Urwin P, Yi L, Martin H, Atkinson H, Gilmartin P M
Centre for Plant Sciences, Leeds Institute for Plant Biotechnology and Agriculture, University of Leeds, Leeds LS2 9JT, UK.
Plant J. 2000 Dec;24(5):583-9. doi: 10.1046/j.1365-313x.2000.00904.x.
The translation of eukaryotic messenger RNA is typically dependent upon the presence of an m7GpppN cap structure at the 5' end of the transcript. However, several animal viruses, including the Picorna viruses, have been shown to exhibit cap-independent translation through the presence of an internal ribosome entry site or IRES. This IRES-mediated cap-independent internal translation initiation has been exploited to generate bicistronic transcripts that function in animal cells. Recently IRES elements have also been identified in a small number of vertebrate, insect and yeast cellular messenger RNAs although no such sequences have been identified in endogenous plant genes and there are no reports of animal virus derived IRES activity in plant cells. Here we have constructed a bicistronic gene containing both green fluorescent protein and luciferase open-reading frames separated by the encephalomyocarditis IRES element under the control of the CaMV 35S promoter. Northern analysis reveals expression of the bicistronic transcript and in vivo imaging of GFP and luciferase activities demonstrates the functional presence of both proteins. Western blot analysis confirms the independent translation of both reporter proteins. These data suggest that insertion of the encephalomyocarditis virus (EMCV) IRES element between two open-reading frames of a plant bicistronic transcript can mediate translation of the second open-reading frame. This activity is more apparent in the leaves, than in the roots, of transgenic seedlings carrying the bicistronic reporter gene construct.
真核生物信使核糖核酸的翻译通常依赖于转录本5'端存在m7GpppN帽结构。然而，包括小核糖核酸病毒在内的几种动物病毒已被证明通过存在内部核糖体进入位点（IRES）来进行不依赖帽的翻译。这种IRES介导的不依赖帽的内部翻译起始已被用于产生在动物细胞中起作用的双顺反子转录本。最近，在少数脊椎动物、昆虫和酵母细胞信使核糖核酸中也鉴定出了IRES元件，尽管在内源植物基因中尚未鉴定出此类序列，也没有关于植物细胞中动物病毒衍生的IRES活性的报道。在这里，我们构建了一个双顺反子基因，其包含绿色荧光蛋白和荧光素酶开放阅读框，由脑心肌炎病毒IRES元件隔开，并受CaMV 35S启动子控制。Northern分析揭示了双顺反子转录本的表达，GFP和荧光素酶活性的体内成像证明了两种蛋白质的功能性存在。蛋白质免疫印迹分析证实了两种报告蛋白的独立翻译。这些数据表明，在植物双顺反子转录本的两个开放阅读框之间插入脑心肌炎病毒（EMCV）IRES元件可以介导第二个开放阅读框的翻译。在携带双顺反子报告基因构建体的转基因幼苗的叶片中，这种活性比在根中更明显。
