水稻中活性氧的检测

Navdeep Kaur; Isha Sharma; Kamal Kirat; Pratap Kumar Pati

doi:10.21769/BioProtoc.2061

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Peer-reviewed

Detection of Reactive Oxygen Species in Oryza sativa L. (Rice)

水稻中活性氧的检测

NK Navdeep Kaur

IS Isha Sharma

KK Kamal Kirat

PP Pratap Kumar Pati email

发布: 2016年12月20日第6卷第24期 DOI: 10.21769/BioProtoc.2061 浏览次数: 22394

评审: Pooja SaxenaAnonymous reviewer(s)

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Abstract

Superoxide ions (O^2-) and hydrogen peroxide (H₂O₂) are the reactive oxygen species (ROS) that play a significant role in regulation of many plant processes. The level of O^2- 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 (O^2-) 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. O^2- 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 O^2- (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

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

Equipment

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

Procedure

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如何引用

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.