搜索

Quantification of Total and Soluble Inorganic Phosphate
植物中总无机磷酸盐和可溶性无机磷酸盐的定量分析   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

A simple, rapid, and sensitive colorimetric microassay for inorganic phosphate (Pi) relies upon the absorption at 660 nm of a molybdenum blue complex that forms upon reduction of an ammonium molybdate-Pi complex in acid. The method for determination of total Pi uses plant tissues that have been ashed at 500 °C, whereas quantification of soluble Pi is performed with tissues extracted under mild acid conditions (which preserves acid-labile phosphate ester bonds).

Materials and Reagents

  1. Plant tissues 
  2. 17.5 M Glacial acetic acid
  3. 12 M Concentrated HCl 
  4. 16 M Concentrated HNO3
  5. Sodium phosphate, monobasic (NaH2PO4) (Bioshop, catalog number: SPM400 )
  6. Ascorbic acid (Bioshop, catalog number: AS0704 )
  7. Ammonium molybdate (Bioshop, catalog number: AMN333 )
  8. Zinc acetate (Sigma-Aldrich, catalog number: Z0625 )
  9. Quartz crucibles (Thermo Fisher Scientific, catalog number: 08-072 series)
  10. Acid extraction solution (see Recipes)
  11. Pi stock (for standard curve) (see Recipes)
  12. Pi assay reagent (see Recipes)

Equipment

  1. Crucible
  2. Drying oven
  3. Repeat pipetor
  4. Isotemp Muffle Furnace (Thermo Fisher Scientific, model: 10-650-14 )
  5. Microcentrifuge
  6. A computer supported microplate spectrophotometer (e.g., Spectromax Plus, Molecular Devices, Sunnyvale)

Procedure

  1. Total Pi (Hurley et al., 2010)
    1. Acid wash crucibles by incubating for at least 1 h in 0.1 N HCl at room temperature, then rinse with dH2O and dry.
    2. Pre-weigh crucibles and place at least 60 mg (fresh weight) of tissue in each.
    3. Dry in oven at 50-80 °C for at least 16 h (e.g., overnight), and then record tissue’s dry weight (mg) in each crucible.
    4. Ash the tissue in the furnace using a temperature ramp program (20 min at 150 °C, 1 h at 250 °C, and 3 h at 500 °C).
    5. Weigh crucible and ash. Add 25 μl of acid extraction solution per mg of ash, mix well, and centrifuge at 11,000 x g for 10 min.
    6. Dilute the supernatant 50-fold in dH2O.
    7. Assay Pi using the Drueckes et al. (1995) protocol as modified for plant tissues (Bozzo et al., 2006) by preparing a standard curve over the range 1-133 nmol of Pi using the following template.

      Well #
      Vol of Pi stock (3.3 mM)
      Volume of dH2O
      Amount of Pi added

      (μl)
      (μl)
      (nmol)
      1A
      0
      40
      0
      1B
      2
      38
      6.6
      1C
      4
      36
      13.2
      1D
      8
      32
      26.4
      1E
      12
      28
      39.6
      1F
      16
      24
      52.8
      1G
      20
      20
      66.0
      1H
      24
      16
      79.2
      2A
      30
      10
      99.9
      2B
      35
      5
      116.6
      2C
      40
      0
      133.2

      1. Pipette 1-40 μl of unknown(s) into adjacent wells(s). Add dH2O to bring each well to 40 μl final volume.
      2. Add 200 μl of Pi assay reagent to each well using a repeat pipetor.
      3. Incubate at 37 °C for 30 min.
      4. Measure A660 values and use the Pi calibration (standard) curve to determine Pi content of unknowns.
      5. Express the data as: nmol Pi mg-1 dry weight.

  2. Soluble Pi (Bozzo et al., 2006)
    1. Extract snap-frozen tissues (1:5, w/v) with 1% (v/v) glacial acetic acid.
    2. Centrifuge samples at 11,000 x g for 10 min.
    3. Assay the supernatant for Pi as described above.
    4. Esterified-Pi is calculated from the difference between total and free Pi concentrations.

Recipes

  1. Acid extraction solution
    30 ml 12 M concentrated HCl
    10 ml 16 M concentrated HNO3
    60 ml dH2O
  2. Pi stock (for standard curve)
    3.3 mM NaH2PO4
  3. Pi assay reagent
    1. Ammonium molybdate reagent
      Ammonium molybdate is added to an aqueous solution of 15 mM zinc acetate to give a 10 mM solution of molybdate. The solution is then adjusted to pH 5.0 with HCl. This solution is stored at 4 °C in the dark and is stable for several months.
    2. Reducing reagent
      A 10% (w/v) solution of ascorbic acid is adjusted to pH 5.0 with NaOH.
      Note: This solution must be prepared fresh daily.
    3. The Pi assay reagent is prepared by mixing one part of the ammonium molybdate reagent with four parts of the reducing reagent (prepare fresh daily).

Acknowledgments

Research in our laboratory has been generously funded by research and equipment grants from The Natural Sciences and Engineering Research Council of Canada (NSERC) and Queen’s Research Chairs program to William Plaxton.

References

  1. Bozzo, G. G., Dunn, E. L. and Plaxton, W. C. (2006). Differential synthesis of phosphate‐starvation inducible purple acid phosphatase isozymes in tomato (Lycopersicon esculentum) suspension cells and seedlings. Plant Cell Environ 29(2): 303-313. 
  2. Drueckes, P., Schinzel, R. and Palm, D. (1995). Photometric microtiter assay of inorganic phosphate in the presence of acid-labile organic phosphates. Anal Biochem 230(1): 173-177. 
  3. Hurley, B. A., Tran, H. T., Marty, N. J., Park, J., Snedden, W. A., Mullen, R. T. and Plaxton, W. C. (2010). The dual-targeted purple acid phosphatase isozyme AtPAP26 is essential for efficient acclimation of Arabidopsis to nutritional phosphate deprivation. Plant Physiol 153(3): 1112-1122.

简介

对于无机磷酸盐(Pi)的简单,快速和灵敏的比色微量测定依赖于在酸中通过钼酸铵-Pi络合物还原后形成的钼蓝络合物在660nm处的吸收。 测定总Pi的方法使用在500℃下灰化的植物组织,而可溶性P 1的定量是在温和酸条件下(其保留酸不稳定的磷酸酯键)提取的组织进行。

材料和试剂

  1. 植物组织
  2. 17.5M冰醋酸
  3. 12 M浓盐酸
  4. 16 M浓HNO 3
  5. 磷酸二氢钠(NaH 2 PO 4)(Bioshop,目录号:SPM400)
  6. 抗坏血酸(Bioshop,目录号:AS0704)
  7. 钼酸铵(Bioshop,目录号:AMN333)
  8. 醋酸锌(Sigma-Aldrich,目录号:Z0625)
  9. 石英坩埚(Thermo Fisher Scientific,目录号:08-072系列)
  10. 酸萃取溶液(参见配方)
  11. Pi料(标准曲线)(参见配方)
  12. Pi测试试剂(见配方)

设备

  1. 坩埚
  2. 干燥炉
  3. 重复移液器
  4. Isotemp Muffle Furnace(Thermo Fisher Scientific,型号:10-650-14)
  5. 微量离心机
  6. 计算机支持的微板分光光度计(例如,Spectromax Plus,Molecular Devices,Sunnyvale)

程序

  1. 总Pi(Hurley等人,2010)
    1. 通过在室温下在0.1N HCl中孵育至少1小时来洗涤酸洗涤坩埚,然后用dH 2 O漂洗并干燥。
    2. 预称量坩埚,并在每个位置至少放置60 mg(鲜重)的纸巾
    3. 在50-80℃的烘箱中干燥至少16小时(例如,过夜),然后在每个坩埚中记录组织的干重(mg)。
    4. 使用温度斜坡程序(在150℃下20分钟,在250℃下1小时,在500℃下3小时)使炉中的组织灰化。
    5. 称重坩埚和灰。 每毫克灰分加入25μl酸提取溶液,充分混合,并以11,000×g离心10分钟。
    6. 在dH 2 O中稀释上清液50倍
    7. 使用Drueckes

      的Assay Pi。 (Bozzo等人,2006)通过使用以下模板制备在1-133nmol Pi的范围内的标准曲线来确定对于植物组织修饰的标准曲线(1995)方案。

      呃#
      Pd原料(3.3mM) dH 2 O 2的体积
      添加的Pi量

      (μl)
      (μl)
      (nmol)
      1A
      0
      40
      0
      1B
      2
      38
      6.6
      1C
      4
      36
      13.2
      1D
      8
      32
      26.4
      1E
      12
      28
      39.6
      1F
      16
      24
      52.8
      1G
      20
      20
      66.0
      1H
      24
      16
      79.2
      2A
      30
      10
      99.9
      2B
      35
      5
      116.6
      2C
      40
      0
      133.2

      1. 移取1-40μl未知物到相邻的孔中。 加入dH sub 2 O以使每孔达到40μl终体积
      2. 使用重复移液器向每个孔中加入200μl的Pi测定试剂
      3. 在37℃孵育30分钟。
      4. 测量A <660> 值,并使用Pi校准(标准)曲线来确定未知物的Pi含量。
      5. 将数据表示为:nmol Pi mg -1 干重
  2. 可溶性Pi(Bozzo等人,2006)
    1. 用1%(v/v)冰醋酸提取速冻组织(1:5,w/v)。
    2. 将样品以11,000xg离心10分钟。
    3. 如上所述测定Pi的上清液
    4. 酯化Pi由总的和游离的Pi浓度之间的差值计算。

食谱

  1. 酸萃取溶液
    30ml 12M浓HCl
    10ml 16M浓HNO 3
    60毫升dH 2 O 2 /
  2. Pi库存(用于标准曲线)
    3.3mM NaH 2 PO 4/dub
  3. Pi测定试剂
    1. 钼酸铵试剂
      将钼酸铵加入到15mM乙酸锌的水溶液中,得到10mM钼酸盐溶液。 然后用HCl将溶液调节至pH5.0。 该溶液在4℃下在黑暗中储存,并可稳定几个月
    2. 还原试剂
      用NaOH将10%(w/v)抗坏血酸溶液调节至pH5.0。
      注意:此解决方案必须每天新鲜烹制。
    3. 通过将一份钼酸铵试剂与四份还原剂(每日新鲜制备)混合,制备Pi测定试剂。

致谢

我们实验室的研究由加拿大自然科学和工程研究委员会(NSERC)的研究和设备赠款以及威廉·普拉克斯顿的女王研究椅计划慷慨资助。

参考文献

  1. Bozzo,G.G.,Dunn,E.L.和Plaxton,W.C。(2006)。 在番茄中的磷酸盐饥饿诱导型紫色酸性磷酸酶同功酶的差异合成(番茄) em>)悬浮细胞和幼苗。 植物细胞环境29(2):303-313。 
  2. Drueckes,P.,Schinzel,R。和Palm,D。(1995)。 在酸不稳定有机磷酸盐存在下的无机磷酸盐的光度微量滴定法。 em> Anal Biochem 230(1):173-177。 
  3. Hurley,B.A.,Tran,H.T.,Marty,N.J.,Park,J.,Snedden,W.A.,Mullen,R.T.and Plaxton,W.C。(2010)。 双靶向紫色酸性磷酸酶同功酶AtPAP26对于拟南芥的有效适应是必需的植物生理学153(3):1112-1122。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2013 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. Knowles, V. and Plaxton, W. (2013). Quantification of Total and Soluble Inorganic Phosphate. Bio-protocol 3(17): e890. DOI: 10.21769/BioProtoc.890.
  2. Hurley, B. A., Tran, H. T., Marty, N. J., Park, J., Snedden, W. A., Mullen, R. T. and Plaxton, W. C. (2010). The dual-targeted purple acid phosphatase isozyme AtPAP26 is essential for efficient acclimation of Arabidopsis to nutritional phosphate deprivation. Plant Physiol 153(3): 1112-1122.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。