Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/16729
標題: Erwinia carotovora 低分子量細菌素Carocin S3 的基因選殖與表現
Gene Cloning and Expression of the Low-Molecular-Weight Bacteriocin Carocin S3 from Erwinia carotovora
作者: 徐志豪
Hsu, Shih-Hao
關鍵字: bacteriocin
細菌素
出版社: 化學系所
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摘要: Erwinia carotovora subsp. carotovora (E.c.c.)是一種會造成許多植物根莖部腐爛的病原菌,目前為止對於此菌種造成的細菌性軟腐病仍找不到有效的防治方法。低分子量細菌素為細菌產生的毒性蛋白,可以抑制親緣相近菌種的生長。在本研究中,我們發現一種由柯隆(cloning)產生的低分子量細菌素。 E.c.c. rif-TO6菌株所產生的低分子量細菌素可以抑制指示菌株E.c.c. 95F-3的生長。在接合生殖的實驗中,我們利用跳躍因子transposon Tn5在Rif-TO6染色體上進行插入突變,並在10000株完成接合生殖的突變株中選擇14株不再產生低分子量細菌素的突變株,其Tn5可能阻斷到生產細菌素的基因。藉由TAIL-PCR的方法增幅並定序各突變株被Tn5阻斷的周圍未知序列,經由NCBI的資料庫(NCBI blast)以DNA或胺基酸序列比對後,我們發現TT6-13突變株被阻斷的基因與colicin D有24%的同質性,此蛋白可能為RNA水解酵素。由定序結果得知此完整基因(carocin S3基因)全長2750 bp,包含caroS3K(ORF1)和caroS3I(ORF2)兩個開放讀取框,而Tn5阻斷在caroS3K 基因1274~1275 bp之間的位置。 pGS3K為具有carocin S3 基因的構築體,將之轉形送入TT6-13和95F-3等無法生產細菌素的菌株,實驗結果顯示帶有pGS3K構築體的菌株均能表現細菌素的活性。 根據NCBI blast的結果,大致可將carocin S3 區分為四個區域:接收體接合區域、未知功能區域、轉位區域和殺手區域。我們發現carocin S1和carocin S3在任何區域都是不相同的。與carocin S2同質性的比較中,在接收體接合區域、未知功能區域和轉位區域的同質性高,但是在殺手區域則完全不同,因此我們推斷這兩種細菌素進入目標細胞的模式一樣,但導致目標細胞死亡的方式是不同的。
Erwinia carotovora subsp. carotovora is a phytopathogenic enterobacterium responsible for the soft-rot disease of many plants species. In spite of economic importance, there is no efficient method has been found to control this disease. The low-molecular-weight bacteriocin is an antibiotic that inhibit the growth of closely related bacteria . In this study, we show a newly bacteriocin that be found from E.c.c. The strain Rif-T06 produces low-molecular-weight bacteriocin(LMWB) to inhibit the growth of indicator strain 95F-3. The transposon Tn5, which harboring drug-resistance marker, was integrated into the genomic DNA of the host cell Rif-T06. Out of the 10000 colonies of possible mutants, 14 colonies were obtained that could not produce LMWB. The TAIL-PCR technology was used to amplify unknown DNA fragments from the Tn5 transposon. TT6-13 was a Tn5 insertional mutant strain, and it has not expressed LMWB. After sequencing, a DNA fragment of 2750 base pair was determinated. It shows that there two complete open reading frames(ORF1 and ORF2) in the Tn5 interrupted gene, and Tn5 was located in ORF1 between 1274-bp and 1275-bp. The 2750-bp gene, named carocin S3 gene, consists of caroS3K(ORF1) and caroS3I(ORF2). Deduction of amino acid sequence, the homology of carocin S3 was similar to the colicin D(24%), which has ribonuclease activity. The construct pGS3K, which contains the carocin S3 gene, has produced bacteriocin activity in E.c.c. TT6-13 and 95F-3 strains. The carocin S3 can divide into four domains: receptor domain(domain I), unknown domain(domain II), translocation domain(domain III) and killing domain(domain IV). In our previous studies, the carocin S3 is different with carocin S1, and is different with carocin S2 in domain IV. Interestingly, the domain I, domain II and domain III are homologous between carocin S3 and carocin S2. It indicate that has the same translocation modle but different attack way.
URI: http://hdl.handle.net/11455/16729
其他識別: U0005-1507200916130000
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