Biolistic Bombardment for Co-expression of Proteins Fused to YFP and mRFP in Leaf Epidermal Cells of Phaseolus vulgaris ‘Red Mexican’
基因枪法在‘墨西哥红’菜豆叶表皮细胞中共表达YEP和mRFP融合蛋白
发布: 2014年01月05日第4卷第1期 DOI: 10.21769/BioProtoc.1019 浏览次数: 13978
评审: Anonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Apr 2013
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Abstract
Biolistic bombardment is based on coating of tungsten or gold particles with DNA and delivery of these “biobullets” into living plant cells under high pressure (Sudowe and Reske-Kunz, 2013). This method enables transient expression of a DNA construct encoding fusion of the protein of interest to a fluorescence protein e.g. GFP for microscopic approaches. Usually it is performed for plants for which infiltration with Agrobacterium tumefaciens does not work efficiently e.g. model plant Arabidopsis thaliana (Ueki et al., 2009). Although transfection rate is relatively low, it is still sufficient to analyze subcellular localization of the protein of interest under a fluorescence microscope. Here we present the protocol that was optimized for Nicotiana benthamiana and also successfully applied to Phaseolus vulgaris (Giska et al., 2013).
Materials and Reagents
- Upper leaves from 2 to 4 weeks old bean plants grown in a greenhouse
- Plasmid DNA 2-4 μg
- 2.5 M CaCl2 (Sigma-Aldrich, catalog number: C3306-500G ) (optional other chlorides e.g. ZnCl2, MgCl2 can be used if it is necessary to avoid calcium ions)
- 0.1 mM Spermidine (Sigma-Aldrich, catalog number: S0266-1G )
- 75% , 96% and 100% ethanol
- Sterile miliQ water
- 1 M spermidine stock solution (see Recipes)
- 0.1 mM spermidine working solution (see Recipes)
Equipment
- Microcentrifuge (Eppendorf MiniSpin)
- Rupture disks 1,100 psi (Bio-Rad Laboratories, catalog number: 165-2329 ) (available selection form 450 to 2,000 psi)
- Microcarriers: Tungsten M17 (Bio-Rad Laboratories, catalog number: 165-2267 )
- Macrocarriers (Bio-Rad Laboratories, catalog number: 165-2257 )
- Stopping Screens (Bio-Rad Laboratories, catalog number: 165-2336 )
- Vacuum pump (Bio-Rad Laboratories) (according to Bio-Rad technical recommendation)
- Vortex
- Forceps
- 1.5 ml microfuge tube (Eppendorf)
- Parafilm
- Petri dishes with wet filter paper at the bottom
- Biolistic PDS-1000/He Particle Delivery System (Bio-Rad Laboratories; www.bio-rad.com)
- Stereomicroscope (Nikon Corporation, model: SMZ 1500 ) with epi-fluorescence equipment (optional)
Procedure
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文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Lichocka, M. (2014). Biolistic Bombardment for Co-expression of Proteins Fused to YFP and mRFP in Leaf Epidermal Cells of Phaseolus vulgaris ‘Red Mexican’. Bio-protocol 4(1): e1019. DOI: 10.21769/BioProtoc.1019.
- Giska, F., Lichocka, M., Piechocki, M., Dadlez, M., Schmelzer, E., Hennig, J. and Krzymowska, M. (2013). Phosphorylation of HopQ1, a type III effector from Pseudomonas syringae, creates a binding site for host 14-3-3 proteins. Plant Physiol 161(4): 2049-2061.
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分类
植物科学 > 植物转化 > 基因枪法
植物科学 > 植物细胞生物学 > 细胞成像
分子生物学 > DNA > 转化
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