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首页> 外文期刊>BMC Biotechnology >An efficient method for stable protein targeting in grasses (Poaceae): a case study in Puccinellia tenuiflora
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An efficient method for stable protein targeting in grasses (Poaceae): a case study in Puccinellia tenuiflora

机译：一种有效稳定蛋白质靶向禾本科（禾本科）的方法：以Puccinellia tenuiflora为例

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Background An efficient transformation method is lacking for most non-model plant species to test gene function. Therefore, subcellular localization of proteins of interest from non-model plants is mainly carried out through transient transformation in homologous cells or in heterologous cells from model species such as Arabidopsis . Although analysis of expression patterns in model organisms like yeast and Arabidopsis can provide important clues about protein localization, these heterologous systems may not always faithfully reflect the native subcellular distribution in other species. On the other hand, transient expression in protoplasts from species of interest has limited ability for detailed sub-cellular localization analysis (e.g., those involving subcellular fractionation or sectioning and immunodetection), as it results in heterogeneous populations comprised of both transformed and untransformed cells. Results We have developed a simple and reliable method for stable transformation of plant cell suspensions that are suitable for protein subcellular localization analyses in the non-model monocotyledonous plant Puccinellia tenuiflora . Optimization of protocols for obtaining suspension-cultured cells followed by Agrobacterium -mediated genetic transformation allowed us to establish stably transformed cell lines, which could be maintained indefinitely in axenic culture supplied with the proper antibiotic. As a case study, protoplasts of transgenic cell lines stably transformed with an ammonium transporter-green fluorescent protein (PutAMT1;1-GFP) fusion were successfully used for subcellular localization analyses in P. tenuiflora . Conclusions We present a reliable method for the generation of stably transformed P. tenuiflora cell lines, which, being available in virtually unlimited amounts, can be conveniently used for any type of protein subcellular localization analysis required. Given its simplicity, the method can be used as reference for other non-model plant species lacking efficient regeneration protocols.
机译：背景技术对于大多数非模型植物物种而言，缺乏有效的转化方法来测试基因功能。因此，来自非模型植物的目的蛋白质的亚细胞定位主要是通过瞬时转化在来自模型物种例如拟南芥属的同源细胞或异源细胞中进行的。尽管对诸如酵母和拟南芥这样的模型生物中表达模式的分析可以提供有关蛋白质定位的重要线索，但是这些异源系统可能并不总是如实地反映其他物种的天然亚细胞分布。另一方面，目的物种在原生质体中的瞬时表达对于详细的亚细胞定位分析（例如，涉及亚细胞分级分离或切片和免疫检测的那些）的能力有限，因为它导致由转化的和未转化的细胞组成的异质群体。结果我们开发了一种简单而可靠的方法来稳定转化植物细胞悬液，该方法适用于非模式单子叶植物Puccinellia tenuiflora中的蛋白质亚细胞定位分析。优化用于获得悬浮培养的细胞，然后通过农杆菌介导的遗传转化的方案使我们能够建立稳定转化的细胞系，该细胞系可以在带有适当抗生素的无菌培养物中无限期地维持。作为一个案例研究，用铵转运蛋白-绿色荧光蛋白（PutAMT1; 1-GFP）融合体稳定转化的转基因细胞系的原生质体已成功用于ten.flora的亚细胞定位分析。结论我们提供了一种可靠的方法来生成稳定转化的藤黄假单胞菌细胞系，其数量几乎不受限制，可方便地用于所需的任何类型的蛋白质亚细胞定位分析。鉴于其简单性，该方法可作为缺乏有效再生方案的其他非模式植物物种的参考。

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