Experimental principle Malondialdehyde (MDA) is a commonly used indicator of membrane lipid peroxidation. Under acidic and high temperature conditions, it can react with thiobarbituric acid (TBA) to produce reddish brown trimethine (3,5,5- Trimethyl oxazole 2,4-dione), its maximum absorption wavelength is 532nm. However, the determination of MDA in plant tissues is disturbed by a variety of substances, the most important of which is soluble sugar. The maximum absorption wavelength of the color reaction product of sugar and TBA is 450 nm and 532 nm. Soluble sugar increases when plants are subjected to stresses such as drought, high temperature, and low temperature. Therefore, when measuring the content of MDA-TBA reaction substances in plant tissues, the interference of soluble sugar must be excluded. The low concentration of iron ions can significantly increase the extinction value of the coloring reactants of TBA and sucrose or MDA at 532 and 450nm, so a certain amount of iron ions is required in the color reaction of sucrose, MDA and TBA, usually iron ions in plant tissues The content is 100 ～ 300μg / g DW, and the final concentration of added Fe3 + is 0.5μmol / L according to the plant sample volume and the volume of the extract.
A. Linear regression method
The extinction value of the color reaction product of MDA and TBA at 450nm wavelength is zero. Different concentrations of sucrose (0 ～ 25mmol / L) and the TBA color reaction product's extinction value at 450nm are positively correlated with the difference between the extinction values ​​at 532nm and 600nm. After preparing a series of concentrations of sucrose and TBA color reaction, Measure the extinction values ​​of the above three wavelengths, find the straight line equation, and calculate the extinction value of sugar at 532nm. The straight line equation of UV-120 ultraviolet-visible spectrophotometer is:
Y532 = ï¹£0.00198 + 0.088D450 â‘
B. Two-component spectrophotometer
According to Lambert-Beer law: D = kCL, when the thickness of the liquid layer is 1 cm, k = D / C, k is called the specific absorption coefficient of the substance. When there are several kinds of light-absorbing substances in a solution, the extinction value at a certain wavelength is equal to the sum of the extinction degrees of the color-developing substances of this mixed solution at that wavelength.
It is known that the specific absorption coefficients of the color reaction products of sucrose and TBA at the wavelengths of 450 nm and 532 nm are 85.40 and 7.40, respectively. MDA has no absorption at the wavelength of 450nm, so the specific absorption coefficient at this wavelength is 0, and the specific absorption coefficient at the wavelength of 532nm is 155. According to the two-component spectrophotometer method, an equation system is established, and the equation is calculated by solving the equation:
C1 (mmol / L) = 11.71D450 â‘¡
C2 (μmol / L) = 6.45 (D532-D600) -0.56D450 ③
Where C1- is the concentration of soluble sugar;
C2- is the concentration of MDA;
D450, D532, and D600 represent the extinction values ​​at the wavelengths of 450nm, 532nm, and 600nm, respectively.
Experimental reagent 10% trichloroacetic acid (TCA); 0.6% thiobarbituric acid, first add a small amount of sodium hydroxide (1mol / L) to dissolve, and then use 10% trichloroacetic acid to make volume; quartz sand.
Experimental equipment: 1 ultraviolet-visible spectrophotometer; 1 centrifuge; 1 electronic balance; 4 10ml centrifuge tubes; 2 sets of mortars; 4 test tubes; graduated straws: 1 10ml, 2ml 1; 1 scissors.
Experimental materials Plant leaves or aging plant organs subjected to drought, high temperature and low temperature stress 1. Experimental materials Plant leaves or aging plant organs subjected to drought, high temperature and low temperature stress
2. Extraction of MDA Weigh 1g of the chopped test material, add 2ml of 10% TCA and a small amount of quartz sand, grind to homogenate, add 8ml of TCA to further grind, centrifuge (4000 × g) for 10min, the supernatant is Sample extract.
3. Color reaction and determination Pipette 2ml of centrifuged supernatant (control plus 2ml of distilled water), add 2ml of 0.6% TBA solution, and react the mixture on a boiling water bath for 15min. After cooling quickly, centrifuge again. The supernatant was taken to measure the extinction at 532nm, 600nm and 450nm wavelengths.
4. Calculate the content
(1) The linear equation method calculates the extinction value Y532 of the sugar in the sample at 532nm according to the formula â‘ , subtract the extinction value of 600nm non-specific absorption by the measured extinction value of 532nm and subtract Y532, and the difference is determined The extinction value of the MDA-TBA reaction product in the sample at 532nm. The MDA concentration in the extract was calculated by converting the extinction coefficient of MDA at 532 nm to 155.
(2) The two-component spectrophotometric method can directly obtain the concentration of MDA in the plant sample extract according to formula â‘¢.
Determine the MDA concentration by any of the above methods, and calculate the MDA content in the sample based on the weight of the plant tissue:
MDA (μmol / g FW) = (MDA concentration (umol / L) * extract volume (ml))) / fresh weight of plant tissue (g)
Note 1. 0.1-0.5% trichloroacetic acid is more suitable for MDA-TBA reaction, if it is higher than this concentration, the non-specific absorption of the reaction solution is high;
2. The heating time of MDA-TBA color reaction is best controlled between 10-15min in boiling water bath. If the time is too short or too long, it will cause the light absorption value at 532nm to decrease;
3. If the liquid to be tested is turbid, the centrifugal force and time can be increased appropriately. It is best to use a low-temperature centrifuge.
4. Low concentration of iron ions can enhance the color reaction between MDA and TBA. When the concentration of iron ions in plant tissue is too low, Fe3 should be supplemented (final concentration is 0.5 nmol·L-1)
5. The product of the color reaction between soluble sugar and TBA also absorbs at 532 nm (the maximum absorption is at 450 nm). When the plant is under drought, high temperature, low temperature and other adversities, the soluble sugar content will increase, and if necessary, the interference of soluble sugar should be excluded.
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