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

Abstract

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。

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.