一种基于单交换原理的地衣芽孢杆菌基因敲除方法及应用

发布时间：2018-03-19 02:30

本文选题：地衣芽孢杆菌　切入点：同源单交换　出处：《中国生物工程杂志》2016年11期 　论文类型：期刊论文

【摘要】：目的:基于同源单交换原理构建地衣芽孢杆菌基因快速敲除方法,提高基因敲除效率。方法:以地衣芽孢杆菌(Bacillus licheniformis)20085内切纤维素酶基因celb为拟敲除对象,利用重叠PCR技术将celb基因内约500bp片段与氯霉素抗性基因(Cmr)相连接,经末端单酶切后电转化至B.licheniformis 20085感受态细胞中,仅通过一次同源单交换,将抗性基因Cmr插入至celb基因内部,实现目的基因的敲除。结果:经过氯霉素抗性筛选和基因组PCR鉴定,成功获得celb基因缺失菌株B.licheniformis 20085Δcelb;发酵验证结果显示,B.licheniformis 20085Δcelb较原始菌株滤纸崩解能力显著降低,其中发酵60h后内切纤维素酶(CMC酶)活力由1.86U/ml降低至0.50U/ml,表明celb基因在地衣芽孢杆菌降解纤维素的过程中起着重要作用。结论:通过重叠PCR技术结合同源单交换原理能够实现地衣芽孢杆菌目的基因的快速敲除,为该菌株甚至其它微生物提供了一种基因功能快速鉴定的手段。
[Abstract]:Objective: to construct a rapid gene knockout method for Bacillus licheniformis based on homologous mono-exchange principle, and to improve the efficiency of gene knockout. Methods: the cellulase gene celb of Bacillus licheniformis)20085 was used as the target for knockout. About 500bp fragment of the celb gene was linked to the chloramphenicol resistance gene Cmr. by overlapping PCR technique, the gene Cmr was inserted into the celb gene by single endonuclease digesting into the competent cells of B.licheniformis 20085. Results: after chloramphenicol resistance screening and genomic PCR identification, celb gene deletion strain B.licheniformis 20085 螖 Celb was successfully obtained, and the fermentation results showed that B. licheniformis 20085 螖 celb was significantly lower than that of the original strain. The cellulase activity decreased from 1.86 U / ml to 0.50 U / ml after fermentation for 60 h, indicating that the celb gene plays an important role in the degradation of cellulose by Bacillus licheniformis. Conclusion: the overlapping PCR technique combined with the principle of homologous mono-exchange can be used in the process of cellulose degradation by Bacillus licheniformis. To achieve rapid knockout of the target gene of Bacillus licheniformis, It provides a method for rapid identification of gene function of the strain and even other microbes.
【作者单位】：齐鲁工业大学生物工程学院;
【基金】：国家自然科学基金(31501413) 山东省科技重大专项(新兴产业)(2015ZDXX0403B03) 泰山学者建设工程专项资助项目
【分类号】：Q78

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