CN116908128B - Kit for detecting nitrate reductase activity and detection method thereof - Google Patents
Kit for detecting nitrate reductase activity and detection method thereof Download PDFInfo
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- 108090000913 Nitrate Reductases Proteins 0.000 title claims abstract description 37
- 230000000694 effects Effects 0.000 title claims abstract description 36
- 238000001514 detection method Methods 0.000 title claims abstract description 14
- 239000000243 solution Substances 0.000 claims abstract description 70
- RUFPHBVGCFYCNW-UHFFFAOYSA-N 1-naphthylamine Chemical compound C1=CC=C2C(N)=CC=CC2=C1 RUFPHBVGCFYCNW-UHFFFAOYSA-N 0.000 claims abstract description 38
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 29
- 238000000034 method Methods 0.000 claims abstract description 22
- 229940124530 sulfonamide Drugs 0.000 claims abstract description 16
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 claims abstract description 14
- 150000003456 sulfonamides Chemical class 0.000 claims abstract description 13
- 239000012086 standard solution Substances 0.000 claims abstract description 11
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 claims abstract description 10
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims abstract description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 27
- 239000006228 supernatant Substances 0.000 claims description 21
- 238000002835 absorbance Methods 0.000 claims description 18
- 238000012360 testing method Methods 0.000 claims description 18
- 239000012153 distilled water Substances 0.000 claims description 17
- 241000196324 Embryophyta Species 0.000 claims description 15
- 238000004321 preservation Methods 0.000 claims description 14
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 12
- 238000006243 chemical reaction Methods 0.000 claims description 12
- 239000002904 solvent Substances 0.000 claims description 9
- 239000003085 diluting agent Substances 0.000 claims description 8
- FDDDEECHVMSUSB-UHFFFAOYSA-N sulfanilamide Chemical compound NC1=CC=C(S(N)(=O)=O)C=C1 FDDDEECHVMSUSB-UHFFFAOYSA-N 0.000 claims description 6
- 102000004190 Enzymes Human genes 0.000 claims description 5
- 108090000790 Enzymes Proteins 0.000 claims description 5
- 239000006004 Quartz sand Substances 0.000 claims description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 5
- 239000012295 chemical reaction liquid Substances 0.000 claims description 5
- 239000004570 mortar (masonry) Substances 0.000 claims description 5
- 238000007865 diluting Methods 0.000 claims description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 235000018417 cysteine Nutrition 0.000 claims description 3
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 3
- 239000008055 phosphate buffer solution Substances 0.000 claims description 3
- 230000035484 reaction time Effects 0.000 claims description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 26
- 229910052757 nitrogen Inorganic materials 0.000 description 13
- 235000010469 Glycine max Nutrition 0.000 description 12
- 244000068988 Glycine max Species 0.000 description 11
- 239000000618 nitrogen fertilizer Substances 0.000 description 5
- 238000005259 measurement Methods 0.000 description 3
- 230000004060 metabolic process Effects 0.000 description 3
- 229910002651 NO3 Inorganic materials 0.000 description 2
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 2
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000003337 fertilizer Substances 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 108010025915 Nitrite Reductases Proteins 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000006860 carbon metabolism Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000008121 plant development Effects 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 230000008636 plant growth process Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/906—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7)
- G01N2333/90688—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7) acting on other nitrogen compounds as donors (1.7)
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Abstract
The invention discloses a kit for detecting plant leaf nitrate reductase activity (Nitratereductaseactivity, NRA) and a detection method thereof, and relates to a kit and a detection method thereof. The invention solves the problem that NRA determination is not ideal in the existing nitrate reductase activity detection method. The kit consists of an extracting solution, a KNO 3 solution, a trichloroacetic acid solution, a sulfonamide reagent, a 2% alpha-naphthylamine reagent, an NADH solution with the concentration of 2mg/mL and a standard solution. The method comprises the following steps: 1. drawing a standard curve; 2. and (5) detecting. The kit has the advantages of good repeatability of detecting the activity of the nitrate reductase and low cost, and can accurately and efficiently detect the actual activity of the nitrate reductase in plants.
Description
Technical Field
The invention relates to a kit and a detection method thereof.
Background
Nitrate reductase is a key enzyme for nitrogen assimilation of plants, is also an inducible enzyme, is related to absorption and utilization of nitrogen fertilizer by crops, and can directly regulate nitrate reduction, thereby regulating nitrogen metabolism and affecting light and carbon metabolism. The nitrate absorbed by plants from soil is catalyzed by nitrate reductase to form nitrite, and the nitrite is converted into ammonium under the action of the nitrite reductase and is finally used for synthesizing protein. In the process of plant growth, if the activity of the nitrate reductase is influenced by the external environment, the synthesis of amino acid and protein in the plant body is correspondingly hindered, so that the adverse effect on plant growth and development is caused. Therefore, the activity of nitrate reductase in the plant body is measured, the absorption and metabolism capacity of the plant to nitrogen nutrition can be correctly reflected, and the method is an effective method for correctly judging the influence of the external environment on the nitrogen metabolism of the plant.
Currently, methods for measuring nitrate reductase activity can be classified into an endogenous substrate method and an exogenous substrate method depending on the reflection medium. The living body method and the ex vivo method are classified according to the material treatment. Endogenous matrix assay results may reflect the reducing power of the nitrate reductase under actual growth conditions, while exogenous matrix assay results may reflect the potential or highest reducing power of the nitrate reductase. The in vivo method has simple steps and is suitable for rapid and multi-suitable group measurement, but has the defect that the measurement of nitrate reductase activity (NitrateReductaseActivity, NRA) is not ideal.
Disclosure of Invention
The invention provides a kit for detecting the activity of nitrate reductase and a use method thereof, and aims to solve the problem that NRA (non-return amplification) determination is not ideal in the existing detection method of the activity of nitrate reductase.
The kit for detecting the activity of the nitrate reductase consists of an extracting solution, a KNO 3 solution with the concentration of 0.1 mol/L, a trichloroacetic acid solution with the mass concentration of 30%, a sulfonamide reagent with the mass concentration of 1%, an alpha-naphthylamine reagent with the mass concentration of 2%, an NADH solution with the concentration of 2 mg/mL and a standard solution;
Wherein the extract is prepared by using phosphate buffer solution with concentration of 0.025 mol/L, pH and value of 8.7 as solvent, and EDTA containing Na 2HPO4·12H2 O of 8.8640 g, K 2HPO4·3H2 O of 0.0570 g, 1.2110 g cysteine and 0.3720 g per liter;
The mass concentration of the sulfonamide reagent is 1 percent: distilled water is taken as a solvent, and each liter of the aqueous solution contains 10 g parts of sulfanilamide and 250 to mL parts of hydrochloric acid with 36 to 38 percent of mass fraction;
the mass concentration of the alpha-naphthylamine reagent is 2 percent: distilled water is used as a solvent, and each liter of the distilled water contains 2 g of alpha-naphthylamine and 10 mL of hydrochloric acid with 36 to 38 mass percent;
The standard solution is NaNO 2 solution with the concentration of 5 mug/mL.
The method for detecting the activity of the nitrate reductase by using the kit comprises the following steps:
1. Drawing a standard curve:
(1) Diluting the standard solution with distilled water to obtain diluted solutions with the concentration of 0.5 mug/mL, 1.0 mug/mL, 2.0 mug/mL, 3.0 mug/mL, 4.0 mug/mL and 5.0 mug/mL, and taking distilled water as a blank control;
(2) Absorbing the diluents with different concentrations in the step 1 into a test tube, sequentially adding a sulfonamide reagent 2 mL with the mass concentration of 1% and an alpha-naphthylamine reagent 2 mL with the mass concentration of 2%, shaking uniformly, and standing for 30min;
(3) Detecting the absorbance at 520 nm, and making a standard curve by taking the absorbance as an ordinate and the concentration of NO 2 - in the diluent as an abscissa;
2. and (3) detection:
(1) Taking a 1.0 g plant sample, placing the plant sample into a mortar, adding 0.3-1.0 mL of an extracting solution and 0.3-1.0 g of quartz sand, grinding and homogenizing, transferring, fixing the volume to 10 mL by using the extracting solution, shaking uniformly, and standing for 5min to obtain a reaction solution;
(2) Taking 4 mL supernatant from the reaction liquid after standing in the step 1, and centrifuging for 15min at 4 ℃ under 4000 r/min to obtain the supernatant of the centrifuge tube;
(3) Taking 4 15 mL test tubes, setting 1 blank tube and 3 repeat tubes, and marking;
(4) Sequentially adding a trichloroacetic acid solution with the mass concentration of 1 mL of 30%, a supernatant of the centrifuge tube in the step 2 of 0.2 mL, a KNO 3 solution with the concentration of 0.1 mol/L of 0.5 mL and an NADH solution with the concentration of 2 mg/mL of 0.3 mL into a blank tube in the step 3, and putting into a water bath at 25 ℃ for heat preservation for 30min;
(5) Sequentially adding the supernatant of the centrifuge tube in the step 2 of 0.2 mL, KNO 3 solution with the concentration of 0.5 mL being 0.1 mol/L and NADH solution with the concentration of 0.3 mL being 2 mg/mL into three repeating pipes in the step 3, putting into a water bath with the temperature of 25 ℃ for heat preservation for 30min, adding 1 mL mass percent trichloroacetic acid solution with the concentration of 30% after heat preservation is finished, and shaking uniformly;
(6) Sequentially adding 2 mL mass percent of sulfonamide reagent and 2 mL mass percent of alpha-naphthylamine reagent into the obtained 4 test tubes, shaking uniformly, and standing for 15min;
(7) The absorbance of the sample was measured at the wavelength of spectrophotometer 520 nm, and the absorbance was substituted into the standard curve in step one, the NO 2 - content was detected from the standard curve, in μg/mL, and then the enzyme activity was expressed as mass of NO 2 - produced per gram fresh weight per hour, calculated as follows:
Wherein C represents the mass of NO 2 - measured according to a standard curve, and the unit is mug; v t represents the total volume of the reaction solution in mL; v s represents the measured sample volume in mL; FW represents fresh weight in g; t represents reaction time, unit h;
When calculating, the result is the average value of three repetitions, namely, the detection of the activity of the nitrate reductase is realized.
The invention adopts an in-vitro method to measure the activity of the nitrate reductase, the reaction conditions are controlled manually, the influence of the nature of the organism is reduced, the repeatability is better, the cost is lower, and the invention can accurately and efficiently measure the real activity of the nitrate reductase in plants.
Drawings
Fig. 1 is a standard graph of example 1.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other.
The first embodiment is as follows: the kit for detecting the activity of the nitrate reductase in the embodiment consists of an extracting solution, a KNO 3 solution with the concentration of 0.1 mol/L, a trichloroacetic acid solution with the mass concentration of 30%, a sulfanilamide reagent with the mass concentration of 1%, an alpha-naphthylamine reagent with the mass concentration of 2%, an NADH solution with the concentration of 2 mg/mL and a standard solution;
Wherein the extract is prepared by using phosphate buffer solution with concentration of 0.025 mol/L, pH and value of 8.7 as solvent, and EDTA containing Na 2HPO4·12H2 O of 8.8640 g, K 2HPO4·3H2 O of 0.0570 g, 1.2110 g cysteine and 0.3720 g per liter;
The mass concentration of the sulfonamide reagent is 1 percent: distilled water is taken as a solvent, and each liter of the aqueous solution contains 10 g parts of sulfanilamide and 250 to mL parts of hydrochloric acid with 36 to 38 percent of mass fraction;
the mass concentration of the alpha-naphthylamine reagent is 2 percent: distilled water is used as a solvent, and each liter of the distilled water contains 2 g of alpha-naphthylamine and 10 mL of hydrochloric acid with 36 to 38 mass percent;
The standard solution is NaNO 2 solution with the concentration of 5 mug/mL.
The second embodiment is as follows: the method for detecting the activity of the nitrate reductase by using the kit according to the first embodiment comprises the following steps:
1. Drawing a standard curve:
(1) Diluting the standard solution with distilled water to obtain diluted solutions with the concentration of 0.5 mug/mL, 1.0 mug/mL, 2.0 mug/mL, 3.0 mug/mL, 4.0 mug/mL and 5.0 mug/mL, and taking distilled water as a blank control;
(2) Absorbing the diluents with different concentrations in the step 1 into a test tube, sequentially adding a sulfonamide reagent 2 mL with the mass concentration of 1% and an alpha-naphthylamine reagent 2 mL with the mass concentration of 2%, shaking uniformly, and standing for 30min;
(3) Detecting the absorbance at 520 nm, and making a standard curve by taking the absorbance as an ordinate and the concentration of NO 2 - in the diluent as an abscissa;
2. and (3) detection:
(1) Taking a 1.0 g plant sample, placing the plant sample into a mortar, adding 0.3-1.0 mL of an extracting solution and 0.3-1.0 g of quartz sand, grinding and homogenizing, transferring, fixing the volume to 10 mL by using the extracting solution, shaking uniformly, and standing for 5min to obtain a reaction solution;
(2) Taking 4 mL supernatant from the reaction liquid after standing in the step 1, and centrifuging for 15min at 4 ℃ under 4000 r/min to obtain the supernatant of the centrifuge tube;
(3) Taking 4 15 mL test tubes, setting 1 blank tube and 3 repeat tubes, and marking;
(4) Sequentially adding 1 mL trichloroacetic acid solution with the mass concentration of 30%, supernatant fluid of the centrifuge tube in the step 2 of 0.2 mL, KNO 3 solution with the concentration of 0.1mol/L of 0.5 mL and NADH solution with the concentration of 2 mg/mL of 0.3 mL into a blank tube in the step 3, and putting into a water bath at 25 ℃ for heat preservation for 30min;
(5) Sequentially adding the supernatant of the centrifuge tube in the step 2 of 0.2 mL, KNO 3 solution with the concentration of 0.5 mL being 0.1 mol/L and NADH solution with the concentration of 0.3 mL being 2 mg/mL into three repeating pipes in the step 3, putting into a water bath with the temperature of 25 ℃ for heat preservation for 30min, adding 1 mL mass percent trichloroacetic acid solution with the concentration of 30% after heat preservation is finished, and shaking uniformly;
(6) Sequentially adding 2 mL mass concentration 1% sulfanilamide reagent and 2 mL mass concentration 2% alpha-naphthylamine reagent into the obtained 4 test tubes (containing 2 mL reaction solutions), shaking uniformly, and standing for 15min;
(7) The absorbance of the sample was measured at the wavelength of spectrophotometer 520 nm, and the absorbance was substituted into the standard curve in step one, the NO 2 - content was detected from the standard curve, in μg/mL, and then the enzyme activity was expressed as mass of NO 2 - produced per gram fresh weight per hour, calculated as follows:
Wherein C represents the mass of NO 2 - measured according to a standard curve, and the unit is mug; v t represents the total volume of the reaction solution in mL; v s represents the measured sample volume in mL; FW represents fresh weight in g; t represents reaction time, unit h;
When calculating, the result is the average value of three repetitions, namely, the detection of the activity of the nitrate reductase is realized.
Example 1 Using the method of the present invention, soybean leaves coated with nitrogen fertilizer of different concentrations (nitrogen fertilizer concentrations of 0 mg/kg, 10 mg/kg, 200 mg/kg, respectively) were examined
The specific method comprises the following steps:
1. Taking soybean leaves respectively applying three nitrogen fertilizers with different concentrations in the same growth period, putting 1.0 g leaves in each group into a mortar, adding 0.5 mL extract and 0.5g quartz sand according to the method of a second specific embodiment, grinding and homogenizing, transferring and fixing the volume to 10 mL by using the extract, shaking uniformly, and standing for 5min to obtain a reaction solution;
2. taking 4 mL supernatant from the reaction liquid in the first step, and centrifuging for 15min at 4 ℃ under 4000 r/min to obtain the supernatant;
3. Taking 12 15 mL test tubes, setting 3 groups of experiments according to nitrogen fertilizers with different concentrations, setting 1 blank tube and 3 repeat tubes in each group, and marking;
4. Sequentially adding 1 mL mass concentration 30% trichloroacetic acid solution, 0.2 mL supernatant of the centrifuge tube in the second step, 0.5 mL concentration 0.1 mol/L KNO 3 solution and 0.3 mL concentration 2 mg/mLNADH solution into the blank tube in the third step, and placing into a water bath at 25 ℃ for heat preservation for 30min;
5. Sequentially adding the supernatant 0.2 mL, the solution 0.5 mL of which the concentration is 0.1 mol/LKNO 3 and the solution 0.3 mL of which the concentration is 2 mg/mLNADH of the centrifuge tube in the second step into a repeating tube in the third step, putting into a water bath at 25 ℃ for heat preservation for 30min, adding the trichloroacetic acid solution 1mL of which the mass concentration is 30% after heat preservation is finished, and shaking uniformly;
6. Sequentially adding 2 mL mass concentration 1% sulfonamide reagent and 2 mL mass concentration 2% alpha-naphthylamine reagent into the 12 test tubes filled with 2 mL reaction solutions, shaking uniformly, and standing for 15min;
7. The absorbance of the sample was measured at a wavelength of 520nm by a spectrophotometer, and the absorbance was substituted into a standard curve, and the NO 2 - content was detected from the standard curve in μg/mL.
The test results show that the activity of the nitrate reductase of the soybean leaves is shown in table 1 under different nitrogen fertilizer dosages. As can be seen from Table 1, there was a significant difference in nitrate reductase activity in the soybean leaves between treatments, and at 200 mg/kg nitrogen levels, the nitrate reductase activity was highest in the soybean leaves, and in this variety of soybean leaves the nitrate reductase activity was significantly increased with increasing nitrogen application.
TABLE 1 Soybean leaf nitrate reductase Activity at different Nitrogen fertilizer usage levels
Note that: the vertical bars of different lowercase letters represent the significance of the difference at the level.
Wherein, the measurement of the experimental standard curve in the present example:
(1) Diluting the standard solution with distilled water to obtain diluted solutions with the concentration of 0.5 mug/mL, 1.0 mug/mL, 2.0 mug/mL, 3.0 mug/mL, 4.0 mug/mL and 5.0 mug/mL, and taking distilled water as a blank control;
(2) Absorbing the diluents with different concentrations in the step 1 into a test tube, sequentially adding a sulfonamide reagent 2 mL with the mass concentration of 1% and an alpha-naphthylamine reagent 2 mL with the mass concentration of 2%, shaking uniformly, and standing for 30min;
(3) Detecting absorbance at 520nm, and preparing a standard curve by taking absorbance as an ordinate and taking the concentration of NO 2 - in the diluent as an abscissa;
The standard graph is shown in fig. 1.
Example 2 detection of Soybean leaves at a nitrogen level of 200 mg/kg by the method of the present invention
The specific method comprises the following steps:
1. taking soybean leaves 1.0g with nitrogen level of 200mg/kg, putting into a mortar, adding 0.5 mL extract and 0.5 g quartz sand according to the method of the second embodiment, grinding, homogenizing, transferring, fixing the volume to 10 mL by using the extract, shaking, and standing for 5min to obtain a reaction solution;
2. taking 4 mL supernatant from the reaction liquid in the first step, centrifuging for 15min at 4 ℃ under 4000 r/min to obtain supernatant;
3. taking 11 15 mL test tubes, setting 1 blank tube and 10 repeated tubes for test, and marking;
4. Sequentially adding 1 mL mass percent of 30% trichloroacetic acid solution, 0.2 mL mass percent of supernatant fluid of the centrifuge tube in the second step, 0.5 mL mass percent of 0.1 mol/LKNO 3 solution and 0.3 mL mass percent of 2 mg/mLNADH solution into the blank tube in the third step, and putting into a water bath at 25 ℃ for heat preservation for 30min;
5. Sequentially adding the supernatant 0.2mL, the solution 0.5 mL of which the concentration is 0.1 mol/LKNO 3 and the solution 0.3 mL of which the concentration is 2 mg/mLNADH of the centrifuge tube in the second step into 10 repeat tubes of the third step, putting into a water bath at 25 ℃ for heat preservation for 30min, adding a trichloroacetic acid solution with the mass concentration of 1 mL of 30% after heat preservation is finished, and shaking uniformly;
6. Sequentially adding 2mL mass concentration 1% sulfonamide reagent and 2mL mass concentration 2% alpha-naphthylamine reagent into the 11 test tubes (containing 2mL reaction solutions), shaking uniformly, and standing for 15min;
7. The absorbance of the sample was measured at a wavelength of 520nm by a spectrophotometer, and the absorbance was substituted into a standard curve, and the NO 2 - content was detected from the standard curve in μg/mL.
The results of 10 replicates of soybean leaves at nitrogen level of 200 mg/kg were tested and tested for outliers and are shown in Table 2.
TABLE 2 parallel determination of Soy leaf nitrate reductase Activity at 200 mg/kg Nitrogen level
TABLE 3 precision of soybean leaf nitrate reductase Activity at 200 mg/kg Nitrogen level
The significance level α was 0.01 and T 0.01,10 =2.41 by using the Grubbs (Grubbs) test method, and as shown in table 3, the abnormal value test T in the test was less than 2.41, so that 10 parallel sample values obtained by the method of the present invention have no abnormal value and the variation Coefficient (CV) of NRA was 1.64%, and thus the nitrate reductase measurement method of the present invention was found to be reliable and good in reproducibility.
Claims (2)
1. A kit for detecting the activity of plant leaf nitrate reductase is characterized in that the kit for detecting the activity of nitrate reductase consists of an extracting solution, a solution with the concentration of 0.1 mol/L KNO 3, a solution with the mass concentration of 30% trichloroacetic acid, a sulfanilamide reagent with the mass concentration of 1%, an alpha-naphthylamine reagent with the mass concentration of 2%, an NADH solution with the concentration of 2 mg/mL and a standard solution;
Wherein the extract is prepared by using phosphate buffer solution with concentration of 0.025 mol/L, pH and value of 8.7 as solvent, and EDTA containing Na 2HPO4·12H2 O of 8.8640 g, K 2HPO4·3H2 O of 0.0570 g, 1.2110 g cysteine and 0.3720 g per liter;
The mass concentration of the sulfonamide reagent is 1 percent: distilled water is taken as a solvent, and each liter of the aqueous solution contains 10 g parts of sulfanilamide and 250 to mL parts of hydrochloric acid with 36 to 38 percent of mass fraction;
the mass concentration of the alpha-naphthylamine reagent is 2 percent: distilled water is used as a solvent, and each liter of the distilled water contains 2 g of alpha-naphthylamine and 10 mL of hydrochloric acid with 36 to 38 mass percent;
The standard solution is NaNO 2 solution with the concentration of 5 mug/mL.
2. A method for detecting the activity of nitrate reductase in plant leaves by using the kit as claimed in claim 1, wherein the method for detecting the activity of nitrate reductase is carried out according to the following steps:
1. drawing of a Standard Curve
(1) Diluting the standard solution with distilled water to obtain diluted solutions with the concentration of 0.5 mug/mL, 1.0 mug/mL, 2.0 mug/mL, 3.0 mug/mL, 4.0 mug/mL and 5.0 mug/mL, and taking distilled water as a blank control;
(2) Absorbing the diluents with different concentrations in the step 1 into a test tube, sequentially adding a sulfonamide reagent 2 mL with the mass concentration of 1% and an alpha-naphthylamine reagent 2 mL with the mass concentration of 2%, shaking uniformly, and standing for 30min;
(3) Detecting the absorbance at 520 nm, and making a standard curve by taking the absorbance as an ordinate and the concentration of NO 2 - in the diluent as an abscissa;
2. Detection of
(1) Taking a 1.0 g plant sample, placing the plant sample into a mortar, adding 0.3-1.0 mL of an extracting solution and 0.3-1.0 g of quartz sand, grinding and homogenizing, transferring, fixing the volume to 10 mL by using the extracting solution, shaking uniformly, and standing for 5min to obtain a reaction solution;
(2) Taking 4 mL supernatant from the reaction liquid after standing in the step1, placing the supernatant into a centrifuge tube, and centrifuging for 15min at 4 ℃ under 4000 r/min to obtain the supernatant of the centrifuge tube;
(3) Taking 4 15 mL test tubes, setting 1 blank tube and 3 repeat tubes, and marking;
(4) Sequentially adding a trichloroacetic acid solution with the mass concentration of 1 mL of 30%, a supernatant of the centrifuge tube in the step 2 of 0.2mL, a KNO 3 solution with the concentration of 0.1 mol/L of 0.5 mL and an NADH solution with the concentration of 2 mg/mL of 0.3 mL into the blank tube in the step 3, and putting into a water bath at 25 ℃ for heat preservation for 30min;
(5) Sequentially adding 0.2mL of supernatant of the centrifuge tube in the step 2, 0.5 mL KNO 3 solution with the concentration of 0.1 mol/L and 0.3 mL NADH solution with the concentration of 2 mg/mL into three repeated tubes in the step 3, preserving heat in a water bath at 25 ℃ for 30min, adding 1 mL trichloroacetic acid solution with the mass concentration of 30% after the heat preservation is finished, and shaking uniformly;
(6) Sequentially adding 2mL mass percent of sulfonamide reagent and 2mL of 2 mass percent of alpha-naphthylamine reagent into the obtained 4 test tubes, shaking uniformly, and standing for 15min;
(7) The absorbance of the sample was measured at the wavelength of spectrophotometer 520 nm, the absorbance was substituted into the standard curve in step one, the NO 2 - content was detected from the standard curve, in μg/mL, and then the enzyme activity was expressed as mass of NO 2 - produced per gram fresh weight per hour, calculated as follows:
，
Wherein C represents the mass of NO 2 - measured according to a standard curve, and the unit is mug; v t represents the total volume of the reaction solution in mL; v s represents the measured sample volume in mL; FW represents fresh weight in g; t represents reaction time, unit h;
When calculating, the result is the average value of three repetitions, namely, the detection of the activity of the nitrate reductase is realized.
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| CN114235522A (en) * | 2021-12-08 | 2022-03-25 | 中国科学院东北地理与农业生态研究所 | Kit and method for detecting ureide content and allantoic acid content in plant |
| CN115290585A (en) * | 2022-08-05 | 2022-11-04 | 北京索莱宝科技有限公司 | Method, reagent and kit for detecting nitrate reductase activity |
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| US20100252450A1 (en) * | 2008-04-09 | 2010-10-07 | Riehl Bill L | Electrode and sensor having carbon nanostructures |
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