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Protein Degradation Assays in Arabidopsis Protoplasts
利用拟南芥原生质体系统检测蛋白质的降解   

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Abstract

Plant transformation and exogenous protein expression is essential for molecular biology and biotechnology. Current approaches of stable plant transformation might be problematic and very time-consuming. Because of this, transient expression in protoplasts has become valuable alternative, being less cost and time-effective at the same time. Excellent for eukaryotic proteins, representing a natural cell habitat, protoplast isolation is widely used in protein interaction visualization techniques, like BiFC (Bimolecular fluorescence complementation) and FRET (Förster resonance energy transfer). In this protocol we present a another use of Arabidopsis protoplast in protein degradation assay, proving its high versatility as a tool in proteomics.

Keywords: Degradation assay(降解法), Transient expression(瞬时表达), Protoplasts(原生质体), ABA signalling(ABA信号), Ethylene biosynthesis(乙烯的生物合成)

Materials and Reagents

  1. 3-week old Arabidopsis plants
  2. Mannitol (BDH Prolabo, catalog number: 25311 )
  3. CaCl2 (POCH, catalog number: M00015143 )
  4. KCl (USB, catalog number: 20598 )
  5. 2-(N-morpholino)ethanesulfonic acid (MES) (LabEmpire, catalog number: MES503 )
  6. NaCl (POCH, catalog number: BA4121116 )
  7. Polyethylene glycol (PEG) 4000 (Sigma-Aldrich, catalog number: 81240 )
  8. Cellulase (SERVA Electrophoresis GmbH, catalog number: 16419 )
  9. Macerozyme (SERVA Electrophoresis GmbH, catalog number: 28302 )
  10. GenEluteTM HP Plasmid Midiprep Kit (Sigma-Aldrich, catalog number: PLD35 )
  11. Benzyloxycarbonyl-L-leucyl-L-leucyl-L-leucinal, Z-Leu-Leu-Leu-al (MG132) (Sigma-Aldrich, catalog number: M7449 )
  12. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )
  13. HEPES buffer (Sigma-Aldrich, catalog number: H3375 )
  14. MgCl2 (USB, catalog number: 18641 )
  15. Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
  16. Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
  17. Protease inhibitors (Roche Diagnostics, catalog number: 11873580001 )
  18. Enzyme solution (see Recipes)
  19. W5 (see Recipes)
  20. W1 (see Recipes)
  21. MMg (see Recipes)
  22. 40% PEG (see Recipes)
  23. Protein isolation buffer (see Recipes)

Equipment

  1. Light microscope (Nikon Corporation)
  2. Hemocythometer (Sigma-Aldrich)
  3. Tape (Scotch® MagicTM)
  4. Stainless steel forceps (Sigma-Aldrich)
  5. Scissors
  6. 10 ml pipette
  7. 50 ml tubes
  8. 90 mm Petri dishes
  9. Amicon Ultra-15 centrifugal filter unit (Millipore)
  10. NanoDrop spectrophotometer
  11. Tabletop centrifuge
  12. Horizontal shaker

Procedure

  1. Protoplast isolation
    1. Using tweezers, 5-6 mature leaves of Arabidopsis, petioles removed, were harvested.
    2. Adaxial surface of leaves was put on a piece of tape and leaves were flattened (Figure 1A-B).
    3. Second piece of tape was applied with limited pressure, trapping leaves between them (Figure 1C). To much pressure will result in damaging leaves, too little pressure will lead to poor epidermis removal effectiveness.
    4. Sandwich was placed with adaxial surface on top and pieces of tape were split by pulling away tape on top side, started at the tip of leaf (Figure 1D-E) (see Note 2).


      Figure 1. Preparation of Arabidopsis leaf for protoplast isolation. A. 3-weeks old Aradodopsis thaliana; B-E. Step by step procedure of leaf epidermis removal with tape.

    5. Tape was cut around leaves and put at room temperature enzyme solution in Petri dish, exposed mesophyll down. Tape should not be immersed in solution, but float on top of it. Mesophyll cells were digested for 60 min at 30 °C with gentle shaking (55 rpm) (see Note 3).
    6. After digestion pieces of tape were removed and cells were left for another 5 min in same conditions (see Note 4).
    7. Using 10 ml pipette protoplasts were transported to 50 ml tubes and put on ice.
    8. Protoplast were centrifuged for 3 min (150 x g, 4 °C) and washed twice with W5 buffer. Be careful not to resuspend protoplast to abruptly. After second wash step protoplast were resuspended in 1 ml of MMg solution. Protoplasts were calculated with help of hemocythometer and diluted to optimal concentration of 2 x 104 cells in 100 μl with MMg solution (see Notes 5-7).
    9. 5-10 μg plasmid DNA coding a tag-protein fusion was alequoted to 2 ml eppendorf tube (see Note 8).
    10. 100 μl of isolated protoplasts in MMg medium were transferred to the tube using a pipette tip with the tip of the tip cut off.
    11. Using a pipette tip with the tip of the tip cut off 110 μl of PEG solution was added. Solution was gently mixed and left for 15 min at RT in horizontal position (see Note 9).
    12. After incubation, 450 μl of W1 buffer was added to the tubes to dilute PEG solution, mixed and centrifuged for 3 min, 300 x g.
    13. Supernatant was removed and harvested protoplasts were resuspended in 300 μl of W1 solution.
    14. Transfected protoplasts were incubated overnight at 22 °C in horizontal position in the dark for protein expression. It is important not to disturb protoplasts at this stage.
    15. From this point protoplasts can be used in various protein analysis techniques (kinase assay, in vivo protein degradation assay, protein subcellular localization, BiFC and FRET analyses) (Ludwikow et al., 2014). Here we show in vivo degradation assay for plant proteins.

  2. In vivo degradation assay in protoplasts
    1. After overnight incubation (as indicated in step A14) in the dark protoplasts were treated with 50 μM MG132 (proteasome inhibitor) or mock treated with 0.1% DMSO for 6 h.
    2. After brief centrifugation (300 x g, RT) supernatant was removed and cells were resuspended and disrupted in 100 μl of protein isolation buffer.
    3. Protein concentration was determined using a NanoDrop spectrophotometer.
    4. Prepared samples were separated by SDS-PAGE and further analyzed by Western blotting.

Representative data



Figure 2. MG132 treatment increases ACC synthase6 protein accumulation in the abi1td protoplasts

Protoplasts isolated from WT Col-0 and the ABI1 knockout line (abi1td) were transformed with 5 µg of DNA plasmid coding for StrepTag-ACS6. Transformed protoplasts were treated with 50 µM MG132 or an equivalent volume of DMSO (mock control) for 6 h prior harvesting. A Western blot with anti-StrepTag antibodies confirms the presence of the StrepTag-ACS6 protein. The Western blot shown is representative of at least three independent experiments. Coomassie staining confirms equal protein loading (Ludwikow et al., 2014).

Notes

  1. This protocol is applicable to Brassica napus protoplast isolation and transformation.
  2. To avoid mesophyll cells damage (visible as dark green spots) don't use too much pressure when applying the tape.
  3. It is not recommended to digest leaves for more than 60 min. Protoplasts yield at this point will not increase, but they lose viability.
  4. If one hour digestion did not freed all mesophyll cells from tape fragments, one can gentle dip tape a few times in enzyme solution, to increase protoplast yield.
  5. Use swinging bucket rotor for centrifugation.
  6. Keep low acceleration and deceleration values during centrifugation.
  7. Resuspend the protoplasts by gently rocking the tube.
  8. Use hemocytometer to achieve accurate and reproducible results.
  9. A large volume of plasmid DNA decreases transformation efficiency, therefore keep the volume around 10 μl. Low transformation efficiency is usually a result of low quality plasmid DNA.

Recipes

  1. Enzyme solution (10 ml)
    1.2% cellulose
    0.4% macerozyme
    0.4 M mannitol
    20 mM KCl
    20 mM MES
    dH2O
    Filter sterilize
    Incubate at 55 °C for 10 min
    Prepare fresh, do not store
  2. W5 (50 ml)
    154 mM NaCl
    125 mM CaCl2
    5 mM KCl
    2 mM MES
    dH2O
    Filter sterilize, autoclave
    Stored at 4 °C
  3. W1 (10 ml)
    0.5 M mannitol
    20 mM KCl
    4 mM MES
    dH2O
    Filter sterilize, autoclave
    Stored at 4 °C, but no longer than 2 weeks
  4. MMg (10 ml)
    0.4 M mannitol
    15 mM MgCl2
    4 mM MES
    dH2O
    Filter sterilize, autoclave
    Stored at 4 °C, but no longer than 2 weeks
  5. 40% PEG (10 ml)
    4 g PEG 4000
    200 mM mannitol
    100 mM CaCl2
    Filter sterilize, autoclave
    Stored at 4 °C, but no longer than 3 weeks
  6. Protein isolation buffer
    20 mM HEPES (pH 7.5)
    10 mM MgCl2
    1 mM DTT
    1 mM PMSF
    Protease inhibitor
    dH2O
    Stored at -20 °C

Acknowledgments

This work was supported by COST Action FA0605 project 682/N-COST/2010/0, the National Science Centre grants (5615/B/P01/2010/39, DEC-2012/05/B/NZ3/00352, DEC-2011/03/N/NZ3/01796) and POLAPGEN grant no. WND-POIG.01.03.01-00-101/08. This protocol was adapted from Wu et al. (2009).

References

  1. Ludwików, A., Ciesla, A., Kasprowicz-Maluśki, A., Mitula, F., Tajdel, M., Galganski, L., Ziolkowski, P. A., Kubiak, P., Malecka, A., Piechalak, A., Szabat, M., Gorska, A., Dabrowski, M., Ibragimow, I. and Sadowski, J. (2014). Arabidopsis protein phosphatase 2C ABI1 interacts with type I ACC synthases and is involved in the regulation of ozone-induced ethylene biosynthesis. Mol Plant 7(6): 960-976.
  2. Wu, F. H., Shen, S. C., Lee, L. Y., Lee, S. H., Chan, M. T. and Lin, C. S. (2009). Tape-Arabidopsis Sandwich - a simpler Arabidopsis protoplast isolation method. Plant Methods 5: 16.

简介

植物转化和外源蛋白质表达是分子生物学和生物技术的必要条件。 目前稳定植物转化的方法可能有问题且非常耗时。 因此,在原生质体中的瞬时表达已经成为有价值的替代方案,同时成本更低和时间有效。 优异的真核蛋白,代表天然细胞栖息地,原生质体分离广泛应用于蛋白质相互作用可视化技术,如BiFC(双分子荧光互补)和FRET(弗尔斯特共振能量转移)。 在本协议中,我们提出了在蛋白质降解测定中使用拟南芥原生质体,证明其作为蛋白质组学工具的高度通用性。

关键字:降解法, 瞬时表达, 原生质体, ABA信号, 乙烯的生物合成

材料和试剂

  1. 3周龄拟南芥植物
  2. 甘露醇(BDH Prolabo,目录号:25311)
  3. CaCl 2(POCH,目录号:M00015143)
  4. KCl(USB,目录号:20598)
  5. 2-(N-吗啉代)乙磺酸(MES)(LabEmpire,目录号:MES503)
  6. NaCl(POCH,目录号:BA4121116)
  7. 聚乙二醇(PEG)4000(Sigma-Aldrich,目录号:81240)
  8. 纤维素酶(SERVA Electrophoresis GmbH,目录号:16419)
  9. Macerozyme(SERVA Electrophoresis GmbH,目录号:28302)
  10. GenElute HP质粒小量制备试剂盒(Sigma-Aldrich,目录号:PLD35)
  11. 苄基氧基羰基-L-亮氨酰-L-亮氨酰-L-亮氨酸,Z-Leu-Leu-Leu-al(MG132)(Sigma-Aldrich,目录号:M7449)
  12. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D8418)
  13. HEPES缓冲液(Sigma-Aldrich,目录号:H3375)
  14. MgCl (USB,目录号:18641)
  15. 二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:D0632)
  16. 苯甲基磺酰氟(PMSF)(Sigma-Aldrich,目录号:P7626)
  17. 蛋白酶抑制剂(Roche Diagnostics,目录号:11873580001)
  18. 酶溶液(见配方)
  19. W5(见配方)
  20. W1(见配方)
  21. MMg(请参阅配方)
  22. 40%PEG(见配方)
  23. 蛋白分离缓冲液(参见配方)

设备

  1. 光学显微镜(尼康公司)
  2. 血球计(Sigma-Aldrich)
  3. 胶带(Scotch ® Magic TM
  4. 不锈钢钳(Sigma-Aldrich)
  5. 剪刀
  6. 10 ml移液器
  7. 50ml管子
  8. 90 mm培养皿
  9. Amicon Ultra-15离心过滤器单元(Millipore)
  10. NanoDrop分光光度计
  11. 台式离心机
  12. 水平振动器

程序

  1. 原生质体分离
    1. 使用镊子,收获拟南芥的5-6个成熟叶,除去叶柄。
    2. 叶的近轴表面放在一条胶带上,叶片变平(图1A-B)
    3. 第二片胶带以有限的压力施加,捕获 叶之间(图1C)。 压力会很大 损害叶子,太小的压力将导致不良的表皮去除   有效性。
    4. 三明治放置在顶面上 并通过拉开上侧的胶带将胶带分开,开始 (图1D-E)(见注2)。


      图1.准备 拟南芥叶用于原生质体分离。 A. 3周龄的Aradodopsis thaliana ; 是。 叶片表皮去除的一步一步程序 磁带
    5. 在叶子周围切割带并在室温下放置   酶溶液在培养皿中,暴露的叶肉向下。 磁带不应该   浸没在溶液中,但漂浮在其顶部。 叶肉细胞 在30℃下轻轻摇动(55rpm)消化60分钟(见注3)
    6. 消化后取出胶带,将细胞在相同条件下再放置5分钟(见注4)。
    7. 使用10ml移液管原生质体转移到50ml管中并置于冰上
    8. 将原生质体离心3分钟(150×g,4℃)并洗涤 两次用W5缓冲液。 小心不要重悬原生质体 突然。 在第二次洗涤步骤后,将原生质体重悬浮于1ml MMg解决方案。 在血细胞计数器的帮助下计算原生质体 并用含有MMg的100μl稀释至最佳浓度为2×10 4个细胞   解决方案(见注释5-7)。
    9. 将5-10μg编码标签 - 蛋白质融合物的质粒DNA与2ml eppendorf管(参见注释8)标记。
    10. 将转移到MMg培养基中的100μl分离的原生质体 该管使用移液管尖端,尖端尖端被切掉
    11. 使用吸头尖端的尖端切断110微升的PEG 溶液。 将溶液温和混合并在室温下放置15分钟 在水平位置(见注9)
    12. 孵育后,加入450μl的W1 将缓冲液加入试管中以稀释PEG溶液,混合 离心3分钟,300×g
    13. 除去上清液并将收获的原生质体重悬于300μlW1溶液中
    14. 将转染的原生质体在22℃下孵育过夜 水平位置在黑暗中的蛋白质表达。 这很重要 在此阶段不扰乱原生质体
    15. 从这一点 原生质体可用于各种蛋白质分析技术(激酶 测定,in vivo 蛋白质降解测定,蛋白质亚细胞 定位,BiFC和FRET分析)(Ludwikow等人,2014)。 这里我们 显示植物蛋白的体内降解测定。

  2. 原生质体中的体内降解测定
    1. 在黑暗中过夜温育(如步骤A14中所示) 用50μMMG132(蛋白酶体抑制剂)或模拟物处理原生质体   用0.1%DMSO处理6小时
    2. 在短暂离心(300×g,RT)后,除去上清液,将细胞重悬浮 在100μl蛋白质分离缓冲液中破碎。
    3. 使用NanoDrop分光光度计测定蛋白质浓度。
    4. 通过SDS-PAGE分离制备的样品,并通过蛋白质印迹进一步分析。

代表数据



图2.MG132处理增加了在abi1td原生质体中的ACC合酶6蛋白积累

用5μg编码StrepTag-ACS6的DNA质粒转化分离自WT Col-0和emI ABI1敲除株(abi1td )的原生质体。转化的原生质体用50μMMG132或等体积的DMSO(模拟对照)处理6小时,然后收获。用抗StrepTag抗体的Western印迹证实了StrepTag-ACS6蛋白的存在。所示的Western印迹是至少三次独立实验的代表。考马斯染色证实蛋白负载相等(Ludwikow等人,2014)。

笔记

  1. 本协议适用于欧洲油菜原生质体分离和转化
  2. 为了避免叶肉细胞损伤(作为深绿色斑点可见),在使用胶带时不要使用太大的压力
  3. 不建议消化叶子超过60分钟。原生质体在这一点上的产量不会增加,但它们失去活力
  4. 如果一小时消化不能从胶带碎片中释放所有叶肉细胞,可以在酶溶液中温和浸渍几次,以增加原生质体产量。
  5. 使用摆动转子进行离心。
  6. 离心时保持低的加速和减速值。
  7. 通过轻轻摇动试管重悬原生质体
  8. 使用血细胞计数器获得准确和可重复的结果
  9. 大量的质粒DNA降低转化效率,因此保持体积在10μl左右。 低转化效率通常是低质量质粒DNA的结果

食谱

  1. 酶溶液(10ml)
    1.2%的纤维素 0.4%macerozyme
    0.4M甘露醇 20 mM KCl
    20 mM MES
    dH 2 2 O 过滤灭菌
    在55℃孵育10分钟
    准备新鲜,不要存放
  2. W5(50ml) 154 mM NaCl 125mM CaCl 2。 5 mM KCl
    2 mM MES
    dH 2 2 O 过滤灭菌,高压灭菌
    储存在4°C
  3. W1(10ml)
    0.5 M甘露醇 20 mM KCl
    4 mM MES
    dH 2 2 O 过滤灭菌,高压灭菌
    储存于4°C,但不超过2周
  4. MMg(10ml)
    0.4M甘露醇 15mM MgCl 2·h/v 4 mM MES
    dH 2 2 O 过滤灭菌,高压灭菌
    储存于4°C,但不超过2周
  5. 40%PEG(10ml) 4 g PEG 4000
    200mM甘露糖 100mM CaCl 2
    过滤灭菌,高压灭菌
    储存在4°C,但不超过3周
  6. 蛋白分离缓冲液
    20mM HEPES(pH7.5) 10mM MgCl 2/
    1 mM DTT
    1mM PMSF
    蛋白酶抑制剂
    dH 2 2 O 储存于-20°C

致谢

这项工作得到COST行动FA0605项目682/N-COST/2010/0,国家科学中心赠款(5615/B/P01/2010/39,DEC-2012/05/B/NZ3/00352,DEC-2011/03/N/NZ3/01796)和POLAPGEN批准号 WND-POIG.01.03.01-00-101/08。 该方案改编自Wu等人。 (2009)。

参考文献

  1. Ludwików,A.,Ciesla,A.,Kasprowicz-Maluśki,A.,Mitula,F.,Tajdel,M.,Galganski,L.,Ziolkowski,PA,Kubiak,P.,Malecka,A.,Piechalak, ,Szabat,M.,Gorska,A.,Dabrowski,M.,Ibragimow,I。和Sadowski, (2014年)。 拟南芥蛋白磷酸酶2C ABI1与I型ACC合酶相互作用,并参与臭氧诱导的 乙烯生物合成。 Mol Plant 7(6):960-976。
  2. Wu,F.H.,Shen,S.C.,Lee,L.Y.,Lee,S.H.,Chan,M.T.and Lin,C.S。(2009)。 Tape-Arabidopsis Sandwich - 一种更简单的拟南芥原生质体分离方法植物方法 5:16.
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Mituła, F., Kasprowicz-Maluśki, A., Michalak, M., Marczak, M., Kuczyński, K. and Ludwików, A. (2015). Protein Degradation Assays in Arabidopsis Protoplasts. Bio-protocol 5(4): e1397. DOI: 10.21769/BioProtoc.1397.
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