摘要
以枯草芽孢杆菌(Bacillus subtilis)168-Tres基因组为模板,PCR扩增得到同源臂基因sleB1和cwlJ1,重叠PCR连接sleB1与卡那霉素抗性(km^r)基因,电转获得B.subtilis 168-Tres△sleB菌株,连接cwlJ1与博来霉素抗性(zeo^r)基因,电转获得B.subtilis 168-Tres△sleB△cwlJ菌株。结果表明,经km^r、zeo^r抗性筛选及PCR鉴定,成功获得sleB、cwlJ,基因双缺失菌株B.subtilis 168-Tres△sleB△cwlJ;发酵结果显示,B.subtilis 168-Tres△sleB△cwlJ与出发菌株的芽孢形成率一致,约为88%;在LB固体培养基和麦芽糖转化生成海藻糖体系中B.subtilis168-Tres的芽孢萌发数为4.8×10~8 CFU/mL,B.subtilis 168-Tres△sleB△cwlJ芽孢未萌发;在麦芽糖转化生成海藻糖体系中,重组菌海藻糖合酶酶活为10.42 U,比原始菌提高了78.7%。敲除sleB、cwlJ基因后,不影响枯草芽孢杆菌生成芽孢的量,但能有效控制芽孢在上述转化体系中的萌发,使芽孢表面稳定展示海藻糖合酶,提高了芽孢的利用率。
Using Bacillus subtilis 168-Tres genomic DNA as templates, the amplification of two homologous arms was obtained by PCR technique, namely sleB1 and cwlJ1. Then sleB1 was connected with kanamycin resistance （krff） gene by using overlapping PCR technique, thus, B. subtilis 168- TresAsleB was obtained; then cwlJ1 and Neomycin resistance （zeor） gene was connected and transformed it into B. subtilis 168- TresAsleB com- petent cells, thereby, the target strain B. subtilis 168-TresAsleBAcwlJ was obtained. Results showed that after krff, zeal and PCR identification, the sleB and cwlJ genes deletion strain B. subtilis 168-TresAsleBAcwlJ was successfully constructed. The verification results of fermentation showed that the sporulation rate ofB. subtilis 168-TresAsleBAcwlJwas consistent with the original strain, and both of them were about 88%. In LB solid medium and converting maltose into trehalose system, the spore germination of B. subtilis 168-Tres was 4.8x108 CFU/ml, however, the spore of B. subtilis 168-TresAsleBAcwlJ did not germinate. In converting maltose into trehalose system, the trehalose synthase enzyme activity of recombinant strain was 10.42 U, compared with original strain; the enzyme activity increased 78.7%. In conclusion, the knockout of sleB and cwlJ genes had no effect on spore formation of B. subtilis 168-Tres, but it can effectively control spores germination in the above-described transformation system, the spore sur- face can stably display trehalose synthase, improved the utilization efficiency of spore.
出处
《中国酿造》
CAS
北大核心
2017年第3期138-143,共6页
China Brewing
基金
国家自然科学基金(31501413)
山东省高校创新项目(J14LE02)
工业发酵微生物教育部重点实验室暨天津市工业微生物重点实验室(天津科技大学)开放基金(2016IM005)
