Nitrate reductase (NR) is a key enzyme in the process of nitrate assimilation, which plays an important role in plant growth and development, and the determination of nitrate reductase activity can be used as a physiological and biochemical index for crop breeding and nutritional diagnosis.The determination of NR can be divided into the in vivo method and the in vitro method. The in vivo method is simple and suitable for rapid, multi-group determination. The in vivo method is simple and suitable for rapid, multi-group determinations. The in vitro method is more complicated but more reproducible.

Operation method

In vivo assay of nitrate reductase activity in objects

Move

I. Material Instrumentation and Reagents

1. Materials: leaves, young spikes of rice or wheat, etc.

2. Instrumentation: vacuum pump; vacuum drier; small beaker; glass stopper; other utensils are the same as in vitro method.

3. reagents and preparation:

Sodium nitrite standard solution ( The same as in vitro method ).

0.1mol-L-1KNO3 ( Preparation method is the same as the isolated method ).

Sulfanilamide 1% (same preparation method as the isolated method);

0.02% naphthyl vinylamine ( Preparation method is the same as the isolated method (same as the isolated method).

Preparation of 30% trichloroacetic acid solution: weigh 30g of trichloroacetic acid, dissolve in water and make volume to 100ml.

Experimental steps

1. Standard curve preparation ( Same as in vitro method ).

2. enzymatic reaction

(1) Weigh crop leaves 1.0 to 2.0 g four, cut into small sections of about 1 cm, divided into four small beakers, with a diameter slightly smaller than the diameter of the beaker of the glass stopper will be all the material pressed to the bottom of the cup, one of them as a control, the other three for the determination of enzyme activity.

(2) Firstly, 1ml of 30% TCA was added to the control cup, then 9ml of 0.1 mol-L-1KNO3 solution was added to each cup, and then immediately put into a desiccator after mixing, and then evacuated for 1min, and then evacuated several times to exclude the gas of the tissue interstitial space, and then the leaves were completely softened and sank to the bottom of the cup, so as to allow the substrate solution to enter into the tissues. Finally, after being sealed by passing nitrogen, the reaction was carried out in the dark at 25°C for half an hour, and then 1 ml of 30% trichloroacetic acid was added to the assay cups (except for the control cup) to terminate the enzyme reaction, respectively.

3. Assay

After each cup was shaken well and left to stand for 2 min, 2 ml of reaction solution was taken into a test tube, 1 ml of 1% sulfanilamide and 1 ml of 0.02% naphthyl vinyl amine were added, and the color was developed by shaking well for 15 min, then poured into centrifugal tubes, and then centrifuged at 4000 r/min for 15 min, and then the supernatant was taken to determine the absorbance (A) at 520 nm. And the nitrite nitrogen content (μg) in the reaction solution was found from the standard curve.

III. Calculation of enzyme activity ( The same as the isolated method ).

Caveat

1. Nitrate reductase is easily inactivated, and the operation should be rapid and carried out at 4℃ when measuring in vitro.

2. Sampling should be carried out on a sunny day, preferably one day in advance of the application of a certain amount of nitrate nitrogen fertilizer, and the sampling site should be consistent.

3. the nitrate reduction process should be carried out in the dark to prevent the reduction of nitrite to ammonia.

4. The time from color development to colorimetry should be consistent, and too long or too short a time for color development will have an effect on the color.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/