Under normal conditions, the free proline content of plants is very low, but when encountering adversities such as drought, low temperature, salinity and alkalinity, free proline accumulates in large quantities, and the accumulation index is related to the plant's stress tolerance. Therefore, proline can be used as a biochemical indicator of plant stress resistance. In this experiment, we will study the principle and method of free proline determination.
Principle
The basic principle of the determination of free proline in plants is to use sulfosalicylic acid to extract free proline from plants, which not only greatly reduces the interference of other amino acids, but also is fast and simple, and is not limited by the state of the sample (dry or fresh). Under acidic conditions, proline reacts with lycopene to form a stable red condensate, which has a maximum absorption peak at 520 nm after extraction with toluene. The concentration of proline is proportional to its absorbance within a certain range.
Operation method
Determination of free proline content in plants
Principle
The basic principle of the determination of free proline in plants is to use sulfosalicylic acid to extract free proline from plants, which not only greatly reduces the interference of other amino acids, but also is fast and simple, and is not limited by the state of the sample (dry or fresh). Under acidic conditions, proline reacts with lycopene to form a stable red condensate, which has a maximum absorption peak at 520 nm after extraction with toluene. The concentration of proline is proportional to its absorbance within a certain range.
Materials and Instruments
Materials: Plant leaves, including fresh and wilted leaves. Move The basic procedure for the determination of free proline content in plants can be divided into the following steps: （1） Take 7 stoppered graduated test tubes and add each reagent according to Table 45-1. After mixing, add a glass stopper and heat in boiling water for 40 min.(2) After removing and cooling, add 5 mL of toluene to each tube and shake well to extract the red color. Let it stand and wait for stratification, then absorb the toluene layer, and use tube No. 1 as the control to extract the red color. The toluene layer was aspirated and colorimetrically measured at 520 nm using tube No. 1 as a control.(3) Plot the standard curve with absorbance value as the vertical coordinate and proline content as the horizontal coordinate, and find the linear regression equation.Table 45-1 Amount of reagent added to each test tube （1） Proline extraction: Take 0.2~0.5 g of cut and mixed wheat leaves from different treatments (dry samples should be reduced according to the moisture content), put them into large test tubes, add 5 mL of 3% nitrosalicylic acid solution, cover the mouth of the tube with a glass ball, and extract them for 10 min in a boiling water bath.(2) Determination: Take out the test tube, after cooling to room temperature, suck up 2 mL of supernatant, add 2 mL of glacial acetic acid and 3 mL of acid station triton color developing solution, heat in boiling water bath for 40 min, and then carry out toluene extraction and colorimetry according to the method of making standard curve.(3) Calculation of results: Find out the proline content in the determination solution from the standard curve. Calculate the percentage of proline content in the sample according to the following formula.Proline content Paul g - g7 (dry or fresh samples)] = Textile methodWhere: knife a proline content in the extract g ~ E _ from the standard curve;V-Total volume of extract, mL;A-volume sucked during the determination, mL;W-sample weight, g. Caveat 1. Leaf chlorosis time is generally 3~4 h, not too long, otherwise the proline accumulation is high, and the sample needs to be diluted for determination. 2. Acid tridecanone coloring solution and proline solution can be used at present. For more product details, please visit Aladdin Scientific website.
Equipment: balance, spectrophotometer, water bath, funnel, 20 mL large test tube, 20 mL stoppered graduated test tube, 5 to 10 mL syringe or dropper.
Reagents:
① 3% sulfosalicylic acid solution;
② Toluene;
③ 2. 5% acid station tritone color development solution;
③2. 5% acid station tritone color developing solution; ④ ice acetic acid and 6 mol-L
(iv) Ice acetic acid and 6 mol-L -1
(i) ice-acetic acid and 6 mol-L-1 phosphoric acid mixed with 3:2 (prepared as solvent, effective for 2~3 days at 4°C);
⑤ Proline standard solution: weigh 25 mg of proline accurately, dissolve it in distilled water and then make it into 250 mL, and its concentration is 100 μg - mL-1.
The concentration is 100 μg - mL -1.
The concentration is 100 μg - mL -1. The concentration is 100 μg - mL -1. Then take 10 mL of this solution and dilute it to 100 mL with unauthorized water, that is 10 μg - mL -1.
-1
This is 10 μg-mL-1 of proline standard solution.
Table 45-1 Reagent Tube number 1 1 2 3 4 5 6 Standard proline amount/mL 0 0.2 0.4 0.8 1.2 1.6 2.0 H2O/mL 2 1.8 1.6 1.2 0.8 0.4 0.4
2 - Determination of free proline content of samples： Reagents Tube number 3 1 2 3 4 5 6 Glacial acetic acid/mL 2 2 2 2 2 2 2 Acid with triacetone color developing solution/mL 3 3 3 3 3 3 3 Proline content//g 0 2 3 8 12 16 20