An Efficient Procedure for Protoplast Isolation from Mesophyll Cells and Nuclear Fractionation in Rice
水稻叶肉细胞原生质体分离和细胞核分级分离的有效方法
发布: 2015年03月05日第5卷第5期 DOI: 10.21769/BioProtoc.1412 浏览次数: 25744
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参见作者原研究论文
The authors used this protocol in:
Aug 1993
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Abstract
Plant protoplasts, a proven physiological and versatile cell system, are widely used in high-throughput analysis and functional characterization of genes. Green protoplasts have been successfully used in investigations of plant signal transduction pathways related to hormones, metabolites and environmental challenges. This protocol, adapted from Zhang et al. (2011), describes a procedure for the isolation of rice protoplasts from green tissue and shows an efficient and rapid method for isolation of nuclei form these protoplasts which are commonly used in a variety of experimental procedures including the isolation of high-molecular-weight DNA (Watson and Thompson, 1986), in vitro DNA synthesis (Roman, 1980), isolation of labeled transcripts for differential screening of cDNA libraries (Somssich et al., 1989), preparation of nuclear extracts for in vitro transcription systems (Roberts and Okita, 1991), isolation of nuclear proteins (Harrison et al., 1992) and studies of protein targeting to the nucleus (Hicks and Raikhel, 1993).
Materials and Reagents
- 12-day-old plants with 1-2 cm leaves before flowering
- Cellulase RS (Yakult Pharmaceutical)
- Macerozyme R-10 (Yakult Pharmaceutical)
- D-Mannitol (Sigma-aldrich, catalog number: M4125 )
- KCl (Sigma-aldrich, catalog number: P9333 )
- MES hydrate (Sigma-aldrich, catalog number: M8250 )
- CaCl2 (Sigma-aldrich, catalog number: C1016 )
- BSA Bovine serum albumin (Sigma-aldrich, catalog number: A2153 )
- Spermidine (Sigma-aldrich, catalog number: S2626 )
- EDTA (Sigma-aldrich, catalog number: EDS )
- Sucrose (Sigma-aldrich, catalog number: 84097 )
- Triton X-100 (Sigma-aldrich, catalog number: X100 )
- DTT (Sigma-aldrich, catalog number: 43815 )
- Enzyme solution (ES) (see Recipes)
- Washing and incubation solution (WS1) (see Recipes)
- Washing solution (WS2) (see Recipes)
- MMG solution (see Recipes)
- Nuclei isolation solution (NIB) (see Recipes)
Equipment
- Razor blades (No18 sterile stainless steel scalpel balde) (Swann Morton, catalog number: 0323 )
- Micro-dissecting forceps (Sigma-aldrich, catalog number: F3767 )
- Nunc petri dishes (diam 90 mm × H 15 mm, surface area size 58 cm2, vented) (Sigma-aldrich, catalog number: Z717223 )
- 50 ml conical tubes (Bioland scientific, catalog number: A02-04 )
- Orbitron Rotator I (115 V) (Boekel Scientific)
- Nylon mesh CX-60 (120 µm) and CX-400 (38 µm) (Carolina Biological Supplies, catalog number: 65-2222N )
- Glass funnel
- Hemocytometer [Mfr Part Number: 3100; Cell depth: 0.100mm +/- 2% (1/10mm); Volume: 0.1 Microliter] (PGC Scientific, catalog number: 1907353 ).
- Stereo Microscope (Olympus, catalog number: SZ51 )
- 25G5/8 gauge needle (Kendall, catalog number: KND8881511235 )
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Jabnoune, M., Secco, D., Lecampion, C., Robaglia, C., Shu, Q. and Poirier, Y. (2015). An Efficient Procedure for Protoplast Isolation from Mesophyll Cells and Nuclear Fractionation in Rice . Bio-protocol 5(5): e1412. DOI: 10.21769/BioProtoc.1412.
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分类
植物科学 > 植物细胞生物学 > 细胞分离
植物科学 > 植物细胞生物学 > 细胞器分离
细胞生物学 > 细胞器分离 > 细胞核
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