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After the plant cells are removed the cell walls by digestive enzyme, the plant protoplasts still have good cell activity. The protoplasts can be used to transiently express proteins of target genes in living plant cells through polyethylene glycol (PEG) mediated transformation. The purpose of this method is to employ the rice protoplasts and Green fluorescent protein (GFP) as an experimental system to observe the protein interactions in vivo. Meanwhile a 505~530 nm emission filter is used in confocal microscope to eliminate the interference of the autofluorescence from plant cells. The phenomenon of plant cell body spontaneous fluorescence can be eliminated by confocal observation.

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Bimolecular Fluorescence Complementation (BIFC) Protocol for Rice Protoplast Transformation
水稻原生质体中的(双分子荧光互补)BiFC实验

植物科学 > 植物细胞生物学 > 细胞成像
作者: Kun Wang
Kun WangAffiliation 1: State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, China
Affiliation 2: Wuhan Botanical Garden, Chinese Academy of Science, Wuhan, China
Bio-protocol author page: a973
Ying Liu
Ying LiuAffiliation: State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, China
Bio-protocol author page: a1825
 and Shaoqing Li
Shaoqing LiAffiliation: State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, China
For correspondence: shaoqingli@whu.edu.cn
Bio-protocol author page: a974
Vol 3, Iss 22, 11/20/2013, 4104 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.979

[Abstract] After the plant cells are removed the cell walls by digestive enzyme, the plant protoplasts still have good cell activity. The protoplasts can be used to transiently express proteins of target genes in living plant cells through polyethylene glycol (PEG) mediated transformation. The purpose of this method is to employ the rice protoplasts and Green fluorescent protein (GFP) as an experimental system to observe the protein interactions in vivo. Meanwhile a 505~530 nm emission filter is used in confocal microscope to eliminate the interference of the autofluorescence from plant cells. The phenomenon of plant cell body spontaneous fluorescence can be eliminated by confocal observation.
Keywords: Bimolecular Fluorescence Complementation(Bimolecular Fluorescence的互补性), Rice(水稻), Protoplast(原生质体)

[Abstract]

Materials and Reagents

  1. Rice (Oryza sativa L.) grain seeds
  2. Mannitol (Sigma-Aldrich)
  3. Morpholinoethane sulfonic acid (MES) (Sigma-Aldrich)
  4. Cellulose R-10 (Yakult Honsha)
  5. Macerozyme R-10 (Yakult Honsha)
  6. Bovine serum albumin (BSA) (Sigma-Aldrich)
  7. Carbenicillin/Ampicillin (AMRESCO)
  8. β-Mercaptoethanol (β-ME) (Amresco)
  9. Polyethtlene glycol (PEG) 4000 (Sigma-Aldrich)
  10. Enzyme solution (see Recipes)
  11. PEG4000 solution (see Recipes)
  12. W5 solution (see Recipes)
  13. MMG solution (see Recipes)

Equipment

  1. Shaker P270 (Chinese Academy of Sciences, Wuhan Scientific Instrument Plant)
  2. Collagen-coated 35-mm-diameter glass-base dish
  3. Vortex XW-80A (JiaPeng Techno)
  4. Nylon mesh (35 μm) (EMD Millipore)
  5. Vacuum pump
  6. 50 ml with round bottom centrifuge tube
  7. 2 ml centrifuge tube
  8. Tabletop centrifuges (Eppendorf 5810R and 5417R )
  9. Collagen-coated 35-mm-diameter glass-base dish (Asahi Techno Glass Corporation)
  10. Confocal microscopy (Olympus, model: FV1000 )
  11. OLYMPUS FV1000 system (Fluoview Ver.1.7b Viewer) (Olympus)

Procedure

  1. Protoplast Preparation
    1. Germinate 100 of the sterilizing rice grain seeds on wet gauze under darkness at 28 °C for about one week.
    2. When the etiolated seedlings grow to about 7~8 cm, collect the etiolated seedlings, and immediately use a sharp blade to cut the seedlings into ~0.5 mm segments, then, have the segments fully immersed in 50 ml 0.6 M mannitol solution for 10 min.
    3. Transfer the seedling fragments into Enzyme Solution.
    4. Using vacuum pump to remove air in the tissues to help them being completely precipitated in the Enzyme Solution.
    5. Keep the tissue-immersed solution in darkness at 28 °C, and agitated at 80 rpm on a shaker for ~4 h.
    6. Wash the nylon net (35 μm) with ddH2O and then wet it with W5 solution for 3-5 min before filtering the protoplast, and then the enzyme digested samples are filtered to a 50 ml centrifuge tube with round bottom. Slightly twist the nylon net to improve the yield.
    7. Centrifuge the filtration at 100 x g for 5 min, discard the supernatant and remove the residual liquid as much as possible with pipette, then add 10 ml pre-cooled W5 solution to resuspend the protoplast pellet by gentle swirling.
    8. Incubate the tube for 30 min on ice (the following operations are under room temperature).
    9. Precipitate the protoplast by centrifugation (100 x g for 5 min). Discard the supernatant and remove the residual liquid, then gently add 1 ml MMG solution to resuspend the protoplasts. Finally, adjust the protoplast density to 2 x 105 cells/ml under microscope (40x).

  2. Protoplast Transformation
    Note: Before protoplast transformation, please prepare the BIFC expression vectors according to the protocols of Walter et al. (2004), and we recommend to refer the information on how to prepare plasmid DNA using the economical CsCl gradient on the website of Sheen lab (http://genetics.mgh.harvard.edu/sheenweb/protocols.html).
    1. Aliquot 100 μl of the protoplasts to 2 ml centrifuge tubes.
    2. For transformation, empty vectors pUC-SPYNE/pUC-SPYCE and bZIP6-YFPN/bZIP6-YFPC are used as negtive and positive controls, respectively. 20 μl of the BIFC vectors (≥ 1-2 μg/μl, 10 μl per vector), negative control and positive control are added to each tube, respectively, and then mix gently.
    3. Add equal volume (120 μl) PEG solution to each tube and mix well.
    4. Incubate the mixture for 15 min for transformation.
    5. Add 480 μl of W5 solution to stop the transformation.
    6. Centrifuge the solution at 100 x g for 2 min, and discard the supernatant.
    7. Add 1 ml W5 solution to gently resuspend the protoplast pellet, and add 1 μl Carbenicillin (50 mg/ml) before transferring the protoplasts to culture plate, culture at room temperature for 16-20 h in darkness to allow expression of the BIFC proteins.
    8. Before confocal observation, the transformed protoplasts should be centrifuged at 100 x g for 2 min and remove most of the supernatant, then resuspend the protoplasts.

  3. Confocal observation
    1. Transfer the protoplast into a collagen-coated 35-mm-diameter glass-base dish for microscopy observation.
    2. Collection of the confocal fluorescence signals was performed on Olympus FV1000 system.
    3. The interference from autofluorescence problem in experiment can be eliminated by optical sectioning generated in confocal microscopy. We choose using excitation with the 488-nm line of an argon laser and a 505~530 nm band-pass emission filter.
    4. Under this observation regime, the positive control show strong yellow fluorescence, and the negative control is black. This confirms all of the operations above are reliable for the BIFC observation.

Recipes

  1. Enzyme solution (10 ml)
    Mannitol (0.6 M)
    1.093 g
    MES (10 mM, pH 5.7)
    1 ml (100 mM stock solution)
    Cellulose R-10 (1.5%)
    0.15 g
    Macerozyme R-10 (0.75%)
    0.075 g
    BSA (0.1%)
    0.01 g
    CaCl2 (1 mM)
    0.1 ml (100 mM stock solution)
    Carbenicilli (0.25 g/ml)
    2 μl
    β-ME
    4 μl
    Add ddH2O to 10 ml
    55 °C 10 min
    Natural cooling (Preparing it when you use)
  2. PEG4000 solution
    Mannitol (0.6 M)
    1.093 g
    CaCl2 (100 mM)
    0.111 g
    PEG4000 (40%)
    4 g
    Add ddH2O to 10 ml
    Using 1 M KOH to adjust the pH to 7.5~8.0
    Aliquot with 1.5 ml centrifuge tube and preserve at -20 °C
  3. W5 solution
    W5 (100 ml)
    154 mM NaCl
    NaCl
    0.9 g
    125 mM CaCl2
    CaCl2
    1.39 g
    5 mM KCl
    KCl
    5 ml 100 mM stock solution
    5 mM glucose
    Glucose
    0.09 g
    2 mM MES
    MES
    2 ml 100 mM stock solution
    Adjust pH to 5.8 with KOH, High temperature and high pressure sterilization for 20 min, room temperature preservation
  4. MMG solution
    MMG solution (10 ml)
    15 mM MgCl2
    MgCl2
    1.5 ml 100 mM stock solution
    4 mM MES 
    MES
    0.4 ml 100 mM stock solution
    0.6 M Mannitol
    Mannitol
    1.093 g
    Adjust pH to 5.8 with KOH, High temperature and high pressure sterilization for 20 min, room temperature preservation

Acknowledgments

This protocol is adapted from Wymer et al. (1999); Walter et al. (2004); Yoo et al. (2007) and Whang (2009).

References

  1. Wymer, C. L., Beven, A. F., Boudonck, K. and Lloyd, C. W. (1999). Confocal microscopy of plant cells. Methods Mol Biol 122: 103-130.
  2. Whang, S. S. (2009). Confocal microscopy study of Arabidopsis embryogenesis using GFP: mTn. J Plant Biol 52(4): 312-318.
  3. Walter, M., Chaban, C., Schutze, K., Batistic, O., Weckermann, K., Nake, C., Blazevic, D., Grefen, C., Schumacher, K., Oecking, C., Harter, K. and Kudla, J. (2004). Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J 40(3): 428-438.
  4. Yoo, S. D., Cho, Y. H. and Sheen, J. (2007). Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2(7): 1565-1572.

材料和试剂

  1. 水稻(<稻>水稻 L.)谷粒种子
  2. 甘露醇(Sigma-Aldrich)
  3. 吗啉代乙烷磺酸(MES)(Sigma-Aldrich)
  4. 纤维素R-10(Yakult Honsha)
  5. Macerozyme R-10(Yakult Honsha)
  6. 牛血清白蛋白(BSA)(Sigma-Aldrich)
  7. 羧苄青霉素/氨苄青霉素(AMRESCO)
  8. β-巯基乙醇(β-ME)(Amresco)
  9. 聚乙二醇(PEG)4000(Sigma-Aldrich)
  10. 酶溶液(见配方)
  11. PEG4000溶液(参见配方)
  12. W5解决方案(参见配方)
  13. MMG解决方案(参见配方)

设备

  1. Shaker P270(中国科学院武汉科学仪器厂)
  2. 胶原包被的35毫米直径的玻璃底皿
  3. Vortex XW-80A(JiaPeng Techno)
  4. 尼龙网(35μm)(EMD Millipore)
  5. 真空泵
  6. 50ml用圆底离心管
  7. 2ml离心管
  8. 台式离心机(Eppendorf 5810R和5417R)
  9. 胶原包被的35mm直径的玻璃基皿(Asahi Techno Glass Corporation)
  10. 共聚焦显微镜(Olympus,型号:FV1000)
  11. OLYMPUS FV1000系统(Fluoview Ver.1.7b Viewer)(Olympus)

程序

  1. 原生质体制备
    1. 在28℃的黑暗条件下,在湿纱布上发芽100粒灭菌米粒种子约1周。
    2. 当培养的幼苗生长至约7〜8cm时,收集培养的幼苗,并立即使用锋利的刀片将幼苗切成约0.5mm的节段,然后将节段完全浸入50ml 0.6M甘露醇溶液中10分钟 。
    3. 将幼苗片段转移到酶溶液中。
    4. 使用真空泵除去组织中的空气,帮助它们在酶溶液中完全沉淀。
    5. 保持组织浸没的解决方案在黑暗中28°C,并在振荡器上以80 rpm搅拌约4小时。
    6. 用ddH 2 O洗涤尼龙网(35μm),然后用W5溶液湿润3-5分钟,然后过滤原生质体,然后将酶消化的样品过滤到50ml离心管 与圆底。 稍微扭曲尼龙网,提高产量。
    7. 在100×g离心5分钟,弃去上清液并用移液管尽可能多地除去残余液体,然后加入10ml预冷却的W5溶液,通过温和涡旋重新悬浮原生质体沉淀。
    8. 在冰上孵育管子30分钟(以下操作在室温下)。
    9. 通过离心(100×g离心5分钟)沉淀原生质体。弃去上清液并去除残余液体,然后轻轻加入1ml MMG溶液以重悬原生质体。最后,在显微镜下(40x)将原生质体密度调节至2×10 5个细胞/ml。

  2. 原生质体转化
    注意:在原生质体转化之前,请根据Walter等人的方案制备BIFC表达载体。 (2004),我们建议参考关于如何使用经济的CsCl梯度在Sheen实验室网站上制备质粒DNA的信息(http://genetics.mgh.harvard.edu/sheenweb/protocols.html )。
    1. 将100μl原生质体分装到2ml离心管中
    2. 为了转化,空载体pUC-SPYNE/pUC-SPYCE和bZIP6-YFPN/bZIP6-YFPC分别用作阴性和阳性对照。 20μl的BIFC载体(≥1-2μg/μl,每个载体10μl), 阴性对照和阳性对照分别加入每个管中,然后轻轻混合。
    3. 每管加入等体积(120μl)PEG溶液,混匀
    4. 孵育混合物15分钟以进行转化
    5. 加入480μlW5溶液以终止转化。
    6. 将溶液以100×g离心2分钟,弃去上清液。
    7. 加入1毫升W5溶液轻轻地重悬原生质体沉淀,加入1微升羧苄青霉素(50毫克/毫升),然后将原生质体转移到培养板,在室温下在黑暗中培养16-20小时,以允许BIFC蛋白的表达。
    8. 在共聚焦观察之前,转化的原生质体应该在100×g离心2分钟并除去大部分上清液,然后重悬原生质体。

  3. 共聚焦观察
    1. 将原生质体转移到胶原包被的35毫米直径的玻璃基皿中进行显微镜观察
    2. 在Olympus FV1000系统上进行共焦荧光信号的收集
    3. 实验中自体荧光问题的干扰可以通过在共聚焦显微镜中产生的光学切片消除。 我们选择使用488-nm线的氩激光器和505〜530nm带通发射滤光器的激发。
    4. 在该观察方案下,阳性对照显示强黄色荧光,阴性对照是黑色。 这确认了上述所有操作对于BIFC观察是可靠的

食谱

  1. 酶溶液(10ml)
    甘露醇(0.6 M)
    1.093克
    MES(10mM,pH 5.7) 1ml(100mM储备液)
    纤维素R-10(1.5%)
    0.15 g
    Macerozyme R-10(0.75%)
    0.075克
    BSA(0.1%)
    0.01 g
    CaCl 2(1mM)
    0.1ml(100mM储备液)
    Carbenicilli(0.25g/ml)
    2微升
    β-ME
    4微升
    将ddH <2> O加到10ml ml/h 55℃10分钟
    自然冷却(使用时准备)
  2. PEG4000溶液
    甘露醇(0.6 M)
    1.093克
    CaCl 2(100mM)
    0.111克
    PEG4000(40%)
    4克
    将ddH <2> O加到10ml ml/h 使用1M KOH将pH调节至7.5〜8.0 用1.5ml离心管等分,并保存在-20°C
  3. W5溶液
    W5(100ml)
    154 mM NaCl NaCl
    0.9克
    125mM CaCl 2。 CaCl <2>
    1.39克
    5 mM KCl
    KCl
    5ml 100mM储备液
    5mM葡萄糖 葡萄糖
    0.09克
    2 mM MES
    MES
    2 ml 100 mM储备液
    用KOH调节pH至5.8,高温高压灭菌20分钟,室温保存
  4. MMG解决方案
    MMG溶液(10ml)
    15mM MgCl 2·h/v MgCl 2
    1.5 ml 100 mM储备液
    4 mM MES
    MES
    0.4ml 100mM储备溶液
    0.6 M甘露醇
    甘露醇
    1.093克
    用KOH调节pH至5.8,高温高压灭菌20分钟,室温保存

致谢

该协议改编自Wymer等人(1999); Walter等人(2004); Yoo (2007)和Whang(2009)。

参考文献

  1. Wymer,C.L.,Beven,A.F.,Boudonck,K.and Lloyd,C.W。(1999)。 植物细胞的共焦显微镜检查 方法Mol Biol 122 :103-130。
  2. Whang,S.S。(2009)。 拟南芥胚胎发生的共聚焦显微镜研究使用GFP:mTn。植物生物学 52(4):312-318。
  3. Walter,M.,Chaban,C.,Schutze,K.,Batistic,O.,Weckermann,K.,Nake,C.,Blazevic,D.,Grefen,C.,Schumacher,K.,Oecking, Harter,K。和Kudla,J。(2004)。 使用双分子荧光互补技术观察活植物细胞中蛋白质相互作用。植物J 40(3):428-438。
  4. Yoo,S.D.,Cho,Y.H。和Sheen,J。(2007)。 拟南芥叶肉原生质体:用于瞬时基因表达分析的多功能细胞系统。 Nat Protoc 2(7):1565-1572。
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How to cite this protocol: Wang, K., Liu, Y. and Li, S. (2013). Bimolecular Fluorescence Complementation (BIFC) Protocol for Rice Protoplast Transformation. Bio-protocol 3(22): e979. DOI: 10.21769/BioProtoc.979; Full Text



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