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Plasma Membrane Preparation from Lilium davidii and Oryza sativa Mature and Germinated Pollen
兰州百合和亚洲水稻成熟和萌发花粉的浆膜制备   

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Abstract

Pollen germination is an excellent process to study cell polarity establishment. During this process, the tip-growing pollen tube will start elongating. The plasma membrane as the selectively permeable barrier that separates the inner and outer cell environment plays crucial roles in this process. This protocol described an efficient aqueous polymer two-phase system followed by alkaline solution washing to prepare Lilium davidii or Oryza sativa plasma membrane with high purity.

Keywords: Mature pollen grains(成熟花粉粒), Germinated pollen grains(萌发花粉粒), Plasma membran(浆膜), Aqueous polymer two-phase system(水性聚合物两相体系), Alkaline solution(碱性溶液)

Background

Pollen plasma membrane contains various proteins that are vital for pollen tube growth and fertilization, such as receptor-like kinases (Wang et al., 2016) and ion channels (Hamilton et al., 2015). Isolating pure plasma membrane (PM) is the premise for the comprehensive PM proteome analysis. There are mainly four methods for PM preparation: differential centrifugation, density gradient centrifugation, preparative free-flow electrophoresis and the aqueous polymer two-phase system. Normally, differential centrifugation is often combined with density gradient centrifugation together to separate the subcellular components according to their size, shape and density. This technique is rapid, but due to the organelle density’s overlap, the resultant PM yield and purity are low (Schindler and Nothwang, 2006). Both free-flow electrophoresis and the aqueous polymer two-phase system separate membrane vesicles according to their surface properties. These two methods can enrich PM pure enough for proteomic analysis (Alexandersson et al., 2007). However, the instrument for the free-flow electrophoresis is complicated to operate (Sandelius et al., 1986). On the contrast, the aqueous polymer two-phase system can be performed easily and rapidly with centrifugation, making this method more convenient for PM preparation. PM enriched by the aqueous polymer two-phase system present in the form of vesicles which contain some cytoplasm contaminations (Alexandersson et al., 2008). Treatment with alkaline solution (100 mM Na2CO3, pH 11.5) can open these vesicles into sheets to release the contaminations (Fujiki et al., 1982).

Materials and Reagents

  1. 1,000 μl pipette tips (Corning, Axygen®, catalog number: T-1000-B )
  2. 20 x 10 cm envelope
  3. 50 ml tube (Corning, catalog number: 430829 )
  4. 60 x 15 mm Petri dish (Corning, catalog number: 430196 )
  5. 150 x 25 mm Petri dish (Corning, catalog number: 430599 )
  6. Gauze
  7. 10 ml tube (Biosharp, catalog number: BS-100-M )
  8. 100 μm cell strainer (Corning, Falcon®, catalog number: 352360 )
  9. 2 ml microtube (SARSTEDT, catalog number: 72.694.005 )
  10. 4 ml ultracentrifuge tube (Beckman Coulter, catalog number: 355603 )
  11. 26.3 ml ultracentrifuge tube (Beckman Coulter, catalog number: 355654 )
  12. Lily mature pollen grains harvest according to Han et al. (2010)
  13. Rice mature pollen grains harvest according to Dai et al. (2007)
  14. Boric acid (H3BO3) (Sigma-Aldrich, catalog number: B9645 )
  15. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
  16. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C2661 )
    Note: This product has been discontinued.
  17. Sucrose (Sigma-Aldrich, catalog number: S7903 )
  18. Calcium nitrate tetrahydrate, Ca(NO3)2·4H2O (Sigma-Aldrich, catalog number: C1396 )
  19. Thiamine hydrochloride (VB1) (Sigma-Aldrich, catalog number: T4625 )
  20. Poly (ethylene glycol), average Mn 4,000 (PEG 4000) (Sigma-Aldrich, catalog number: 81240 )
  21. 3-(N-Morpholino)propanesulfonic acid (MOPS) (Sigma-Aldrich, catalog number: M1254 )
  22. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E6758 )
  23. DL-Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
  24. Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
  25. cOmplete, EDTA-free protease inhibitor cocktail tablets (Roche Diagnostics, catalog number: 04693132001 )
  26. L-ascorbic acid (VC) (Sigma-Aldrich, catalog number: A7506 )
  27. Poly (vinylpolypyrrolidone) (PVPP) (Sigma-Aldrich, catalog number: 77627 )
  28. Potassium phosphate tribasic (K3PO4) (Sigma-Aldrich, catalog number: P5629 )
  29. Polyethylene glycol, average mol wt 3,350 (PEG 3350) (Sigma-Aldrich, catalog number: P4338 )
  30. Dextran T-500 (Pharmacia, catalog number: 17-0320-01 )
  31. Sodium carbonate (Na2CO3) (Sigma-Aldrich, catalog number: S7795 )
  32. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A1933 )
  33. Lily pollen germination medium (see Recipes)
  34. Rice pollen germination medium (see Recipes)
  35. Homogenate buffer (see Recipes)
  36. Plasma membrane isolation buffer (see Recipes)
  37. Aqueous polymer two-phase system (see Recipes)
  38. Dilution buffer (see Recipes)
  39. Washing buffer (see Recipes)

Equipment

  1. Pipette (Gilson, model: P1000N )
  2. Vortex
  3. Balance
  4. Centrifuge (Beckman Coulter, model: J2-HS )
  5. Homogenizer (MP Biomedicals, model: FastPrep®-24 )
  6. Ultra-centrifuge (Beckman Coulter, model: OptimalTM L-80XP )
  7. Microscope with 5x and 10x objective (Carl Zeiss, model: Axio Imager 1 )

Software

  1. ZEN lite software (2012, blue edition)

Procedure

  1. Mature pollen grain collection
    1. Use 20 x 10 cm envelope to collect the mature pollen grains (MPGs) at noon (11:00 AM-1:00 PM) when the anthers are dehiscing.
    2. Transfer the MPGs to 50 ml tubes.
    3. Use the MPGs immediately or add allochroic silica gels to them and then store at -80 °C.

  2. In vitro pollen germination
    The initiation procedures of in vitro germination are different for lily and rice pollen. For lily pollen, there should be a pretreatment to wash out the thick lipid coat, while the thin-walled rice pollen does not need this wash.
    1. Lilium davidii pollen germination
      Note: This procedure was modified from Ren et al. (1998) and Prado et al. (2004).
      1. Transfer the -80 °C stored MPGs to -20 °C in the dark for 2 weeks before the in vitro germination.
      2. Put 0.5 g MPGs into a dark, humid environment at 4 °C for 10 h.

      Note: The dark, humid environment is custom made as follows: Put MPGs in a Petri dish (60 x 15 mm) (Figure 1A), covered with gauze (Figure 1B), and then placed them in a larger Petri dish (150 x 25 mm) with 50 ml ddH2O (Figure 1C). This arrangement only permitted gauze contact with the ddH2O, do not let the MPGs contact the ddH2O directly.


      Figure 1. Set up of the humid environment. A. Put 0.5 g lily MPGs in a 60 x 15 mm Petri dish; B. Cover the Petri dish with four layers of gauzes; C. Put the set of B into a 150 x 25 mm Petri dish with 50 ml ddH2O.

      1. Wash the MPGs twice with 5 ml germination medium in a 10 ml tube and discard the flow-through after centrifuging at room temperature at 4,500 x g for 5 min.
      2. Put the washed MPGs (Figure 2A) into 100 ml germination medium in a Petri dish (60 x 15 mm), and then incubate them in the dark at 28 °C for 2 h with gentle shaking at 75 rpm/min.


        Figure 2. Morphology of Lilium davidii and Oryza sativa MPGs and GPGs. A. Lilium davidii MPGs; B. Lilium davidii MPGs in vitro germinated for 2 h; C. Oryza sativa MPGs; D. Oryza sativa MPGs in vitro germinated for 15 min. Bars = 50 μm.

      3. Use microscope with 5x objective to observe the germination rate, make sure the germination rate is more than 90% and the pollen tube is approximately 500 μm long (Figure 2B).
      4. Collect the germinated pollen grains (GPGs) by using 100 μm cell strainers, discard the flow-through that contains the ungerminated MPGs.
      5. Use the GPGs immediately or store them (transfer the mass retained by the strainer to 50 ml tubes, do not add any solution) at -80 °C.
    2. Oryza sativa pollen germination
      Note: This procedure was modified from Dai et al. (2007).
      1. Put 0.5 g freshly collected MPGs (Figure 2C) immediately into 100 ml germination medium in a Petri dish and culture them at room temperature with gentle shaking for about 15 min.
        Note: Rice MPGs must be freshly collected, if not, they will lose the activity for germination.
      2. Use microscope with 10x objective for observation to get synchronously germinated pollen tubes (Figure 2D).
        Note: If pollen germination is not synchronized, or the germination rate is less than 80%, the material should not be used for GPGs PM proteomic analysis. 
      3. Collect the GPGs through centrifugation at 4 °C, 1,000 x g, 5 min.
      4. Use the GPGs immediately or store at -80 °C.

  3. Microsomal vesicles isolation
    1. Use 6 ml homogenate buffer to suspend 0.5 g pollens (MPGs or GPGs) in a 50 ml beaker, and then transfer the suspension to three 2 ml microtubes with equal volume.
      Note: Lily MPGs should be washed as step B1c before starting step C1.
    2. Homogenize the pollens in 2 ml microtubes by using the FastPrep®-24 at the speed of 6.5 m/sec for 120 sec.
      Note: For rice pollens, using the speed of 6.5 m/sec for 30 sec is enough.
    3. Centrifuge the suspension at 4 °C, 1,500 x g for 5 min to remove cell debris, and then transfer the supernatants to new tubes.
    4. Centrifuge the resulting supernatants at 4 °C, 12,000 x g for 20 min to remove mitochondria, then transfer the supernatants to new tubes.
    5. Centrifuge the resulting supernatants at 4 °C, 31,000 x g for 15 min to remove other organelle contaminants, then transfer the supernatants to ultracentrifuge tubes.
    6. Ultracentrifuge the resulting supernatants at 4 °C, 100,000 x g for 1 h, the resulting pellets are the total microsomal vesicles (MSVs).

  4. Plasma membrane enrichment
    Aqueous polymer two-phase system is an effective tool to enrich plasma membrane.
    The following protocol was designed according to the principle of Schindler and Nothwang (2006).
    1. Suspend the MSVs in 1.2 ml plasma membrane (PM) isolation buffer.
    2. Put 1.0 g suspended MSVs into the 8.0 g aqueous polymer two-phase system, mix by vortex and then centrifuge at 4 °C, 4,200 x g for 30 min.
      Note: The aqueous polymer two-phase system were prepared by using the electronic balance, because the concentrations of the components PEG 3350 and Dextran T-500 were mass ratios (see Recipes).
    3. Transfer 3.0 ml of the top layer into another 8.0 g aqueous polymer two-phase system, mix and then centrifuge at 4 °C, 4,200 x g for 30 min.
    4. Repeat step D3 once.
    5. Transfer 3.0 ml of the top layer to an ultracentrifuge tube, dilute it 3-5 folds with dilution buffer, centrifuge at 4 °C, 200,000 x g for 1 h. The resulting pellets are PM vesicles.
    6. Suspend the resulting pellets with 50 μl dilution buffer for immediate use or store this suspension at -80 °C.

  5. Plasma membrane purification
    Alkaline solution can change the PM vesicles into PM sheets to remove the imbedded cytoplasm. The following protocol was mainly according to Fujiki et al. (1982).
    1. Dilute the PM vesicles with the washing buffer to protein concentration at 0.02~1.00 μg/μl.
    2. Keep in an ice-bath for 30 min with occasionally vortexing at every 10 min.
    3. Centrifuge at 4 °C, 50,000 x g for 1 h. Discharge the supernatants and the resulting pellets are the purified PM.
    4. Wash the purified PM pellets gently with cold ddH2O, don’t suspend the pellets, and then discard the ddH2O.
      Note: Usually, starting with 0.5 g pollen can get about 20 μg purified PM proteins. Their purity can be tested through Western blot by using antibodies for PM-specific P-type H+-ATPase and some organelle specific proteins, such as nuclear protein histone H1.
    5. Use the purified PM immediately or store the pellets at -80 °C.

Data analysis

Digital images of lily and rice germinated pollens were obtained using an upright light microscope (Axio Imager 1, Carl Zeiss, Germany) and ZEN lite software (2012, blue edition).

Notes

Don’t over homogenize the pollens. Too much cell debris will reduce the capacity of aqueous polymer two-phase system for plasma membrane purification. The homogenization will be fine when 80% pollens (8 out of 10 pollens) are broken observed under microscope.

Recipes

Note: Use MilliQ water to prepare the following solutions, do not need to autoclave or sterilize by filtration.

  1. Lily pollen germination medium
    1.6 mM H3BO3
    1.0 mM KCl
    500 μM CaCl2
    15% (w/v) sucrose
  2. Rice pollen germination medium
    40 mg/L H3BO3
    3 mM Ca(NO3)2·4H2O
    3 mg/L VB1
    10% (w/v) PEG4000
    250 mM sucrose
  3. Homogenate buffer
    250 mM sucrose
    Note: For lily pollen, use 15% (w/v) sucrose.
    50 mM MOPS, pH 7.8
    1 mM EDTA
    1 mM DTT
    1 mM PMSF
    1x protease inhibitor cocktail
    Note: For lily pollen, add 5 mM ascorbic acid, 0.6% (w/v) PVPP, 1% (m/v) PMSF.
  4. Plasma membrane isolation buffer
    250 mM sucrose
    5 mM potassium phosphate, pH 7.8
    1 mM DTT
    1 mM PMSF
  5. Aqueous polymer two-phase system
    6.5% (w/w) PEG3350
    6.5% (w/w) Dextran T-500
    250 mM sucrose
    5 mM KCl
    5 mM potassium phosphate, pH 7.8
    1 mM DTT
    Note: For lily, use 6.3% (w/w) PEG3350 and 6.3% (w/w) Dextran T-500.
  6. Dilution buffer
    250 mM sucrose
    50 mM MOPS/KOH, pH 7.8
    1 mM DTT
    1 mM PMSF
  7. Washing buffer
    100 mM sodium carbonate, pH 11.5

Acknowledgments

This protocol was modified from Han et al. (2010) and Yang and Wang (2017). This work was supported by the Chinese Ministry of Science and Technology (grant No. 2013CB945101) and the China Postdoctoral Science Foundation (grant No. 2016M591284).

References

  1. Alexandersson, E., Gustavsson, N., Bernfur, K., Karlsson, A., Kjellbom, P. and Larsson, C. (2008). Purification and proteomic analysis of plant plasma membranes. Methods Mol Biol 432: 161-173.
  2. Alexandersson, E., Gustavsson, N., Bernfur, K., Kjellbom, P. and Larsson, C. (2007). Plasma membrane proteomics. In: Šamaj, J. and Thelen, J. J. (Eds). Plant Proteomics. Springer Berlin Heidelberg, pp: 186-206.
  3. Dai, S., Chen, T., Chong, K., Xue, Y., Liu, S. and Wang, T. (2007). Proteomics identification of differentially expressed proteins associated with pollen germination and tube growth reveals characteristics of germinated Oryza sativa pollen. Mol Cell Proteomics 6(2): 207-230.
  4. Fujiki, Y., Hubbard, A. L., Fowler, S. and Lazarow, P. B. (1982). Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J cell biol 93(1): 97-102.
  5. Hamilton, E. S., Jensen, G. S., Maksaev, G., Katims, A., Sherp, A. M. and Haswell, E. S. (2015). Mechanosensitive channel MSL8 regulates osmotic forces during pollen hydration and germination. Science 350(6259): 438-441.
  6. Han, B., Chen, S., Dai, S., Yang, N. and Wang, T. (2010). Isobaric tags for relative and absolute quantification- based comparative proteomics reveals the features of plasma membrane-associated proteomes of pollen grains and pollen tubes from Lilium davidii. J Integr Plant Biol 52(12): 1043-1058.
  7. Prado, A. M., Porterfield, D. M. and Feijo, J. A. (2004). Nitric oxide is involved in growth regulation and re-orientation of pollen tubes. Development 131(11): 2707-2714.
  8. Ren, D. T., Han, S. C., Yan, L. F. and Yen, L. F. (1998). Actin and myosin during pollen germination. Chin Sci Bull 43: 690-694.
  9. Sandelius, A. S., Penel, C., Auderset, G., Brightman, A., Millard, M. and Morre, D. J. (1986). Isolation of highly purified fractions of plasma membrane and tonoplast from the same homogenate of soybean hypocotyls by free-flow electrophoresis. Plant Physiol 81(1): 177-185.
  10. Schindler, J. and Nothwang, H. G. (2006). Aqueous polymer two-phase systems: effective tools for plasma membrane proteomics. Proteomics 6(20): 5409-5417.
  11. Wang, T., Liang, L., Xue, Y., Jia, P. F., Chen, W., Zhang, M. X., Wang, Y. C., Li, H. J. and Yang, W. C. (2016). A receptor heteromer mediates the male perception of female attractants in plants. Nature 531(7593): 241-244.
  12. Yang, N. and Wang, T. (2017). Comparative proteomic analysis reveals a dynamic pollen plasma membrane protein map and the membrane landscape of receptor-like kinases and transporters important for pollen tube growth and interaction with pistils in rice. BMC Plant Biol 17(1): 2.

简介

花粉萌发是研究细胞极性的一个很好的过程。在此过程中,尖端生长的花粉管将开始延长。作为分离内外细胞环境的选择性渗透屏障的质膜在该过程中起关键作用。该方案描述了一种有效的含水聚合物两相体系,接着进行碱性溶液洗涤以制备高纯度的百合百合或水稻质膜。

背景 花粉质膜包含对花粉管生长和受精至关重要的各种蛋白质,例如受体样激酶(Wang等人,2016)和离子通道(Hamilton等人, em>,2015)。分离纯质膜(PM)是综合PM蛋白质组分析的前提。 PM制备主要有四种方法:差速离心,密度梯度离心,制备型自由流动电泳和含水聚合物两相体系。通常,差速离心通常与密度梯度离心合并,以根据其大小,形状和密度分离亚细胞组分。这种技术是快速的,但是由于细胞器密度的重叠,所得的PM产率和纯度都很低(Schindler和Nothwang,2006)。自由流动电泳和水性聚合物两相系统根据其表面性质分离膜囊泡。这两种方法可以富集PM足够纯化蛋白质组学分析(Alexandersson等人,2007)。然而,用于自由流动电泳的仪器操作复杂(Sandelius等人,1986)。相比之下,通过离心可以容易且快速地进行含水聚合物两相体系,使得该方法对PM制备更方便。 PM以含有一些细胞质污染物的囊泡形式存在的水性聚合物两相体系富集(Alexandersson等人,2008)。用碱性溶液(100mM Na 2 CO 3,pH11.5)处理可以将这些囊泡打开以释放污染物(Fujiki等人, >,1982)。

关键字:成熟花粉粒, 萌发花粉粒, 浆膜, 水性聚合物两相体系, 碱性溶液

材料和试剂

  1. 1,000μl移液器吸头(Corning,Axygen ®,目录号:T-1000-B)
  2. 20 x 10厘米信封
  3. 50ml管(Corning,目录号:430829)
  4. 60 x 15毫米培养皿(康宁,目录号:430196)
  5. 150 x 25毫米培养皿(康宁,目录号:430599)
  6. 纱布
  7. 10ml管(Biosharp,目录号:BS-100-M)
  8. 100μm细胞过滤器(Corning,Falcon ®,目录号:352360)
  9. 2 ml微管(SARSTEDT,目录号:72.694.005)
  10. 4 ml超速离心管(Beckman Coulter,目录号:355603)
  11. 26.3ml超速离心管(Beckman Coulter,目录号:355654)
  12. 百合成熟花粉根据Han等人收获。(2010)
  13. 根据Dai等人(2007)
    收获水稻成熟花粉
  14. 硼酸(H 3 O 3 BO 3)(Sigma-Aldrich,目录号:B9645)
  15. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
  16. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:C2661)
    注意:本产品已停产。
  17. 蔗糖(Sigma-Aldrich,目录号:S7903)
  18. 硝酸钙四水合物,Ca(NO 3 3)2·4H 2 O(Sigma-Aldrich,目录号:C1396)
  19. 盐酸硫胺素(VB 1)(Sigma-Aldrich,目录号:T4625)
  20. 聚(乙二醇),平均M n 4 000(PEG 4000)(Sigma-Aldrich,目录号:81240)
  21. 3-(N-吗啉代)丙磺酸(MOPS)(Sigma-Aldrich,目录号:M1254)
  22. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E6758)
  23. DL-二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:D0632)
  24. 苯基甲磺酰氟(PMSF)(Sigma-Aldrich,目录号:P7626)
  25. cOmplete,不含EDTA的蛋白酶抑制剂鸡尾酒片(Roche Diagnostics,目录号:04693132001)
  26. L-抗坏血酸(VC)(Sigma-Aldrich,目录号:A7506)
  27. 聚(乙烯基聚吡咯烷酮)(PVPP)(Sigma-Aldrich,目录号:77627)
  28. 磷酸三钾(K 3 3 PO 4)(Sigma-Aldrich,目录号:P5629)
  29. 聚乙二醇,平均摩尔重量3350(PEG 3350)(Sigma-Aldrich,目录号:P4338)
  30. 葡聚糖T-500(Pharmacia,目录号:17-0320-01)
  31. 碳酸钠(Na 2 CO 3)(Sigma-Aldrich,目录号:S7795)
  32. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A1933)
  33. 百合花粉萌发培养基(见食谱)
  34. 水稻花粉发芽培养基(见食谱)
  35. 均质缓冲液(见配方)
  36. 血浆膜隔离缓冲液(见配方)
  37. 水性聚合物两相体系(见配方)
  38. 稀释缓冲液(见配方)
  39. 洗涤缓冲液(见配方)

设备

  1. 移液器(Gilson,型号:P1000N)
  2. 涡流
  3. 平衡
  4. 离心机(Beckman Coulter,型号:J2-HS)
  5. 均质器(MP Biomedicals,型号:FastPrep -24)
  6. 超离心机(Beckman Coulter,型号:Optimal TM L-80XP)
  7. 具有5x和10x物镜的显微镜(Carl Zeiss,型号:Axio Imager 1)

软件

  1. ZEN lite软件(2012,蓝版)

程序

  1. 成熟花粉收集
    1. 当花药开裂时,使用20 x 10厘米信封收集中午(11:00 AM-1:00 PM)的成熟花粉(MPG)。
    2. 将MPG转移至50ml管。
    3. 立即使用MPG或添加异位硅胶,然后存放在-80°C。

  2. 体外花粉萌发
    萌发的起始程序对于百合和水稻花粉是不同的。对于百合花粉,应该进行预处理,以清除厚的脂质外套,而薄壁的水稻花粉不需要这种洗涤。
    1. 百合百合花粉发芽
      注意:此过程已从Ren等人修改。 (1998)和Prado等人(2004)。
      1. 将-80°C储存的MPG在黑暗中转移至-20°C,持续2周,然后再进行萌发。
      2. 将0.5g MPG放入黑暗,潮湿的环境中,在4℃下持续10小时

      注意:黑暗,潮湿的环境如下定制:将MPG放入培养皿(60 x 15 mm)(图1A)中,用纱布覆盖(图1B),然后将其放入较大的培养皿(150×25mm)和50ml ddH 2 O(图1C)。这种安排只允许与ddH O进行纱布接触,不要让MPG直接与ddH 2接触。


      图1.设置潮湿环境。 A.将0.5 g百合MPG放入60 x 15 mm培养皿中; B.用四层纱布覆盖培养皿; C.将B组放入具有50ml ddH 2 O的150×25mm培养皿中。

      1. 在10ml管中用5ml发芽培养基洗涤MPG两次,并在室温下以4,500×g离心5分钟后弃去流经。
      2. 将洗涤的MPG(图2A)放入培养皿(60×15mm)的100ml发芽培养基中,然后在28℃下在黑暗中孵育2小时,以75rpm/min的温和振荡。 >

        图2. Lilium davidii Oryza sativa MPGs和GPG。 A.百合药物 MPGs; B.Lilium davidii 体外的MPGs发芽2小时; ;; sat;;;;;;;;;;;;;豌豆sat iva sat sat>> g g g。。。。。。。。。。。。。。。。。。。。。。。棒=50μm。

      3. 使用5x目标的显微镜观察发芽率,确保发芽率大于90%,花粉管长约500μm(图2B)。
      4. 通过使用100μm的细胞过滤器收集发芽的花粉粒(GPG),丢弃含有未发芽的MPG的流通。
      5. 立即使用GPG或储存(将过滤器保留的质量转移至50ml管,不要添加任何溶液)-80°C。
    2. 花粉萌发 注意:此过程由Dai等人修改(2007)。
      1. 将0.5g新鲜收集的MPG(图2C)立即放入培养皿中的100ml发芽培养基中,并在室温下轻轻振荡培养约15分钟。
        注意:水稻MPG必须新鲜收集,否则,将失去萌发的活动。
      2. 使用具有10x目标的显微镜观察以获得同步发芽的花粉管(图2D) 注意:如果花粉发芽不同步,或发芽率低于80%,则不应将该物质用于GPGs蛋白质组学分析。
      3. 通过在4℃,1,000×g,5分钟离心收集GPG。
      4. 立即使用GPG或储存于-80°C。

  3. 微粒体囊泡分离
    1. 使用6ml匀浆缓冲液将0.5g花粉(MPG或GPG)悬浮在50ml烧杯中,然后将悬浮液转移到等体积的三个2ml微管中。
      注意:在开始步骤C1之前,应将L1ily MPG洗涤为步骤B1c。
    2. 以6.5米/秒的速度使用FastPrep -24,将2毫升微管中的花粉均质化120秒。
      注意:对于水稻花粉,使用6.5米/秒的速度30秒就可以了。
    3. 将悬浮液在4℃,1,500×g离心5分钟以除去细胞碎片,然后将上清液转移到新管中。
    4. 将得到的上清液在4℃离心12,000×g 20分钟以除去线粒体,然后将上清液转移到新管中。
    5. 将所得上清液在4℃,31,000μg/g下离心15分钟以除去其它细胞器污染物,然后将上清液转移到超速离心管中。
    6. 将所得上清液在4℃,100,000xg下超速离心1小时,得到的颗粒是总微粒体囊泡(MSV)。

  4. 血浆膜富集
    水性聚合物两相体系是富集质膜的有效工具 以下协议是按照辛德勒和诺华王(2006)的原则设计的。
    1. 将MSVs悬浮于1.2ml质膜(PM)隔离缓冲液中
    2. 将1.0g悬浮的MSV放入8.0g含水聚合物两相体系中,通过涡旋混合,然后在4℃,4,200×g离心30分钟。
      注意:通过使用电子天平制备含水聚合物两相体系,因为组分PEG 3350和葡聚糖T-500的浓度是质量比(参见食谱)。
    3. 将3.0ml顶层转移到另一个8.0g水性聚合物两相体系中,混合,然后在4℃,4,200×g离心30分钟。
    4. 重复步骤D3一次。
    5. 将3.0ml的顶层转移到超速离心管中,用稀释缓冲液稀释3-5倍,在4℃离心20,000×g 1小时。所得颗粒为PM泡
    6. 用50μl稀释缓冲液悬浮所得的小丸,立即使用,或将此悬浮液储存于-80°C
  5. 血浆膜纯化
    碱性溶液可以将PM泡沫层改为PM片,以去除嵌入的细胞质。以下方案主要根据Fujiki等人(1982)。
    1. 用洗涤缓冲液稀释PM泡,蛋白浓度为0.02〜1.00μg/μl
    2. 保持在冰浴中30分钟,每10分钟偶尔会涡旋
    3. 在4℃,50,000×g离心1小时。排出上清液,得到的颗粒是纯化的PM。
    4. 用冷的ddH 2 O 2轻轻洗涤纯化的PM颗粒,不要悬浮颗粒,然后丢弃ddH 2 O。
      注意:通常,从0.5g花粉开始可以得到约20μg纯化的PM蛋白。它们的纯度可以通过蛋白质印迹通过使用用于PM特异性P型H +的抗体和一些细胞器特异性蛋白质,例如作为核蛋白组蛋白H1。
    5. 立即使用纯化的PM或将颗粒储存在-80°C。

数据分析

使用直立光学显微镜(Axio Imager 1,Carl Zeiss,Germany)和ZEN lite软件(2012,蓝版)获得百合和稻萌发花粉的数字图像。

笔记

不要使花粉过度均匀化。太多的细胞碎片会降低水性聚合物两相体系的质膜纯化能力。在显微镜下观察到80%花粉(10个花粉中有8个)被破坏时,均匀化将很好。

食谱

注意:使用MilliQ水制备以下溶液,不需要通过过滤进行高压灭菌或消毒。

  1. 百合花粉发芽培养基
    1.6mM H 3 BO 3
    1.0 mM KCl
    500μMCaCl 2
    15%(w/v)蔗糖
  2. 水稻花粉发芽培养基
    40mg/L H 3/3> 3
    3mM Ca(NO 3 3)2·4H 2 O
    3mg/L VB 1
    10%(w/v)PEG4000
    250 mM蔗糖
  3. 均质缓冲液
    250 mM蔗糖 注意:对于百合花粉,请使用15%(w/v)蔗糖。
    50 mM MOPS,pH 7.8
    1 mM EDTA
    1 mM DTT
    1 mM PMSF
    1x蛋白酶抑制剂鸡尾酒
    注意:对于百合花粉,加入5mM抗坏血酸,0.6%(w/v)PVPP,1%(m/v)PMSF。
  4. 血浆膜分离缓冲液
    250 mM蔗糖 5mM磷酸钾,pH7.8。
    1 mM DTT
    1 mM PMSF
  5. 水性聚合物两相体系
    6.5%(w/w)PEG3350
    6.5%(w/w)葡聚糖T-500
    250 mM蔗糖 5 mM KCl
    5mM磷酸钾,pH7.8。
    1 mM DTT
    注意:对于百合,使用6.3%(w/w)PEG3350和6.3%(w/w)葡聚糖T-500。
  6. 稀释缓冲液
    250 mM蔗糖 50mM MOPS/KOH,pH 7.8
    1 mM DTT
    1 mM PMSF
  7. 洗涤缓冲液
    100mM碳酸钠,pH 11.5

致谢

这个协议由Han等人(2010)和Yang和Wang(2017)进行了修改。这项工作得到了中国科技部(授权号:2013CB945101)和中国博士后科学基金(授权号:2016M591284)的支持。

参考

  1. Alexandersson,E.,Gustavsson,N.,Bernfur,K.,Karlsson,A.,Kjellbom,P.和Larsson,C。(2008)。植物血浆膜的纯化和蛋白质组学分析。方法Mol Biol 432:161-173。
  2. Alexandersson,E.,Gustavsson,N.,Bernfur,K.,Kjellbom,P.和Larsson,C.(2007)。 Plasma membrane proteomics。 In:Šamaj,J。和Thelen,JJ(Eds)。植物蛋白质组学。柏林海德堡柏林斯伯格,pp:186-206。
  3. Dai,S.,Chen,T.,Chong,K.,Xue,Y.,Liu,S.and Wang,T(2007)。与花粉萌发和管生长相关的差异表达蛋白质的蛋白质组学鉴定揭示了发芽的水稻花粉的特征。/a>分子细胞蛋白质组学 6(2):207-230。
  4. Fujiki,Y.,Hubbard,AL,Fowler,S。和Lazarow,PB(1982)。通过碳酸钠处理分离细胞内膜:应用于内质网。 J细胞生物素93(1):97-102。 />
  5. Hamilton,ES,Jensen,GS,Maksaev,G.,Katims,A.,Sherp,AM和Haswell,ES(2015)。< a class ="ke-insertfile"href ="http://www.ncbi 。机械敏感通道MSL8调节花粉水合和发芽过程中的渗透力。 350(6259):438-441。
  6. Han,B.,Chen,S.,Dai,S.,Yang,N。和Wang,T。(2010)。用于相对和绝对定量的比较蛋白质组学的等压标签揭示了百合花粉花粉粒和花粉管的质膜相关蛋白质组的特征, em> .J Integr Plant Biol 52(12):1043-1058。
  7. Prado,A.M.,Porterfield,D.M.and Feijo,J.A。(2004)。 一氧化氮涉及花粉管的生长调节和重新取向。 开发 131(11):2707-2714。
  8. Ren,DT,Han,SC,Yan,LF和Yen,LF(1998)。花粉发芽期间的肌动蛋白和肌球蛋白。 Chin Sci Bull 43:690-694。
  9. Sandelius,AS,Penel,C.,Auderset,G.,Brightman,A.,Millard,M。和Morre,DJ(1986)。< a class ="ke-insertfile"href ="http: .ncbi.nlm.nih.gov/pubmed/16664771"target ="_ blank">通过自由流动电泳从大豆下胚轴的相同匀浆中分离高纯度的质膜和质膜的部分。 Physiol 81(1):177-185。
  10. Schindler,J.and Nothwang,H.G。(2006)。 水性聚合物两相系统:用于质膜蛋白质组学的有效工具。蛋白质组学 6(20):5409-5417。
  11. Wang,T.,Liang,L.,Xue,Y.,Jia,PF,Chen,W.,Zhang,MX,Wang,YC,Li,HJ and Yang,WC(2016)。  A受体杂合物介导植物中女性引诱剂的男性感知。 >自然 531(7593):241-244。
  12. Yang,N.和Wang,T.(2017)。比较蛋白质组学分析显示动态花粉质膜蛋白图谱和受体样激酶和转运蛋白的膜景观对于花粉管生长和水稻雌蕊相互作用是重要的。 BMC植物生物学 17 (1):2.
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Yang, N., Han, B., Liu, L., Yang, H. and Wang, T. (2017). Plasma Membrane Preparation from Lilium davidii and Oryza sativa Mature and Germinated Pollen. Bio-protocol 7(10): e2297. DOI: 10.21769/BioProtoc.2297.
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