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標題: 枯草桿菌 (Bacillus subtilis) 中豐原素合成基因的研究
Analysis of the Genes Encoding the Biosynthesis of Fengycin in Bacillus subtilis F29-3
作者: 陳奇良
Chen, Chyi-Liang
關鍵字: Bacillus subtilis
Antifungal antibiotic
Lipopeptide antibiotic
出版社: 植物學系
摘要: 枯草桿菌F29-3為革蘭氏陽性菌,好氧,具游動性,菌體呈桿狀並能產生孢子 以及對抗絲狀真菌的抗生素,豐原素(fengycin).利用轉位子Tn917之突變 作用,總共獲得20株不生產豐原素的突變株.這20株突變基因經選殖,限制 脢圖譜及DNA序列之分析,而被歸類為11種.利用其中一個突變基因(pBTS7) 的序列作為探針,足構築於pHC79中的枯草桿菌F29-3的cosmid基因庫中篩 選得一選殖株,名為pFC660.分析並比較pFC660與所選殖的11種突變基因的 限制脢圖譜以及Southern hybridization的結果,發現有8種突變株的 Tn917所插入的位置在pFC660內.這些突變基因的序列與 GenBank的DNA序 列比較後,發現有4種突變基因與一些胜呔合成脢有很高的相似性.另外有4 種突變基因與脂肪酸的生合成及代謝有關的基因相類似.經DNA序列的分 析,發現pFC660之B2區域的全長為11,459 bp, 其中含有一不完整的基因,'' fenA,及一個完整的基因,fenB.FenB蛋白的大小為 143.6 kDa,與一些胜呔 合成脢的胺基酸結合區有21.0-46.2%的相似度,而 ''FenA蛋白的大小 為227.1-kDa. '
Bacillus subtilis F29-3 is a gram-positve, rod-shaped,aerobic, spore-forming, motile bacterium. This organism is known to produce an antifungal antibiotic, fengycin. A total of twenty B. subtilis F29-3 mutants defective in fengycin bio- synthesis was obtained by Tn917 mutagenesis. Those sequence flanking the Tn917 insertion site in these twenty mutants were cloned, mapped and sequenced. Results showed that Tn917 was inserted in eleven different locations on the chromosome. One of the clones, pBTS7, was selected and used as a probe to screen for genes encoding fengycin biosynthesis in a cosmid library established with pHC79. A clone, pFC660 which hybridized to the probe, was selected. Mapping study revealed that eight mutants had insertion within the DNA fragment cloned in pFC660. Among them, four had Tn917 inserted in regions which encoded peptide sequences similar to part of peptide synthetases. Another four mutants had Tn917 inserted in the regions encoding the peptide sequences that were similar to part of the enzymes required for lipid synthesis. The 12-kb BamHI fragment of pFC660 was sequences. The length of this fragment was 11,459 bp long. This fragment contains a 3,825-bp peptide synthetase gene, fenB which encodes a protein of 143.6-kDa with 21.0-46.2% similarity to the amino acid-binding domains of different peptide synthetases. The 12-kb BamHI fragment also contains an incomplete open reading frame, ''fenA, which encodes a peptide synthetase of 227.1 kDa. Protein analysis revealed that fenB indeed encodes a 143-kDa protein and intact fenA may encode a protein of approximately 500 kDa. The results obtained from this dissertation suggest that a large number of genes in B. subtilis F29-3 is involved in fengycin biosynthesis.
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