- 學位論文
臘狀芽孢桿菌C1L菌株之轉位子誘變及抗真菌相關基因選殖
Transposon mutagenesis of antagonistic bacterium Bacillus cereus C1L and the cloning of genes related to antifungal activity
摘要
本實驗室由百合根圈分離到一株臘狀芽孢桿菌(Bacillus cereus) C1L菌株，對多種植物病原真菌具有拮抗作用。由於目前對臘狀芽孢桿菌C1L菌株是否會產生已知的抗生物質，或是亦會產生其他抗生物質瞭解甚少，因此擬由轉位子誘變方式篩選失去對真菌拮抗作用之C1L誘變株，以進一步獲得與臘狀芽孢桿菌C1L菌株產生抗生物質有關的基因。本研究以紅黴素誘導Tn917ac1轉位子於C1L菌株中轉位，獲得850個誘變株，並將誘變株與十字花科蔬菜黑斑病菌（Alternaria brassicicola）對峙培養，篩選出35株拮抗能力減弱的誘變株；接著再以煙草赤星病菌（Alternaria longipes）進行篩選，獲得5株抗真菌能力顯著減弱的誘變株。進行南方雜合分析確定Tn917ac1插入這5個誘變株的全基因體中，其中只有1個誘變株（M-902）帶有單個Tn917ac1。當與四個病原真菌（A. brassicicola, A. longipes, Botrytis cinerea, B. elliptica）對峙培養時，誘變株M-902均表現減弱之抑菌能力，培養上清液對此四種真菌孢子之發芽亦表現減弱之抑菌能力。Tn917ac1週邊序列分析指出Tn917ac1插入一質體上，南方雜合分析亦顯示，誘變株M-902的質體在電泳膠體上有位移的現象，且Tn917ac1插入一開放解讀框架-orf1及其預測啓動子間。orf1緊鄰另一開放解讀框架-orf2，可能為同一個操縱組（operon）中的結構基因；以A. brassicicola做為測菌株進行互補試驗，結果顯示，包含預測之啟動子，orf1及orf2的載體，或者包含單一orf2 (含啓動子)在對孢子發芽之抑菌試驗中，可使誘變株M-902部分回復對孢子發芽之抑菌功能，以單一orf1 (含啓動子)無法使誘變株M-902回復對A. brassicicola之抑菌功能，由此結果推測orf2可能與C1L菌株抗生物質的產生相關。
並列摘要
Bacillus cereus C1L, an antagonistic bacterium, was originally isolated from the rhizosphere of Fomorsa lily. In order to identify the genes related to the antagonistic ability of B. cereus C1L, a transponson insertion mutant library was constructed. Totally, 850 mutants were obtained. Alternaria brassicicola and Alternaria longipes were used in mutant screening on potato dextrose agar and a mutant, M-902, with significant decrease on antifungal activity was selected. M-902 loses its inhibitory activity against mycelial growth of A. brassicicola, A. longipes, Botrytis cinerea, and Botrytis elliptica ; in addition, the culture supernatant of M-902 decreases spore germination rate. Sequence analysis revealed that the Tn917ac1 was inserted into a plasmid pC1L8 of B. cereus C1L wild-type strain. Southern blot analysis showed a band shift of pC1L8 of B. cereus M-902. The Tn917ac1 insertion site was located downstream a putative promoter of open reading frames-orf1 and orf2。These two ORFs may constitute a two-gene operon. In a complementay test using A. brassicicola as the test fungus, the vector containing predicted promoter, orf1 and orf2, or orf2 singly showed a partial recovery of the inhibition on conidial germination, but orf1 singly could not recover the inhibition on conidial germination. Thus, orf 2 related to antibiotic production of strain C1L was presumed.