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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2016, Vol. 36 Issue (11): 63-69    DOI: 10.13523/j.cb.20161109
技术与方法     
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
韩海红, 汪俊卿, 王腾飞, 肖静, 韩登兰, 王瑞明
齐鲁工业大学生物工程学院 济南 250353
Method and Application of Gene Knockout Based Single Cross in Bacillus licheniformis 20085
HAN Hai hong, WANG Jun qing, WANG Teng fei, XIAO Jing, HAN Deng lan, WANG Rui ming
Qilu University of Technology, Biological Engineering, Jinan 250353, China
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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。
关键词： 基因敲除;  同源单交换;  地衣芽孢杆菌;  celb基因    
Abstract:

Bacillus licheniformis 20085 has fast breeding, adaptability and strong lignocellulose degradation characteristics than other lignocellulose-degrading microorganisms, it also plays an important role in the process of degradation of natural lignocellulosic, however, it could not be as lignin degradation strain of bio-pulping due to it also has a strong ability to degrade cellulose.An endo-1,4-β-D-glucanohydrolase (CMCase) gene celb in Bacillus licheniformis 20085 was intended to knockout, about 500bp DNA fragment celb1 in celb gene with the chloramphenicol resistance gene (Cmr) connected by using overlapping PCR technique, thus, after a single enzyme digestionat the end of fragment, recombinant gene fragment was transformed into B. licheniformis 20085 competent cells. Through a single homologous exchange, the resistance gene Cmr was inserted into the celb gene, thereby, the target gene was knocked out successfully. After chloramphenicol resistance experiments, PCR determination, The celb gene deletion strain B. licheniformis 20085Δcelb successfully was obtained; The verification results offer mentation shows, the ability of the filter paper disintegration of B. licheniformis 20085Δcelb reduced significantly than the original strain. B. licheniformis 20085Δcelb than the original strain disintegrating paper capacity significantly reduced; After 60h fermentation, the activity of CMCase was decreased from 1.86U/ml to 0.50U/ml, and it shown that celb gene in Bacillus licheniformis plays an important role in the process of degradation of cellulose. Conclusion:The combination between the principle of single cross of homologous recombination and overlapping PCR technology enables a targeted gene to achieve quick knockout in Bacillus licheniformis, or even provides a new means for the rapid identification of gene function for other microorganisms.
Key words: Bacillus licheniformis    Single-crossover    celb gene    Gene knockout
收稿日期: 2016-05-04 出版日期: 2016-11-25
ZTFLH:  Q78  
基金资助:

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
通讯作者: 王瑞明,ruiming3k@163.com     E-mail: ruiming3k@163.com
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引用本文:

韩海红, 汪俊卿, 王腾飞, 肖静, 韩登兰, 王瑞明. 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[J]. 中国生物工程杂志, 2016, 36(11): 63-69.

HAN Hai hong, WANG Jun qing, WANG Teng fei, XIAO Jing, HAN Deng lan, WANG Rui ming. Method and Application of Gene Knockout Based Single Cross in Bacillus licheniformis 20085. China Biotechnology, 2016, 36(11): 63-69.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20161109    或    https://manu60.magtech.com.cn/biotech/CN/Y2016/V36/I11/63

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