本文選題：地衣芽孢桿菌　切入點：同源單交換　出處：《中國生物工程雜志》2016年11期 　論文類型：期刊論文

【摘要】：目的:基于同源單交換原理構(gòu)建地衣芽孢桿菌基因快速敲除方法,提高基因敲除效率。方法:以地衣芽孢桿菌(Bacillus licheniformis)20085內(nèi)切纖維素酶基因celb為擬敲除對象,利用重疊PCR技術(shù)將celb基因內(nèi)約500bp片段與氯霉素抗性基因(Cmr)相連接,經(jīng)末端單酶切后電轉(zhuǎn)化至B.licheniformis 20085感受態(tài)細胞中,僅通過一次同源單交換,將抗性基因Cmr插入至celb基因內(nèi)部,實現(xiàn)目的基因的敲除。結(jié)果:經(jīng)過氯霉素抗性篩選和基因組PCR鑒定,成功獲得celb基因缺失菌株B.licheniformis 20085Δcelb;發(fā)酵驗證結(jié)果顯示,B.licheniformis 20085Δcelb較原始菌株濾紙崩解能力顯著降低,其中發(fā)酵60h后內(nèi)切纖維素酶(CMC酶)活力由1.86U/ml降低至0.50U/ml,表明celb基因在地衣芽孢桿菌降解纖維素的過程中起著重要作用。結(jié)論:通過重疊PCR技術(shù)結(jié)合同源單交換原理能夠?qū)崿F(xiàn)地衣芽孢桿菌目的基因的快速敲除,為該菌株甚至其它微生物提供了一種基因功能快速鑒定的手段。
[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.
【作者單位】： 齊魯工業(yè)大學(xué)生物工程學(xué)院;
【基金】：國家自然科學(xué)基金(31501413) 山東省科技重大專項(新興產(chǎn)業(yè))(2015ZDXX0403B03) 泰山學(xué)者建設(shè)工程專項資助項目
【分類號】：Q78

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