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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.

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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

植物科学 > 植物转化 > 农杆菌介导的转化方法
作者: Xiyan Li
Xiyan LiAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: lixiyan@stanford.edu
Bio-protocol author page: a13
7/20/2011, 29599 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.95

[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)

[Abstract] 通常,我们可以利用烟草的瞬时表达系统来观察感兴趣蛋白的亚细胞定位(通过与绿色荧光蛋白构成融合蛋白)。同时,还可以检测蛋白的表达量。这一个过程是通过农杆菌作为一个工具将目的基因整合到到烟草的细胞内的。

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.

材料与试剂

 

1.       携带表达载体的农杆菌菌株(通常表达载体由35S启动子驱动)

2.       2-4周的烟草植株

3.       LB培养基

4.       乙酰丁香

5.       2-(N-) 乙磺酸

6.       抗生素

7.       注射器

 

工具

 

1.       50ml离心管

2.       光谱仪

3.       紫外灯

4.       荧光显微镜

 

步骤:

 

1.       挑取单克隆于5mlLB液体培养中,28-30°C震荡培养。通常,LB中加入100ug/ml庆大霉素(农杆菌株GV3101携带抗性),50ug/ml大观霉素(载体携带)。

2.       1ml过夜培养的农杆菌转接到25mlLB液体培养基中(加有与1相同的抗生素,另外加入高压灭菌的乙酰丁香酮)。

3.       检测过夜培养的菌液OD600的值。

4.       5000g15分钟集菌,用重悬液重悬菌体,最终OD6000.4

5.       室温放置2-3h后注射烟草。

6.       侵染液装入5ml注射器内,用拇指按压注射器反板将液体从叶片下表皮注射到烟草叶片内(勿使用子叶)。注射后,烟草叶片会出现湿润的现象。

7.       注射后2-5天,在便携式长波长紫外灯下检测GFP荧光信号(只适用于荧光很强的叶片)。

8.       通过荧光显微镜或者激光共聚交荧光显微镜检测GFP信号。同时,可以提取蛋白,检测蛋白的含量。

 

配方

 

1.       加有相应抗生素的LB液体培养基(一种抗生素是菌株携带,一种为载体携带)。

2.       乙酰丁香酮(100mM 溶于乙醇,-20°C储存)。

3.       1M MgCl2

4.       重悬液(10mM MgCl2,10mM   2-(N-) 乙磺酸(pH5.6)高温高压灭菌15分钟,100uM 乙酰丁香酮,高温高压灭菌)

5.       乙酰丁香酮(来自Aldrich):

又名3,5-二甲基-4-羟基苯乙酮,或4-羟基-3,5-二甲基苯乙酮

分子式 C10H12O4

相对分子量 196.20

证书号2478-38-8

德国注册号1966119

欧盟号 2196105

MDL MFCD00008748

 

参考文献

 

1.        Li X., Chanroj S., Wu Z., Romanowsky S.M., Harper J.F., Sze H. (2008). A distinct endosomal Ca2+/Mn2+ pump affects root growth through the secretory process. Plant Physiology 147(4): 1675-89. 

 

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How to cite this protocol: Li, X. (2011). Infiltration of Nicotiana benthamiana Protocol for Transient Expression via Agrobacterium. Bio-protocol Bio101: e95. DOI: 10.21769/BioProtoc.95; Full Text



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