Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22137
標題: 枯草桿菌(Bacillus subtilis)F29-3豐原素之表現及其基因節構的分析
Studies of the expression of fengycin and structural analysis of a gene responsible for fengycin synthesis of Bacillus subtilis F29-3
作者: 李憲明
Lee, Hsien-ming
關鍵字: Bacillus subtilis
枯草桿菌豐原素
Fengycin
出版社: 植物學系
摘要: 枯草桿菌(Bacillus subtilis)為革蘭氏陽性菌,特徵為桿狀、好氧、有鞭 毛,並能產生孢子。豐原素為枯草桿菌F29-3所產生的一種抗生素,能抑 制真菌及某些細菌的生長。為了對豐原素做進一步的了解,本論文利用高 壓色層分析及生物分析,探討豐原素的合成與枯草桿菌生理之關係,並以 核甘酸定序法研究其基因組成。首先,萃取純化豐原素,將萃取物進行高 壓色層分析及薄層色層分析,與前人所萃取之豐原素圖譜相同,表示所萃 取之豐原素可做為進一步分析時之比對標準。為了探討豐原素的生成情形 ,將F29-3一至九天的萃取物,進行高壓色層分析及生物分析,發現豐原 素於第五天開始生成;而以相同的方法分析fen-突變株及不產生豐原素的 菌株MI113,則沒有豐原素的生成。同時,經由F29-3生長曲線分析,發現 豐原素生成的同時,也是F29-3由衰退期進入第二個生長高峰的時期,表 示二者之間有很高的相關性。另外,由互補測試得知,選殖質體 pFC660 中一段2.7 kb BamHI-SalI區域,對於豐原素的合成相當重要,所以,本 實驗即進行此一區域的核甘酸定序。目前已定出1.7 kb左右核甘酸序列, 其中含有一個可能的開放讀碼架構(fenA),長度為843 bp,在fenA的啟始 碼前並找到一個可能為核醣體結合位置,由相對的氨基酸序列推測 fenA 的產物大小為37.1 kDa,經由比對的結果顯示fenA的氨基酸序列與 gramicidin S synthetase 2 (GrsB)類似,由於gramicidin S 是以 multienzyme thiotemplate mechanism 的機制進行,所以推測豐原素可 能依同樣的模式合成。由前人的互補測試及跳躍因子(transposon)的插入 位置顯示,除了fenA以外應有其它的基因存在。所以必須再分析選殖體 (pFC660)上其餘的核甘酸序列,以找出其他基因,才能進一步的了解豐原 素的基因結構及其調節機制。
Bacillus subtilis is a rod-shaped, aerobic, spore-forming, motil, grame-positive bacterium. Bacillus subtilis F29-3 produces fengycin, which is anatagonistic to fungi and to some bacterial strains. The major goal of this thesis is to study the expression of fengycin in cells and the sequence analysis of a DNA fragment resposible for fengycin systhesis. Fengycin was purified by HPLC and was used as a standard for the subsequent studies. The extract of the cultures of Bacillus subtilis F29-3 , fen-mutants, and strain MI113 which did not produce fengycin, were analyzed by HPLC. The expression of fengycin began at the fifth day and was detectable after nine days of culturing. On the other hand, fen- mutants and MI113 did not express the antibiotics. Growth study showed that cells entered stationary phase at the sixteenth hour after inoculation. The viable counts decreased to approximately 100,000 cfu/ml after 24 hours. The cell number increased again at day 5. The rise of cell population coincided with the expression of fengycin to form a second growth phase. Complementation test demenstrated that the 2.7 kb BamHI-SalI region of fen gene genomic clone, pFC660 contained genes for fengycin synthesis. Sequense analysis revealed a 843 bp open reading frame(fenA) located in the 2.7 kb BamHI-SalI fragement. The amino acid sequence encoded by fenA was similar to the amino acid sequence of gramicidin S synthetase 2. Since the gramicidin S is synthesized by multienzyme thiotemplate mechanism, this is a reasion to believe that fengycin may be synthesized by the same mechanism.
URI: http://hdl.handle.net/11455/22137
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