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[Cre lox重组系统的lox位点对转基因植物中无启动子bar基因表达的影响]

[Effect of lox-sites of the Cre lox recombination system on promoterless bar gene expression in transgenic plants].

作者信息

Shcherbak N L, Belokurova V B, Getsko I O, Komarnitskiĭ I K, Kuchuk N V

出版信息

Tsitol Genet. 2006 Jan-Feb;40(1):3-9.

Abstract

We demonstrate that localization of lox site between the right border of T-DNA and promoterless bar gene (RB-lox-bar-) led to its highly efficient expression in transgenic plants of Nicotiana tabacum and N. africana. Plasmid vectors used in gene integration experiments contained neomycin phosphotransferase II (npt II) gene under nos promoter as well. Transgenic plants were selected according to their capacity to grow on the medium with kanamycin and then they were tested on the selective medium containing phosphinothricin. 80% of transgenic plants expressed bar gene at the level similar to that in plants transformed with the bar gene under widely used constitutive promoter. Transformation of plants with the plasmid vector containing only promoterless bar gene near T-DNA right border (RB-bar-) and with the vector containing lox site and promoterless bar gene in the middle of the construction (-lox-bar-) led to obtaining no more than 4.5% of transgenic plants resistant to phosphinothricin. PCR analyses confirmed both the absence of tandem repeats and of plasmid recombination resulting in transference of bar gene under promoter in plasmid vector. Nos-terminator situated between the lox site and the right border of T-DNA did not decrease bar gene expression.

摘要

我们证明,lox位点定位于T-DNA右边界与无启动子的bar基因之间(RB-lox-bar-),导致其在烟草和非洲烟草的转基因植株中高效表达。基因整合实验中使用的质粒载体还包含在nos启动子控制下的新霉素磷酸转移酶II(npt II)基因。根据转基因植株在含卡那霉素培养基上的生长能力进行筛选,然后在含草丁膦的选择培养基上进行检测。80%的转基因植株中bar基因的表达水平与用广泛使用的组成型启动子驱动bar基因转化的植株相似。用仅在T-DNA右边界附近含有无启动子bar基因的质粒载体(RB-bar-)以及在构建体中间含有lox位点和无启动子bar基因的载体(-lox-bar-)转化植株,获得的对草丁膦有抗性的转基因植株不超过4.5%。PCR分析证实既不存在串联重复,也不存在导致质粒载体中启动子下bar基因转移的质粒重组。位于lox位点和T-DNA右边界之间的nos终止子不会降低bar基因的表达。

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