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Detection of Reactive Oxygen Species in Oryza sativa L. (Rice)
水稻中活性氧的检测   

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Abstract

Superoxide ions (O2-) and hydrogen peroxide (H2O2) are the reactive oxygen species (ROS) that play a significant role in regulation of many plant processes. The level of O2- ions is determined qualitatively using nitrobluetetrazolium (NBT) assay while the H2O2 is qualitatively estimated using 3,3-diaminobenzidine (DAB) and 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA) assay. Further the aqueous content of H2O2 is estimated quantitatively using ferrous oxidation-xylenol orange (FOX) assay.

Keywords: Rice(水稻), Reactive oxygen species(活性氧), Superoxide(超氧化物), Hydrogen peroxide(过氧化氢), Nitrobluetetrazolium(四氮唑蓝), 3,3-diaminobenzidine(3,3-二氨基联苯胺)

Background

Superoxide ions (O2-) and hydrogen peroxide (H2O2) are the vital reactive oxygen molecules that play a central role in many processes involved in plant growth and development including abiotic stress tolerance. To get better insights into the ROS mediated regulation of these processes, qualitative and quantitative estimation of different types of ROS is of significant importance. O2- is produced by the transfer of electrons from NADPH to oxygen (O2) mediated by the NADPH oxidase enzyme system. These ions are estimated in rice seedlings using NBT assay which is based upon the principle of reduction of yellow coloured NBT into dark blue coloured insoluble formazan by O2- (Kaur et al., 2016).

H2O2 is another reactive oxygen molecule that acts as an important signaling molecule regulating different plant processes. The content of H2O2 is estimated qualitatively in rice seedlings using DAB and H2DCFDA assay (Kaur et al., 2016). DAB assay is based upon the principle of formation of deep brown polymerization product on the reaction of DAB with H2O2 while H2DCFDA assay is based upon the principle of fluorescent microscopy. When non fluorescent H2DCFDA binds to ROS (predominantly H2O2), it gets converted into highly fluorescent 2’,7’-dichlorofluorescein (DCF). DCF gives a green coloured fluorescence when excited with a laser beam of excitation 488 nm using confocal microscope. Further, the quantitative measurement of aqueous H2O2 is carried out using ferrous oxidation-xylenol orange (FOX) method (Kaur et al., 2016). FOX assay is based upon the principle of oxidation of ferrous ions by H2O2 to ferric ions. Ferric ions then bind with xylenol orange to give a coloured complex having absorption maxima at 560 nm.

Materials and Reagents

  1. Petri dish (35 mm) (Tarsons, catalog number: 460035 )
  2. Microscopic glass slides
  3. Glue or nail enamel
  4. Whatman filter paper No.1 (Thermo Fisher Scientific, Fisher Scientific, catalog number: 09-805 )
  5. Amber Eppendorf (Capacity: 2 ml) (Tarsons, catalog number: 500013 )
  6. Glass tubes
  7. Tubes (Capacity:15 ml and 50 ml) (Tarsons, catalog numbers: 546021 [15 ml]; 546041 [50 ml])
  8. Disposable cuvettes (Sigma-Aldrich, catalog number: Z330361 )
    Note: This product has been discontinued.
  9. Leaf of 14 days old fresh rice seedlings
  10. Tri-sodium citrate dihydrate (HiMedia Laboratories, catalog number: RM1415 )
  11. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  12. Absolute ethanol
  13. Activated charcoal (HiMedia Laboratories, catalog number: PCT1001 )
  14. Trichloroacetic acid (Sigma-Aldrich, catalog number: T6399 )
  15. Liquid nitrogen
  16. 30 % hydrogen peroxide solution (H2O2) (Sigma-Aldrich, catalog number: H1009 )
  17. Hydrochloric acid (HCl) (Molychem, catalog number: 23540 )
  18. Diaminobenzidine (DAB) (Sigma-Aldrich, catalog number: D8001 )
  19. Nitrobluetetrazolium (NBT) (HiMedia Laboratories, catalog number: MB107 )
  20. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 1310-73-2 )
  21. 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA) (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: D399 )
  22. Dimethyl sulphoxide (Minimum assay: 99.0%) (S D Fine-Chem, catalog number: 38216 )
  23. Ammonium ferrous sulfate (HiMedia Laboratories, catalog number: GRM1026 )
  24. Sulfuric acid (HiMedia Laboratories, catalog number: AS016 )
  25. Xylenol orange (LobaChemie, catalog number: 06507 )
  26. Methanol (HPLC grade) (Minimum Assay: 99.7%) (HiMedia Laboratories, catalog number: AS061 )
  27. Butylated hydroxytoluene (HiMedia Laboratories, catalog number: GRM797 )
  28. NBT solution (see Recipes)
  29. DAB solution (see Recipes)
  30. H2DCFDA solution (see Recipes)
  31. Ferrous oxidation-xylenol orange (FOX) reagent (see Recipes)
  32. Standard H2O2 solutions (see Recipes)

Equipment

  1. Pipette (Corning, model: Lambda Plus)
  2. Vacuum infiltration equipment (Dessicator connected to vacuum pump) (Vacuum pump: Rocker 300 , Rocker Scientific, model: Rocker 300]; Dessicator vacuum: tarsons 403010 [Tarsons, model: 403010 ] )
  3. Water bath (Polyscience, model: WB02S )
  4. Stereomicroscope (Olympus, model: SZ61 )
  5. Confocal microscope (Nikon A1R, Laser scanning confocal microscope system)
  6. Centrifuge (REMI, model: C-24 PLUS )
  7. Spectrophotometer (PerkinElmer, model: Lambda 25 )
    Note: This product has been discontinued.
  8. Weighing balance (Citizen Scale, model: CY220 )
  9. pH meter (Systronics, model: µ361 )
  10. Pestle mortar

Procedure

  1. NBT assay (For detection of O2-) (Video 1)

    Video 1. Procedure for qualitative estimation of superoxide ions (O2-) using NBT assay in rice

    1. Cut the second leaf of rice seedlings at the base of stem into small pieces (approximately 1 cm) (Figure 1).


      Figure 1. Pieces of rice seedlings approximately 1 cm in length

    2. Dip the cut pieces immediately into 6 mM NBT solution (2 ml) prepared in sodium citrate (pH 6.0) in a Petri dish (35 mm) using a tweezer.
    3. Vacuum infiltrate the dipped samples for 10 min at 60 KPa pressure and then incubate at room temperature for 10 min under room light.
      Note: If vacuum infiltration unit is not available, dip and incubate the samples for 8 h at room temperature under light.
    4. After incubation, dip the samples in absolute ethanol and then keep them in a water bath (100 °C) till the chlorophyll is removed from the samples completely (usually occurs in 2 h) (Figure 2).


      Figure 2. Pieces of rice seedlings approximately 1 cm in length after removal of chlorophyll

    5. Cool and dip the samples in 20% glycerol.
    6. Capture the images using a stereomicroscope by keeping the samples on a slide.
    7. Appearance of dark blue colour indicates the presence of O2- (Figure 3).

  2. DAB assay (For detection of H2O2)
    1. Cut the second leaf of rice seedlings at the base of stem into small pieces (approximately 1 cm).
    2. Dip the cut pieces immediately into DAB solution (1 mg/ml) prepared in double distilled water (2 ml) (pH = 3.8) in a Petri dish (35 mm) using tweezers.
    3. Vacuum infiltrate the dipped samples for 10 min at 60 KPa pressure and then incubate at room temperature for 10 min under room light.
      Note: If vacuum infiltration unit is not available, dip and incubate the samples for 8 h at room temperature under light.
    4. After incubation, dip the samples in the absolute ethanol and then keep them in a water bath (100 °C) till the chlorophyll is removed from the samples completely (usually occurs in 2 h).
    5. Cool and dip the samples in 20% glycerol.
    6. Capture the images using a stereo microscope by keeping the samples on a slide.
    7. Appearance of brown coloured product indicates the presence of H2O2 (Figure 4).

  3. H2DCFDA assay (For detection of H2O2)
    1. Cut the second leaf of rice seedlings at the base of stem into small pieces (approximately 1 cm).
    2. Dip the cut pieces immediately into 10 µM H2DCFDA solution in a Petri dish (35 mm) using tweezers.
    3. Vacuum infiltrate the dipped samples for 5 min at 60 KPa pressure and then incubate at room temperature for 10 min in the dark.
      Note: If vacuum infiltration unit is not available, dip the samples for 2 h at room temperature.
    4. After incubation, wash the samples three times properly with autoclaved double distilled water.
    5. Dip the samples in 20% glycerol after washing.
    6. For confocal analysis, put the sample on a slide and fix a thin cover slip over it using nail enamel or glue.
    7. Observe the samples under a confocal microscope using laser beam of wavelength of excitation 488 nm.
    8. Green fluorescence indicates the presence of H2O2 and red represents the autofluorescence of chlorophyll.

  4. Quantification of aqueous H2O2 content
    1. Homogenize 0.5 g fresh seedlings in activated charcoal (0.1 g) prepared in 5 ml of 5% trichloroacetic acid using a pestle and mortar with liquid nitrogen.
    2. Filter the homogenate using a Whatman filter (No.1) and collect the filtrate in 2 ml amber Eppendorf tubes (H2O2 is light sensitive).
    3. Centrifuge the filtrate at 5,000 x g for 10 min in the tubes.
      Note: Either centrifuge the filtrate from one sample in two 2 ml Eppendorf tubes and then pool the supernatant together or use a 5 ml amber Eppendorf tube.
    4. Collect 0.2 ml of the supernatant in a glass tube and add 1 ml FOX reagent to it.
    5. Mix the reaction mix properly and incubate at room temperature for 15 min.
    6. Estimate the content of aqueous H2O2 by recording the absorbance at 560 nm using a disposable plastic cuvette and calculate the concentration using a standard curve.
    7. The concentration is calculated using the beer-lambert law, i.e.
                  A = ε x b x c
      Where,
      A = absorbance,
      ε is the wavelength-dependent molar absorptivity coefficient,
      b = path length,
      c = concentration.
    8. For the standard curve preparation, take 0.2 ml of different concentrations of H2O2, add 1 ml of FOX reagent and proceed as similar to the samples (see Recipe 5).
      Note: The absorbance should be taken at 560 nm wavelength.

Data analysis

  1. NBT assay
    Observation: Appearance of dark blue colour indicates presence of O2- (Figure 3).


    Figure 3. Stereomicroscope image of NBT assay of rice leaf showing the presence of superoxide ions (O2-) indicated by presence of blue coloured formazan. A. Negative control; B. Rice sample showing the presence of superoxide ions (O2-).

  2. DAB assay
    Observation: Appearance of brown coloured product indicates presence of H2O2 (Figure 4).


    Figure 4. Stereomicroscope image of DAB assay of rice leaf showing the presence of hydrogen peroxide (H2O2) indicated by presence of brown coloured product. A. Negative control; B. Rice sample showing the presence of Hydrogen peroxide (H2O2).

  3. H2DCFDA assay
    Observation: Green fluorescence indicates presence of H2O2 and red signifies autofluorescence of chlorophyll (Figure 5).


    Figure 5. Confocal H2DCFDA staining images. 1. Red colour indicates chlorophyll; 2. Green colour indicates ROS predominantly hydrogen peroxide; 3. Overlay of ROS and chlorophyll.

Notes

  1. DAB will dissolve in double distilled water only at acidic pH (pH = 3.8), so set the pH of water with HCl before adding DAB. It will take a little longer for dissolution.
  2. During H2DCFDA assay, samples should be minimally exposed to light.
  3. After H2DCFDA exposure, proceed immediately for confocal microscopy.
  4. All solutions for aqueous hydrogen peroxide estimation should be prepared fresh and should be used within 2 h.

Recipes

  1. NBT solution
    6 mM NBT prepared in 10 mM of sodium citrate (pH = 6)
    Adjust the pH using either 1 N HCl or 1 N NaOH
  2. DAB solution
    1 mg/ml DAB solution prepared in autoclaved double distilled water (pH = 3.8)
    Adjust the pH using 1 N HCl
  3. H2DCFDA solution
    Prepare 10 mM of H2DCFDA in dimethyl sulphoxide (DMSO)
    Dilute it to 10 µM using autoclaved double distilled water
  4. Ferrous oxidation-xylenol orange (FOX) reagent (see Table 1)
    Prepare the following stock solutions:
    Reagent a: 25 mM ammonium ferrous sulfate prepared in 2.5 M sulfuric acid
    Reagent b: 0.25 M xylenol orange prepared in HPLC-grade methanol
    Reagent c: 9.69 mg of butylated hydroxytoluene prepared in 90 ml of HPLC-grade methanol

    Table 1. Preparation of FOX reagent
    Stock Solutions
    Volume
    Reagent a
    1 ml
    Reagent b
    50 µl
    Reagent c
    90 ml
    Autoclaved double distilled water
    8.95 ml
    Total
    100 ml

  5. Standard H2O2 solutions
    1. 5 % trichloroacetic acid (TCA) solution
      Dissolve 5 g of TCA in 100 ml of autoclaved double distilled water
    2. 100 µM H2O2 stock solution in 5% trichloroacetic acid
    3. Standard H2O2 solutions (see Table 2)
      Notes:
      1. Different concentrations of H2O2 should be prepared in 5% trichloroacetic acid.
      2. The range of concentrations to be used for preparing standard curve depends upon the range of hydrogen peroxide content present in the given sample.

      Table 2. Preparation of standard H2O2 solutions
      Concentration
      of H2O2 (in µM)
      Volume of H2O2 to be
      used from stock (in µl)
      Volume of TCA to be used
      from stock (in µl)
      FOX reagent
      (in ml)
      BLANK
      0
      200
      1
      10
      20
      180
      1
      20
      40
      160
      1
      30
      60
      140
      1
      40
      80
      120
      1
      50
      100
      100
      1
      60
      120
      80
      1
      70
      140
      60
      1
      80
      160
      40
      1
      90
      180
      20
      1
      100
      200
      0
      1

Acknowledgments

This protocol has been adopted and modified from DeLong et al. (2003), Kristiasen et al. (2009) and Wu et al. (2010). Authors acknowledge the Department of Biotechnology (DBT), Government of India for financial support (Project No. BT/PR13965/BRB/10/883/2010).

References

  1. DeLong, J. M., Prange, R. K., Hodges, D. M., Forney, C. F., Bishop, M. C. and Quilliam, M. (2002). Using a modified ferrous oxidation-xylenol orange (FOX) assay for detection of lipid hydroperoxides in plant tissue. J Agr Food Chem 50(2): 248-254.
  2. Kaur, N., Dhawan, M., Sharma, I. and Pati, P. K. (2016). Interdependency of reactive oxygen species generating and scavenging system in salt sensitive and salt tolerant cultivars of rice. BMC Plant Biol 16(1): 131.
  3. Kristiansen, K. A., Jensen, P. E., Moller, I. M. and Schulz, A. (2009). Monitoring reactive oxygen species formation and localisation in living cells by use of the fluorescent probe CM-H(2)DCFDA and confocal laser microscopy. Physiol Plant 136(4): 369-383.
  4. Wu, G. L., Cui, J., Tao, L. and Yang, H. (2010). Fluroxypyr triggers oxidative damage by producing superoxide and hydrogen peroxide in rice (Oryza sativa). Ecotoxicology 19(1): 124-132.

简介

超氧化物离子(O 2 - )和过氧化氢(H 2 O 2 O 2)是起重要作用的活性氧(ROS)在许多植物过程的调节。使用硝基四氮唑(NBT)测定定性地测定O 2 - 离子的水平,同时使用3,3 - 二氨基联苯胺(DAB)和2',7'-二氯二氢荧光素二乙酸酯(H 2 N 2 DCFDA)测定。此外,使用亚铁氧化 - 二甲苯酚橙(FOX)测定定量地估计H 2 O 2 O 2的含水量。

背景 超氧化物离子和过氧化氢是在许多情况下起主要作用的活性氧分子涉及植物生长发育的过程包括非生物胁迫耐受性。为了更好地了解ROS对这些过程的调节,对不同类型ROS的定性和定量估计具有重要意义。通过由NADPH氧化酶系统介导的电子从NADPH转移到氧(O 2),产生O 2。使用NBT测定法在水稻幼苗中估计这些离子,其基于通过O将黄色NBT还原成深蓝色不溶性甲the的原理(Kaur等人,2016)。
  H 2是另一种作为调节不同植物过程的重要信号分子的活性氧分子。在使用DAB和H 2 DCFDA测定的水稻幼苗中定性地估计H 2 O 2 O 2的含量(Kaur等人,2016)。 DAB测定是基于DAB与H 2 O 2 O 2的反应形成深棕色聚合产物的原理,而H 2 N 2 DCFDA测定是基于荧光显微镜的原理。当非荧光H 2 DCFDA与ROS(主要是H 2 O 2 O 2)结合时,其被转化为高度荧光的2',7'-二氯荧光素(DCF)。当使用共焦显微镜激发488nm激光束时,DCF产生绿色荧光。此外,使用亚铁氧化 - 二甲苯酚橙(FOX)方法(Kaur等人)进行H 2 O 2 O 2水溶液的定量测量。 ,2016)。 FOX测定是基于将亚铁离子通过H 2 O 2氧化成三价铁离子的原理。然后铁离子与二甲酚橙结合,得到在560nm具有最大吸收的着色络合物。

关键字:水稻, 活性氧, 超氧化物, 过氧化氢, 四氮唑蓝, 3,3-二氨基联苯胺

材料和试剂

  1. 培养皿(35毫米)(Tarsons,目录号:460035)
  2. 显微镜玻璃片
  3. 胶或指甲油
  4. Whatman滤纸No.1(Thermo Fisher Scientific,Fisher Scientific,目录号:09-805)
  5. 琥珀Eppendorf(容量:2毫升)(Tarsons,目录号:500013)
  6. 玻璃管
  7. 管(容量:15ml和50ml)(Tarsons,目录号:546021 [15ml]; 546041 [50ml])
  8. 一次性比色皿(Sigma-Aldrich,目录号:Z330361)
    注意:本产品已停产。
  9. 14日龄新鲜水稻幼苗的叶子
  10. 柠檬酸三钠二水合物(HiMedia Laboratories,目录号:RM1415)
  11. 甘油(Sigma-Aldrich,目录号:G5516)
  12. 绝对乙醇
  13. 活性炭(HiMedia Laboratories,目录号:PCT1001)
  14. 三氯乙酸(Sigma-Aldrich,目录号:T6399)
  15. 液氮
  16. 30%过氧化氢溶液(H 2 O 2 O 2)(Sigma-Aldrich,目录号:H1009)
  17. 盐酸(HCl)(Molychem,目录号:23540)
  18. 二氨基联苯胺(DAB)(Sigma-Aldrich,目录号:D8001)
  19. 硝基四唑(NBT)(HiMedia Laboratories,目录号:MB107)
  20. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:1310-73-2)
  21. 2',7'-二氯二氢荧光素二乙酸酯(H 2 N 2 DCFDA)(Thermo Fisher Scientific,Molecular Probes TM,目录号:D399)
  22. 二甲基亚砜(最小测定:99.0%)(S D Fine-Chem,目录号:38216)
  23. 硫酸亚铁(HiMedia Laboratories,目录号:GRM1026)
  24. 硫酸(HiMedia Laboratories,目录号:AS016)
  25. 二甲苯酚橙(LobaChemie,目录号:06507)
  26. 甲醇(HPLC级)(Minimun测定:99.7%)(HiMedia Laboratories,目录号:AS061)
  27. 丁基化羟基甲苯(HiMedia Laboratories,目录号:GRM797)
  28. NBT解决方案(见配方)
  29. DAB解决方案(请参阅配方)
  30. H 2 2 DCFDA溶液(参见食谱)
  31. 亚铁氧化 - 二甲苯酚橙(FOX)试剂(见食谱)
  32. 标准H 2 O 2解决方案(参见食谱)

设备

  1. 移液器(康宁,型号:Lambda Plus)
  2. 真空渗透设备(与真空泵连接的Dessicator)(真空泵:Rocker 300,Rocker Scientific,型号:Rocker 300); Dessicator真空:tarsons 403010 [Tarsons,型号:403010])
  3. 水浴(Polyscience,型号:WB02S)
  4. 立体显微镜(Olympus,型号:SZ61)
  5. 共焦显微镜(Nikon A1R,激光扫描共焦显微镜系统)
  6. 离心机(REMI,型号:C-24 PLUS)
  7. 分光光度计(PerkinElmer,型号:Lambda 25)
    注意:本产品已停产。
  8. 称重平衡(公民规模,型号:CY220)
  9. pH计(Systronics,型号:μ361)
  10. 杵砂浆

程序

  1. NBT测定(用于检测O 2 - )(视频1)

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    视频1.使用NBT测定在水稻中定性估计超氧化物离子(O2-)的程序
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    1. 将茎的基部的水稻幼苗的第二叶切成小块(约1厘米)(图1)

      图1.长度约1厘米的水稻幼苗片

    2. 立刻蘸碎片 将样品冷却并浸入20%甘油中

    3. 将样本保留在幻灯片上,使用立体显微镜捕获图像
    4. 深蓝色的外观表示O 2 - 的存在(图3)。

  2. DAB测定(用于检测H 2 O 2 O 2)
    1. 将茎的基部的水稻幼苗的第二叶切成小块(约1厘米)
    2. 将切片立即放入使用镊子的培养皿(35mm)中在双蒸水(2ml)(pH = 3.8)中制备的DAB溶液(1mg/ml)中。
    3. 真空在60KPa压力下浸渍浸渍的样品10分钟,然后在室温下室温孵育10分钟。
      注意:如果没有真空浸渗装置,请在室温下轻轻浸泡并孵育样品8小时。
    4. 孵育后,将样品浸在无水乙醇中,然后将其保存在水浴(100℃)中,直到完全从样品中除去叶绿素(通常在2小时内)。
    5. 将样品冷却并浸入20%甘油中
    6. 使用立体显微镜拍摄图像,方法是将样本保存在幻灯片上。
    7. 褐色产品的外观表示存在H 2 O 2 O 2(图4)。

  3. H 2 O 2 DCFDA测定(用于检测H 2 O 2 O 2)
    1. 将茎的基部的水稻幼苗的第二叶切成小块(约1厘米)
    2. 使用镊子将切片立即浸入培养皿(35 mm)的10μMH 2 N 2 DCFDA溶液中。
    3. 真空在60KPa压力下浸渍浸渍样品5分钟,然后在室温下在黑暗中孵育10分钟。
      注意:如果没有真空渗透装置,请将样品在室温下浸泡2小时。
    4. 孵化后,用高压灭菌双蒸水将样品三次洗涤三次
    5. 洗涤后将样品浸入20%甘油。
    6. 对于共聚焦分析,将样品放在载玻片上,并使用指甲油或胶水将薄的盖子滑过其上
    7. 使用激光波长488 nm的激光束在共聚焦显微镜下观察样品
    8. 绿色荧光指示H 2 O 2 O 2的存在,红色表示叶绿素的自发荧光。

  4. 含水H 2 O 2含量的量化
    1. 用0.5ml 5%三氯乙酸制备的活性炭(0.1g)中的0.5g新鲜幼苗均匀,使用研杵和用液氮的研钵。
    2. 使用Whatman过滤器(1号)过滤匀浆物,并将滤液收集在2ml琥珀色的Eppendorf管中(H 2 O 2 O 2光敏)。
    3. 将滤液以5,000×g离心管中10分钟。
      注意:将两个2ml Eppendorf管中的一个样品的滤液离心,然后将上清液一起或使用5ml琥珀色的Eppendorf管。
    4. 在玻璃管中收集0.2ml上清液,并向其中加入1ml FOX试剂
    5. 混合反应混合物,室温孵育15分钟
    6. 通过使用一次性塑料比杯记录560nm处的吸光度并使用标准曲线计算浓度来估算H 2 O 2 O 2水溶液的含量。
    7. 浓度使用啤酒 - 朗伯定律计算,即。
                   A =εx b x c
      哪里,
      A =吸光度,
      ε是波长依赖的摩尔吸光系数,
      b =路径长度,
      c =浓度
    8. 对于标准曲线制备,取0.2ml不同浓度的H 2 O 2 O 2,加入1ml FOX试剂,并与样品相似(参见方案5) 。
      注意:吸光度应在560 nm波长下进行。

数据分析

  1. NBT分析
    观察:深蓝色的外观表示存在O 2 - (图3)。


    图3.水稻NBT测定的立体显微镜图像,显示存在蓝色甲状结晶表明的超氧化物离子(O 2 - )。 A.阴性对照; B.显示超氧化物离子存在的水稻样品(O 2 -
  2. DAB分析
    观察:褐色产品的外观表示H 2 O 2 O 2的存在(图4)。


    图4.显示存在由棕色产品表示的过氧化氢(H 2 O 2 O 2)的水稻的DAB测定的立体显微镜图像。强> A.负控制; B.显示过氧化氢(H 2 O 2 O 2)存在的水稻样品。

  3. H 2 DCFDA分析
    观察:绿色荧光表明存在H 2 O 2 O 2,红色表示叶绿素的自动荧光(图5)。


    图5.共聚焦H 2 DCFDA染色图像。 1.红色表示叶绿素;绿色表示ROS主要是过氧化氢; 3.覆盖ROS和叶绿素。

笔记

  1. DAB仅在酸性pH(pH = 3.8)下溶解在双蒸水中,所以在添加DAB之前,用HCl设定pH值。解散需要一点时间。
  2. 在H 2 O 2 DCFDA测定期间,样品应最低限度地暴露于光。
  3. 在二级DCFDA曝光后,立即进行共焦显微镜检查。
  4. 所有用于过氧化氢水溶液估算的溶液应该是新鲜的,应在2小时内使用

食谱

  1. NBT解决方案
    在10mM柠檬酸钠(pH = 6)中制备的6mM NBT
    使用1N HCl或1N NaOH调节pH值
  2. DAB解决方案
    在高压灭菌的双蒸水(pH = 3.8)中制备1mg/ml DAB溶液 使用1N HCl调节pH值
  3. H 2/2 DCFDA溶液
    在二甲基亚砜(DMSO)中制备10mM H 2 DCFDA
    使用高压蒸馏双蒸水将其稀释至10μM
  4. 亚铁氧化 - 二甲苯酚橙(FOX)试剂(见表1)
    准备以下股票解决方案:
    试剂a:在2.5M硫酸中制备的25mM硫酸亚铁铵// 试剂b:在HPLC级甲醇中制备的0.25M二甲苯酚橙 试剂c:在90ml HPLC级甲醇中制备9.69mg丁基化羟基甲苯
    表1. FOX试剂的制备
    库存解决方案

    试剂一个
    1 ml
    试剂b
    50μl
    试剂c
    90 ml
    高压灭菌双蒸水
    8.95 ml
    总计
    100 ml

  5. 标准H 2 O 2 解决方案
    1. 5%三氯乙酸(TCA)溶液
      将5g TCA溶于100ml的高压灭菌双蒸水中
    2. 在5%三氯乙酸中的100μMH 2 O 2种储备溶液
    3. 标准H 2 O 2溶液(参见表2)
      注意:
      1. 不同浓度的H 2 O 2 O 2应该在5%三氯乙酸中制备。
      2. 用于制备标准曲线的浓度范围取决于给定样品中存在的过氧化氢含量的范围。

      表2.标准H 2 O 2 的制备 sub> 解决方案
      集中
      2 2 (以μM计)
      H 2 O 2
      从库存(以μl计)使用
      要使用的TCA的数量
      从库存(以磅计)
      FOX试剂
      (以ml为单位)
      空白
      0
      200
      1
      10
      20
      180
      1
      20
      40
      160
      1
      30
      60
      140
      1
      40
      80
      120
      1
      50
      100
      100
      1
      60
      120
      80
      1
      70
      140
      60
      1
      80
      160
      40
      1
      90
      180
      20
      1
      100
      200
      0
      1


致谢

该协议已经从DeLong等人采用和修改。 (2003),Kristiasen等人。 (2009)和Wu等人。 (2010)。作者承认印度政府生物技术部(DBT)的财政支持(项目号:BT/PR13965/BRB/10/883/2010)。

参考文献

  1. DeLong,JM,Prange,RK,Hodges,DM,Forney,CF,Bishop,MC和Quilliam,M。(2002)。  使用修饰的亚铁氧化二甲苯酚橙(FOX)测定法检测植物组织中的脂质氢过氧化物。 em> 50(2):248-254。
  2. Kaur,N.,Dhawan,M.,Sharma,I。和Pati,PK(2016)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/peter/27286833"target ="_ blank">水稻盐敏感和耐盐品种中活性氧产生和清除系统的相互依赖关系。BMC Plant Biol 16(1):131。
  3. Kristiansen,KA,Jensen,PE,Moller,IM and Schulz,A。(2009)。  通过使用荧光探针CM-H(2')DCFDA和共聚焦激光显微镜监测活细胞中活性氧的形成和定位。 Physiol Plant 136(4):369-383。
  4. Wu,GL,Cui,J.,Tao,L.and Yang,H。(2010)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/普罗米特/19644752"target ="_ blank">氟吡虫啉通过在水稻(Oryza sativa)中产生超氧化物和过氧化氢来触发氧化损伤。生态毒理学 19(1) :124-132。
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引用:Kaur, N., Sharma, I., Kirat, K. and Pati, P. K. (2016). Detection of Reactive Oxygen Species in Oryza sativa L. (Rice). Bio-protocol 6(24): e2061. DOI: 10.21769/BioProtoc.2061.
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