地衣芽胞杆菌FLP/FRT基因编辑系统的构建及验证
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十三五国家重点研发计划 (No. 2016YFD0401400),国家自然科学基金青年基金项目 (No. 31401674),江苏省研究生科研与实践创新计划项目 (No. SJCX18_0616),江苏高校品牌专业建设工程项目,江苏省科技项目 (No. BE2016628),青蓝工程,江苏省渔业科技类项目 (No. Y2018-26),无锡市科技发展资金项目 (No. CLE02N1713) 资助。


Development and verification of an FLP/FRT system for gene editing in Bacillus licheniformis
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The Key Research Project of “13th 5-year Plan” of China (No. 2016YFD0401400), National Natural Science Foundation of China Youth Fund (No. 31401674), Postgraduate Research & Practice Innovation Program of Jiangsu Province (No. SJCX18_0616), Top-notch Academic Programs Project of Jiangsu Higher Education Institutions, Science and Technology Project of Jiangsu (No. BE2016628), Cyan Engineering, Jiangsu Fishery Science and Technology Project (No. Y2018-26), Wuxi Science and Technology Development Fund Project (No. CLE02N1713).

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    摘要:

    地衣芽胞杆菌有效的基因编辑工具有限,为了拓展和丰富其基因编辑手段,在地衣芽胞杆菌中构建一个抗性标记可重复使用的FLP/FRT基因编辑系统,并通过敲除α-淀粉酶基因amyL、蛋白酶基因aprE及敲入外源透明颤菌血红蛋白基因vgb对该系统进行初步验证。首先以温敏质粒pNZT1为载体分别构建amyL和aprE基因敲除质粒pNZTT-AFKF和pNZTT-EFKF,两个敲除质粒各自包含针对目标基因的同源臂、抗性基因及同向的FRT位点;将敲除质粒转化地衣芽胞杆菌并经过两次同源交换过程实现目标基因的敲除;最后导入一个FLP重组酶表达质粒通过FLP/FRT系统的重组作用介导抗性基因的回收。为进一步验证本系统的实用性及编辑效率,构建了包含透明颤菌血红蛋白编码基因vgb表达盒及基因组丙酮酸甲酸裂解酶编码基因pflB敲除盒的重组质粒pNZTK-PFTF-vgb,并以此进行外源基因vgb在基因组上的定向敲入。结果显示,成功敲除amyL及aprE并回收了抗性标记卡那霉素基因,敲除后淀粉酶活和蛋白酶活分别减少95.3%和80.4%;vgb基因成功整合入基因组pflB位点并回收了抗性标记四环素基因,并利用荧光定量PCR技术检测到vgb的整合表达。文中首次建立了一个适用于地衣芽胞杆菌的、抗性标记可重复使用的FLP/FRT基因编辑系统,并进行了基因敲除及基因敲入验证,为地衣芽胞杆菌遗传改造提供了良好的方法参考。

    Abstract:

    Few tools of gene editing have been developed in Bacillus licheniformis at present. In order to enrich the tools, an FLP/FRT gene editing system that can repeatedly use a single selectable marker was constructed in Bacillus licheniformis, and the system was verified by knocking out an alpha amylase gene (amyL), an protease gene (aprE) and knocking in an exogenous Vitreoscilla hemoglobin gene (vgb). First, knock-out plasmids pNZTT-AFKF of amyL and pNZTT-EFKF of aprE were constructed using thermosensitive plasmid pNZT1 as a carrier. The two knock-out plasmids contained respective homology arms, resistance genes and FRT sites. Then the knock-out plasmids were transformed into Bacillus licheniformis and the target genes were replaced by respective deletion cassette via twice homologous exchange. Finally, an expression plasmid containing FLP recombinase reading frane was introduced and mediated the excision of resistance marker. In order to expand the practicability of the system, knock-in plasmid pNZTK-PFTF-vgb was constructed, with which knock-in of vgb at pflB site was carried out successfully. The results showed that amyL and aprE were successfully knocked out and the marker kanamycin cassette exactly excised. The activities of amylase and protease of deletion mutants were reduced by 95.3% and 80.4% respectively. vgb was successfully knocked in at pflB site and the marker tetracycline cassette excised. The expression of integrated vgb was verified via real-time PCR. It is the first time to construct an FLP/FRT system for gene editing in Bacillus licheniformis, which could provide an effective technical means for genetic modification.

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李宗文,李由然,顾正华,丁重阳,张梁,徐沙,石贵阳. 地衣芽胞杆菌FLP/FRT基因编辑系统的构建及验证[J]. 生物工程学报, 2019, 35(3): 458-471

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  • 收稿日期:2018-08-12
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  • 在线发布日期: 2019-03-22
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