细胞的细胞大分子运输检测在植物利用基因枪法
Summary
高分子植物细胞之间的贩运，可以通过瞬时表达利益荧光标记的蛋白质，分析其内部和细胞间共聚焦显微镜的分布进行评估。
Abstract
在这里，我们提出一个简单而快速的的协议来检测和评估，在植物细胞与细胞大分子运输的程度。在这个协议中，一个荧光标记的利益蛋白在植物组织中瞬时表达其编码的DNA构建的基因枪交付。标签蛋白内和细胞间的分布是由共聚焦显微镜分析。我们详细描述了这一技术，提供一步一步的协议，以检测和评估symplastic蛋白运输在3个植物物种的程度，拟南芥，烟草benthamiana和N. tabacum（烟草）。
Protocol
背景 Symplastic运输大分子通过植物细胞间的连接，胞间连丝，是许多植物病理学家和生物学家的兴趣。例如，一些病毒蛋白是已知的，规范plasmodesmal大小排斥的限制，以使病毒运动1-3。此外，一些内源性蛋白，其中重要的发展监管，是假设从细胞到细胞的移动，大概是通过胞间连丝，功能非细胞自主4。因此，一个可靠的方法来识别和可视化植物细胞的大分子之?…
Discussion
symplastic运输检测成功的关键是获得高转化效率，这使得生产的统计学意义，并轻松地探测信号集群。这样就可以实现使用健康，植株健壮收获的叶子，并准备由一个纯粹和浓缩DNA的制备涂层的金粒子。
使用相同的成长阶段的叶子也是检测的可靠性至关重要。 Plasmodesmal光圈差异调节，取决于对组织7-11的发展阶段。许多植物的叶发展basipetally，从顶点到基地，超过其<su…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们的工作是由美国国立卫生研究院/ NIGMS，美国国家科学基金会，美国农业部/ NifA的，和Bard补助到VC支持。
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Gold microparticles, 1.0 μm in diameter | Bio-Rad | 165-2262 | ||
| Gold microparticles, 0.6 μm in diameter | Bio-Rad | 165-2263 | ||
| Spermidine | Sigma | S0266-1G | ||
| Tefzel tubing | Bio-Rad | 165-2441 | ||
| Helios cartridge preparatory station | Bio-Rad | 165-2420 | ||
| Tubing cutter | Bio-Rad | 165-2422 | ||
| Helios gene gun | Bio-Rad | 165-2432 | ||
| Helium gas regulator | Bio-Rad | 165-2413 |
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Citar este artigo
Ueki, S., Meyers, B. L., Yasmin, F., Citovsky, V. A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment. J. Vis. Exp. (42), e2208, doi:10.3791/2208 (2010).