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標題: 改良式芽孢桿菌屬細菌轉型及再生效率提升技術
An improved method for efficient transformation and regeneration of diverse Bacillus species
作者: 邵宇恆
Yu-Heng Shao
關鍵字: 芽孢桿菌屬細菌;PEG 原生質體轉型法;溶菌酶;Bacillus;PEG-mediated protoplast transformation;Lysostaphin
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芽孢桿菌屬菌株為革蘭氏陽性菌,可在環境逆境下產生內生孢子而存活,此屬細菌目前有廣泛的農業及與工業應用價值。由前人研究得知具有生物防治能力的芽孢桿菌屬菌株在系統分類學上多分屬於 Bacillus subtilis group,然而只有少數的馴化菌株可進行遺傳分析。為能有效分析野生型芽孢桿菌屬細菌功能性基因體,提高轉型效率,本研究使用三株已獲得全基因體序列之生物防治菌株 ( Bacillus mycoides BM02、Bacillus pumilus PMB102、及 Bacillus amyloliquefaciens PMB01) 為實驗材料,改良Chang 等人於 1979 年發表之 PEG 原生質體轉型法 (protoplast transformation) 進行逢機基因突變。在優化的原生質體轉型法中,細菌以終濃度 2 μg/ml 之 lysozyme 與 lysostaphin 溶菌酶混合液處理 2 至 3 小時可有效提高原生質體形成率,後續以 PEG6000 進行質體 DNA轉型時,額外添加終濃度為 3.3 mM 過濾滅菌之 ATP,再將轉型菌株培養於含抗生素之DM3固態培養基 [洋菜濃度為 2% (w/v)] 則可提昇轉型及原生質體再生效率。以優化之原生質體轉型法分別將帶有跳躍子 Tn-YLB-1 之 pMarB 質體送入 B. mycoides BM02、B. pumilus PMB102 與 B. amyloliquefaciens PMB01 中,所得之逢機突變菌株庫以 96 孔微孔盤建立快速篩選平台,以測試跳躍子隨機突變效率及穩定性;下一步可製作專一性基因突變菌株,用以分析二次代謝物生合成基因與細菌生物防治能力的相關性。另外,運用優化之轉型法將綠螢光蛋白 (Green fluorescent protein, GFP) 表現質體 pAD43-25 送入 B. mycoides BM02 及 B. pumilus PMB102 中,可於螢光顯微鏡下觀察表現綠色螢光之轉型株,後續可用於原位 (in situ) 觀察細菌在植物根系分布情形。結果顯示,利用優化之原生質體轉型法,明顯提升田間分離之三株芽孢桿菌屬細菌轉型效率,並說明此轉型方法可將候選基因轉型至遺傳特性不明之芽孢桿菌屬細菌內表現。

Genetic analyses are the key to unraveling gene functions in microorganisms, particularly in the post-genomics era when the draft sequence of a microbial genome can be done in a few days. Forward genetics employing random and specific mutations are commonly used to characterize gene functions, e.g. the genes associated with the biological control activity in Bacillus spp. Nevertheless, genetic manipulations in the field-isolated Bacillus strains are challenging. In this research, we aim to develop a feasible genetic protocol to study the functions of biocontrol-associated genes in 3 biocontrol-active Bacillus strains, namely B. mycoides BM02, B. pumilus PMB102, and B. amyloliquefaciens PMB01. The first step in the protocol is to set up a stable protoplast transformation method. Based on the standard PEG protoplast transformation method, we optimized protoplast generation by treating bacteria with 2 peptidoglycan degrading enzymes— lysostaphin (2 μg/ml) and lysozyme (2 μg/ml)—for 2 to 3 hours, followed by PEG-mediated protoplast transformation with tester plasmid DNAs and regeneration on 2% (w/v) DM3 agar supplemented with appropriate antibiotics. During the trials, the efficiency of protoplast formation was monitored by microscopic observation and optical absorbance at 660 nm, and the putative transformants were confirmed by colony PCR. Previous studies showed that the treatment of 3.3-mM ATP during protoplast transformation reduces endonuclease activity. Similar results were obtained by treating plasmid DNA with Bacillus whole-cell extract supplemented with ATP. The optimized protoplast transformation protocol was used to generate random mutations by transforming Bacillus spp. with the transposon-harboring plasmid pMarB. For high throughput screening of putative mutants, we employed a microtitration assay system using starch agar (for amylase activity) to test the efficiency of mutation. In addition, the protoplast transformation efficiency was monitored by transforming GFP plasmid pAD43-25 and visualized under an epifluorescent microscope. Our results revealed that this optimized transformation method increases protoplast transformation efficiency in the 3 field-isolated Bacillus strains, indicating it is an applicable protocol for transforming candidate genes to other genetically uncharacterized Bacillus strains.
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