Transient Gene Expression for the Characteristic Signal Sequences and the Estimation of the Localization of Target Protein in Plant Cell
植物细胞中特征信号序列的瞬时基因表达及靶蛋白定位的估判
发布: 2018年02月20日第8卷第4期 DOI: 10.21769/BioProtoc.2738 浏览次数: 8854
评审: Arsalan DaudiJian ChenAlexandros Alexandratos
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参见作者原研究论文
The authors used this protocol in:
Oct 2013
Abstract
We have proposed and tested a method for characterization of the signal sequences and determinations of target protein localization in a plant cell. This method, called the AgI-PrI, implies extraction of protoplasts from plant tissues after agroinfiltration. The suggested approach combines the advantages of two widely used methods for transient gene expression in plants–agroinfiltration and transfection of isolated protoplasts. The AgI-PrI technic can be applied to other plant species.
Keywords: Agroinfiltration (农杆菌渗透)
Protoplast isolation (原生质体分离)
Tobacco (烟草)
Transient expression (瞬态表达)
Signal sequences (信号序列)
Subcellular localization (亚细胞定位)
Background
To date, the following techniques for transient expression of genes in plants have been developed and widely used: agroinfiltration, biolistics of plant explants and transfection of protoplasts using polyethylene glycol or electroporation. The effectiveness of these approaches has been clearly demonstrated. Each strategy for transient expression of genes in plants, along with benefits, has its limitations and disadvantages, such as the difficulties in the fine imaging of recombinant reporter proteins in plant cell compartments owing to intricate shapes of plant epidermal cells (agroinfiltration), a low efficiency of transformation and the necessity of specialized equipment and auxiliary material (for biolistics), as well as complex preparatory procedures required for a high yield of viable protoplasts and their effective transfection. This is the reason for developing and testing new methods for transient expression of genes in a plant cell, preferably by improving the experimental protocols and preserving the physiological significance of the results of the studies. Since the cellular localization of proteins in living organisms, including plants, is closely interrelated with their functions, a fine visualization of proteins in living cells becomes an important tool for assessing the functions of the proteins.
Materials and Reagents
- Pipettes (Corning, Costar®, catalog number: 4101 )
- Inoculation loop
- Petri dishes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 101IRR )
- Syringe without a needle (B. Braun Melsungen, catalog number: 4645103C )
- Scalpel
- Nylon mesh, pore size, 40 µm (Sterile Cell Strainers, Corning, catalog number: 431750 )
- 10-ml tubes (Corning, Axygen®, catalog number: SCT-10ML )
- Agrobacterium tumefaciens strain GV3101 (Mohamed et al., 2004; strain is available in the collection of the Institute of Plant Physiology and can be provided to researchers for experiments)
- Nicotiana benthamiana (Sheludko et al., 2007; seeds are available in the collection of the Institute of Plant Physiology and can be provided to researchers for experiments)
- LB medium (MP Biomedicals, catalog number: 113002042 )
- Rifampicin (Fisher Scientific, catalog number: BP26791 )
- Gentamicin (Thermo Fisher Scientific, GibcoTM, catalog number: 15750060 )
- Kanamycin (Thermo Fisher Scientific, GibcoTM, catalog number: 11815024 )
- Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888 )
- Tris-HCl, pH 7.0 (Roche Diagnostics, catalog number: 10812846001 )
- Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C1016 )
- Acetosyringone (Abcam, catalog number: ab146533 )
- MES (Sigma-Aldrich, catalog number: 69892 )
- Magnesium sulfate heptahydrate (MgSO4·7H2O) (Merck, catalog number: 1058860500 )
- Calcium chloride dihydrate (CaCl2·2H2O) (AMRESCO, catalog number: 0556-500G )
- Ammonium phosphate monobasic (NH4H2PO4) (Sigma-Aldrich, catalog number: A3048 )
- Sorbitol (Sigma-Aldrich, catalog number: S1876 )
- Potassium hydroxide (KOH) (AppliChem, catalog number: 211514 )
- Cellulase Onozuka R10 (Kinki Yakult)
- Pectinase Macerozyme R10 (Kinki Yakult)
- Driselase (Sigma-Aldrich, catalog number: D9515 )
- Calcium nitrate tetrahydrate (Са(NО3)2·4H2O) (Sigma-Aldrich, catalog number: C2786 )
- Potassium phosphate monobasic (KH2PO4) (Fisher Scientific, catalog number: P285 )
- Magnesium sulfate (MgSO4) (Acros Organics, catalog number: AC413485000 )
- Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
- Ferric chloride (FеСl3) (Sigma-Aldrich, catalog number: 157740 )
- MS medium (Sigma-Aldrich, catalog number: M5519 )
- Sucrose (MP Biomedicals, catalog number: 04802536 )
- Solution 1 (see Recipes)
- Solution 2 (see Recipes)
- Solution 3 (see Recipes)
- Solution 4 (see Recipes)
- Solution 5 (see Recipes)
- Knop’s solution (see Recipes)
- Agroinfiltration buffer (see Recipes)
Equipment
- Incubator Shaker (Biosan, model: ES-20 , catalog number: BS-010111-AAA)
- Centrifuge (Eppendorf)
- Transilluminator (Vilber, model: ETX-F26.M , catalog number: Vilber Lourmat 2131 2600 1)
- Microscope Axio Imager Z2 (ZEISS, model: Axio Imager Z2 ) equipped with digital camera (ZEISS, model: AxioCam MRc5 ), filter set No. 38 (38 Endow GFP shift free (EX BP 470 nm/40 nm, BS FT 495 nm, EM BP 525 nm/50 nm), ZEISS, catalog number: 000000-1031-346 ) and module ApoTome (ZEISS, model: ApoTome )
Software
- ZEN, AxioVision 4.8 (ZEISS)
Procedure
文章信息
版权信息
© 2018 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Berestovoy, M. A., Tyurin, A. A., Kabardaeva, K. V., Sidorchuk, Y. V., Fomenkov, A. A., Nosov, A. V. and Goldenkova-Pavlova, I. V. (2018). Transient Gene Expression for the Characteristic Signal Sequences and the Estimation of the Localization of Target Protein in Plant Cell. Bio-protocol 8(4): e2738. DOI: 10.21769/BioProtoc.2738.
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分类
植物科学 > 植物生理学 > 植物生长
分子生物学 > DNA > 转化
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