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.

参考文献

[1] Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci USA , 1990, 87(12): 4576-4579.
[2] Allers T, Mevarech M. Archaeal genetics—the third way. Nat Rev Genet , 2005, 6(1): 58-73.
[3] Kelman Z, White MF. Archaeal DNA replication and repair. Curr Opin Microbiol , 2005, 8(6): 669-676.
[4] Wu Z, Liu J, Yang H, Xiang H. DNA replication origins in archaea. Front Microbiol , 2014, 5: 179.
[5] Norais C, Hawkins M, Hartman AL, Eisen JA, Myllykallio H, Allers T. Genetic and physical mapping of DNA replication origins in Haloferax volcanii . PLoS Genet , 2007, 3(5): e77.
[6] Allers T, Ngo HP. Genetic analysis of homologous recombination in Archaea: Haloferax volcanii as a model organism. Biochem Soc Trans , 2003, 31: 706-710.
[7] Liu HL, Han J, Liu XQ, Zhou J, Xiang H. Development of pyrF -based gene knockout systems for genome-wide manipulation of the archaea Haloferax mediterranei and Haloarcula hispanica . J Genet Genomics , 2011, 38(6): 261-269.
[8] Liu HL, Wu ZF, Li M, Zhang F, Zheng HJ, Han J, Liu JF, Zhou J, Wang SY, Xiang H. Complete genome sequence of Haloarcula hispanica , a Model Haloarchaeon for studying genetics, metabolism, and virus-host interaction. J Bacteriol , 2011, 193(21): 6086-6087.
[9] Wagner M, van Wolferen M, Wagner A, Lassak K, Meyer BH, Reimann J, Albers SV. Versatile genetic tool box for the crenarchaeote sulfolobus acidocaldarius. Front Microbiol , 2012, 3: 214.
[10] Zhang CY, Guo L, Deng L, Wu YX, Liang YX, Huang L, She QX. Revealing the essentiality of multiple archaeal pcna genes using a mutant propagation assay based on an improved knockout method. Microbiology , 2010, 156(11): 3386-3397.
[11] Bridger SL, Clarkson SM, Stirrett K, DeBarry MB, Lipscomb GL, Schut GJ, Westpheling J, Scott RA, Adams MWW. Deletion strains reveal metabolic roles for key elemental sulfur-responsive proteins in Pyrococcus furiosus . J Bacteriol , 2011, 193(23): 6498-6504.
[12] Sato T, Fukui T, Atomi H, Imanaka T. Targeted gene disruption by homologous recombination in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. J Bacteriol , 2003, 185(1): 210-220.
[13] Hartman AL, Norais C, Badger JH, Delmas S, Haldenby S, Madupu R, Robinson J, Khouri H, Ren QH, Lowe TM, Maupin-Furlow J, Pohlschroder M, Daniels C, Pfeiffer F, Allers T, Eisen JA. The complete genome sequence of Haloferax volcanii DS2, a model archaeon. PLoS One , 2010, 5(3): e9605.
[14] Cline SW, Lam WL, Charlebois RL, Schalkwyk LC, Doolittle WF. Transformation methods for halophilic archaebacteria. Can J Microbiol , 1989, 35(1): 148-152.
[15] Allers T, Ngo HP, Mevarech M, Lloyd RG. Development of additional selectable markers for the halophilic archaeon Haloferax volcanii based on the leuB and trpA genes. Appl Environ Microbiol , 2004, 70(2): 943-953.
[16] Bitan-Banin G, Ortenberg R, Mevarech M. Development of a gene knockout system for the halophilic archaeon Haloferax volcanii by use of the pyrE gene. J Bacteriol , 2003, 185(3): 772-778.
[17] Holmes M, Pfeifer F, Dyall-Smith M. Improved shuttle vectors for Haloferax volcanii including a dual-resistance plasmid. Gene , 1994, 146(1): 117-121.
[18] Allers T, Barak S, Liddell S, Wardell K, Mevarech M. Improved strains and plasmid vectors for conditional overexpression of his-tagged proteins in Haloferax volcanii . Appl Environ Microbiol , 2010, 76(6): 1759-1769.
[19] Liu HT, Rudolf J, Johnson KA, McMahon SA, Oke M, Carter L, McRobbie AM, Brown SE, Naismith JH, White MF. Structure of the DNA repair helicase XPD. Cell , 2008, 133(5): 801-812.
[20] Lehmann AR. The xeroderma pigmentosum group D ( XPD ) gene: one gene, two functions, three diseases. Genes Dev , 2001, 15(1): 15-23.
[21] Winkler GS, Araújo SJ, Fiedler U, Vermeulen W, Coin F, Egly JM, Hoeijmakers JHJ, Wood RD, Timmers HTM, Weeda G. TFIIH with inactive XPD helicase

编辑推荐

Metrics

www.chinagene.cn
备案号：京ICP备09063187号-4
总访问:,今日访问:,当前在线:

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

PDF (PC)

可视化

被引次数

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

[1]	刘梅珍, 王立人, 李咏梅, 马雪云, 韩红辉, 李大力. 利用CRISPR/Cas9技术构建基因编辑大鼠模型[J]. 遗传, 2023, 45(1): 78-87.
[2]	郭雨萱, 严顺平, 王应祥. 重组酶RAD51和DMC1功能保守和分化研究进展[J]. 遗传, 2022, 44(5): 398-413.
[3]	耿喜宁, 芦特, 杜康, 杨珺, 康向阳. 不同基因型毛白杨同源重组变异研究[J]. 遗传, 2021, 43(2): 182-193.
[4]	林珉婷, 赖璐璐, 赵淼, 林必玮, 姚香平. 利用CRISPR/Cas9 AAV系统构建纹状体Slc20a2基因敲除小鼠模型[J]. 遗传, 2020, 42(10): 1017-1027.
[5]	李帆, 余蓉培, 孙丹, 王继华, 李绅崇, 阮继伟, 单芹丽, 陆平利, 汪国鲜. 抑制植物减数分裂重组的分子机理[J]. 遗传, 2019, 41(1): 52-65.
[6]	全绒, 李国玲, 莫健新, 钟翠丽, 李紫聪, 顾婷, 郑恩琴, 刘德武, 蔡更元, 吴珍芳, 张献伟. RNA干扰猪NHEJ通路修复因子对HR效率的影响[J]. 遗传, 2018, 40(9): 749-757.
[7]	张桂珊, 杨勇, 张灵敏, 戴宪华. 机器学习方法在CRISPR/Cas9系统中的应用[J]. 遗传, 2018, 40(9): 704-723.
[8]	唐浚博, 曹浩伟, 许蕊, 张丹丹, 黄娟. 果蝇睾丸基因敲除突变体的构建及表型分析[J]. 遗传, 2018, 40(6): 478-487.
[9]	梁彩娇, 孟繁梅, 艾云灿. 基于CRISPR/Cas系统的噬菌体基因组编辑[J]. 遗传, 2018, 40(5): 378-389.
[10]	刘春霞, 耿立召, 许建平. 植物基因组编辑检测方法[J]. 遗传, 2018, 40(12): 1075-1091.
[11]	黄敏,杨业然,孙晓艳,张婷,郭彩霞. RAD51调控REV1参与DNA双链断裂修复[J]. 遗传, 2018, 40(11): 1007-1014.
[12]	贺燕,谢梦女,余立,任真,朱芳,符淳. 范可尼贫血基因在卵泡发育中的调节作用[J]. 遗传, 2017, 39(6): 469-481.
[13]	李国玲,钟翠丽,莫健新,全绒,吴珍芳,李紫聪,杨化强,张献伟. 动物基因组定点整合转基因技术研究进展[J]. 遗传, 2017, 39(2): 98-109.
[14]	王伟, 王玉霜, 黄兰兰, 简子健, 王新华, 刘守仁, 皮文辉. siRNA干扰绵羊胚胎成纤维细胞Lig4基因增加同源重组载体重连修复效率[J]. 遗传, 2016, 38(9): 831-839.
[15]	张峰华,王厚鹏,黄思雨,熊凤,朱作言,孙永华. 两种密码子优化的Cas9编码基因在斑马鱼胚胎中基因敲除效率的比较[J]. 遗传, 2016, 38(2): 144-154.