一种基于线性DNA片段同源重组的嗜盐古菌高效基因敲除系统

doi:10.16288/j.yczz.14-366

遗传 ›› 2015, Vol. 37 ›› Issue (4): 388-395.doi: 10.16288/j.yczz.14-366

一种基于线性DNA片段同源重组的嗜盐古菌高效基因敲除系统

王小利,姜闯,刘建华,刘喜朋

上海交通大学生命科学技术学院,微生物代谢国家重点实验室,上海 200240

收稿日期:2014-10-24 出版日期:2015-04-20 发布日期:2015-03-12
通讯作者: 刘喜朋,博士,副教授,研究方向：微生物遗传学。E-mail: xpliu@sjtu.edu.cn E-mail:650914@sjtu.edu.cn
作者简介:王小利,硕士,专业方向：DNA修复。E-mail: 650914@sjtu.edu.cn
基金资助:
国家自然科学基金项目(编号31371260)资助

An efficient genetic knockout system based on linear DNA fragment homologous recombination for halophilic archaea

Xiaoli Wang,Chuang Jiang,Jianhua Liu,Xipeng Liu

State Key Laboratory of Microbial Metabolism , School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China

Received:2014-10-24 Online:2015-04-20 Published:2015-03-12

摘要/Abstract

摘要： 随着功能基因组学研究的深入发展,基因敲除技术日益成为基因功能研究的重要手段。嗜盐古菌Haloferax volcanii易于培养,是研究古菌基因功能的良好模式菌株。虽然现已开发了多种嗜盐古菌的遗传操作系统,但基因敲除成功率不十分理想。这些遗传操作方法基于pyrE筛选标记,利用携带同源片段的环状质粒与基因组同源片段间的两次同源重组,敲除目的基因。由于基于环状质粒和pyrE筛选标记的经典同源重组敲除方法在二次重组时,普遍存在回复到野生型菌株的可能,导致二次重组子中敲除目的基因的阳性菌株比例较低。为了克服传统同源重组技术的上述缺陷,文章建立了基于线性DNA片段的同源重组技术。该方法通过一次重组在目标基因的下游引入一段上游同源片段和pyrE标记,从而限定二次重组的发生部位只能在两段上游同源片段之间,发生二次重组的重组子理论上都敲除了目标基因。利用该方法,文章成功敲除了嗜盐古菌Haloferax volcanii的xpd2基因,阳性克隆率达65%。这种线性DNA片段重组法为嗜盐古菌的基因敲除提供了一种高效策略,便于嗜盐古菌的基因改造。

关键词: 嗜盐古菌, 基因敲除, 同源重组, 线性DNA重组

Abstract: With the development of functional genomics, gene-knockout is becoming an important tool to elucidate gene functions in vivo. As a good model strain for archaeal genetics, Haloferax volcanii has received more attention. Although several genetic manipulation systems have been developed for some halophilic archaea, it is time-consuming because of the low percentage of positive clones during the second-recombination selection. These classical gene knockout methods are based on DNA recombination between the genomic homologous sequence and the circular suicide plasmid, which carries a pyrE selection marker and two DNA fragments homologous to the upstream and downstream fragments of the target gene. Many wild-type clones are obtained through a reverse recombination between the plasmid and genome in the classic gene knockout method. Therefore, it is necessary to develop an efficient gene knockout system to increase the positive clone percentage. Here we report an improved gene knockout method using a linear DNA cassette consisting of upstream and downstream homologous fragments, and the pyrE marker. Gene deletions were subsequently detected by colony PCR analysis. We determined the efficiency of our knockout method by deleting the xpb2 gene from the H. volcanii genome, with the percentage of positive clones higher than 50%. Our method provides an efficient gene knockout strategy for halophilic archaea.

Key words: halophilic archaea, gene knockout, homologous recombination, linear DNA recombination

王小利,姜闯,刘建华,刘喜朋. 一种基于线性DNA片段同源重组的嗜盐古菌高效基因敲除系统[J]. 遗传, 2015, 37(4): 388-395.

Xiaoli Wang,Chuang Jiang,Jianhua Liu,Xipeng Liu. An efficient genetic knockout system based on linear DNA fragment homologous recombination for halophilic archaea[J]. HEREDITAS(Beijing), 2015, 37(4): 388-395.

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一种基于线性DNA片段同源重组的嗜盐古菌高效基因敲除系统

An efficient genetic knockout system based on linear DNA fragment homologous recombination for halophilic archaea

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