Soil non-symbiotic nitrogen-fixing microorganism detection method based on 15N2 tracer method

Abstract

The invention discloses a soil non-symbiotic nitrogen fixation microorganism detection method based on a 15N2 tracer method, and belongs to the technical field of biological nitrogen fixation and microbial ecology. The method comprises the following steps: (1) carrying out a 15N2 isotope microcosm experiment; (2) determination of gas and soil 15N abundance in a microcosm experiment; (3) separating stable isotope labeled DNA and non-labeled DNA; and (4) identifying the enrichment degree of the 15N-DNA and analyzing the flora structure. On the basis of a 15N2 tracer method, a stable isotope nucleic acid probe technology and a 15N2 isotope direct labeling technology are integrated, the active nitrogen-fixing microorganisms and the ecological functions of the active nitrogen-fixing microorganisms can be researched at the same time, and technical support is provided for defining the types of the soil active nitrogen-fixing microorganisms in a natural ecological system and making contribution to nitrogen input. The method is simple and efficient in operation process, and has the advantages of high sensitivity and rapidness.

Description

[0001] The present invention belongs to the technical field of biological nitrogen fixation and microbial ecology, and specifically relates to a method based on 15 N2 tracer method for detecting non-symbiotic nitrogen-fixing microorganisms in soil. Background Art

[0002] Biological nitrogen fixation is the process by which nitrogen-fixing microorganisms convert atmospheric nitrogen (N2) into ammonia nitrogen (NH3) or other nitrogen compounds that can be used by plants through nitrogenase. Biological nitrogen fixation is the main source of nitrogen in natural ecosystems and plays a key role in the balance of nitrogen cycle in global ecosystems.

[0003] At present, the identification of nitrogen-fixing microorganisms mainly relies on culture medium screening and molecular biology techniques, such as functional gene (nifH) PCR amplification, DNA sequencing, etc. These methods can identify nitrogen-fixing microorganisms to a certain extent, but there are some limitations. For example, the pure culture system can only explain the important physiological processes of a single microbial strain and cannot fully cover the complex and diverse microbial communities in the natural environment; the amplification and sequencing of the nifH gene can only represent the number of potential nitrogen-fixing bacteria in the soil, and cannot directly indicate that they are involved in the nitrogen fixation process. Stable isotope nucleic acid probe technology uses stable isotopes to trace microbial genomic DNA in complex environments, realizing the transformation from the study of single microbial physiological processes to the study of microbial community physiological ecology, and can target the discovery of important microbial resources at a higher and more complex overall level. However, this method cannot directly quantify the ecological functions of functional microorganisms.

[0004] Traditional methods for studying the rate of biological nitrogen fixation are mainly 15 N2 natural abundance method, 15 Indirect determination methods such as N2 isotope dilution method and nitrogen balance method have great uncertainty due to the lack of accurate conversion factors or the need to consider various factors such as nitrogen source and nitrogen loss. The most direct method to determine nitrogen fixation is 15 N2 direct labeling method, this method does not require a conversion coefficient and is not interfered by external nitrogen sources, so it can accurately determine the nitrogen fixation rate. 15 N2 is expensive and only used 15 The N2 direct labeling method is costly.

[0005] In view of the above problems, the present invention provides a 15 The N2 tracer method is a method for detecting non-symbiotic nitrogen-fixing microorganisms in soil. 15 Based on the N2 tracer method, the stable isotope nucleic acid probe technology and 15 N2 isotope direct labeling technology can simultaneously explore active nitrogen-fixing microorganisms and their ecological functions, and provide technical support for clarifying the types of active nitrogen-fixing microorganisms in soil in natural ecosystems and their contribution to nitrogen input. Summary of the invention

[0006] The purpose of the present invention is to provide a 15 The N2 tracer method for detecting non-symbiotic nitrogen-fixing microorganisms in soil can solve the problems mentioned in the background technology.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A kind of 15 The soil non-symbiotic nitrogen-fixing microorganism detection method of the N2 tracer method specifically includes the following steps:

[0009] (1) 15 N2 isotope microcosm experiment, take 6g (dry weight) of soil sample and put it into the headspace bottle for 15 N2 isotope microcosm experiment. The headspace bottle is sealed with a butyl rubber stopper, an aluminum cap and a sealing film, and a mixed gas (98atom% 15 N2:UHP-O2:UHP-He=78:10:12, v / v). The culture bottle containing the mixed gas was placed at 25°C and incubated in the dark for 7 days, and then the soil was mixed and weighed to 0.3g to extract soil DNA;

[0010] (2) Gas and soil in microcosm experiments 15 Determination of N abundance: The mixed gas in the headspace bottle after 7 days of incubation was extracted to measure the mixed gas 15 N abundance, and then the soil samples were dried at 60℃ to measure soil total nitrogen and 15 N abundance, the biological nitrogen fixation rate of soil samples is calculated as follows:

[0011]

[0012] Among them, ANF rate is the biological nitrogen fixation rate, atom% 15 N excess soil represents the atomic percentage of the soil sample relative to the control soil sample, and TN soil represents the total nitrogen in the soil, atom% 15 N excess gas represents the atomic percentage excess of the gas sample relative to the control gas sample, and t represents the incubation time.

[0013] (3) separating stable isotope-labeled and unlabeled DNA, separating the heavy layer DNA by ultra-high speed centrifugation based on cesium chloride density gradient solution, adding cesium chloride and GB buffer solution to the DNA sample extracted in step (1), respectively, and performing ultra-high speed density centrifugation, and further performing layered purification on the centrifuged sample to obtain DNA solutions of different density levels;

[0014] (4) Conduct 15The enrichment degree of N-DNA was identified and the bacterial community structure was analyzed. The nifH gene fluorescence quantitative PCR was performed on the DNA samples after layer purification in step (3) to analyze the quantitative change pattern of nifH in DNA with different buoyancy densities and compare it with the 14 Compared with the N control treatment, the displacement of nifH functional gene in different buoyancy density gradient zones can be accurately determined. 15 The location and enrichment of N-DNA in different buoyancy density zones. The DNA in the enriched area and the control area were sequenced by high-throughput sequencing of the nifH gene, and finally compared. 14 N and 15 The microbial community structure of N-layer DNA samples was analyzed to identify active nitrogen-fixing microorganisms.

[0015] Preferably, the sample used in step (1) is collected from a non-leguminous grassland area to exclude the symbiotic nitrogen fixation caused by the symbiosis between rhizobia and leguminous plants. After the sample is collected, it is transported to the laboratory at 4°C and stored at -20°C for future use.

[0016] Preferably, in step (1), the moisture content of the soil sample is adjusted to 60% WHC with deionized water, and pre-incubated for 24 h at 25° C. in the dark to restore the activity of nitrogen-fixing bacteria.

[0017] Preferably, in step (1), a mixed gas (98atom% 14 N2:UHP-O2:UHP-He=78:10:12, v / v) was used as the control group, and the mixed gas (98atom% 15 N2:UHP-O2:UHP-He=78:10:12, v / v) was used as the experimental group.

[0018] Preferably, in step (3), a 5.1 mL ultracentrifuge tube is filled with GB buffer (0.1 M Tris-HCl, pH 8; 0.1 M KCl; 1.0 mM EDTA), 6.0 μg DNA and 1.725 g mL -1 CsCl, the refractive index of the mixed sample should be 1.4015±0.0002. The centrifuge tube was placed in an ultra-high speed centrifuge at 20°C and 45k rpm for 48 hours. After the centrifugation, the centrifuge was further divided into 16 layers using an ultra-high speed density gradient centrifuge automatic stratification device, and the refractive index of each layer of DNA was measured. After each layer of DNA was precipitated with PEG 6000 and purified with 70% (v / v) ethanol, it was diluted with 30 mL of nuclease-free water.

[0019] Preferably, in step (4) 15 The relative abundance of N-labeled ASVs and 14 The ratio of the relative abundance of N-labeled ASVs greater than 2 was considered active ASVs.

[0020] Beneficial effects:

[0021] 1) The present invention adopts isotope labeling technology, combines macrobiology and microbiology from different perspectives, closely links the structure and function of nitrogen-fixing microorganisms, and can simultaneously detect active nitrogen-fixing microorganisms and their ecological functions;

[0022] 2) The present invention can directly measure the biological nitrogen fixation rate of soil samples for different grasslands and soil types, and can accurately identify active nitrogen-fixing microorganisms in soil samples.

[0023] 3) The operation process of the present invention is simple and efficient, with high sensitivity and rapidity, and provides a new quantitative analysis method for soil biology research. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of isotope labeling of the present invention; 1 is a syringe, 2 is a three-way valve, 3 is a butyl rubber stopper, 4 is an aluminum cap, 5 is a needle, 6 is a headspace bottle, and 7 is the soil to be tested.

[0025] Figure 2 This is the quantitative result of nifH gene in each layer of DNA sample after layered purification in the isotope microcosm experiment of Example.

[0026] Figure 3 This is the result of bacterial genus level differences in heavy-layer DNA fragments in the isotope microcosm experiment. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. Specific embodiments are only used to further describe the present invention in detail and do not limit the scope of protection of the present invention. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0028] The present invention provides a 15 The soil non-symbiotic nitrogen-fixing microorganism detection method of the N2 tracer method specifically includes the following steps:

[0029] In one embodiment, during the peak plant growth season on the Qinghai-Tibet Plateau (July-August), a 30m×30m concentric circle is randomly arranged in Gangcha County, and three sample lines are arranged at an angle of 120 degrees on the concentric circle. 0-10cm soil is collected from non-leguminous grassland areas at equal distances on each sample line. After the soil is collected, it is uniformly sieved with a 2mm sieve to remove impurities, and then transported to the laboratory at 4°C in sealed bags, and the soil nitrogen-fixing microorganisms are identified and the rate is determined according to the following steps.

[0030] (1) Conduct 15N2 isotope microcosm experiment. Take 6g of soil sample (dry weight) ( Figure 1 Mark 7) into the headspace bottle ( Figure 1 The soil sample was adjusted to 60% WHC with deionized water and pre-incubated at 25°C in the dark for 24 h before the headspace bottle was sealed with a butyl rubber stopper ( Figure 1 Mark 3), aluminum cover ( Figure 1 Marked 4 in the middle) and sealed with sealing film, vacuumed and injected with mixed gas (98atom% 15 N2:UHP-O2:UHP-He=78:10:12, v / v). The culture bottle containing the mixed gas was placed at 25℃ and incubated in the dark for 7 days, then the soil was mixed and weighed 0.3g to extract soil DNA, and stored at -20℃ for later use; the mixed gas (98atom% 14 N2:UHP-O2:UHP-He=78:10:12, v / v) was used as the control group, and five groups were repeated. (2) Gas and soil in the microcosm experiment 15 Determination of N abundance. The mixed gas in the headspace bottle after 7 days of incubation was extracted to measure the mixed gas 15 N abundance, and then the soil samples were dried at 60℃ to measure soil total nitrogen and 15 N abundance was determined by MAT253 stable isotope mass spectrometry. 15 The biological nitrogen fixation rate of soil samples is calculated as follows:

[0031]

[0032] Among them, ANF rate is the biological nitrogen fixation rate, atom% 15 N excess soil represents the atomic percentage of the soil sample relative to the control soil sample, and TN soil represents the total nitrogen in the soil, atom% 15 N excess gas represents the atomic percentage excess of the gas sample relative to the control gas sample, and t represents the incubation time.

[0033] (3) Separation of stable isotope labeled and unlabeled DNA:

[0034] First, prepare the ultra-high-speed density gradient centrifugation reagent. Preparation method of Gadient Buffer (GB buffer contains 0.1M Tris-HCl (pH=8.0), 0.1M KCl, 1.0mM EDTA): Add 50mL 1M Tris-HCl, 3.75g KC1 and 1.0mL 0.5M EDTA in 400ml deionized water, dilute to 500mL with deionized water, filter at 0.2mm and sterilize. Cesium chloride solution (gradient separation of nucleic acids DNA with different density gradients): Dissolve 603g cesium chloride in a final volume of 500ml GB buffer (can be heated to 30°C to promote the dissolution of cesium chloride). At room temperature of 20°C, its density is about 1.88-1.89mg·mL -1 ; 70% ethanol: add 370mL 95% ethanol to 130mL deionized water; PEG 6000 solution: dissolve 150g PEG 6000 and 46.8g NaCl in deionized water and make up to 500mL, filter and sterilize;

[0035] Then perform DNA ultra-high-speed density gradient centrifugation. Add 4.4 mL of cesium chloride, 1.1 mL of GB buffer and 6.0 μg of DNA sample to a 15 ml centrifuge tube in sequence. Use cesium chloride solution and GB buffer to adjust the refractive index of the mixed sample to 1.4015±0.0002, and use an AR200 refractometer to measure the refractive index of the mixed solution. Use a syringe to transfer the centrifuged solution sample to a 5.1 mL ultracentrifuge tube, and use a special sealing gun to seal the centrifuge tube. Ultra-high-speed centrifuge operating parameters: centrifugation time (48h), centrifugation temperature (20°C), centrifugation speed (45k rpm), centrifuge start acceleration parameters (maximum), centrifuge stop parameter settings (no brake);

[0036] Finally, DNA density gradient stratification and purification were performed. After centrifugation, the ultra-high-speed density gradient centrifuge liquid automatic stratification device was further used to divide the centrifuge liquid into 16 layers, each layer was 300 μL (the last layer was discarded), and the refractive index of each layer of DNA was measured. 550 mL of PEG 6000 solution was added to the different buoyancy density gradient DNA solutions, and the solution was mixed by inverting several times, and then allowed to stand at room temperature for 2 hours or heated at 37°C for 1 hour, and centrifuged at 13000×g for 30 minutes at 15-20°C, and the supernatant was removed; 500 μL of 70% ethanol was added to wash the DNA precipitate, centrifuged for 10 minutes, and the supernatant was removed; the above steps were repeated to further remove cesium chloride and remove PEG 6000 as much as possible. After each layer was precipitated with PEG 6000 and purified with 70% (v / v) ethanol, the DNA precipitate was dried at room temperature, and after ensuring that there was no liquid in the DNA precipitate, it was dissolved in 30 mL of nuclease-free water.

[0037] (4) Conduct 15Identification of N-DNA enrichment and analysis of bacterial community structure:

[0038] first, 15 Identification of the enrichment degree of N-DNA. Fluorescence quantitative PCR of nifH gene was performed on the purified DNA samples to analyze the quantitative changes of nifH in DNA with different buoyancy densities and compare them with 14 Compared with the N control treatment, the displacement of nifH functional gene in different buoyancy density gradient zones can be accurately determined. 15 The position and enrichment of N-DNA in different buoyancy density zones are used to evaluate whether the heavy-layer microorganisms on the marker are active nitrogen-fixing bacteria. Figure 2 As shown, it can be seen that the soil non-symbiotic nitrogen-fixing bacteria were successfully labeled;

[0039] Then the microbial community structure was identified. 15 The N2-labeled and screened heavy buoyancy density DNA was subjected to high-throughput sequencing of the nifH gene. 14 N and 15 Microbial community structure of N-layer DNA samples, 15 The relative abundance of N-labeled ASVs and 14 ASVs with a relative abundance ratio of N-labeled ASVs greater than 2 were considered active ASVs. Figure 3 As shown, it can be seen that Mesorhizobium, Bradyrhizobium and Rhodovulum are active nitrogen-fixing bacterial genera in alpine grassland ecosystems, among which Mesorhizobium is the most important nitrogen-fixing microorganism.

Claims

1. A method based on 15 The method for detecting non-symbiotic nitrogen-fixing microorganisms in soil by N2 tracing method is characterized in that: The following steps are involved: (1) Take the soil sample and put it into the headspace bottle. 15 N2 isotope microcosm experiment. The headspace bottle is sealed with a butyl rubber stopper, an aluminum cap and a sealing film, and a mixed gas (98atom% 15 N2:UHP-O2:UHP-He=78:10:12, v / v). The culture bottle containing the mixed gas was placed in the dark under suitable conditions for incubation for 7 days, and then the soil was mixed to extract soil DNA; (2) Extract the mixed gas from the headspace bottle after 7 days of incubation and use it to measure the mixed gas 15 N abundance, and then the soil samples were dried at 60℃ to measure soil total nitrogen and 15 N abundance; (3) using an ultra-high speed centrifugation method based on a cesium chloride density gradient solution to separate the heavy layer DNA, respectively adding cesium chloride and GB buffer solution to the DNA sample extracted in step (1), performing ultra-high speed density centrifugation, and further performing layered purification on the centrifuged sample to obtain DNA solutions of different density levels; (4) Conduct 15 The enrichment degree of N-DNA was identified and the bacterial community structure was analyzed. The nifH gene fluorescence quantitative PCR was performed on the DNA samples after layer purification in step (3) to analyze the quantitative change pattern of nifH in DNA with different buoyancy densities and compare it with the 14 Compared with the N control treatment, the displacement of nifH functional gene in different buoyancy density gradient zones can be accurately determined. 15 The location and enrichment of N-DNA in different buoyancy density zones. The DNA in the enriched area and the control area were sequenced by high-throughput sequencing of the nifH gene, and finally compared. 14 N and 15 The microbial community structure of N-layer DNA samples was analyzed to identify active nitrogen-fixing microorganisms.

2. A method according to claim 1 based on 15 The method for detecting non-symbiotic nitrogen-fixing microorganisms in soil by N2 tracing method is characterized in that: The sample used in step (1) is collected from a non-leguminous grassland area to exclude the symbiotic nitrogen fixation caused by the symbiosis between rhizobia and leguminous plants. After collection, the samples were transported to the laboratory at low temperature.

3. A method according to claim 1 based on 15 The method for detecting non-symbiotic nitrogen-fixing microorganisms in soil by N2 tracing method is characterized in that: Step (1) The soil sample is adjusted to an optimum moisture content with deionized water and pre-incubated for 24 hours in suitable dark conditions to restore the activity of nitrogen-fixing bacteria.

4. A method according to claim 1 based on 15 The method for detecting non-symbiotic nitrogen-fixing microorganisms in soil by N2 tracing method is characterized in that: In step (1), a mixed gas (98atom% 14 N2:UHP-O2:UHP-He=78:10:12, v / v) was used as the control group, and the mixed gas (98atom% 15 N2:UHP-O2:UHP-He=78:10:12, v / v) was used as the experimental group.

5. A method according to claim 1 based on 15 The method for detecting non-symbiotic nitrogen-fixing microorganisms in soil by N2 tracing method is characterized in that: Step (3) A 5.1 mL ultracentrifuge tube was filled with GB buffer (0.1 M Tris-HCl, pH 8; 0.1 M KCl; 1.0 mM EDTA), 6.0 μg DNA, and 1.725 g mL -1 CsCl, the refractive index of the mixed sample should be 1.4015±0.0002. The centrifuge tube was placed in an ultra-high speed centrifuge at 20°C and 45k rpm for 48 hours. After the centrifugation, the centrifuge was further divided into 16 layers using an ultra-high speed density gradient centrifuge automatic stratification device, and the refractive index of each layer of DNA was measured. After each layer of DNA was precipitated with PEG 6000 and purified with 70% (v / v) ethanol, it was diluted with 30 mL of nuclease-free water.

6. A method according to claim 1 based on 15 The method for detecting non-symbiotic nitrogen-fixing microorganisms in soil by N2 tracing method is characterized in that: In step (4) 15 The relative abundance of N-labeled ASVs and 14 The ratio of the relative abundance of N-labeled ASVs greater than 2 was considered active ASVs.

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