| 一个含LoxP-FRT重组酶位点及易于操作的植物表达双元载体pYBA100 |
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| 引用本文： | 闫晓红,王慧,叶艳英,曾钢,马荣才,米福贵,姚磊.一个含LoxP-FRT重组酶位点及易于操作的植物表达双元载体pYBA100[J].分子植物育种,2012(3):371-379. |
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| 作者姓名： | 闫晓红 王慧 叶艳英 曾钢 马荣才 米福贵 姚磊 |
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| 作者单位： | 内蒙古农业大学生态环境学院;北京市农林科学院农业生物技术研究中心;内蒙古师范大学生命科学与技术学院;江西农业大学;福建农林大学 |
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| 基金项目： | 北京市农林科学院科技创新能力建设专项(KJCX201102003);国家“863”计划项目(2009AA10Z104-2);国家国际科技合作项目(2010DFB33740)共同资助 |
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| 摘 要： | 在农杆菌(Agrobacterium tumefaciens)介导的植物遗传转化中,常常需要用到植物表达双元载体。为便于基因工程操作并能在获得转基因植物后方便将标记基因删除,我们构建了植物表达双元载体pYBA100,以满足用于生产安全转基因植物的需要。本研究通过融合PCR方法,尽可能去除所有非必需的序列,将pYBA载体最小化。pYBA载体在大肠杆菌中为多拷贝,骨架为3.69kb。我们构建的含NptⅡ植物筛选标记基因的载体pYBA100仅为5.37kb,多克隆位点为22个,方便基因工程操作。载体pYBA100的T-DNA植物筛选基因表达框两侧预留有LoxP-FRT融合位点,方便在获得转基因植物后通过Cre或FLP重组酶将植物筛选标记基因删除。pYBA100载体能于大肠杆菌和农杆菌中自我复制,并成功转化了拟南芥,符合农杆菌介导的植物转基因要求。离体删除实验结果证明,载体pYBA100能经Cre重组酶删除植物标记基因表达框。
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| 关 键 词： | LoxP-FRT融合位点 植物表达双元载体 农杆菌介导 植物遗传转化 标记基因删除 |
pYBA100:An Ease-of-use Binary Vector with LoxP-FRT Recombinase Site for Plant Transformation |
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| Yan Xiaohong,Wang Hui,Ye Yanying,Zeng Gang,Ma Rongcai,Mi Fugui, Yao Lei.pYBA100:An Ease-of-use Binary Vector with LoxP-FRT Recombinase Site for Plant Transformation[J].Molecular Plant Breeding,2012(3):371-379. |
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| Authors: | Yan Xiaohong Wang Hui Ye Yanying Zeng Gang Ma Rongcai Mi Fugui Yao Lei |
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| Institution: | 1 Inner Mongolia Agricultural University,Huhehaote,010018;2 Beijing Ago-Biotechnology Research Center,Beijing Academy of Agriculture and Forestry Sciences,Beijing,100097;3 Inner Mongolia Normal University,Huhehaote,010022;4 Jiangxi Agricultural University,Nanchang,330045;5 Fujian Agriculture and Forestry University,Fuzhou,350002 |
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| Abstract: | In Agrobacterium-mediated plant gene transformation,we often have to use plant expressive binary vectors.In order to facilitate genetic engineering manipulation and application in plant transformation,as well as to easily excise the selected marker genes after obtaining transgenic plants,we constructed a versatile plant binary vector pYBA100,which meets the demands for producing bio-safety transgenic plants.The fusion PCR method was used in vector construction for eliminating all the nonessential sequences.The pYBA is highly copied vector in Escherichia coli,and the backbone is minimized to 3.69 kb.The pYBA100 vector,which contains a NptⅡ plant selected marker gene cassette,is only 5.37 kb and its unique multiple cloning sites is 22,which is convenient for genetic engineering manipulation.Both sides of the selected marker cassette of T-DNA have the LoxP-FRT combing sites,which can be conveniently excised by the plant selected marker cassette via Cre or FLP recombinase after obtaining the transgenic plants.The pYBA100 vector can replicate in Escherichia coli and Agrobacterium,and has been successful transformed to Arabidopsis thaliana.It is consistent with the requirements of Agrobacterium-mediated plant transformation.In vitro assay,the plant selected marker cassette of pYBA100 is demonstrated to be excised by Cre recombinase. |
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| Keywords: | LoxP-FRT combining site Binary vector Agrobacterium-mediated Plant gene transformation Eliminate selected marker genes |
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