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【目的】比较苏云金芽胞杆菌与枯草芽胞杆菌在碱性培养条件下生长情况,明确clpP基因在碱刺激条件下的作用。【方法】采用同源重组技术敲除苏云金芽胞杆菌HD73菌株clpP基因,通过在不同pH下生长曲线的测定明确了clpP基因缺失突变体对碱性环境的敏感性,测定clpP基因的缺失对芽胞形成率、芽胞萌发效率和盐胁迫的影响。【结果】苏云金芽胞杆菌在碱刺激后,当培养基pH值为8.9-9.1时可以恢复生长,而枯草芽胞杆菌在pH值为8.2-8.4时可以恢复生长,说明苏云金芽胞杆菌对碱性环境适应能力更强,这有助于作为病原菌的Bt适应昆虫中肠的碱性环境。clpP基因缺失对芽胞形成率和萌发效率没有明显的影响。在将培养基中NaOH终浓度调节至30 mmol/L NaOH时,clpP基因缺失突变体的生长较出发菌株慢。说明ClpP在苏云金芽胞杆菌对碱性环境的适应过程中具有重要作用。
【Objective】 The purpose of this study was to compare the growth of Bacillus thuringiensis and Bacillus subtilis in alkaline culture and to clarify the role of clpP gene in alkaline stimulation. 【Method】 The homologous recombination technique was used to knock out the clpP gene of Bacillus thuringiensis HD73 strain. The sensitivity of clpP gene deletion mutant to alkaline environment was determined by measuring the growth curve at different pH. The clpP gene deletion Rate, sprouting germination efficiency and salt stress. 【Result】 After Bacillus thuringiensis stimulated with alkali, the growth of Bacillus thuringiensis could be resumed when the pH was 8.9-9.1, while Bacillus subtilis could resume growth at pH 8.2-8.4, which indicated that Bacillus thuringiensis could adapt to alkaline environment It is more capable, which helps Bt as a pathogen to adapt to the alkaline environment of insects’ midgut. The absence of clpP gene had no significant effect on the rate of sporulation and germination efficiency. The clpP deletion mutant grew slower than the original strain when the final NaOH concentration in the medium was adjusted to 30 mmol / L NaOH. This indicates that ClpP plays an important role in the adaptation of Bacillus thuringiensis to the alkaline environment.