A type of nitrate nitrogen in biological tissues 15 Method for determining N abundance

Abstract

The present invention provides a method for treating nitrate nitrogen in biological tissues 15 The method for determining nitrogen abundance belongs to the technical field of element quantitative detection. The determination method provided by the present invention overcomes the problem of low accuracy or even inability to detect in the prior art determination method. The determination method of the present invention can avoid the adverse effects caused by concentrated sulfuric acid reagents and can successfully detect the nitrate nitrogen in biological tissues. 15 N abundance can be accurately detected even for arbuscular mycorrhizae that have high detection requirements.

Description

Technical Field

[0001] The present invention relates to the technical field of element quantitative detection, in particular to a method for detecting nitrate nitrogen in biological tissues. 15 Method for determining nitrogen abundance. Background Art

[0002] At present, in agricultural science research, the stable isotopes used are mainly 13 C. 15 N, 18 O and 2 H, among which the most widely used is 15 Because these elements are essential nutrients for organisms, research on their metabolism is a major area of ​​agricultural research. Furthermore, due to the uniqueness, sole use, and irreplaceable nature of isotope tracing methods, the application of isotopes has permeated nearly every field of agricultural research, including animal and plant nutrition and physiology, resources and environment, agricultural product traceability, molecular biology research, and agricultural product traceability. The application of isotopes has greatly promoted the development of agricultural research.

[0003] In the past decade or so, as the research projects and fields of isotope tracing have increased and become wider, the demand for markers has increased dramatically, and the requirements for analytical methods (pretreatment) have also become higher and higher.

[0004] For solid samples 15 For nitrogen analysis, there are mainly combustion method (dry method) and Kjeldahl method (wet method). The dry method uses elemental analyzer + mass spectrometry (EA+MS). The testing principle is: the sample is burned at high temperature to convert various forms of nitrogen into NO2, which is reduced to N2 by copper, and then separated and purified by chromatographic column. The nitrogen content is determined by TCD, and then enters the mass spectrometer through the interface for isotope analysis. The wet method uses Kjeldahl method + dual injection + mass spectrometry (K+DI+MS). The testing principle is: the sample is digested with concentrated sulfuric acid to convert various forms of nitrogen (excluding NO3 and NO2) into NH3 and form (NH4)2SO4. It is distilled and titrated by Kjeldahl nitrogen analyzer to determine the nitrogen content. After concentration, it reacts with LiBrO under vacuum conditions to convert NH 4+ The converted nitrogen is then fed into a dual-path sampling system, where it is analyzed by a mass spectrometer for isotope analysis. However, the inventors discovered in their previous research that the concentrated sulfuric acid digestion method is prone to dry-out, which can affect the accuracy of biological sample test results.

[0005] In the previous research, the inventors paid special attention to Arbuscular mycorrhizae (AM) to study the NO 3-To investigate the utilization efficiency of different nitrogen sources, different nitrogen sources were added to the mycelial chamber of the symbiotic system of AM fungus Glomus intraradices and Agrobacterium tumefaciens-transformed carrot roots. 15 N-KNO3 was added to the mycelium chamber to make 15 N-NO3 - It is absorbed by hyphae and transported to mycorrhizal tissues, and then the amount of 15 However, in the experiment, it was found that even the abundance of N markers could not be detected by directly digesting mycorrhizal tissue with sulfuric acid. 15 N-NO3 labeled mycorrhizal tissue 15 N abundance. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for treating nitrate nitrogen in biological tissues. 15 The method for determining N abundance is used to solve the problem of low accuracy or even inability to detect in existing determination methods.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for treating nitrate nitrogen in biological tissues 15 The method for determining N abundance includes the following steps:

[0009] 1) pre-freezing the biological tissue and then freeze-drying it to obtain a dried biological tissue;

[0010] 2) Mixing dried biological tissue, salicylic acid, and concentrated sulfuric acid and allowing to stand to obtain a mixture A;

[0011] 3) adding copper sulfate and potassium sulfate to mixture A to obtain mixture B;

[0012] 4) digesting the mixture B to obtain a digestive fluid;

[0013] 5) distilling the digestion liquid and receiving it with sulfuric acid solution to obtain a receiving liquid;

[0014] 6) performing rotary evaporation on the receiving solution to obtain a concentrated solution;

[0015] 7) Chlorosulfonic acid and DMSO-d6 were added to the concentrated solution to obtain 15 N abundance determination samples;

[0016] 8) 15 N abundance determination samples were subjected to nuclear magnetic resonance spectroscopy.

[0017] Preferably, the pre-freezing temperature is -70 to -90°C, and the pre-freezing time is 8 to 14 hours.

[0018] Preferably, the freeze drying is vacuum freeze drying, the freeze drying temperature is -40 to -60°C, and the time is 12 to 36 hours.

[0019] Preferably, the mass ratio of the dried biological tissue to salicylic acid is 1:1000-2000.

[0020] Preferably, the mass fraction of the concentrated sulfuric acid is 70-98.3%;

[0021] The mass volume ratio of the dried biological tissue to concentrated sulfuric acid is 1 mg: 10-20 ml;

[0022] The standing time is 20 to 40 minutes.

[0023] Preferably, the mass ratio of the dried biological tissue to copper sulfate is 1:200-400;

[0024] The mass ratio of the dried biological tissue to potassium sulfate is 1:2000-4000.

[0025] Preferably, the digestion temperature is 300-500°C;

[0026] During the digestion process, digestion is continued for 20 to 40 minutes after the liquid at the bottom of the container becomes clear.

[0027] Preferably, the distillation in step 5) is achieved by a Kjeldahl nitrogen analyzer, the distillate outlet of the Kjeldahl nitrogen analyzer is placed in a standard sulfuric acid solution, and the concentration of the standard sulfuric acid solution is 0.4 to 0.6 mol / L;

[0028] The digestive tube of the Kjeldahl nitrogen analyzer needs to be fed with a NaOH aqueous solution, the concentration of which is 5 to 15 mol / L.

[0029] Preferably, the temperature of the rotary evaporation is 50-70°C, the speed is 40-80 rpm, and the rotary evaporation is performed to 0.5-1.0% of the volume of the receiving liquid;

[0030] The volume ratio of the concentrated solution to chlorosulfonic acid is 1:0.3-0.8;

[0031] The volume ratio of the concentrated solution to DMSO-d6 is 1:0.05-0.15.

[0032] Preferably, the biological tissue is from plants and / or fungi.

[0033] Technical effects and advantages of the present invention:

[0034] The determination method of the present invention can avoid the adverse effects of concentrated sulfuric acid reagent and can successfully detect the nitrate nitrogen in biological tissues. 15 N abundance can be accurately detected even for arbuscular mycorrhizae that have high detection requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is the test result of the standard sample;

[0036] Figure 2 Add to the mycelium chamber 15 N-KNO3+Gly treatment group;

[0037] Figure 3 Add to the mycelium chamber 15 N-KNO3+(NH4)2SO4 treatment group;

[0038] Figure 4 Add to the mycelium chamber 15 N-KNO3+urea treatment group;

[0039] Figure 5 Add to the mycelium chamber 15 N-KNO3+H2O treatment group. DETAILED DESCRIPTION

[0040] The present invention provides a method for treating nitrate nitrogen in biological tissues 15 The method for determining N abundance includes the following steps:

[0041] 1) pre-freezing the biological tissue and then freeze-drying it to obtain a dried biological tissue;

[0042] 2) Mixing dried biological tissue, salicylic acid, and concentrated sulfuric acid and allowing to stand to obtain a mixture A;

[0043] 3) adding copper sulfate and potassium sulfate to mixture A to obtain mixture B;

[0044] 4) digesting the mixture B to obtain a digestive fluid;

[0045] 5) distilling the digestion liquid and receiving it with sulfuric acid solution to obtain a receiving liquid;

[0046] 6) performing rotary evaporation on the receiving solution to obtain a concentrated solution;

[0047] 7) Chlorosulfonic acid and DMSO-d6 were added to the concentrated solution to obtain 15 N abundance determination samples;

[0048] 8) 15 N abundance determination samples were subjected to nuclear magnetic resonance spectroscopy.

[0049] In the present invention, the biological tissue is preferably from plants and / or fungi, more preferably from arbuscular mycorrhizae (AM); the biological tissue in the present invention is preferably cleaned before pre-freezing; the pre-freezing temperature is preferably -70 to -90°C, more preferably -75 to -85°C, and the pre-freezing time is preferably 8 to 14 hours, more preferably 9 to 12 hours.

[0050] In the present invention, freeze drying is performed after pre-freezing, and vacuum freeze drying is preferably used for freeze drying. The freeze drying temperature is preferably -40 to -60°C, and the freeze drying time is preferably 12 to 36 hours, more preferably 20 to 26 hours. The freeze drying is preferably performed using a vacuum freeze dryer. Before the freeze drying is performed, the vacuum freeze dryer is preferably pre-cooled. The pre-cooling temperature is preferably -40 to 60°C, and the pre-cooling time is preferably 20 to 40 minutes. During the drying process, the centrifuge tube cover is preferably opened.

[0051] In the present invention, dried biological tissue, salicylic acid, and concentrated sulfuric acid are mixed, the mass fraction of the concentrated sulfuric acid is preferably 70-98.3%, more preferably 75-85%, the mass ratio of the dried biological tissue to salicylic acid is preferably 1:1000-2000, more preferably 1:1300-1700; the mass volume ratio of the dried biological tissue to concentrated sulfuric acid is preferably 1 mg:10-20 ml, more preferably 1 mg:13-17 ml, the mixing process is preferably accompanied by shaking, and the shaking time is preferably 15-30 s; after the mixing, the mixture is allowed to stand for the purpose of eliminating the foam, and the standing time is preferably 20-40 min, more preferably 25-35 min.

[0052] In the present invention, copper sulfate and potassium sulfate need to be added to mixture A, and the copper sulfate and potassium sulfate are in a solid state. The amount of copper sulfate and potassium sulfate added is calculated based on the mass of the dry biological tissue. The mass ratio of the dry biological tissue to copper sulfate is preferably 1:200-400, more preferably 1:250-350, and the mass ratio of the dry biological tissue to potassium sulfate is preferably 1:2000-4000, more preferably 1:2500-3500.

[0053] In the present invention, mixture B is digested, and the digestion temperature is preferably 300-500°C, more preferably 350-450°C, and the digestion is preferably carried out in a digestion furnace; during the digestion process, it is preferably digested until the liquid at the bottom of the container becomes clear and then continued to digest for 20-40 minutes, and the continued digestion time is more preferably 15-35 minutes. After digestion, it is preferably cooled, and the cooling is preferably natural cooling to room temperature.

[0054] In the present invention, distillation is performed after the digestion, and the distillation is preferably achieved by a Kjeldahl nitrogen analyzer. The distillate pipe mouth of the Kjeldahl nitrogen analyzer is preferably placed in a standard sulfuric acid solution, the concentration of the standard sulfuric acid solution is preferably 0.4-0.6 mol / L, and the volume of the standard sulfuric acid solution is preferably 20-30 ml; the digestion tube of the Kjeldahl nitrogen analyzer is preferably passed through a NaOH aqueous solution, the concentration of the NaOH aqueous solution is preferably 5-15 mol / L, more preferably 8-12 mol / L, and the distillation is preferably stopped after the receiving liquid reaches 120-160 ml.

[0055] In the present invention, after distillation, the receiving liquid is subjected to rotary evaporation, and the rotary evaporation is performed using a rotary evaporator. The temperature of the rotary evaporation is preferably 50-70°C, more preferably 55-65°C, and the speed of the rotary evaporation is preferably 40-80rpm, more preferably 60-70rpm. The rotary evaporation is preferably rotary evaporated to 0.5-1.0% of the volume of the receiving liquid, more preferably 0.7-0.8%. In a specific embodiment of the present invention, the operation of the rotary evaporation is as follows: the receiving liquid is transferred to the rotary bottle of the rotary evaporator and rinsed with distilled water, and the residual receiving liquid is transferred to the rotary bottle, the condensing device and the vacuum device are turned on, the temperature is set, the speed is adjusted from low to high, stabilized at medium speed, and rotary evaporation is performed. In this specific embodiment, the model of the rotary evaporator is RE-5299 rotary evaporator.

[0056] In the present invention, chlorosulfonic acid and DMSO-d6 are added to the concentrate, and the volume ratio of the concentrate to chlorosulfonic acid is preferably 1:0.3-0.8, more preferably 1:0.4-0.6; the volume ratio of the concentrate to DMSO-d6 is preferably 1:0.05-0.15, more preferably 1:0.08-0.12, to obtain 15 N abundance determination samples; 15 The pH of the sample for N abundance determination is preferably below 1.0; 15 After the N abundance determination sample is tested using a nuclear magnetic resonance spectroscopy detection method, the present application does not specifically limit the parameters in the nuclear magnetic resonance spectroscopy detection process, and conventional parameters that can be used for detection can be used.

[0057] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0058] Example 1

[0059] The cleaned 15The N-KNO3-labeled AM fungal mycorrhizal tissue was placed in a centrifuge tube and then placed in a -80°C refrigerator for 12 hours. The mycorrhizal tissue was placed in a vacuum freeze dryer that had been pre-cooled at -50°C for 30 minutes for drying. The centrifuge tube was opened during the drying process. After drying at -50°C for 24 hours, it was taken out and weighed on an electronic balance to an accuracy of 1 mg. The tube was transferred to a clean and dried digestion tube. 1.5 g of salicylic acid and 15 mL of 98% concentrated sulfuric acid were added to the digestion tube, shaken for 20 seconds, and then allowed to stand for 30 minutes to allow the foam to disappear. 0.3 g of copper sulfate and 3 g of potassium sulfate were added to the digestion tube, shaken, and placed in a digestion furnace. The digestion furnace temperature was set to 400°C and heated until the bottom of the digestion tube was clear and transparent. The tube was digested for another 30 minutes, taken out, and cooled.

[0060] The cooled digestion liquid was connected to the adjusted Kjeldahl nitrogen analyzer, and the outlet of the effluent tube was immersed in 25 mL of 0.5127 mol / L sulfuric acid standard solution. Then 40 mL of 10 mol / L NaOH solution was introduced into the digestion tube and distillation was started. The distillate was stopped after the receiving liquid (containing 25 mL of sulfuric acid standard solution) in the conical flask reached 150 mL. The receiving liquid was transferred to the rotary flask of the rotary evaporator and the conical flask was rinsed with distilled water. The residual receiving liquid was transferred to the rotary flask, and the condensing device and the vacuum device were turned on. The temperature was set to 60 ° C, the speed was adjusted from low to high, and stabilized at medium speed. The concentrated solution volume was 1 mL, 0.5 mL of chlorosulfonic acid and 0.1 mL of DMSO-d6 were added to the concentrated solution, and mixed to obtain 15 The pH value of the sample was 0.9.

[0061] Example 2

[0062] The cleaned 15 The AM fungus-cotton mycorrhizal tissue labeled with N-KNO3 was placed in a centrifuge tube and then placed in a -80°C refrigerator for 14 hours; the mycorrhizal tissue was placed in a vacuum freeze dryer that had been pre-cooled at -60°C for 20 minutes for drying. The centrifuge tube was opened during the drying process and taken out after drying at -50°C for 24 hours. It was weighed on an electronic balance to an accuracy of 1 mg and transferred to a clean and dried digestion tube. 1.8 g of salicylic acid and 20 mL of 98% concentrated sulfuric acid were added to the digestion tube, shaken for 30 seconds, and then allowed to stand for 40 minutes to allow the foam to disappear. 0.25 g of copper sulfate and 0.25 g of potassium sulfate were added to the digestion tube, shaken, and placed in a digestion furnace. The digestion furnace temperature was set to 350°C and heated until the bottom of the digestion tube was clear and transparent. Digestion was continued for 25 minutes, removed, and cooled.

[0063] The cooled digestion liquid was connected to the adjusted Kjeldahl nitrogen analyzer, and the outlet of the liquid tube was immersed in 25mL0.5127mol / L sulfuric acid standard solution. Then 40mL 10mol / LNaOH solution was introduced into the digestion tube and distillation was started. After the receiving liquid (containing 25mL sulfuric acid standard solution) in the conical flask reached 150mL, it was stopped. The receiving liquid was transferred to the rotary flask of the rotary evaporator and the conical flask was rinsed with distilled water. The residual receiving liquid was transferred to the rotary flask, and the condensing device and the vacuum device were turned on. The temperature was set to 55℃, the speed was adjusted from low to high, and stabilized at 70rpm. The concentrated solution was evaporated to a volume of 1mL, 0.4mL chlorosulfonic acid and 0.1mL DMSO-d6 were added to the concentrated solution, and mixed to obtain 15 N abundance determination samples.

[0064] Example 3

[0065] The cleaned 15 The AM fungal mycorrhizal tissue labeled with N-KNO3 was placed in a centrifuge tube and then placed in a -80°C refrigerator for 8 hours; the mycorrhizal tissue frozen overnight was placed in a vacuum freeze dryer that had been pre-cooled at -50°C for 30 minutes for drying. The centrifuge tube was opened during the drying process and taken out after drying at -50°C for 20 hours. It was weighed on an electronic balance to an accuracy of 1 mg and transferred to a clean and dry digestion tube. 1 g of salicylic acid and 10 mL of 98% concentrated sulfuric acid were added to the digestion tube, shaken for 25 seconds, and then allowed to stand for 20 minutes until the foam disappeared. 0.2 g of copper sulfate and 4 g of potassium sulfate were added to the digestion tube, shaken, and placed in a digestion furnace. The digestion furnace temperature was set to 500°C and heated until the bottom of the digestion tube was clear and transparent. Digestion was continued for 20 minutes, taken out, and cooled.

[0066] The cooled digestion liquid was connected to the adjusted Kjeldahl nitrogen analyzer, and the outlet tube was immersed in 25 mL of 0.5127 mol / L sulfuric acid standard solution. Then 40 mL of 10 mol / L NaOH solution was introduced into the digestion tube and distillation was started. After the receiving liquid in the conical flask (containing 25 mL of sulfuric acid standard solution) reached 150 mL, the distillation was stopped. The receiving liquid was transferred to the rotary flask of the rotary evaporator and the conical flask was rinsed with distilled water. The residual receiving liquid was transferred to the rotary flask, and the condensing device and the vacuum device were turned on. The temperature was set to 70 ° C, the speed was adjusted from low to high, and stabilized at medium speed. The concentrated solution volume was 1 mL, 0.3 mL of chlorosulfonic acid and 0.15 mL of DMSO-d6 were added to the concentrated solution, and the mixture was obtained after mixing. 15 N abundance determination samples.

[0067] Experimental Example 1

[0068] Set up the mycelium chamber and add 15 N-KNO3+Gly, 15N-KNO3+(NH4)2SO4, 15 N-KNO3+urea, 15 Four experimental groups of N-KNO3+H2O were obtained by the method of Example 1. 15 The samples were added to the washed and dried NMR tubes and the NMR in the mycorrhizal tissue was detected by NMR mass spectrometry. 15 N abundance.

[0069] Preparation of standard sample: weigh 20 mg ( 15 Dissolve 10 mg (NH4)2SO4 and 10 mg (NH4)2SO4 in 0.5 mL chlorosulfonic acid, add 0.1 mL DMSOd6, mix well, and put into a nuclear magnetic resonance tube for detection.

[0070] NMR mass spectrometer setup: 34 μL of a clear solution of (NH4)2SO4 in H2SO4 was dissolved in 600 μL of 99.9% DMSOd6 (a solvent used to provide a frequency locking signal to stabilize the spectrometer field) containing 0.05% (vol / vol) TMS reference.

[0071] 1 The H spectra were acquired on a 9.2-TVarian INOVA spectrometer with a 5-mm z-axis PFG dual broadband probe operating at 400 MHz.

[0072] The spectra were acquired using approximately 1,400 transients with a pulse width of 90° (10.8 μs), a spectral width of 5042 Hz, a pulse delay of 1.0 s, and an acquisition time of 1.6 s at 25 °C.

[0073] The T1 relaxation time of NH4 protons is 0.4s.

[0074] Relative to the TMS resonance at 0.0 ppm, it was observed 1 H- 14 N triplet resonance and 1 H- 15 The triplet resonance of N is centered at 7.2 ppm and the observed 1 H- 15 The N coupling is 53Hz and 74Hz respectively.

[0075] 1 H- 15 The integrated area of ​​the N doublet resonance divided by the sum of the doublet and triplet resonance areas gives the total N in the tissue. 15 N indicates percentage.

[0076] The test results of the standard sample are as follows Figure 1 As shown, from Figure 1 Three can be observed in14 There is one peak between each of the two N peaks. 15 N peak, according to the standard sample 14 N / 15 The N content was compared and the results were obtained. 15 The nitrogen content is 95.1% ± 1.7 of the standard sample, and the accuracy is reliable.

[0077] The test results of the experimental group are as follows Figures 2 to 5 As shown by Figures 2 to 5 It can be seen that three 14 N peak and two 15 The peak of N.

[0078] In mycorrhizal tissue 15 The N abundance data are shown in Table 1 below:

[0079] Table 1 Mycorrhizal tissue 15 N abundance

[0080]

[0081] As shown in Table 1, the mycorrhizal tissue of the control group without nitrogen source added in the mycelial chamber 15 The N abundance was the highest, at 1.317%, and the (NH4)2SO4 addition treatment group had the lowest, at only 0.168%.

[0082] Experimental Example 2

[0083] Set up the mycelium chamber to add different nitrogen 15 N-glutamine, 15 N-(NH4)2SO4, 15 N-urea, 15 N-KNO3、NH4 15 The five experimental groups of N-NO3 were prepared by prolonging the mycorrhizal tissue culture time, and four groups of 15 The samples were added to the washed and dried NMR tubes and the NMR in the mycorrhizal tissue was detected by NMR mass spectrometry. 15 N abundance, parameter settings are the same as in Experiment 1, and the results are shown in Table 2 below:

[0084] Table 2 Mycorrhizal tissue 15 N abundance

[0085]

[0086] As shown in Table 2, 15 N abundance has increased significantly, indicating that the analytical method is accurate and reliable. 15 N-NH4 15 N, can also analyze 15N-NO3 15 N.

[0087] From the above examples, it can be seen that the determination method provided by the present invention can avoid the adverse effects caused by concentrated sulfuric acid reagent and can successfully detect the nitrate nitrogen in biological tissues. 15 N abundance can be accurately detected even for arbuscular mycorrhizae with high detection requirements, and can also be analyzed 15 N-NH4 15 N, can also analyze 15 N-NO3 15 N.

[0088] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A kind of nitrate nitrogen in biological tissue 15 The method for determining N abundance is characterized by: The following steps are included: 1) pre-freezing the biological tissue and then freeze-drying it to obtain a dried biological tissue; 2) Mixing dried biological tissue, salicylic acid, and concentrated sulfuric acid and allowing to stand to obtain a mixture A; 3) adding copper sulfate and potassium sulfate to mixture A to obtain mixture B; 4) digesting the mixture B to obtain a digestive fluid; 5) distilling the digestion liquid and receiving it with sulfuric acid solution to obtain a receiving liquid; 6) performing rotary evaporation on the receiving solution to obtain a concentrated solution; 7) Chlorosulfonic acid and DMSO-d6 were added to the concentrate to obtain a pH less than 1. 15 N abundance determination samples; 8) 15 The samples were subjected to NMR spectroscopy at a frequency of 400 MHz. 1 H- 14 N triplet resonance and 1 H- 15 The doublet resonance result of N protons will be 1 H- 15 The integrated area of ​​the N doublet resonance divided by the sum of the doublet and triplet resonances gives the nitrate nitrogen in the mycorrhizal tissue. 15 N abundance; The mass ratio of the dried biological tissue to salicylic acid is 1:1000-2000; The mass fraction of the concentrated sulfuric acid is 70-98.3%; The mass volume ratio of the dried biological tissue to concentrated sulfuric acid is 1 mg: 10-20 ml; The standing time is 20 to 40 minutes; The rotary evaporation temperature is 50-70°C, the rotation speed is 40-80 rpm, and the rotary evaporation is performed to 0.5-1.0% of the volume of the receiving liquid; The volume ratio of the concentrated solution to chlorosulfonic acid is 1:0.3-0.8; The volume ratio of the concentrate to DMSO-d6 is 1:0.05-0.15; The mass ratio of the dried biological tissue to copper sulfate is 1:200-400; The mass ratio of the dried biological tissue to potassium sulfate is 1:2000-4000; The biological tissue is arbuscular mycorrhiza.

2. The nitrate nitrogen in biological tissue according to claim 1 15 The method for determining N abundance is characterized by: The pre-freezing temperature is -70 to -90°C, and the pre-freezing time is 8 to 14 hours.

3. The nitrate nitrogen in biological tissue according to claim 1 or 2 15 The method for determining N abundance is characterized by: The freeze drying adopts vacuum freeze drying, the freeze drying temperature is -40 to -60°C, and the time is 12 to 36 hours.

4. The biological tissue nitrate nitrogen according to claim 1 15 The method for determining N abundance is characterized by: The digestion temperature is 300-500°C; During the digestion process, digestion is continued for 20 to 40 minutes after the liquid at the bottom of the container becomes clear.

5. The nitrate-nitrogen in biological tissues according to claim 4 15 The method for determining N abundance is characterized by: Step 5) the distillation is achieved by a Kjeldahl nitrogen analyzer, the distillate outlet of the Kjeldahl nitrogen analyzer is placed in a standard sulfuric acid solution, the concentration of the standard sulfuric acid solution is 0.4 to 0.6 mol / L; The digestive tube of the Kjeldahl nitrogen analyzer needs to be fed with a NaOH aqueous solution, the concentration of which is 5 to 15 mol / L.

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