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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 (7): 64-71    DOI: 10.13523/j.cb.20160710
技术与方法     
构建尿嘧啶磷酸核糖转移酶基因缺失菌株实现Gluconobacter suboxydans基因组无痕修饰
杜红燕, 李天明, 刘金雷, 冯惠勇
河北科技大学生物科学与工程学院 石家庄 050018
Construct the Uracil Phosphoribosyl Transferase Gene Mutant Strain in Gluconobacter suboxydans for Seamless Genome Editing
DU Hong-yan, LI Tian-ming, LIU Jin-lei, FENG Hui-yong
College of Biological Science and Engineering, Hebei University of Science and Technology, Shijiazhuang 050018, China
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摘要：

弱氧化葡糖酸杆菌（Gluconobacter suboxydans）可高效不完全氧化多种糖类和醇类等多羟基化合物，生成相应的醛类、酮类和有机酸类等产物，是一类重要的工业微生物。利用同源重组技术对基因组进行修饰改造是工业育种的有效手段。传统方法多选用抗生素为筛选标记，存在诸多缺陷。构建尿嘧啶磷酸核糖转移酶基因缺失菌株以实现Gluconobacter suboxydans基因组无痕修饰为目标，将自杀质粒pMD18-Jqupp电转化至野生型Gluconobacter suboxydans J12中，通过四环素抗性和5-氟尿嘧啶双重筛选，获得敲除了编码尿嘧啶磷酸核糖转移酶的基因upp的突变株。经生理验证表明，该突变株在含0.5mg/ml 5-氟尿嘧啶的培养基上生长，而回补upp基因后，在0.5mg/ml 5-F氟尿嘧啶的培养基上不生长。说明获得的G.suboxydans-upp突变株能以upp基因作为负向筛选标记，通过两次同源重组，实现Gluconobacter suboxydans基因组无痕修饰与改造，为今后代谢工程改造Gluconobacter suboxydans获得有价值的工业菌种奠定基础。
关键词： 尿嘧啶磷酸核糖转移酶;  基因敲除;  无痕修饰;  弱氧化葡糖酸杆菌    
Abstract:

Gluconobacter suboxydans is an important industrial microbiology and can incompletely and efficiently oxidize a great variety of carbohydrates, alcohols and other polyols to form the corresponding aldehydes, ketones and organic acids and other products. The use of homologous recombination technology to transform genomic modification is an effective means of industrial breeding. There are many defects when use antibiotic marker as the screening marker in traditional methods. The construction of uracil phosphoribosyl transferase gene deletion strain is to achieve Gluconobacter suboxydans seamless genome editing. The suicide plasmid pMD18-Jqupp is transformed into wild type Gluconobacter suboxydans J12 and the mutant which knocked encoding uracil phosphoribosyl transferase gene was screened by tetracycline resistance and 5-fluorouracil. The physiological validation results show that the mutant strain can grow on medium containing 0.5mg/ml 5-fluorouracil, but the mutant covering upp gene can't grow on medium containing 0.5mg/ml 5-fluorouracil. It proves that the upp gene can be used as a negative selection marker in G.suboxydans-upp mutants to achieve Gluconobacter suboxydans genome modification and transformation by twice homologous recombination. It provides the foundation of changing Gluconobacter suboxydans to obtain valuable industrial strains using metabolic engineering in the future.
Key words: Gluconobacter suboxydans    Gene knockout    Scarless modify    Uracil phosphoribosyl transferase
收稿日期: 2016-01-18 出版日期: 2016-03-16
ZTFLH:  Q78  
基金资助:

国家科技支撑计划资助项目（2015BAD15B0501）
通讯作者: 冯惠勇     E-mail: fenghuiyong@163.com
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引用本文:

杜红燕, 李天明, 刘金雷, 冯惠勇. 构建尿嘧啶磷酸核糖转移酶基因缺失菌株实现Gluconobacter suboxydans基因组无痕修饰[J]. 中国生物工程杂志, 2016, 36(7): 64-71.

DU Hong-yan, LI Tian-ming, LIU Jin-lei, FENG Hui-yong. Construct the Uracil Phosphoribosyl Transferase Gene Mutant Strain in Gluconobacter suboxydans for Seamless Genome Editing. China Biotechnology, 2016, 36(7): 64-71.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20160710    或    https://manu60.magtech.com.cn/biotech/CN/Y2016/V36/I7/64

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