A bifunctional transposon mini-Tn5gfp-km which can be used to select for promoter fusions and report gene expression levels in Agrobacterium tumefaciens.
A bifunctional transposon mini-Tn5gfp-km which can be used to select for promoter fusions and report gene expression levels in Agrobacterium tumefaciens.
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DOI：
10.1111/j.1574-6968.1999.tb08704.x
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发表时间：
1999-10
影响因子：
2.1
通讯作者：
Xie Tang;Bai F Lu;Shen Q. Pan
中科院分区：
文献类型：
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作者：
Xie Tang;Bai F Lu;Shen Q. Pan
构建了一个含有绿色荧光蛋白(GFP)和新霉素磷酸转移酶II(NptII)两个开放阅读框的无启动子人工操纵子的mini-Tn5转座子衍生物mini-Tn5gfp-Km。用该转座子诱变根癌农杆菌时，所有在卡那霉素存在下选择的突变体都有GFP的表达，这可以方便地用荧光仪进行监测。转座子似乎是双功能的，既可以提供选择功能，也可以提供报告功能。即使是GFP表达水平最低的突变株也对卡那霉素产生了抗药性。这表明，该转座子可以用于选择弱启动子和强启动子下游的插入，只要插入本身是非致死的。该系统被用来鉴定根癌农杆菌对酸性pH反应上调的基因。只对20个菌落进行筛选，鉴定出两个启动子是由低pH值诱导的，一个启动子是由乙酰丁香酮在最低pH 5.5的最低培养基中特异性诱导的。
A mini-Tn5 transposon derivative, mini-Tn5gfp-km, has been constructed which contained a promoter-less artificial operon consisting of two open reading frames, green fluorescent protein (GFP) and neomycin phosphotransferase II (NptII). When this transposon was used to mutagenize Agrobacterium tumefaciens, all the mutants selected in the presence of kanamycin exhibited GFP expression, which could be conveniently monitored by a fluorometer. The transposon appeared to be bifunctional and could provide both selection and reporter functions. Even the mutants showing minimal levels of GFP expression were still resistant to kanamycin. This suggests that this transposon can be used to select for insertions downstream of both weak and strong promoters, as long as the insertions themselves are non-lethal. This system was used to identify A. tumefaciens genes that were upregulated in response to acidic pH. Screening only 20 colonies led to identification of two promoters that were specifically induced by low pH and one promoter that was specifically induced by acetosyringone in a minimal medium of pH 5.5.