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| 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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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.
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Received: 04 May 2016
Published: 25 November 2016
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