十二、X. campestris 之雙組件系統 77
十三、X. campestris 訊號傳遞 domain 78
十四、X. campestris 訊號傳遞蛋白 81
十五、X. campestris 之雙組件系統 85
十六、相關網站 87

陸、 圖 88

一、algR 基因突變株載體構築流程圖 88
二、algZ 基因突變株載體構築流程圖 89
三、algR 基因互補株構築流程圖 90
四、不同菌株於 LB 培養基中之生長曲線圖 91
五、不同菌株於 XOLN 培養基中之生長曲線圖 92
六、胞外酵素澱粉酶 (amylase) 之定性分析 93
七、胞外酵素纖維素酶 (cellulase) 之定性分析 94
八、P20H 突變株及互補株之 swarming motility assay 95
九、溶菌斑測試 (spot test) 96
十、pETalgR 蛋白表現載體構築流程圖 97
十一、AlgR protein 於 BL21 (DE3) 誘導表現之SDS-PAGE 電泳分析圖 98
十二、Pseudomonas AlgR 蛋白調控胞外多醣 (EPS) 生合成路徑圖 99
十三、Pseudomonas AlgR 蛋白結合之 DNA 多序列排比 100
十四、fabA-lacZ transcriptional fusion construct 構築流程圖 101
十五、fabA-lacZ transcriptional fusion construct pFYfabA 之於不同轉殖株之啟動子活性分析 102
十六、gltD-lacZ transcriptional fusion construct 構築流程圖 103
十七、gltD-lacZ transcriptional fusion construct pFYgltD 之於不同轉殖株之啟動子活性分析 104
十八、plcN-lacZ transcriptional fusion construct 構築流程圖 105
十九、plcN-lacZ transcriptional fusion construct pFYplcN 之於不同轉殖株之啟動子活性分析 106
二十、phoR-lacZ transcriptional fusion construct 構築流程圖 107
二十一、phoR-lacZ transcriptional fusion construct pFYphoR 之於不同轉殖株之啟動子活性分析 108
二十二、slp-lacZ transcriptional fusion construct 構築流程圖 109
二十三、slp-lacZ transcriptional fusion construct pFYslp 之於不同轉殖株之啟動子活性分析 110
二十四、pilA1-lacZ transcriptional fusion construct 構築流程圖 111
二十五、pilA1-lacZ transcriptional fusion construct pFYpilA1之於不同轉殖株之啟動子活性分析 112
二十六、lacZ transcriptional fusion constructs 於不同轉殖株之啟動子活性分析比較 113
二十七、X. campestris 第四型纖毛合成相關基因 114
二十八、AlgR 對 pilA1 基因兩種可能之轉錄調控方式 115
二十九、AlgR 於不同基因可能之轉錄調控方式 116
三十、P20H strain 細胞內 PilA1 蛋白表現量之 Western blot 分析 117
三十一、two-component system 訊號傳遞 118

柒、 參考文獻 121

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