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
发布: 2016年09月05日第6卷第17期 DOI: 10.21769/BioProtoc.1913 浏览次数: 29125
评审: Tie LiuHideyuki TakahashiZhanwu Dai
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参见作者原研究论文
The authors used this protocol in:
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
- Centrifuge tubes (1.5 ml) (Corning, Axygen®, catalog number: MCT-150-C-S )
- Gossypium barbadense seeds
- Soil mix (vermiculite:humus = 1:1) (http://www.xing-nong.cn/ProductList.Asp?SortID=207)
- 1/2 MS media salt without sugar and agar (Hopebio, catalog number: HB8469-12 )
- Sodium phosphate dibasic anhydrous (Na2HPO4) (Sinopharm Chemical Reagent, catalog number: 20040618 )
- Sodium phosphate monobasic anhydrous (NaH2PO4) (Sinopharm Chemical Reagent, catalog number: 20040818 )
- ddH2O
- Liquid nitrogen
- Ice
- Ninhydrin (C9H4O3·H2O) (Sinopharm Chemical Reagent, catalog number: 30130212 )
- Trichloroacetic acid (TCA) (Sinopharm Chemical Reagent, catalog number: 80132618 )
- 5-sulphosalicylic acid dihydrate (Sinopharm Chemical Reagent, catalog number: 10021516 )
- L-proline (Genview, catalog number: FL259 )
- Thiobarbituric acid (TBA) (Sigma-Aldrich, catalog number: T5500 )
- Sodium hydroxide (NaOH) (Sinopharm Chemical Reagent, catalog number: 10019762 )
- EDTA-2Na (Sigma-Aldrich, catalog number: 27285 )
- L-methionine (Sigma-Aldrich, catalog number: V900487 )
- Nitroblue tetrazolium (NBT) (Beijing Dingguo Changsheng Biotechnology, catalog number: JN213 )
- Riboflavin (Sigma-Aldrich, catalog number: 47861 )
- Guaiacol (Sigma-Aldrich, catalog number: G5502 )
- 30% hydrogen peroxide (H2O2) (Sinopharm Chemical Reagent, catalog number: 10011208 )
- Ethanol (Sinopharm Chemical Reagent, catalog number: 10009259 )
- Glutathione reduced (GSH) (Sigma-Aldrich, catalog number: G4251 )
- 1-chloro-2, 4-dinitrobenzene (CDNB) (Sigma-Aldrich, catalog number: C38702 )
- Acetic acid (Sinopharm Chemical Reagent, catalog number: 10000208 )
- 1/2 MS solution (see Recipes)
- Na2HPO4 stock solution (see Recipes)
- NaH2PO4 stock solution (see Recipes)
- PBS (pH 7.0) (see Recipes)
- 2.5% acid-ninhydrin (see Recipes)
- 3% sulphosalicylic acid (see Recipes)
- 10% TCA (see Recipes)
- 0.25% TBA (see Recipes)
- PBS (pH 7.8) (see Recipes)
- 1 mM EDTA-2Na (see Recipes)
- 130 mM methionine (see Recipes)
- 750 μM NBT (see Recipes)
- 20 μM riboflavin (see Recipes)
- 0.2% guaiacol (see Recipes)
- PBS (pH 6.5) (see Recipes)
- 5 mM GSH (see Recipes)
- 1.5 mM CDNB (see Recipes)
Equipment
- Plant growth chamber (25 °C, 16 h/8 h photoperiod, 10,000 lux, 50-60% humidity)
- Mortar and pestle
- Plastic pots (8 cm diameter and 12 cm depth) (http://www.lyyyb.com/yingyangbo.html)
- Electronic balance (Sartorius, model: BAS124S-CW )
- ddH2O purification system (Xiamen RSJ Scientific Instruments, model: Spring-S10 )
- Ice making machine (Xueke, model: IMS-40 )
- Centrifuge (Eppendorf, model: 5810 R )
- Spectrophotometer (Analytik Jena, model: ScanDrop® 250)
- Water bath (Changzhou Aohua Instrument, model: HH-1 )
- Pipette, 2-20 µl (Eppendorf, Eppendorf Research® plus, model: 3120000038 )
- Pipette, 20-200 µl (Eppendorf, Eppendorf Research® plus, model: 3120000054 )
- Pipette, 100-1,000 µl (Eppendorf, Eppendorf Research® plus, model: 3120000062 )
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
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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分类
植物科学 > 植物生理学 > 非生物胁迫
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