Plant-specific promoter sequences carry elements that are recognised by the eubacterial transcription machinery
Plant-specific promoter sequences carry elements that are recognised by the eubacterial transcription machinery
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DOI：
10.1023/a:1015620016472
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发表时间：
2002-06-01
影响因子：
3
通讯作者：
Appel, B
中科院分区：
文献类型：
--
作者：
Jacob, D;Lewin, A;Appel, B
在进化过程中，来自原核生物和真核生物的启动子元件在其序列和结构方面发展不同，这意味着通常真核启动子序列转移到原核生物中不会引起有效的基因表达。然而，已经有关于来自花椰菜花叶病毒（CaMV）的35 S启动子在细菌中的功能的报道。因此，我们决定实验研究植物启动子序列在各种细菌中指导基因表达的能力。因此，我们测试了来自马铃薯、烟草、CaMV、根癌农杆菌和A. rhizogenes在五种不同的真细菌物种（大肠杆菌、小肠结肠炎耶尔森氏菌（Yersinia enterocolitica）、A.根瘤菌、恶臭假单胞菌和不动杆菌属BD 413）。为了监测细菌中植物特异性启动子的强度，我们在这些启动子与哈维氏弧菌荧光素酶基因的编码区之间创建了融合体，并测量了细菌中的发光。在50%的分析组合中观察到异源基因表达。然后，我们定位了由植物特异性启动子之一，S. tuberosum，在这些细菌物种中。映射的转录起始位点的位置表明，植物启动子本身的序列被细菌转录器识别。通过对ST-LS 1启动子的定点突变和突变对E.杆菌利用这些突变体在我们的报告基因分析中，我们可以定位在大肠杆菌中作为-10区的ST-LS 1启动子序列。杆菌我们的研究结果表明，启动子序列的特异性远低于一般认为的。这对于我们了解基因表达系统的进化和构建优化的表达载体是非常重要的。
During evolution the promoter elements from prokaryotes and eukaryotes have developed differently with regard to their sequence and structure, implying that in general a transfer of eukaryotic promoter sequences into prokaryotes will not cause an efficient gene expression. However, there have been reports on the functionality of the 35S promoter from cauliflower mosaic virus (CaMV) in bacteria. We therefore decided to experimentally investigate the capability of plant promoter sequences to direct gene expression in various bacteria. Accordingly, we tested ten different plant-specific promoters from Solanum tuberosum, Nicotiana tabacum, CaMV, Agrobacterium tumefaciens, and A. rhizogenes for their ability to initiate transcription in five different eubacterial species (Escherichia coli, Yersinia enterocolitica, A. tumefaciens, Pseudomonas putida, and Acinetobacter sp. BD413). To monitor the strength of the plant-specific promoters in bacteria we created fusions between these promoters and the coding region of the luciferase genes from Vibrio harveyi and measured the luminescence in the bacteria. Heterologous gene expression was observed in 50% of the combinations analysed. We then mapped the transcription start site caused by one of the plant-specific promoters, the ST-LS1 promoter from S. tuberosum, in these bacterial species. The location of the mapped transcription start site indicated that the sequences of the plant promoter themselves were recognised by the bacterial transcription apparatus. The recognition of plant-specific promoter sequences by the bacterial RNA polymerase was further confirmed by site-directed mutagenesis of the ST-LS1 promoter and the analysis of the effects of the mutations on the strength of gene expression in E. coli. Using these mutants in our reporter assays we could localise the sequences of the ST-LS1 promoter serving as -10 region in E. coli. The results of our study show that promoter sequences are much less specific than is generally assumed. This is of great importance for our knowledge about the evolution of gene expression systems and for the construction of optimised expression vectors.