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[Bio101] Infiltration of Nicotiana benthamiana Protocol for Transient Expression via Agrobacterium
[Bio101] 农杆菌注射烟草叶片进行体内瞬时表达的方法   

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Abstract

Transient expression in tobacco plant (Nicotiana benthamiana) is used to determine the subcellular location of a protein of interest when tagged with a reporter such as green fluorescent protein (GFP), or to mass produce proteins without making transgenic plants. The root tumor bacteria, Agrobacteria, are used as medium to introduce the target gene expression cassette into benthamiana mesophyll cells.

Keywords: Transient expression(瞬时表达), Tobacco(烟草), Agrobacterium(根癌农杆菌), Fluorescence(荧光), Leaves(叶)

Materials and Reagents

  1. Agrobacterium strain hosting a plant expression construct (usually driven by Cauliflower mosaic virus 35S promoter)
  2. Healthy Nicotiana benthamiana (N. benthamiana) plants 2-4 weeks
  3. MES
  4. MgCl2 stock
  5. Antibiotics
  6. Acetosyringone
  7. LB media with appropriate antibiotics (see Recipes)
  8. Acetosyringone stock (see Recipes)
  9. MES-K (see Recipes)
  10. Resuspension solution (see Recipes)
  11. Acetosyringone datasheet (Sigma-Aldrich) (see Recipes)

Equipment

  1. Centrifuge for 50 ml tubes
  2. Spectrometer
  3. Syringe
  4. UV lamp (optional)
  5. Fluorescence microscope (optional)
  6. Confocal laser scanning microscope (optional)

Procedure

  1. Inoculate one single colony of Agrobacterium in 5 ml LB with appropriate antibiotics. Grow overnight at 28-30 °C.
    Note: I usually use 100 μg/ml gentamicin (maintain the virulence of Agrobacterium strain GV3101) and 50 μg/ml spectinomycin (selective marker for shuttle vector) for most of the shuttle vectors.
  2. Use 1 ml of the overnight culture to inoculate 25 ml LB (with same antibiotics, plus 20 μM acetosyringone added after autoclaving and immediately before use) and grow overnight.
  3. Measure the A600 of overnight culture. 
  4. Precipitate the bacteria (5,000 x g, 15 min), resuspend the pellet in Resuspension Solution. The final A600 should be adjusted to 0.4. 
  5. Leave on the bench (room temperature) for 2-3 h (or overnight) before infiltration.
  6. Perform the infiltration with 5 ml syringe. Simple press the syringe (no needle) on the underside of the leaf (Note: Avoid cotyledons), and exert a counter-pressure with finger on the other side. Successful infiltration is often observed as a spreading “wetting” area in the leaf.
  7. (Optional) Check the GFP fluorescence by a portable long-wavelength UV lamp 2-5 days after infiltration. This only applies to strong expression of GFP signal (as green from red background).
  8. Observe the fluorescence labeled protein under a fluorescent microscope or confocal laser scanning microscope. Or harvest leaves for protein purification.

Recipes

  1. LB media with appropriate antibiotics
    Usually two antibiotics used: one to maintain Agrobacteria virulence, one for the shuttle vector
  2. Acetosyringone stock
    100 mM in ethanol, stored at -20 °C
  3. MES-K (0.5 M) (pH 5.6)
    First make 0.5 M MES, adjust pH with KOH to 5.6
  4. Resuspension solution
    10 mM MgCl2
    10 mM MES-K (pH 5.6)
    Autocloave 15 min
    100 μM acetosyringone (note: Added after autoclaving and immediately before using)
  5. Acetosyringone datasheet
    Synonyms     3’, 5’-Dimethoxy-4’-hydroxyacetophenone
    Synonyms     Acetosyringone
    4’-Hydroxy-3’, 5’-dimethoxyacetophenone
    Molecular Formula              C10H12O4
    Molecular Weight                196.20
    CAS Number                       2478-38-8
    Beilstein Registry Number   1966119
    EG/EC Number                   2196105
    MDL number                      MFCD00008748

References

  1. Li, X., Chanroj, S., Wu, Z., Romanowsky, S. M., Harper, J. F. and Sze, H. (2008). A distinct endosomal Ca2+/Mn2+ pump affects root growth through the secretory process. Plant Physiol 147(4): 1675-1689.

简介

当用例如绿色荧光蛋白(GFP)的报告物标记时,烟草植物(本塞姆氏烟草)中的瞬时表达用于确定目标蛋白质的亚细胞定位,或大规模生产蛋白质而不进行转基因 植物。 根瘤细菌,土壤杆菌用作培养基以将靶基因表达盒导入本氏体叶肉细胞中。

关键字:瞬时表达, 烟草, 根癌农杆菌, 荧光, 叶

材料和试剂

  1. 承载植物表达构建体(通常由花椰菜花叶病毒35S启动子驱动)的土壤杆菌
  2. 健康本塞姆氏烟草(<本生烟草)植物2-4周
  3. MES
  4. MgCl 2原料
  5. 抗生素
  6. 乙酰丁香酮
  7. 含适当抗生素的LB培养基(见配方)
  8. Acetosyringone股票(见食谱)
  9. MES-K(见配方)
  10. 重悬溶液(见配方)
  11. 乙酰丁香酮数据表(Sigma-Aldrich)(参见Recipes)

设备

  1. 离心50毫升管
  2. 光谱仪
  3. 注射器
  4. UV灯(可选)
  5. 荧光显微镜(可选)
  6. 共聚焦激光扫描显微镜(可选)

程序

  1. 用合适的抗生素接种5ml LB中的单个菌落的土壤杆菌。 在28-30℃生长过夜。
    注意:对于大多数穿梭载体,我通常使用100μg/ml庆大霉素(保持土壤杆菌菌株GV3101的毒力)和50μg/ml壮观霉素(穿梭载体的选择标记)。
  2. 使用1毫升的过夜培养物接种25毫升LB(与相同的抗生素,加20微升乙酰丁香酮在高压灭菌后和即将使用前加入),并生长过夜。
  3. 测量过夜培养物的A <600>。
  4. 沉淀细菌(5,000×g/g,15分钟),将沉淀重悬于重悬溶液中。最后的A 600 应调整为0.4。
  5. 在浸润前放置在台上(室温)2-3小时(或过夜)。
  6. 用5 ml注射器进行浸润。简单地按下叶片下面的注射器(无针)(注意:避免子叶),并用手指在另一侧施加反压力。成功的渗透通常被观察为叶中的扩散"润湿"区域。
  7. (可选)在浸润后2-5天,通过便携式长波紫外灯检查GFP荧光。这仅适用于GFP信号的强表达(如来自红色背景的绿色)。
  8. 在荧光显微镜或共聚焦激光扫描显微镜下观察荧光标记的蛋白质。或收获叶子用于蛋白质纯化

食谱

  1. 含适当抗生素的LB培养基 通常使用两种抗生素:一种用于维持农杆菌毒力,一种用于穿梭载体
  2. Acetosyringone股票
    100mM于乙醇中,贮存于-20℃
  3. MES-K(0.5M)(pH 5.6) 首先制备0.5M MES,用KOH调节pH至5.6
  4. 重悬溶液
    10mM MgCl 2/
    10mM MES-K(pH5.6) 自动抓取15分钟
    100μM乙酰丁香酮(注意:高压灭菌后添加,并在使用前立即使用)
  5. Acetosyringone数据表
    同义词   3',5'-二甲氧基-4'-羟基苯乙酮
    同义词     Acetosyringone
    4'-羟基-3',5'-二甲氧基苯乙酮
    分子式               C 10 H 12 4
    分子量                  196.20
    CAS编号                         2478-38-8
    Beilstein注册表号   1966119
    EG/EC编号                     2196105
    MDL编号                        MFCD00008748

参考文献

  1. Li,X.,Chanroj,S.,Wu,Z.,Romanowsky,S.M.,Harper,J.F。和Sze,H。(2008)。 不同的内体Ca 2 + /Mn 2+ 泵通过分泌过程影响根生长。 植物生理学 147(4):1675-1689。
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
引用:Li, X. (2011). Infiltration of Nicotiana benthamiana Protocol for Transient Expression via Agrobacterium. Bio-protocol Bio101: e95. DOI: 10.21769/BioProtoc.95;
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Sarah Fernandes
University of Cape Town
Hello

Please could you advise me as to how to prepare slides for N benthamiana for fluorescent microscopy. Is it necessary to use a microtome or can one peel the epidermis away? My sample preparation will be made a bit more challenging as I will be dehydrating the plants for about a week.

Many thanks
8/15/2017 12:12:31 PM Reply
Marek Szecowka
University of Cambridge

It depends on what you are trying to see. For observation of fluorescent proteins usually you can use small piece of a leaf and suspend it in watter. You don't have to remove an epidermis.

9/14/2017 6:20:35 AM