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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   

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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

  1. Upper leaves from 2 to 4 weeks old bean plants grown in a greenhouse
  2. Plasmid DNA 2-4 μg
  3. 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)
  4. 0.1 mM Spermidine (Sigma-Aldrich, catalog number: S0266-1G )
  5. 75% , 96% and 100% ethanol
  6. Sterile miliQ water
  7. 1 M spermidine stock solution (see Recipes)
  8. 0.1 mM spermidine working solution (see Recipes)

Equipment

  1. Microcentrifuge (Eppendorf MiniSpin)
  2. Rupture disks 1,100 psi (Bio-Rad Laboratories, catalog number: 165-2329 ) (available selection form 450 to 2,000 psi)
  3. Microcarriers: Tungsten M17 (Bio-Rad Laboratories, catalog number: 165-2267 )
  4. Macrocarriers (Bio-Rad Laboratories, catalog number: 165-2257 )
  5. Stopping Screens (Bio-Rad Laboratories, catalog number: 165-2336 )
  6. Vacuum pump (Bio-Rad Laboratories) (according to Bio-Rad technical recommendation)
  7. Vortex
  8. Forceps
  9. 1.5 ml microfuge tube (Eppendorf)
  10. Parafilm
  11. Petri dishes with wet filter paper at the bottom
  12. Biolistic PDS-1000/He Particle Delivery System (Bio-Rad Laboratories; www.bio-rad.com)
  13. Stereomicroscope (Nikon Corporation, model: SMZ 1500 ) with epi-fluorescence equipment (optional)

Procedure

  1. Microcarrier aliquots preparation
    1. Weigh out 50 mg tungsten into 1.5 ml microfuge tube.
      Note: Tungsten particles, 1.1 μm diameter, easily stick to the plastic. We recommend weighing out of the tungsten directly into microfuge cap, formerly cut out from the tube, to avoid wasting of metal powder.
    2. For surface sterilization of tungsten add 1 ml 96% ethanol and vortex vigorously for 2 min and spin for 5 sec in microcentrifuge.
    3. Discard ethanol and resuspend tungsten particles in 1 ml miliQ water.
    4. Vortex vigorously and spin for 5 sec in microcentrifuge.
    5. Discard water and repeat washing with water three times.
    6. Add 1 ml water and prepare 50 μl aliquots; continuous vortexing during aliquoting is necessary to sustain uniform sampling. Aliquots can be stored at -20 °C.

  2. Coating of tungsten particles with plasmid DNA
    1. Microcarriers should be coated with DNA at the day of scheduled bombardment. For two shots (10 μl each) prepare the following mixture in a 1.5 ml microfuge tube:
      Note: It is recommended to vortex the mixture continuously during whole procedure for uniform precipitation of plasmid DNA onto tungsten particles.
      1. 12.5 μl tungsten suspension.
      2. Add 5 μl 0.1 mM spermidine.
      3. Plasmid DNA at final concentration ca. 2-4 μg (the amount of plasmid DNA can be increased up to 5-10 μg when working with large plasmids e.g. binary vectors).
      4. For co-expression of two constructs mix DNA in ratio 1:1 to the total 2-4 μg.
      5. Add 12 μl 2.5 M CaCl2 and allow the mixture to precipitate for 1 min at room temperature.
    2. Pellet microcarriers by spinning for 2 sec in microcentrifuge.
    3. Discard the supernatant and wash the tungsten with fifty μl 75% ethanol, after short spin remove supernatant and repeat washing with 100% ethanol two times. Spin for 5 sec in microcentrifuge after each washing step.
    4. Add twenty μl 100% ethanol and disperse the aggregates by vortexing and pipetting. If large aggregates are still visible, sonicate the samples.
      Note: Large aggregates may cause extensive damage of plant tissue during bombardment which significantly reduces transfection rate.

  3. Macrocarriers preparation according to manufacturer’s protocol, useful link:
    http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_9075.pdf
    1. Place macrocarrier inside macrocarrier holder using forceps. The edge of the macrocarrier should be securely placed at the bottom of the macrocarrier holder. When correctly inserted, it does not stick out and creates a flat surface at the bottom of the holder.
    2. Load 10 μl microcarriers coated with DNA on macrocarrier (Figure 1A). Try to spread them out through the center of macrocarrier surface with the use of a pipette tip. Avoid aggregation of tungsten particles in one place.
    3. Allow the ethanol to evaporate, leave the macrocarries for 10 min in a dry place.


      Figure 1. A. Macrocarrier coated with microcarriers (arrow), inserted into macrocarrier holder. B. Stopping screen (arrow) placed inside fixed next of macrocarrier launch assembly. C. Placement of inverted macrocarrier holder (arrow), with dried microcarriers facing down, inside fixed next of macrocarrier launch assembly. D. Placement of macrocarrier cover lid (arrow) onto assembled fixed nest.

  4. Bombardment
    1. Cut leaves just before microbombardment and place them in a Petri dish with the abaxial side exposed for bombardment.
      Perform bombardment according to the manufacturer’s protocol, useful link:
      http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_9075.pdf
    2. Turn on the biolistic device, vacuum pump, open helium tank with the main valve, set the pressure with regulator to 200 dpi over the selected rupture disk burst pressure (using 1,100 psi rupture disk set it at 1,300-1,400 psi).
    3. Load the rupture disk into the retaining cup using forceps; screw retaining cup with the rupture disk onto the gas acceleration tube using left-to-right motion. It must be tightened sufficiently; otherwise the rupture disk may slip out as the gas acceleration tube fills with helium.
    4. Load the macrocarrier holder and stopping screen into the macrocarrier launch assembly (Figure 1B, 1C, 1D).
    5. Place the macrocarrier launch assembly into the bombardment chamber, set the distance gap between rupture retaining cup (Figure 2, we usually placed it at second position from the top) and close the door.
    6. Place the opened Petri dish with plant sample on the target shelf inserted into second position from the bottom of the sample chamber (Figure 2).


      Figure 2. Front view of the PDS/He unit loaded with plant sample

    7. Hold vacuum at desired level (minimum 5 inches of mercury, we set it at 27.5).
    8. Bombard sample: keep the fire button pressed until rupture disk bursts and then release the fire button.
    9. Release the vacuum for the sample chamber.
    10. Remove the plant sample from the chamber.
    11. Unload macrocarrier launch assembly and rupture disk retaining cup.
    12. Close the main valve on helium tank and remove the helium form the system: set vacuum at 5 inches of Hg, keep the fire button pressed until the helium pressure in helium regulator reaches 0 level.
    13. Turn off biolistic device and vacuum pump.
    14. After bombardment place the leaves onto wet filter paper in Petri dishes and seal them with Parafilm. Keep in a dark place at room temperature for ca. 18 h. Usually 90-100% of transfected cells exhibit fluorescence of both fluorophores. It is recommended to examine first the tissue under fluorescence stereomicroscope to cut out leaf fragments with the highest density of transfected cells.

Recipes

  1. Prepare 1 M spermidine stock solution in miliQ water. Sterilize the solution by filtration trough 0.22 μm syringe filter and store at  -80 °C.
  2. Prepare 0.1 mM spermidine working solution by dilution of 1 M stock with sterile miliQ water. Prepare aliquots of 100 μl each and store them at -20 °C for up to 1 year.

Acknowledgments

This protocol is based on the procedure described by Giska et al. (2013).

References

  1. 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.
  2. Sudowe, S. and Reske-Kunz, A. B. eds. (2013). Biolistic DNA delivery methods and protocols. Methods Mol Biol Vol 940.
  3. Ueki, S., Lacroix, B., Krichevsky, A., Lazarowitz, S. G. and Citovsky, V. (2009). Functional transient genetic transformation of Arabidopsis leaves by biolistic bombardment. Nat Protoc 4(1): 71-77.

简介

生物轰击基于用DNA涂覆钨或金颗粒,并在高压下将这些"生物小球"递送到活的植物细胞中(Sudowe和Reske-Kunz,2013)。 该方法使得能够瞬时表达编码感兴趣的蛋白质与荧光蛋白例如GFP的融合物的DNA构建体用于显微方法。 通常,其对于用土壤杆菌根瘤土壤渗入不能有效工作的植物进行,例如模拟植物拟南芥(Ueki et al。 ,2009)。 虽然转染率相对较低,但仍然足以在荧光显微镜下分析目标蛋白的亚细胞定位。 在这里,我们提出针对 Nicotiana benthamiana 优化的方案,并成功地应用于 Phaseolus vulgaris (Giska ,2013)。

材料和试剂

  1. 在温室中生长的2至4周龄豆类植物的上叶
  2. 质粒DNA 2-4微克
  3. 将2.5M CaCl 2(Sigma-Aldrich,目录号:C3306-500G)(任选的其它氯化物,例如ZnCl 2,MgCl 2 可用于需要避免钙离子的情况)
  4. 0.1mM亚精胺(Sigma-Aldrich,目录号:S0266-1G)
  5. 75%,96%和100%乙醇
  6. 无菌miliQ水
  7. 1 M亚精胺储备溶液(见配方)
  8. 0.1 mM亚精胺工作溶液(见配方)

设备

  1. 微量离心机(Eppendorf MiniSpin)
  2. 破裂盘1,100psi(Bio-Rad Laboratories,目录号:165-2329)(可选择形式为450至2,000psi)
  3. 微载体:Tungsten M17(Bio-Rad Laboratories,目录号:165-2267)
  4. 大载体(Bio-Rad Laboratories,目录号:165-2257)
  5. 停止屏幕(Bio-Rad Laboratories,目录号:165-2336)
  6. 真空泵(Bio-Rad Laboratories)(根据Bio-Rad技术推荐)
  7. 涡流
  8. 镊子
  9. 1.5ml微量离心管(Eppendorf)
  10. parafilm
  11. 培养皿底部有湿滤纸
  12. Biolistic PDS-1000/He颗粒递送系统(Bio-Rad Laboratories; www.bio-rad.com
  13. 立体显微镜(尼康公司,型号:SMZ 1500)与外荧光设备(可选)

程序

  1. 微载体等分试样制备
    1. 称量50mg钨到1.5ml微量离心管中 注意:直径1.1μm的钨颗粒容易粘到塑料上。 我们建议将钨直接放入微量盖,先前从管中切出,以避免浪费金属粉末。
    2. 对于钨的表面灭菌,加入1ml 96%乙醇并剧烈涡旋2分钟,并在微量离心机中旋转5秒。
    3. 弃去乙醇,并在1ml miliQ水中重悬钨颗粒
    4. 大力涡旋并在微量离心机中旋转5秒
    5. 弃去水,并重复用水洗涤三次。
    6. 加入1毫升水,准备50微升等分试样; 在等分期间连续涡旋对于维持均匀取样是必要的。 等分试样可储存于-20°C
  2. 用质粒DNA包被钨颗粒
    1. 微载体应在计划轰击当天用DNA包被。 对于两次注射(每次10μl),在1.5ml微量离心管中制备以下混合物:
      注意:建议在整个过程中连续旋转混合物以使质粒DNA均匀沉淀到钨颗粒上。
      1. 12.5μl钨悬浮液
      2. 加入5μl0.1mM亚精胺
      3. 最终浓度约1μg的质粒DNA。 2-4μg(当使用大质粒(例如二进制载体)时,质粒DNA的量可增加至5-10μg)。
      4. 为了共表达两种构建体,以1:1的比例混合DNA至总共2-4μg
      5. 加入12μl2.5M CaCl 2,并使混合物在室温下沉淀1分钟。
    2. 通过在微量离心机中旋转2秒来沉淀微载体
    3. 弃去上清液,用50μl75%乙醇洗涤钨,短暂旋转后,除去上清液,并用100%乙醇重复洗涤两次。每次洗涤步骤后在微量离心机中旋转5秒
    4. 加入二十微升100%乙醇,并通过涡旋和移液分散聚集体。如果仍然可以看见大的聚集体,则对样品进行超声处理 注意:大的聚集体在轰击期间可能会对植物组织造成严重损害,从而显着降低转染率。

  3. 大载体根据制造商的方案制备,有用的链接:
    http://www.bio-rad.com/webroot /web/pdf/lsr/literature/Bulletin_9075.pdf
    1. 使用镊子将macrocarrier放在macrocarrier持有人内。大载波的边缘应该被牢固地放置在大载波载体的底部。正确插入时,它不会伸出,并在支架底部形成平坦表面。
    2. 加载10微升载体上载有微载体的微载体(图1A)。尝试通过使用移液管尖端通过宏载体表面的中心扩散它们。避免钨颗粒聚集在一个地方。
    3. 让乙醇蒸发,在干燥的地方留下10分钟的macrocarries

      图1。 A.涂有微载体(箭头)的大载体,插入大载体支架中。 B.停止屏幕(箭头)放置在固定的宏载体发射组件的下一个。 C.倒置的巨载体支架(箭头),干燥的微载体面朝下,在宏载体发射组件的固定下面。 D.将大载体盖子盖(箭头)放置到组装的固定座上。

  4. 轰击
    1. 在微轰击之前切叶,并将它们放置在培养皿中,其中轴侧暴露用于轰击。
      根据制造商的协议进行轰击,有用的链接:
      http://www.bio-rad.com/webroot /web/pdf/lsr/literature/Bulletin_9075.pdf
    2. 打开生物弹道装置,真空泵,用主阀打开氦气箱,用调节器将压力设置为在所选破裂盘爆破压力(使用1,100psi破裂盘设置为1,300-1,400psi)下为200dpi。
    3. 使用镊子将破裂盘装入保持杯;带有破裂盘的螺旋保持杯安装到气体加速管上,使用从左到右的运动。必须充分拧紧;否则当气体加速管充满氦气时,破裂盘可能会滑出。
    4. 将大载体支架和停止屏幕装载到宏载体发射组件(图1B,1C,1D)
    5. 将宏载体发射组件放入轰击室,设置破裂保持杯之间的距离间隙(图2,我们通常将其放置在距离顶部第二个位置)并关闭门。
    6. 将打开的带植物样品的培养皿放在从样品室底部插入第二位置的目标架上(图2)。


      图2.装有植物样品的PDS/He单元的前视图

    7. 保持真空在所需的水平(最低5英寸汞柱,我们设置在27.5)
    8. Bombard样品:按下火按钮,直到爆破片爆裂,然后释放火按钮
    9. 释放样品室的真空。
    10. 从室中取出植物样品。
    11. 卸载宏载体启动组件和破裂盘保持杯。
    12. 关闭氦气箱上的主阀,从系统中取出氦气:将真空度设定在5英寸汞柱,按住灭火按钮,直到氦气调节器中的氦气压力达到0级。
    13. 关闭生物弹道装置和真空泵
    14. 轰击后将叶子放在培养皿中的湿滤纸上并用石蜡膜密封。 保持在黑暗的地方在室温下约ca. 18小时。 通常90-100%的转染细胞显示两种荧光团的荧光。 建议首先在荧光立体显微镜下检查组织,以切出具有最高密度的转染细胞的叶片段。

食谱

  1. 在miliQ水中制备1M亚精胺储备溶液。 通过0.22μm注射器式过滤器过滤灭菌溶液, -80℃。
  2. 通过用无菌miliQ水稀释1M储备液制备0.1mM亚精胺工作溶液。 准备100微升的等分试样,并将其存储在-20°C长达1年

致谢

该协议基于Giska等人(2013)描述的程序。

参考文献

  1. Giska,F.,Lichocka,M.,Piechocki,M.,Dadlez,M.,Schmelzer,E.,Hennig,J.and Krzymowska,M。(2013)。 HopQ1的磷酸化,一种来自丁香假单胞菌的III型效应物,创造了一种 主机14-3-3蛋白的结合位点。 植物生理学 161(4):2049-2061。
  2. Sudowe,S。和Reske-Kunz,A.B。 (2013年)。 生物体DNA递送方法和方案方法 Mol Biol Vol 940.
  3. Ueki,S.,Lacroix,B.,Krichevsky,A.,Lazarowitz,S.G.and Citovsky,V。(2009)。 通过生物射弹轰击拟南芥叶片的功能性瞬时遗传转化。 4(1):71-77。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. 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.
  2. 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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