臺灣博碩士論文加值系統

本研究利用已知的熱休克調節基因hrcA C端之部份DNA序列為探針，於十字花科黑腐病菌 (Xanthomonas campestris pv. campestris 17) 基因庫中選殖出hrcA基因的N端DNA片段，並發現hrcA基因上游為DNA修補基因recN。 X. campestris的hrcA密碼區有1,053 bp，可轉譯出一包含350個氨基酸之鏈，推測其蛋白分子量為38.3 kDa， PI值為5.87。 X. campestris的HrcA氨基酸序列與Caulobacter crescentus的HrcA有36 % 的相同性 (identity)，與Bradyrhizobium japonicum的HrcA有34 % 的相同性。在X. campestris的hrcA轉譯啟始密碼ATG上游6~13 bp處有一個類似ribosomal binding site，在轉譯終止密碼TAA下游有一個類似terminator的序列。將已發表的HrcA蛋白進行演化親源分析，結果X. campestris的HrcA與同為革蘭氏陰性菌的C. crescentus、 B. japonicum及Agrobacterium tumefaciens的HrcA親源關係最接近。將這些HrcA的氨基酸序列進行alignment，結果發現在HrcA蛋白N端前100個氨基酸序列有高度的保守性，此區域可能與HrcA的功能或結構有重要的關係。 以包含hrcA的DNA片段為探針進行Northern blotting分析，結果顯示經熱休克處理的RNA樣品在1.1 kb處有明顯訊號，與hrcA密碼區大小相近，因此推測X. campestris的hrcA可能為monocistronic operon。 Primer extension分析結果發現hrcA轉錄起始點位於轉譯起始點上游第20 bp的Cytosine位置。在轉錄起點上游可發現 -10與 -35序列與其它菌種的hrcA啟動子的 -10與 -35序列相似。 將hrcA與recN基因間的EcoRI─SphI片段選殖於啟動子選殖載體pFY7上進行hrcA啟動子活性測試，結果顯示所選殖的片段有啟動子活性，但熱休克處理後其活性維持定量，並無增加。 將hrcA選殖於泛寄主載體pHC8的衍生質體上，得到pHC8-hrcA-6His，可於X. campestris菌體內表現C端帶有His tag的HrcA蛋白。 為大量表現HrcA蛋白，將N端缺失114 bp的hrcA選殖於表現載體pQE31上，得到pQE31hrcA質體，此質體可於Escherichia coli菌體內大量表現。分析已完成genome project的菌種，發現古生菌genome中沒有hrcA基因出現，真細菌除了親源最接近古生菌的Aquifex aeolicus及E. coli、Haemophilus influenzae外，其它菌種genome中皆有hrcA基因，因此推測hrcA基因出現於古生菌與真細菌分化後的初期。

The hrcA and its flanking sequences of Xanthomonas campestris pv. campestris 17 were cloned from genomic library using the DNA fragment carrying the 3'' end of hrcA sequence as a probe. After subcloning and sequencing the X. campestris hrcA and its upstream sequence were determined. Sequence analysis showed that the 1,053 bp hrcA can code for a protein of 350 amino acids with a predicted molecular mass of 38.3 kDa and PI of 5.87. Comparison of the amino acid sequences with those of other HrcA showed 36% identity to that of the HrcA of Caulobacter crescentus and 34% identity to that of the HrcA of Bradyrhizobium japonicum. The putative ribosomal binding site is located 6 to 13 bp upstream from the translational start codon ATG of hrcA, and the putative terminator is located 7 to 23 bp downstream from the translational stop codon TAA. Evolutionary analysis of the relationship of various HrcA sequences appeared that X. campestris is most closely related to C. crescentus, B. japonicum and Agrobacterium tumefaciens. The first 100 amino acids of HrcA are most consensus suggesting that this region maybe important in function. Northern blotting analysis detected an mRNA with a size similar to that of the X. campestris hrcA coding region suggesting the hrcA gene-containing transcript may be monocistronic. Primer extension showed that the nucleotide cytosine locating at 20 nt upstream from the translational start codon of the X. campestris hrcA is the possible transcriptional initiation site. The putative promoter region was cloned into promoter-proving vector pFY7 for analysis. The hrcA promoter activity was detectable under nonstressed condition but was not inducible by heat shock treatment.
In addition, the DNA fragment containing the full length hrcA and the N-terminal deleted hrcA were cloned for protein expression. Based on sequences available in database, hrcA present in many eubacteria but not in Escherichia coli, Haemophilus influenzae, Aquifex aeolicus and archaebacteria. Therefore, it is suggested that hrcA genes evolved in eubacteria after diverging from archaebacteria evolution.

中文摘要....................................................... 1
英文摘要....................................................... 3
前言........................................................... 5
材料與方法.................................................... 13
I. 材料................................................... 13
一、菌種、噬菌體及質體................................ 13
二、藥品及酵素........................................ 13
三、引子 (Primers) ................................... 13
四、培養基及緩衝液.................................... 14
II. 實驗方法.............................................. 14
一、微生物培養........................................ 14
二、DNA抽取........................................... 14
1. 染色體DNA 抽取................................. 14
2. 質體DNA抽取................................... 15
3. 單股DNA誘導及抽取............................. 16
三、限制切割DNA.................................... 16
四、DNA洋菜膠電泳..................................... 17
五、DNA回收........................................... 17
六、質體選殖與構築.................................... 18
七、5''端去磷酸根反應................................... 19
八、轉型作用.......................................... 19
1. Calcium chloride方法 .......................... 19
2. Electroporation方法 ........................... 20
九、探針製備........................................... 20
1. Random primer方法.................................. 20
2. 5'' End labeling方法.................................. 21
十、Sephadex G-50 column.................................... 21
十一、DNA定序.............................................. 21
1. 定序反應............................................ 21
2. 定序凝膠電泳........................................ 22
十二、Southern blotting..................................... 23
十三、Colony hybridization................................. 23
十四、Reprobe.............................................. 24
十五、RNA抽取.............................................. 24
十六、Northern blotting..................................... 25
1. Formaldehyde gel electrophoresis..................... 25
2. Hybridization...................................... 25
十七、Slot blotting測mRNA表現量............................ 25
十八、mRNA primer extension偵測轉錄起始點................... 26
十九、PCR.................................................. 26
二十、T-vector製備......................................... 27
二十一、SDS PAGE............................................ 27
二十二、蛋白誘導及純化..................................... 28
二十三、熱休克處理......................................... 28
二十四、螢光測定hrcA promoter活性......................... 29
結果與討論........................................................ 30
一、 hrcA之選殖............................................... 30
二、 hrcA基因之定序及比對分析................................. 30
三、 Northern blotting偵測hrcA轉錄產物大小及操縱子組成........ 32
四、 Primer extension決定hrcA轉錄起始點及啟動子位置........... 33
五、 hrcA promoter活性測試.................................... 34
六、 HrcA蛋白表現............................................. 36
七、 hrcA演化起源分析......................................... 38
八、 hrcA未來研究展望......................................... 39
參考文獻.......................................................... 41
圖表.............................................................. 50
附錄一、縮寫字對照表...................... ......................... 78
附錄二、培養基、藥品及緩衝液配方.................................... 80
附錄三、換算單位及計算公式......................................... 90
附錄四、pQE31hrcA質體及其表現的HrcA蛋白之序列..................... 94
附錄五、pHC8-hrcA-6His質體及其表現的HrcA蛋白之序列................ 97
附錄六、hrcA演化起源分析所用之序列參考資料......................... 99

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