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首页> 外文期刊>Molecular biotechnology >Use of the GFP reporter as a vital marker for Agrobacterium-mediated transformation of sugar beet (Beta vulgaris L.).
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Use of the GFP reporter as a vital marker for Agrobacterium-mediated transformation of sugar beet (Beta vulgaris L.).

机译：GFP报告基因作为农杆菌介导的甜菜转化的重要标志物的用途。

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Molecular approaches to sugar beet improvement will benefit from an efficient transformation procedure that does not rely upon exploitation of selectable marker genes such as those which confer antibiotic or herbicide resistance upon the transgenic plants. The expression of the green fluorescent protein (GFP) signal has been investigated during a program of research that was designed to address the need to increase the speed and efficiency of selection of sugar beet transformants. It was envisaged that the GFP reporter could be used initially as a supplement to current selection regimes in order to help eliminate "escapes" and perhaps eventually as a replacement marker in order to avoid the public disquiet associated with antibiotic/herbicide-resistance genes in field-released crops. The sgfp-S65T gene has been modified to have a plant-compatible codon usage, and a serine to threonine mutation at position 65 for enhanced fluorescence under blue light. This gene, under the control of the CaMV 35S promoter, was introduced into sugar beet via Agrobacterium-mediated transformation. Early gene expression in cocultivated sugar beet cultures was signified by green fluorescence several days after cocultivation. Stably transformed calli, which showed green fluorescence at a range of densities, were obtained at frequencies of 3-11% after transferring the inoculated cultures to selection media. Cocultivated shoot explants or embryogenic calli were regularly monitored under the microscope with blue light when they were transferred to media without selective agents. Green fluorescent shoots were obtained at frequencies of 2-5%. It was concluded that the sgfp-S65T gene can be used as a vital marker for noninvasive screening of cells and shoots for transformation, and that it has potential for the development of selectable marker-free transgenic sugar beet.
机译：改进甜菜的分子方法将得益于有效的转化程序，该程序不依赖选择标记基因的开发，例如那些赋予转基因植物抗生素或除草剂抗性的标记基因。绿色荧光蛋白（GFP）信号的表达已在一项旨在解决提高甜菜转化子选择速度和效率需求的研究计划中进行了研究。可以设想，GFP报道基因起初可被用作当前选择机制的补充，以帮助消除“逃逸”，并可能最终被用作替代标记，以避免与野外与抗生素/除草剂抗性基因有关的公众不安。释放的农作物。 sgfp-S65T基因经过修饰，具有植物相容的密码子用法，并在65位具有丝氨酸至苏氨酸突变，可增强蓝光下的荧光。该基因在CaMV 35S启动子的控制下，通过农杆菌介导的转化被引入甜菜。共培养数天后，绿色荧光表示共培养的甜菜培养物中的早期基因表达。将接种的培养物转移至选择培养基后，以3-11％的频率获得了稳定转化的愈伤组织，该密度在一定密度范围内显示绿色荧光。当将共培养的芽外植体或胚发生的愈伤组织转移至不含选择剂的培养基中时，在蓝光下定期对其进行监控。以2-5％的频率获得绿色荧光芽。结论是，sgfp-S65T基因可以用作非侵入性筛选转化细胞和芽的重要标记，并且它具有开发无选择标记的转基因甜菜的潜力。

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