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Measurements of Proline and Malondialdehyde Content and Antioxidant Enzyme Activities in Leaves of Drought Stressed Cotton
干旱胁迫棉花叶片中脯氨酸和丙二醛含量以及抗氧化酶活性的测定   

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Plant & Cell Physiology
May 2015

Abstract

Drought stress negatively affects cotton plant growth and induces various biochemical and physiological responses in cotton plants. Proline content and antioxidant enzymes are thought to be associated with maintaining the structure of cellular components or with protecting cellular function. Study of cotton plant responses towards drought stress and investigation of the mechanism of drought tolerance are helpful to develop drought tolerant cotton plants. Here, we describe a protocol to investigate cotton plant response towards drought stress through measurements of biochemical parameters including antioxidant enzyme activities, proline content and malondialdehyde (MDA) content.

Materials and Reagents

  1. Centrifuge tubes (1.5 ml) (Corning, Axygen®, catalog number: MCT-150-C-S )
  2. Gossypium barbadense seeds
  3. Soil mix (vermiculite:humus = 1:1) (http://www.xing-nong.cn/ProductList.Asp?SortID=207)
  4. 1/2 MS media salt without sugar and agar (Hopebio, catalog number: HB8469-12 )
  5. Sodium phosphate dibasic anhydrous (Na2HPO4) (Sinopharm Chemical Reagent, catalog number: 20040618 )
  6. Sodium phosphate monobasic anhydrous (NaH2PO4) (Sinopharm Chemical Reagent, catalog number: 20040818 )
  7. ddH2O
  8. Liquid nitrogen
  9. Ice
  10. Ninhydrin (C9H4O3·H2O) (Sinopharm Chemical Reagent, catalog number: 30130212 )
  11. Trichloroacetic acid (TCA) (Sinopharm Chemical Reagent, catalog number: 80132618 )
  12. 5-sulphosalicylic acid dihydrate (Sinopharm Chemical Reagent, catalog number: 10021516 )
  13. L-proline (Genview, catalog number: FL259 )
  14. Thiobarbituric acid (TBA) (Sigma-Aldrich, catalog number: T5500 )
  15. Sodium hydroxide (NaOH) (Sinopharm Chemical Reagent, catalog number: 10019762 )
  16. EDTA-2Na (Sigma-Aldrich, catalog number: 27285 )
  17. L-methionine (Sigma-Aldrich, catalog number: V900487 )
  18. Nitroblue tetrazolium (NBT) (Beijing Dingguo Changsheng Biotechnology, catalog number: JN213 )
  19. Riboflavin (Sigma-Aldrich, catalog number: 47861 )
  20. Guaiacol (Sigma-Aldrich, catalog number: G5502 )
  21. 30% hydrogen peroxide (H2O2) (Sinopharm Chemical Reagent, catalog number: 10011208 )
  22. Ethanol (Sinopharm Chemical Reagent, catalog number: 10009259 )
  23. Glutathione reduced (GSH) (Sigma-Aldrich, catalog number: G4251 )
  24. 1-chloro-2, 4-dinitrobenzene (CDNB) (Sigma-Aldrich, catalog number: C38702 )
  25. Acetic acid (Sinopharm Chemical Reagent, catalog number: 10000208 )
  26. 1/2 MS solution (see Recipes)
  27. Na2HPO4 stock solution (see Recipes)
  28. NaH2PO4 stock solution (see Recipes)
  29. PBS (pH 7.0) (see Recipes)
  30. 2.5% acid-ninhydrin (see Recipes)
  31. 3% sulphosalicylic acid (see Recipes)
  32. 10% TCA (see Recipes)
  33. 0.25% TBA (see Recipes)
  34. PBS (pH 7.8) (see Recipes)
  35. 1 mM EDTA-2Na (see Recipes)
  36. 130 mM methionine (see Recipes)
  37. 750 μM NBT (see Recipes)
  38. 20 μM riboflavin (see Recipes)
  39. 0.2% guaiacol (see Recipes)
  40. PBS (pH 6.5) (see Recipes)
  41. 5 mM GSH (see Recipes)
  42. 1.5 mM CDNB (see Recipes)

Equipment

  1. Plant growth chamber (25 °C, 16 h/8 h photoperiod, 10,000 lux, 50-60% humidity)
  2. Mortar and pestle
  3. Plastic pots (8 cm diameter and 12 cm depth) (http://www.lyyyb.com/yingyangbo.html)
  4. Electronic balance (Sartorius, model: BAS124S-CW )
  5. ddH2O purification system (Xiamen RSJ Scientific Instruments, model: Spring-S10 )
  6. Ice making machine (Xueke, model: IMS-40 )
  7. Centrifuge (Eppendorf, model: 5810 R )
  8. Spectrophotometer (Analytik Jena, model: ScanDrop® 250)
  9. Water bath (Changzhou Aohua Instrument, model: HH-1 )
  10. Pipette, 2-20 µl (Eppendorf, Eppendorf Research® plus, model: 3120000038 )
  11. Pipette, 20-200 µl (Eppendorf, Eppendorf Research® plus, model: 3120000054 )
  12. Pipette, 100-1,000 µl (Eppendorf, Eppendorf Research® plus, model: 3120000062 )

Procedure

  1. Soak cotton seeds in water overnight at 28 °C to promote seed germination.
    Note: Seeds germinate after 24 h.
  2. Next day, sow germinated seeds into pots filled with soil mix (vermiculite:humus = 1:1), one seed per pot.
    Note: Pots with small holes at the bottom are important to leak surplus water.
  3. Maintain plantlets in a plant growth chamber with controlled conditions of 25 °C and 16 h light/8 h dark photoperiod at a light intensity of 10,000 lux during the experiment. Regularly water the pots with liquid 1/2 MS media (300 ml per pot) to provide nutrition to the plants.
    Note: The regular supplement of 1/2 MS media in the soil is required for plant growth in pots. After 7~10 days for two cotyledons to fully expand under the controlled conditions, this is a suitable time to perform the infiltration operation of virus-induced gene silencing (VIGS) assay in cotton (Chen et al., 2015; Gao et al., 2011; Gu et al., 2014).
  4. After 3-4 weeks, pots with two-three-leaves plantlets are watered every day for a period of seven days to saturation.
    Note: Surplus water could leak out from holes at the bottom, and a condition of saturated moisture in the soil provides similar water content before drought treatment.
  5. For drought treatment, plantlets are then deprived of water for 35 days, followed by re-watering once. Seven days after the re-watering treatment, cotton plants with at least one un-wilting green young leaf as well as a living shoot apex is regarded as “survival” (Figure 1A). Survival rate is calculated as the ratio of number of survived plants over the total number of treated plants (Figure 1B). Well-watered plantlets under normal growth conditions serve as controls.
  6. After 14 days of water-withhold treatment, take one or two young leaves for biochemical parameter analysis.
    Note: For crude protein/enzyme extraction, the leaves are collected from the same position (second to third leaf from the top) to avoid initial variance among different lines.
  7. Crude protein/enzyme extract
    1. Weigh 0.2 g of fresh leaf with primary veins from one or two young leaves, grind them with a mortar and pestle in liquid nitrogen (Figure 2A).
      Note: Usually, the two youngest leaves collected are enough, and the extra are discarded. Young leaf with a size of 3.5 x 3.5 cm is about 0.1-0.12 g, while a leaf with 4.0 x 4.0 cm is about 0.16-0.19 g.
    2. Homogenize the leaf powder by adding 3 ml of 100 mM PBS buffer (pH 7.8) (Figure 2B).
      Note: Unless otherwise specified, all solutions except for NaH2PO4 and Na2HPO4 stock solutions in this protocol are prepared fresh on the day and stored at room temperature during the assay. To simplify the protocol here, use PBS instead of 3% sulphosalicylic acid and 0.25% thiobarbituric acid made in 10% trichloroacetic acid to extract proline and malondialdehyde, respectively. PBS buffer will be frozen after adding the liquid nitrogen. Homogenize the sample in PBS buffer when it begins to thaw in 3-5 min.
    3. Transfer the homogenate to two 1.5 ml centrifuge tubes (Figure 2C) and centrifuge at 10,000 x g for 20 min at 4 °C.
      Note: During crude protein/enzyme extraction, make sure all samples are kept under 4 °C or on ice.
    4. Transfer the supernatant (Figure 2D) to new centrifuge tubes for further analysis.
      Note: Keep the extract on ice before storing. Aliquot crude protein/enzyme extraction into 1.5 ml centrifuge tubes and drop the tubes in liquid nitrogen to quickly freeze the samples, and then store them at -80 °C. Each aliquot is only used once with one cycle of thawing.
    5. Measure the concentration of crude protein (mg/ml) in the supernatant with Analytik Jena ScanDrop 250 by a spectrophotometric method with Formula Warburg-Christian (protein): protein concentration (mg/ml) = 1.55 x A280 - 0.76 x A260 (Simonian and Smith, 2006) (Figure 3).
      Note: Because stray light can affect the linearity of absorbance versus concentration, samples with absorbance > 2.0 should be diluted further in the PBS buffer to obtain absorbance < 2.0 (Simonian and Smith, 2006).
  8. Determination of proline content
    1. Reagents preparation (see Recipes)
      100 mM PBS (pH 7.0)
      2.5% acid-ninhydrin
      3% sulphosalicylic acid
    2. Prepare reaction solution (for 40 reactions) which contains 10 ml 3% sulphosalicylic, 10 ml acetic acid and 20 ml 2.5% acid-ninhydrin.
    3. Add 50 μl crude protein/enzyme extract from each sample into 1 ml reaction solution in a 1.5 ml centrifuge tube. Reaction solution with 50 μl 100 mM PBS (pH 7.8) serves as a reference.
    4. Boil the reaction mixture in a boiling water bath for 15 min.
      Note: The reaction mixture turns red after boiling.
    5. Cool down the reaction mixture on ice for 5 min.
    6. Pipette 200 μl of the reaction mixture and measure the absorbance at 520 nm with Analytik Jena ScanDrop 250.
    7. Prepare L-proline standard curve for quantification according to the above steps 8b-f (Figure 4).
    8. Determine proline content of testing sample against the standard curve of L-proline.
    9. Normalize proline content to protein content in sample with the unit “μg/mg protein”.
    10. Figure 5A shows the proline content decreased in GbMYB5-silenced cotton under drought stress.
  9. Determination of malondialdehyde (MDA) content
    1. Reagents preparation (see Recipes)
      10% TCA
      0.25% TBA
    2. Add 100 μl crude protein/enzyme extract from each sample into 1 ml 0.25% TBA solution in a 1.5 ml centrifuge tube. A total of 1 ml 0.25% TBA solution with 100 μl 100 mM PBS (pH 7.8) serves as a reference.
    3. Boil the reaction mixture in a boiling water bath for 15 min.
      Note: The reaction mixture turns red after boiling.
    4. Cool down reaction mixture on ice for 5 min.
    5. Pipette 200 μl of the reaction mixture and measure the absorbance at 532 nm and 600 nm with Analytik Jena ScanDrop 250.
    6. Formula: MDA (nmol/mg protein) = (A532-A600) x Vr x (V/Vt)/155 x 1,000/Cp
      A532: the absorbance at 532 nm
      A600: the absorbance at 600 nm
      Vr: the volume of reaction mixture
      V: total volume of crude enzyme solution
      Vt: volume of crude enzyme used in the testing tube
      Cp: crude protein concentration (mg/ml)
      155: the extinction coefficient of MDA-TBA at 532 (mM-1 cm-1)
    7. Figure 5B shows the MDA content increased in GbMYB5- silenced cotton under drought stress.
  10. SOD activity test
    1. Reagents preparation (see Recipes)
      100 mM PBS (pH 7.8)
      1 mM EDTA-2Na
      130 mM methionine
      750 μM NBT
      20 μM riboflavin
    2. Prepare reaction solution (for 30 reactions) which contains 30 ml 100 mM PBS (pH 7.8), 0.6 ml 1 mM EDTA-2Na, 2 ml 130 mM Met, 2 ml 750 μM NBT, and 2 ml 20 μM Riboflavin.
    3. Add 50 μl crude enzyme solution from each sample into 1 ml reaction solution in a 1.5 ml centrifuge tube. Reaction solution with 50 μl 100 mM PBS (pH 7.8) but no crude enzyme under dark and light condition serve as controls I and control II, respectively.
    4. Put the tubes as well as Control II under light, uniformly, with the light intensity of 4,000 lux for 10-15 min. By contrast, keep Control I in the dark.
    5. Move the tubes away from the light quickly.
    6. Measure the absorbance at 560 nm in the dark with Analytik Jena ScanDrop 250. Use Control I as reference.
    7. Formula: SOD total activity (unit: u/mg protein) = [(Ack-As) x V]/(0.5 x Ack x Vt)/Cp
      Ack: the absorbance at 560 nm of Control II (exposed to light with no crude enzyme)
      As: the absorbance at 560 nm of candidate sample tube
      V: total volume of crude enzyme solution
      Vt: volume of crude enzyme used in the testing tube
      Cp: crude protein concentration (mg/ml)
      0.5: One unit of SOD is defined as the amount of enzyme that inhibits 50% nitroblue tetrazolium photoreduction.
    8. Figure 5C shows the activities of SOD decreased in GbMYB5-silenced cotton under drought stress.
  11. POD activity test
    1. Reagents preparation (see Recipes)
      100 mM PBS (pH 7.0)
      0.2% guaiacol
      30% H2O2
    2. Prepare reaction solution (for 50 reactions) by adding 28 μl 0.2% guaiacol in 50 ml 100 mM PBS (pH 7.0), heat and stir well, add 19 μl 30% H2O2 after cooling.
    3. Add 50 μl 100 mM PBS (pH 7.8) and 1 ml of the reaction solution into a cuvette for reference (see the operation video).
      Note: Use a pipettor and tip to remove the solutions from the cuvette before measuring the next sample.

      Video 1. Enzyme activity determination by spectrophotometer ScanDrop 250 (Take POD for example). This smart phone video provides a whole procedure of enzyme activity determination by spectrophotometer ScanDrop 250: how to organize the working batch, how to store all reaction solutions and crude extracts, how to set the measurement parameters in the software, how to add testing crude extracts and reaction buffer, etc. The enzyme activity determination of CAT and GST is the same as the POD.

      Note: In order to make a homogeneous solution for enzyme reaction, it is importance to add the crude extracts (small volume) in prior to the reaction buffer (big volume) into the cuvette.
    4. Add 50 μl crude enzyme solution into a cuvette, insert the cuvette into the holder, then add 1 ml of the reaction solution into the cuvette and immediately record the dynamic absorbance at 470 nm with Analytik Jena ScanDrop 250 at every 15 sec for 1 min, looking for steady average alteration.
      Note: The best linear regression between absorbance at 470 nm and reaction time is within 75 sec (Figure 6).
    5. Formula: POD activity (unit: u/mg protein) = △A470 x (V/Vt)/(0.01 x t)/Cp
      △A470: the change of absorbance at 470 nm during every 15 sec
      V: total volume of crude enzyme solution
      Vt: volume of crude enzyme used in the testing tube
      t: reaction time (min)
      Cp: crude protein concentration (mg/ml)
      0.01: One unit of POD is defined as the amount of enzyme that increases 0.01 of absorbance at 470 nm per minute.
    6. Figure 5D shows the activities of POD decreased in GbMYB5-silenced cotton under drought stress.
  12. CAT activity assay
    1. Reagents preparation
      100 mM PBS (pH 7.0)
      30% H2O2
    2. Prepare reaction solution (for 50 reactions) by adding 77.5 μl 30% H2O2 in 50 ml 100 mM PBS (pH 7.0).
    3. Add 50 μl crude enzyme into a cuvette, insert the cuvette into the holder, then add 1 ml of the reaction solution into the cuvette and immediately record the dynamic absorbance at 240 nm with Analytik Jena ScanDrop 250 at every 15 sec for 1 min, looking for steady average alteration. Reaction solution with 50 μl 100 mM PBS (pH 7.8) serves as a reference.
    4. Formula: CAT activity (unit: u/mg protein) = △A240 x (V/Vt)/(0.1 x t)/Cp
      △A240: the change of absorbance at 240 nm during every 15 sec
      V: total volume of crude enzyme solution
      Vt: volume of crude enzyme used in the testing tube
      t: reaction time (min)
      Cp: crude protein concentration (mg/ml)
      0.1: One unit of CAT is defined as the amount of enzyme that decreases 0.1 of absorbance at 240 nm per minute.
    5. Figure 5E shows the activities of CAT decreased in GbMYB5-silenced cotton under drought stress.
  13. GST activity assay
    1. Reagents preparation (see Recipes)
      100 mM PBS (pH 6.5)
      5 mM GSH
      1.5 mM CDNB
    2. Prepare reaction solution (for 50 reactions) by adding 20 ml 5 mM GSH and 40 ml 1.5 mM CDNB.
    3. Add 50 μl crude enzyme solution into a cuvette, insert the cuvette into the holder, then add 1 ml of the reaction solution into the cuvette and immediately record the dynamic absorbance at 340 nm with Analytik Jena ScanDrop 250 at every 15 sec for 1 min, looking for steady average alteration. Reaction solution with 50 μl 100 mM PBS (pH 7.8) serves as a control and ddH2O serves as a reference.
    4. Formula: GST activity (unit: u/mg protein) = (△A340s - △A340ck) x (V/Vt)/t/Cp
      △A340s: the change of A340 of candidate sample tube during every 15 sec
      △A340ck: the change of A340 of control during every 15 sec
      V: total volume of crude enzyme solution
      Vt: volume of crude enzyme used in the testing tube
      t: reaction time (min)
      Cp: crude protein concentration (mg/ml)
      One unit of GST is defined as the amount of enzyme that increases 1 of absorbance at 340 nm per min.
    5. Figure 5F shows the activities of GST decreased in GbMYB5-silenced cotton under drought stress.

Representative data



Figure 1. Phenotypes (A) and survival rates (B) of GbMYB5-silenced cotton plantlets under water-withholding treatment. A. The phenotype of cotton plantlets in response to water-withhold treatment. WT: wild-type cotton plantlets; VIGS: cotton plantlets agroinfiltrated with the vectors pCLCrVA-GbMYB5 and pCLCrVB to silence the GbMYB5 gene (Chen et al., 2015); CK: cotton plantlets agroinfiltrated with the empty vectors pCLCrVA and pCLCrVB. 0 d: the beginning of water-withholding treatment; 35 d: 35 d post water-withholding treatment; Control: normal growth conditions. B. The survival rates of cotton plantlets after 35 d of water-withholding treatment. Control: normal growth conditions; Drought: 35 d post water-withholding treatment; The survival values are presented as means ± SE from three biological replicates with eight plants per replicate (**P < 0.01; t-test).


Figure 2. Crude protein/enzyme extraction from cotton leaves of 14 d post water-withholding treatment. A. Grind leaf tissue with a mortar and pestle in liquid nitrogen. B. Homogenize the leaf powder with PBS buffer. C. Tissue homogenates in centrifuge tubes before centrifugation. D. Supernatants and pellets after centrifugation.


Figure 3. Protein quantification with spectrophotometer ScanDrop 250. A. Start machine ScanDrop 250 and its software FlashSoft Pro. B. Select “Standard MP” measurement position and “Bio method” module in the Module window, set “Flash number” (3),“Number of accumulation” (10) and activate “Dark correction”. C. Select the “Serial measurement”, enter the number of samples under “Cycle” in the Standard MP window. D. Activate the desired Formula Warburg-Christian (protein) by ticking the box in the “Bio” window, then press start button. E. Insert cuvette for reference (100 mM PBS buffer, pH = 7.8) according to the instruction. F. Measure the absorbance of the crude solutions according to the instruction. The key points of operation are marked with red circles.


Figure 4. The standard curve between the absorbance at 520 nm and L-proline content. Series content of L-proline (0, 5, 10, 15, 20, 25, and 30 μg) dissolved in 0.5 ml ddH2O were added into 1 ml of reaction solution to measure their absorbance at 520 nm. A linear regression was observed between the absorbance values at 520 nm and L-proline contents at 0-30 μg (R2 = 0.9935). The values are means of two independent experiments.


Figure 5. Analysis of metabolites and enzyme activities in GbMYB5-silenced cotton under drought stress. A. Proline content decreased in GbMYB5-silenced cotton under drought stress. B. MDA content increased in GbMYB5- silenced cotton under drought stress. Activities of SOD (C), POD (D), CAT (E), and GST (F) decreased in GbMYB5-silenced cotton under drought stress. CK: cotton plantlets agroinfiltrated with the empty vectors pCLCrVA and pCLCrVB; VIGS: cotton plantlets agroinfiltrated with the vectors pCLCrVA-GbMYB5 and pCLCrVB to silence the GbMYB5 gene. Values were means ± SE of biological replicates (n ≥ 5) (*P < 0.05; **P < 0.01; t-test).


Figure 6. Linear regression between absorbance at 470 nm and reaction time. The absorbance value of POD was automatically collected in a 15 sec interval and up to 150 sec. The blue line and red line represent the linear regression between absorbance of POD at 470 nm and reaction time within 150 sec or 60-75 sec, respectively. The tests of 4 POD samples (4 sub-panels) shows the best linear regression between absorbance at 470 nm and reaction time is within 75 sec.

Recipes

Note: The pH value of the PBS buffer should be verified with pH meter before using.

  1. 1/2 MS solution
    Dissolve 2.47 g 1/2 MS media salt in 1,000 ml ddH2O
  2. 200 mM Na2HPO4 stock solution
    Dissolve 53.65 g Na2HPO4·7H2O in 1,000 ml ddH2O
    Keep in 4 °C before using
  3. 200 mM NaH2PO4 stock solution
    Dissolve 27.8 g NaH2PO4·H2O in 1,000 ml ddH2O
    Keep in 4 °C before using
  4. 100 mM PBS (pH 7.0)
    61 ml 200 mM Na2HPO4 stock solution
    39 ml 200 mM NaH2PO4 stock solution
    100 ml ddH2O
  5. 2.5% acid-ninhydrin
    Dissolve 1.250 g ninhydrin in 30 ml acetic acid and 20 ml 100 mM PBS (pH 7.0) stirring in 70 °C water bath
    Keep in 4 °C before using
  6. 3% sulphosalicylic acid
    Dissolve 3.496 g 5-sulphosalicylic acid dihydrate in 100 ml ddH2O
  7. 10% TCA
    Dissolve 10 g TCA in 100 ml ddH2O
  8. 0.25% TBA
    Dissolve 0.125 g TBA in 5 ml 1 mol/L NaOH
    Add into 45 ml 10% TCA
    Keep in 4 °C before using
  9. 100 mM PBS (pH 7.8)
    91.5 ml 200 mM Na2HPO4 stock solution
    8.5 ml 200 mM NaH2PO4 stock solution
    100 ml ddH2O
  10. 1 mM EDTA-2Na
    Dissolve 0.037 g EDTA-2Na in 100 ml ddH2O
  11. 130 mM methionine
    Dissolve 0.970 g methionine in 50 ml 100 mM PBS (pH 7.8)
    Keep in 4 °C before using
  12. 750 μM NBT
    Dissolve 0.031 g NBT in 50 ml 100 mM PBS (pH 7.8)
    Keep in the dark
  13. 20 μM Riboflavin
    Dissolve 0.007 g riboflabin in 100 ml ddH2O
    Keep in the dark
  14. 0.2% guaiacol
    Dissolve 0.1 g guaiacol in 0.5 ml ethanol
    Add into 50 ml 100 mM PBS (pH 7.0)
    Keep in the dark
  15. 100 mM PBS (pH 6.5)
    31.5 ml 200 mM Na2HPO4 stock solution
    68.5 ml 200 mM NaH2PO4 stock solution
    100 ml ddH2O
  16. 5 mM GSH
    Dissolve 0.077 g GSH in 50 ml 100 mM PBS (pH 6.5)
  17. 1.5 mM CDNB
    Dissolve 0.015 g CDNB in 5 ml ethanol
    Add into 45 ml 100 mM PBS (pH 6.5)

Acknowledgments

This protocol was adapted from the research article: Chen et al. (2015). This work was supported by the National Natural Science Foundation of China [grant No.31371930]; National Science and Technology Major Project for Transgenic Breeding, China [grant No.2014ZX0800501B]; the Independent Innovation of Agricultural Sciences in Jiangsu Province, China [grant No.CX(15)1005].

References

  1. Chen, T., Li, W., Hu, X., Guo, J., Liu, A. and Zhang, B. (2015). A cotton MYB transcription factor, GbMYB5, is positively involved in plant adaptive response to drought stress. Plant Cell Physiol 56(5): 917-929.
  2. Gao, X., Wheeler, T., Li, Z., Kenerley, C. M., He, P. and Shan, L. (2011). Silencing GhNDR1 and GhMKK2 compromises cotton resistance to Verticillium wilt. Plant J 66(2): 293-305.
  3. Gu, Z., Huang, C., Li, F. and Zhou, X. (2014). A versatile system for functional analysis of genes and microRNAs in cotton. Plant Biotechnol J 12(5): 638-649.
  4. Simonian, M. H. and Smith, J. A. (2006). Spectrophotometric and colorimetric determination of protein concentration. Curr Protoc Mol Biol Chapter 10: Unit 10 11A.

简介

干旱胁迫对棉花植物生长有不利影响,并在棉花植物中诱导各种生化和生理反应。 脯氨酸含量和抗氧化酶被认为与保持细胞组分的结构或保护细胞功能相关。 棉花植物对干旱胁迫的反应研究和对干旱耐受机制的研究有助于开发耐旱棉花植物。 在这里,我们描述一个协议调查棉花植物对干旱胁迫的反应,通过测量的生化参数,包括抗氧化酶活性,脯氨酸含量和丙二醛(MDA)含量。

材料和试剂

  1. 离心管(1.5ml)(Corning,Axygen ,目录号:MCT-150-C-S)
  2. 种子
  3. 土壤混合物(蛭石:腐殖质= 1:1)( http://www.xing-nong.cn/ProductList.Asp?SortID=207
  4. 不含糖和琼脂的1/2 MS培养基盐(Hopebio,目录号:HB8469-12)
  5. 磷酸氢二钠无水(Na 2 HPO 4)(Sinopharm Chemical Reagent,目录号:20040618)
  6. 磷酸二氢钠无水(NaH 2 PO 4)(Sinopharm Chemical Reagent,目录号:20040818)
  7. ddH sub 2 O
  8. 液氮

  9. 水合茚三酮(C 9 H 4 O 3 O 3·H 2 O)(Sinopharm Chemical Reagent,目录号:30130212 )
  10. 三氯乙酸(TCA)(Sinopharm Chemical Reagent,目录号:80132618)
  11. 5-磺基水杨酸二水合物(Sinopharm Chemical Reagent,目录号:10021516)
  12. L-脯氨酸(Genview,目录号:FL259)
  13. 硫代巴比妥酸(TBA)(Sigma-Aldrich,目录号:T5500)
  14. 氢氧化钠(NaOH)(Sinopharm Chemical Reagent,目录号:10019762)
  15. EDTA-2Na(Sigma-Aldrich,目录号:27285)
  16. L-甲硫氨酸(Sigma-Aldrich,目录号:V900487)
  17. Nitroblue tetrazolium(NBT)(BEIJING DINGGUO CHANGSHEND BIOTECHNOLOGY,目录号:JN133)
  18. 核黄素(Sigma-Aldrich,目录号:47861)
  19. Guaiacol(Sigma-Aldrich,目录号:G5502)
  20. 30%过氧化氢(H 2 O 2)(Sinopharm Chemical Reagent,目录号:10011208)
  21. 乙醇(Sinopharm Chemical Reagent,目录号:10009259)
  22. 谷胱甘肽还原(GSH)(Sigma-Aldrich,目录号:G4251)
  23. 1-氯-2,4-二硝基苯(CDNB)(Sigma-Aldrich,目录号:C38702)
  24. 乙酸(Sinopharm Chemical Reagent,目录号:10000208)
  25. 1/2 MS解决方案(参见配方)
  26. (参见配方)。
  27. NaH 2(PO 4)储备溶液(参见配方)。
  28. PBS(pH 7.0)(参见配方)
  29. 2.5%酸 - 茚醇(见配方)
  30. 3%磺基水杨酸(参见配方)
  31. 10%TCA(参见配方)
  32. 0.25%TBA(参见配方)
  33. PBS(pH 7.8)(参见配方)
  34. 1mM EDTA-2Na(参见配方)
  35. 130 mM甲硫氨酸(参见配方)
  36. 750μMNBT(参见配方)
  37. 20μM核黄素(参见配方)
  38. 0.2%愈创木酚(参见配方)
  39. PBS(pH 6.5)(参见配方)
  40. 5 mM GSH(参见配方)
  41. 1.5 mM CDNB(参见配方)

设备

  1. 植物生长室(25℃,16小时/8小时光周期,10,000勒克斯,50-60%湿度)
  2. 砂浆和杵
  3. 塑料盆(直径8厘米,深12厘米)( http://www。 lyyyb.com/yingyangbo.html
  4. 电子天平(Sartorius,型号:BAS124S-CW)
  5. ddH 2 O纯化系统(厦门RSJ Scientific Instruments,型号:Spring-S10)
  6. 制冰机(Xueke,型号:IMS-40)
  7. 离心机(Eppendorf,型号:5810R)
  8. 分光光度计(Analytik Jena,型号:ScanDrop 250)
  9. 水浴(常州奥华仪器,型号:HH-1)
  10. 移液管,2-20μl(Eppendorf,Eppendorf Research plus,型号:3120000038)
  11. 移液管,20-200μl(Eppendorf,Eppendorf Research plus,型号:3120000054)
  12. 移液管,100-1000μl(Eppendorf,Eppendorf Research plus,型号:3120000062)

程序

  1. 将棉花种子在28℃下浸泡过夜,以促进种子发芽。
    注意:种子在24小时后发芽。
  2. 第二天,母猪将种子发芽到装有土壤混合物(蛭石:腐殖质= 1:1)的盆中,每盆一个种子。
    注意:底部有小孔的罐对于泄漏多余的水是非常重要的。
  3. 在植物生长室中在25℃和16小时光照/8小时黑暗光周期的控制条件下在实验期间以10,000勒克司的光强度维持苗。用液体1/2 MS培养基(每盆300ml)定期给盆施水以向植物提供营养。
    注意:在土壤中的1/2 MS培养基的常规补充物是在盆中植物生长所需的。 7〜10天,使两个子叶在受控条件下完全膨胀。这是在棉花中进行病毒诱导基因沉默(VIGS)测定的浸润操作的合适时间(Chen等人,2015; Gao等人,2011; Gu等人,2014)。
  4. 3-4周后,每天给具有两三叶植物的盆浇水七天,直至饱和。
    注意:剩余的水可能会从底部的孔中泄漏出来,而且土壤中饱和水分的条件在干旱处理前提供相同的含水量。
  5. 对于干旱处理,然后使小植物脱水35天,然后再浇水一次。在再浇水处理后7天,将具有至少一个不萎朽的绿色幼叶以及活的枝顶的棉花植物视为"存活"(图1A)。存活率计算为存活植物数相对于处理植物总数的比率(图1B)。在正常生长条件下的充满水的小植物用作对照。
  6. 经过14天的停水处理后,取一或两片幼叶进行生化参数分析 注意:对于粗蛋白质/酶提取,从相同位置收集叶子(从顶部第二至第三叶),以避免不同品系之间的初始差异。
  7. 粗蛋白/酶提取物
    1. 从一个或两个幼叶中称取0.2g具有主脉的新鲜叶,用研钵和杵在液氮中研磨(图2A)。
      注意:通常,收集的两个最小的叶子就足够了,额外的叶子被丢弃。尺寸为3.5×3.5cm的幼叶为约0.1-0.12g,而具有4.0×4.0cm的幼叶为约0.16-0.19g。
    2. 通过加入3ml的100mM PBS缓冲液(pH 7.8)(图2B)使叶粉均匀化 注意:除非另有说明,否则除了NaH 2 PO 4和Na 2 SO 4以外的所有溶液,该方案中的储备溶液在一天新鲜制备并在测定期间在室温下储存。为了简化这里的协议,使用PBS代替3%磺基水杨酸和0.25%硫代巴比妥酸在10%三氯乙酸中提取分别提取脯氨酸和丙二醛。加入液氮后,PBS缓冲液将被冷冻。当样品在3-5分钟内开始解冻时,将样品在PBS缓冲液中匀浆。
    3. 将匀浆转移到两个1.5ml离心管(图2C),并在10,000xg在4℃离心20分钟。
      注意:在粗蛋白质/酶提取过程中,确保所有样品都保持在4°C或冰上。
    4. 将上清液(图2D)转移到新的离心管进行进一步分析。
      注意:储存前将提取物放在冰上。将粗蛋白/酶提取物倒入1.5ml离心管中,并将管放入液氮中以快速冷冻样品,然后将其储存在-80℃。每个等分试样只使用一次解冻一个周期。
    5. 用Analytik Jena ScanDrop 250通过分光光度法用式Warburg-Christian(蛋白质)测量上清液中粗蛋白质的浓度(mg/ml):蛋白质浓度(mg/ml)= 1.55×A280-0.76×A260(Simonian和Smith,2006)(图3)。
      注意:因为杂散光可以影响吸光度对浓度的线性,所以吸光度> 2.0应在PBS缓冲液中进一步稀释, 2.0(Simonian and Smith,2006)。
  8. 脯氨酸含量的测定
    1. 试剂准备(参见配方)
      100mM PBS(pH7.0)
      2.5%酸 - 茚醇
      3%磺基水杨酸
    2. 制备含有10ml 3%磺基水杨酸,10ml乙酸和20ml 2.5%酸 - 茚三酮的反应溶液(用于40个反应)。
    3. 添加50微升粗蛋白/酶提取物从每个样品到1毫升反应溶液在1.5毫升离心管。 将具有50μl100mM PBS(pH 7.8)的反应溶液用作参考
    4. 将该反应混合物在沸水浴中煮沸15分钟。 注意,沸腾后反应混合物变成红色。
    5. 在冰上冷却反应混合物5分钟
    6. 吸取200μl反应混合物,并用Analytik Jena ScanDrop 250测量520nm处的吸光度。
    7. 根据上述步骤8b-f(图4)制备L-脯氨酸标准曲线进行定量
    8. 相对于L-脯氨酸的标准曲线确定测试样品的脯氨酸含量。
    9. 将脯氨酸含量相对于样品中的蛋白质含量标准化,单位为"μg/mg蛋白质"。
    10. 图5A显示在干旱胁迫下,在GbMYB5-沉默棉中脯氨酸含量降低。
  9. 测定丙二醛(MDA)含量
    1. 试剂准备(参见配方)
      10%TCA
      0.25%TBA
    2. 将100μl来自每个样品的粗蛋白/酶提取物加入到1.5ml离心管中的1ml 0.25%TBA溶液中。将含有100μl100mM PBS(pH7.8)的1ml 0.25%TBA溶液作为参照
    3. 将该反应混合物在沸水浴中煮沸15分钟。 请注意,沸腾后反应混合物变为红色。
    4. 将反应混合物在冰上冷却5分钟
    5. 吸取200μl反应混合物,并用Analytik Jena ScanDrop 250测量532nm和600nm处的吸光度。
    6. 公式:MDA(nmol/mg蛋白质)=(A532-A600)×Vr×(V/Vt)/155×1,000/Cp
      A532:在532nm处的吸光度
      A600:在600nm处的吸光度
      Vr:反应混合物的体积
      V:粗酶溶液的总体积 Vt:试管中使用的粗酶的体积
      Cp:粗蛋白质浓度(mg/ml)
      155:MDA-TBA在532(mM <-1 cm -1 )的消光系数
    7. 图5B显示在干旱胁迫下,在GbMYB5 - 沉默棉中MDA含量增加。
  10. SOD活性测试
    1. 试剂准备(参见配方)
      100mM PBS(pH7.8)
      1mM EDTA-2Na
      130mM甲硫氨酸 750μMNBT
      20μM核黄素
    2. 制备含有30ml 100mM PBS(pH7.8),0.6ml 1mM EDTA-2Na,2ml 130mM Met,2ml750μMNBT和2ml20μM核黄素的反应溶液(30次反应)。
    3. 从每个样品中加入50μl粗酶溶液到1.5ml离心管中的1ml反应溶液中。 在暗处和光照条件下,用50μl100mM PBS(pH7.8),但没有粗酶的反应溶液分别用作对照I和对照II。
    4. 将管以及对照II在光下,均匀地,具有4,000lux的光强度10-15分钟。 相反,将Control I保持在黑暗中。
    5. 快速移动管远离光。
    6. 使用Analytik Jena ScanDrop 250在黑暗中测量560 nm处的吸光度。使用Control I作为参考
    7. 式:SOD总活性(单位:u/mg蛋白质)= [(Ack-As)×V] /(0.5×Ack×Vt)/Cp
      Ack:对照II(没有粗酶的光照)在560nm处的吸光度 As:候选样品管在560nm处的吸光度
      V:粗酶溶液的总体积 Vt:试管中使用的粗酶的体积
      Cp:粗蛋白质浓度(mg/ml)
      0.5:一个单位的SOD定义为抑制50%氮蓝四唑光还原的酶量。
    8. 图5C显示在干旱胁迫下,在GbMYB5-沉默棉中SOD的活性降低。
  11. POD活性测试
    1. 试剂准备(参见配方)
      100mM PBS(pH7.0)
      0.2%愈创木酚 30%H 2 O 2 O 2
    2. 通过加入28μl0.2%愈创木酚在50ml 100mM PBS(pH7.0)中制备反应溶液(50次反应),加热并充分搅拌,加入19μl30%H 2 O 2 。
    3. 加入50μl100 mM PBS(pH 7.8)和1 ml的反应溶液到比色杯参考(见操作视频)。
      注意:在测量下一个样品之前,使用移液器和吸头从试管中取出溶液。

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      视频1.通过分光光度计ScanDrop 250测定酶活性(以POD为例)。 此智能手机视频提供了一个由分光光度计ScanDrop 250确定酶活性的整个过程:如何组织工作批次,如何存储所有反应溶液和粗提取物,如何在软件中设置测量参数,如何加入测试粗提物和反应缓冲液等.CAT和GST的酶活性测定与POD相同。
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      注意:为了制备均匀的酶反应溶液,重要的是在反应缓冲液(大体积)之前将粗提取物(小体积)加入比色杯。
    4. 加入50μl粗酶溶液和1ml反应溶液到比色杯中,立即记录在Analytik Jena ScanDrop 250在470nm处的动态吸光度,每15秒1分钟,寻找稳定的平均变化。
      注意:在470nm处的吸光度和反应时间之间的最佳线性回归在75秒内(图6)。
    5. 式:POD活性(单位:u/mg蛋白质)=△A470×(V/Vt)/(0.01×t)/Cp
      △A470:每15秒内在470nm处的吸光度变化
      V:粗酶溶液的总体积 Vt:试管中使用的粗酶的体积
      t:反应时间(min)
      Cp:粗蛋白质浓度(mg/ml)
      0.01:POD的一个单位定义为在470nm /分钟下增加0.01吸光度的酶量
    6. 图5D显示在干旱胁迫下,在GbMYB5-沉默棉中POD的活性降低。
  12. CAT活性测定
    1. 试剂准备
      100mM PBS(pH7.0)
      30%H 2 O 2 sub
    2. 通过在50ml 100mM PBS(pH 7.0)中加入77.5μl30%H 2 O 2 sub制备反应溶液(50次反应)。
    3. 加入50μl粗酶的1ml反应溶液在每个1.5ml离心管,并立即 用Analytik Jena ScanDrop 250在15分钟内记录240nm处的动态吸光度1分钟,寻找稳定的平均变化。将具有50μl100mM PBS(pH 7.8)的反应溶液用作参考
    4. 式中:CAT活性(单位:u/mg蛋白质)=△A240×(V/Vt)/(0.1×t)/Cp
      △A240:每15秒内在240nm处的吸光度变化
      V:粗酶溶液的总体积 Vt:试管中使用的粗酶的体积
      t:反应时间(min)
      Cp:粗蛋白质浓度(mg/ml)
      0.1:一个单位的CAT定义为在240nm /分钟下降低0.1的吸光度的酶量
    5. 图5E显示在干旱胁迫下,在GbMYB5-沉默棉中CAT的活性降低。
  13. GST活性测定
    1. 试剂准备(参见配方)
      100mM PBS(pH 6.5)
      5 mM GSH
      1.5mM CDNB
    2. 通过加入20ml 5mM GSH和40ml 1.5mM CDNB制备反应溶液(50次反应)
    3. 加入50μl粗酶溶液到1ml的反应溶液在每个1.5ml离心管中,立即记录在340nm的动态吸光度与Analytik Jena ScanDrop 250每15秒1分钟,寻找稳定的平均变化。将具有50μl100mM PBS(pH 7.8)的反应溶液用作对照,并将ddH 2 O用作参考。
    4. 式:GST活性(单位:u/mg蛋白质)=(△A340s-△A340ck)×(V/Vt)/t/Cp
      △A340s:每15秒内候选样品管的A340的变化
      △A340ck:每15秒内控制的A340的变化
      V:粗酶溶液的总体积 Vt:试管中使用的粗酶的体积
      t:反应时间(min)
      Cp:粗蛋白质浓度(mg/ml)
      一个单位的GST定义为在340nm/min下增加1的吸光度的酶量
    5. 图5F显示在干旱胁迫下,在GbMYB5-沉默棉中GST的活性降低。

代表数据



图1.在保水处理下的GbMYB5阳性棉花植株的表型(A)和存活率(B)。 A.棉花幼苗响应水的表型 - 持有治疗。 WT:野生型棉苗; VIGS:用载体pCLCrVA- GbMYB5 和pCLCrVB进行农杆菌浸润的棉花植株以沉默GbMYB5 基因(Chen等人, CK:用空载体pCLCrVA和pCLCrVB农杆菌浸润的棉花植株。 0 d:停水处理的开始; 35 d:停水处理后35 d;对照:正常生长条件。 B.棉花植株35 d后停水处理的存活率。对照:正常生长条件;干旱:35 d后停水处理;生存值以三次生物学重复的平均值±SE表示,每次重复8株植物(**

<0.01; - 测试)。


图2.在停水处理后14天的棉花叶的粗蛋白/酶提取 A.用研钵和杵在液氮中研磨叶组织。 B.用PBS缓冲液匀浆叶粉末。 C.离心前离心管中的组织匀浆。 D.离心后的上清液和沉淀

图3.用分光光度计ScanDrop 250进行蛋白质定量。 A.启动机器ScanDrop 250及其软件FlashSoft Pro。 B.在模块窗口中选择"标准MP"测量位置和"生物方法"模块,设置"闪光次数"(3),"累积次数"(10)并激活"暗校正"。 C.选择"串行测量",在标准MP窗口中的"周期"下输入样品数。 D.通过勾选"Bio"窗口中的框,激活所需的公式Warburg-Christian(蛋白质),然后按启动按钮。 E.根据说明插入用于参考的比色皿(100mM PBS缓冲液,pH = 7.8)。 F.根据说明测量粗溶液的吸光度。操作的关键点用红色圆圈标记。


图4.在520nm处的吸光度与L-脯氨酸含量之间的标准曲线。溶解于L-赖氨酸中的L-脯氨酸(0,5,10,15,20,25和30μg)的系列含量将0.5ml ddH 2 O加入1ml反应溶液中以测量它们在520nm处的吸光度。在520nm处的吸光度值和0-30μg的L-脯氨酸含量之间观察到线性回归(R sup 2 = 0.9935)。这些值是两次独立实验的平均值。


图5.在干旱胁迫下在GbMYB5-沉默棉中的代谢物和酶活性的分析。在GbMYB5-沉默棉中,脯氨酸含量降低干旱胁迫。 B.在干旱胁迫下,在GbMYB5 - 沉默棉中MDA含量增加。在干旱胁迫下,GbMYB5沉默棉花中SOD(C),POD(D),CAT(E)和GST(F)的活性降低。 CK:用空载体pCLCrVA和pCLCrVB农杆菌浸润的棉花植株; VIGS:用载体pCLCrVA- GbMYB5 和pCLCrVB进行农杆菌渗透的棉花植株,以使GbMYB5 基因沉默。值是生物学重复的平均值±SE(n≥5)(* P <0.05; ** <0.01 P <0.01;测试)。


图6.在470nm处的吸光度与反应时间之间的线性回归。在15秒的间隔和高达150秒内自动收集POD的吸光度值。蓝线和红线分别表示470nm处POD的吸光度和150秒或60-75秒内的反应时间之间的线性回归。 4个POD样品(4个子图)的测试显示在470nm处的吸光度与反应时间之间的最佳线性回归在75秒内。


食谱

注意:在使用之前,应使用pH计来验证PBS缓冲液的pH值。

  1. 1/2 MS溶液
    将2.47g 1/2 MS介质盐溶解在1,000ml ddH 2 O中
  2. 200mM Na 2 HPO 4储备溶液 在1000ml ddH 2 O中溶解53.65g Na 2 HPO 4 7H 2 O 2·6H 2 O·/ 在使用
    前保存在4°C
  3. 200mM NaH 2 PO 4储备溶液
    在1000ml ddH 2 O中溶解27.8g NaH 2 PO 4 H·H 2 O·
    在使用
    前保存在4°C
  4. 100mM PBS(pH7.0)
    61ml 200mM Na 2 HPO 4储液
    39ml 200mM NaH 2 PO 4储备溶液
    100毫升ddH 2 O 2 /
  5. 2.5%酸 - 茚醇
    将1.250g茚三酮溶于30ml乙酸和20ml 100mM PBS(pH7.0)中,在70℃水浴中用sterist溶解。
    在使用
    前保存在4°C
  6. 3%磺基水杨酸 将3.496g 5-磺基水杨酸二水合物溶解在100ml ddH 2 O中
  7. 10%TCA
    将10克TCA溶解在100毫升ddH 2 O中
  8. 0.25%TBA
    将0.125g TBA溶解在5ml 1mol/L NaOH中
    加入45ml 10%TCA
    在使用
    前保存在4°C
  9. 100mM PBS(pH7.8)
    91.5ml 200mM Na 2 HPO 4储液
    8.5ml 200mM NaH 2 PO 4储备溶液
    100毫升ddH 2 O 2 /
  10. 1mM EDTA-2Na
    将0.037g EDTA-2Na溶解在100ml ddH 2 O中
  11. 130mM甲硫氨酸 将0.970g甲硫氨酸溶解在50ml 100mM PBS(pH7.8)中
    在使用
    前保存在4°C
  12. 750μMNBT
    将0.031g NBT溶于50ml 100mM PBS(pH7.8)中
    保持阴暗
  13. 20μM核黄素
    将0.007g核黄素在100ml ddH 2 O中溶解 保持阴暗
  14. 0.2%愈创木酚 将0.1g愈创木酚溶于0.5ml乙醇中 加入50ml 100mM PBS(pH 7.0)中 保持阴暗
  15. 100mM PBS(pH 6.5)
    31.5ml 200mM Na 2 HPO 4储液
    68.5ml 200mM NaH 2 PO 4储备液
    100毫升ddH 2 O 2 /
  16. 5 mM GSH
    将0.077g GSH溶于50ml 100mM PBS(pH6.5)中
  17. 1.5mM CDNB
    将0.015g CDNB溶解在5ml乙醇中 加入45ml 100mM PBS(pH6.5)中

致谢

该协议改编自研究文章:Chen 。 (2015)。 这项工作得到了中国国家自然科学基金[资助No.31371930]的支持。 国家科技重大转基因育种重大项目,中国[批准号:204ZX0800501B]; 江苏省农业科学自主创新,中国[补编No.CX(15)1005]。

参考文献

  1. Chen,T.,Li,W.,Hu,X.,Guo,J.,Liu,A.and Zhang,B。(2015)。  棉花MYB转录因子,GbMYB5 积极参与植物对干旱胁迫的适应性反应。 植物细胞生理学 56(5):917-929。
  2. Gao,X.,Wheeler,T.,Li,Z.,Kenerley,CM,He,P.and Shan,L。(2011)。  沉默 GhNDR1 和 GhMKK2 会损害棉花对轮枝孢的抗性。 em> Plant J 66(2):293-305。
  3. Gu,Z.,Huang,C.,Li,F.and Zhou,X.(2014)。  用于棉花中基因和微小RNA的功能分析的通用系统。 Plant Biotechnol J 12(5):638-649。 >
  4. Simonian,MH和Smith,JA(2006)。  分光光度法 和蛋白质浓度的比色测定。 Curr Protoc Mol Biol 第10章:单位10 11A。
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引用:Chen, T. and Zhang, B. (2016). Measurements of Proline and Malondialdehyde Content and Antioxidant Enzyme Activities in Leaves of Drought Stressed Cotton. Bio-protocol 6(17): e1913. DOI: 10.21769/BioProtoc.1913.
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Muhammad Sameeullah
Institute of Plant Science and Resources
Dear Baolong Zhang,
Under recipes, for the preparation of 3% sulphosalicylic acid Dissolve 3.496 g 5-sulphosalicylic acid dihydrate in 100 ml ddH2O.
My question is why 3.496g 5-sulphosalicylic acid dihydrate is required to make 3%
5/30/2017 1:42:03 AM Reply