A Simple and Rapid Method for Optimizing Particle Bombardment Parameters Through Transient Expression of Gus Reporter Gene in Onion Epidermal Layer Cells
A Simple and Rapid Method for Optimizing Particle Bombardment Parameters Through Transient Expression of Gus Reporter Gene in Onion Epidermal Layer Cells
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发表时间：
2009
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通讯作者：
J. Sandhu;S. Gosal
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作者：
J. Sandhu;S. Gosal
通过分析编码$-葡萄糖醛酸酶（GUS）的可视报告基因在洋葱表皮细胞中的瞬时表达，建立了在转基因植株发育前测定转化效率和基因转移参数的方法。用含有花椰菜花叶病毒（CaMV 35 S）启动子控制的GUS基因的质粒pWRG 2426，通过包被钨粒子，单独轰击洋葱表皮层。在粒子轰击后24小时内，通过计数显示靛蓝色的细胞岛的数量来评估GUS基因表达，并且强度的测量基于轰击细胞中的相对靛蓝色发展，其代表转基因表达的效率和转化参数。虽然最佳的稳定转化条件可能不同于瞬时表达，因为在轰击瞬时表达中得分为阳性的细胞可能不分化产生稳定的转化子，但是转基因表达研究可以用作在实验的初步阶段测量转化参数的效率的工具。
Protocol to gauge the efficiency of transformation and gene transfer parameters prior to development of transgenic plants, has been developed by analyzing the transient expression of visual reporter gene encoding $-glucuronidase (GUS) in onion epidermal cells. The onion epidermal layerfs) were independently bombarded by coating tungsten particles with plasmid pWRG2426 containing GUS gene under the control of Cauliflower Mosaic Virus (CaMV 35S) promoter. The GUS gene expression was assessed within 24 h following particle bombardment by counting the number of islands of cells showing indigo coloration and the measure of intensity was based on relative indigo color development among the bombarded cells, which is representation of efficiency of transgene expression and transformation parameters. Although the optimum stable transformation conditions may be different from transient expression, because cells that score positive in transient expression upon bombardment may not devide to produce stable transformant, however transgene expression studies can be used as a tool to gauge efficiency of transformation parameters at preliminary stage of the experiment.