Experimental determination of nitrate reductase activity in plants by the in vitro method
Experimental determination of nitrate reductase activity in plants by the in vitro method
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
Experimental determination of nitrate reductase activity in plants by the in vitro method
Principle
Nitrate reductase (NR) catalyzes the reduction of nitrate to nitrite in plants, and the resulting nitrite is quantitatively combined with p-aminobenzenesulfonic acid (or p-aminobenzenesulfonamide) and α-naphthylamine (or naphthylethylene diamine) to produce a red azo compound under acidic conditions. The reaction is as follows: the generated red azo compound has a maximum absorption peak at 520nm, which can be measured spectrophotometrically. Nitrate reductase activity can be expressed by the amount of nitrite nitrogen produced. It is generally expressed in units of Nμg-g-1Fw-h-1.
Materials and Instruments
Rice Wheat Leaf Move I. Material Instrumentation and Reagents For more product details, please visit Aladdin Scientific website.
Nitrate-Nitrogen Standard Master Mix Phosphate Buffer Aminobenzenesulfonamide
Freezing Centrifuge Spectrophotometer Electronic Analytical Balance Refrigerator Constant Temperature Bath Mortar and pestle Scissors Centrifuge Tubes Plugged Test Tubes Pipettes Earwash Balls
1. Materials: leaves, young spikes of rice or wheat, etc.
2. Instruments and equipment: freezing centrifuge; spectrophotometer; electronic analytical balance; refrigerator; constant temperature water bath; mortar; scissors; centrifuge tubes; stoppered test tubes; pipettes; ear washer.
3. Reagents and preparation.
Preparation of 1μg-ml-1 nitrite nitrogen standard master batch: accurately weigh analytically pure NaNO2 0.9857g, dissolve it in distilled water, then suck up 5ml to 1000ml, i.e. 1μg-ml-1 nitrite nitrogen standard solution.
Preparation of 0.1mol-L-1pH7.5 phosphate buffer: weigh Na2HPO4-12H2O 30.0905g and NaH2PO4-2H2O 2.4965g, add distilled water and dissolve to 1000ml.
Preparation of 1% p-aminobenzene sulfonamide (W∕V) solution: weigh 1.0g of sulfonamide dissolved in 100 ml of 3 mol-L-1HCl (25 ml of concentrated hydrochloric acid and distilled water to 100 ml is 3 mol-L-1HCl).
0.02% (W∕V) naphthalene vinylamine solution: weigh 0.0200g of naphthalene vinylamine dissolved in 100ml of distilled water, stored in a brown bottle.
0.1 mol-L-1KNO3 solution: weigh 2.5275g KNO3 dissolved in 250ml 0.1 mol-L-1 pH7.5 phosphate buffer.
0.025 mol-L-1pH8.7 phosphate buffer preparation: weigh 8.8640g Na2HPO4-12H2O, 0.0570g
K2HPO4-3H2O were dissolved in 1000 ml of non-ionized water.
Extraction buffer preparation: 0.1211g cysteine, 0.0372g EDTA were weighed and dissolved in 100 ml of 0.025 mol-L-1 pH 8.7 phosphate buffer.
2mg-ml-1NADH (coenzyme I) solution was prepared: 4mg NADH was dissolved in 2ml 0.1mol-L-1pH7.5 phosphate buffer (prepared before use).
Experimental steps
1. Nitrite nitrogen standard curve
(1) Take seven 15 ml graduated test tubes, numbered, according to the following table to prepare the content of 0-2.0 μg of nitrite nitrogen standard solution.
Add the reagents in the table, shake well, hold at 25 ℃ for 30min, and then use tube 0 as a blank control, determine the absorbance (A) value at 520nm .
(2) Standard curve
The standard curve was drawn with the nitrite nitrogen content (μg) of tubes No. 1-6 as the horizontal coordinate and the absorbance value as the vertical coordinate.
2. Determination of nitrate reductase activity in samples
(1) Enzyme extraction
Weigh 0.5g of fresh samples, cut in a mortar and put in a low-temperature refrigerator frozen for 30min, take out and put in an ice bath with a small amount of quartz sand and 4ml of extraction buffer, grinding homogenization, transferred to a centrifuge tube, centrifuged at 4℃, 4000r/min for 15min, the supernatant is the enzyme extract. 
(2) Enzymatic reaction
Take 0.4 ml of enzyme solution in a 10 ml test tube, add 1.2 ml of 0.1 mol- L-1KNO3 phosphate buffer and 0.4 ml of NADH solution, mix well, and keep warm for 30 min in a water bath at 25 ℃, and the control is not added with NADH solution, but with 0.4 ml of 0.1 mol-L-1pH7.5pH2.5pH2.5pH2.5pH2.5pH2.5pH2.5pH2.5pH2. 1 pH 7.5 phosphate buffer instead.
(3) Enzyme activity assay
Immediately after the end of the holding period, 1 ml of 1 % sulfanilamide solution was added to terminate the enzyme reaction, and then 1 ml of 0.02 % naphthyl vinyl amine solution was added, the color was developed for 15 min and then centrifuged at 4000 r/min for 15 min, and a blank tube was used as a control, and the supernatant was taken to determine the absorbance (A) value at 520 nm.
Calculation of enzyme activity
According to the measured absorbance (A) value of the sample, find out the content of nitrate nitrogen in the reaction solution from the standard curve, and calculate the enzyme activity in the sample according to the following formula: 