Method for accurately quantifying activity of soil nitrogen cycle microbial enzyme
Through DIA metaproteomic sequencing and mass spectrometer analysis, the problem of inaccurate quantification of soil nitrogen cycle microbial enzyme activity in the prior art was solved, and the accurate quantification of soil nitrogen cycle microbial enzyme activity was achieved, providing a more accurate understanding of the soil nitrogen cycle process.
Patent Information
- Application Number
- CN202510569329.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-01
- Publication Date
- 2025-08-15
AI Technical Summary
Existing metagenomic and metatratonicomic technologies cannot accurately quantify the activity of soil nitrogen circulation microbial enzymes, and cannot truly reflect the actual metabolic activities and functional status of microbial communities, especially when RNA instability leads to inaccurate results when microbial activity is high.
DIA metaproteomic sequencing method was used to extract total soil protein by configuring appropriate soil protein extract, combining timsTOF Pro2 mass spectrometer and Spectronaut 19 software to collect and analyze DIA data, establish a protein sequence database related to microbial nitrogen cycle, and achieve accurate quantification of protease types and abundance.
The accurate quantification of the activity of soil nitrogen circulation microbial enzymes can more truly reflect the functional status and dynamic changes of microbial communities, overcome the limitations of traditional methods, and provide a more accurate understanding of the soil nitrogen circulation process.
Smart Images
Figure CN120490314A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of environmental research and relates to a method for accurately quantifying soil nitrogen cycle microbial enzyme activity. Background Art
[0002] Soil, as an important sink of active nitrogen and an important source of N2O, has attracted extensive attention in the environmental field. Nitrogen cycle microorganisms drive the conversion of NH4 in soil through the nitrification process (ammonia oxidation process), denitrification process, nitrate reduction to ammonium (DNRA) process and nitrogen assimilation process catalyzed by biological enzymes. + 、NO3 - Accurately quantifying the mutual conversion between organic nitrogen and by-product N2O in nitrogen metabolism can help reveal the deeper soil nitrogen cycle mechanism of different ecosystems, help optimize agricultural fertilization management and improve nitrogen use efficiency, and at the same time has important scientific value and practical significance for formulating precise greenhouse gas emission reduction strategies.
[0003] Research on the genetic inventory of nitrogen-cycling microorganisms in soil has a long history. In 2005, Tringe et al. used metagenomics to analyze microbial DNA, systematically comparing microbial communities from diverse environments (including soil, ocean, and acid mine drainage) for the first time. They detected functional genes related to the nitrogen cycle (such as nitrogen fixation and nitrification), marking the beginning of the application of high-throughput sequencing technology to the genomics of soil nitrogen-cycling microorganisms (Chen et al., 2015). However, metagenomics, based on DNA-level analysis, has limitations for studying soil organisms and genetic inventory and is only suitable for regional surveys. The subsequent emergence of RNA-based metatranscriptome analysis provides a closer look at the immediate activity of nitrogen-cycling microorganisms in soil. In 2008, Tim Urich and other scholars extracted total RNA from soil microorganisms and combined it with high-throughput sequencing (454 pyrosequencing) to analyze the active functions of soil microbial communities, including the expression patterns of nitrogen cycle-related genes (such as nitrification and denitrification genes) (Urich et al., 2008). This was the first landmark study to conduct macrotranscriptome analysis directly in the soil environment.
[0004] Although the development of transcriptomics has greatly facilitated the study of microbial enzyme activity, due to the instability of RNA, in some cases, such as when microbial activity is high, transcriptomics cannot truly and accurately quantify microbial enzyme activity. Therefore, existing metagenomics, metatranscriptomics, qPCR, and RT-qPCR technologies can only study the gene inventory and transcription of soil nitrogen cycling microorganisms, and cannot directly reveal the dynamic changes in the microbial nitrogen cycle process in the soil from the level of functional proteins. Compared with DNA and RNA, proteins are the direct executors of function. Proteomics technology directly detects proteins expressed in environmental samples and can more realistically reflect the actual metabolic activities and functional status of microbial communities. However, due to the complex soil environment, the susceptibility of proteins to enzymatic denaturation, and the low proportion of nitrogen cycling microorganisms, the method for quantifying proteins in the soil nitrogen cycle process has not yet been clarified. Summary of the Invention
[0005] To address these technical limitations, the present invention provides a method for accurately quantifying the enzyme activity of soil nitrogen cycle microorganisms. This method, based on DIA metaproteomics sequencing, 1) adds a method for effectively extracting total soil protein, thereby improving the detection rate of effective protein; and 2) develops a method for detecting and quantifying the enzyme activity of microorganisms involved in the nitrogen cycle in soil.
[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:
[0007] A method for accurately quantifying the activity of microbial enzymes involved in the soil nitrogen cycle comprises the following steps: first, preparing an appropriate soil protein extract solution and adding the protein extract solution to the soil from which total protein extraction is desired at an appropriate extract / soil ratio. The reaction is terminated by high-temperature steaming, and total soil protein is extracted and enriched according to the experimental procedures. The extracted total protein is then quantified, subjected to electrophoresis, enzymatic hydrolysis, and desalination, and analyzed using a timsTOF Pro2 mass spectrometer via LC-MS / MS. DIA data is collected in diaPASEF mode and peak areas are calculated. A database of protein sequences and microbial sequences related to the microbial nitrogen cycle is established. The DIA data is searched and analyzed using Spectronaut 19, and the identified protease species and abundances are output.
[0008] The protein extraction reagent involves drugs: sodium dodecyl sulfate, Tris-HCl, sodium chloride, magnesium chloride, dithiothiol, EDTA, and phenylmethylsulfonyl fluoride.
[0009] The sample is any soil sample involving microorganisms, such as crop soil, wetland soil, sediment, etc.
[0010] The protein was quantified using Pierce TM BCA protein detection kit was used for BCA method quantification.
[0011] The protein electrophoresis adopts SDS-PAGE electrophoresis method.
[0012] The microbial nitrogen cycle protein database is established based on NCBI, Ncyc, and Uniprot.
[0013] The mass spectrometer model is: timsTOF Pro2 mass spectrometer (Bruker Daltonik, Bremen, Germany) connected in series with an UltiMate 3000 system (Thermo Fisher Scientific, MA, USA). BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 This is a schematic diagram of the experimental process of a method for accurately quantifying soil nitrogen cycle microbial enzyme activity provided by the present invention.
[0015] Figure 2 This is a BCA line graph of the concentration of concentrated soil protein extraction provided by an embodiment of the present invention.
[0016] Figure 3 This is a comparison chart of quantitative nitrogen cycle microbial enzyme activity groups in a method for accurately quantifying soil nitrogen cycle microbial enzyme activity provided by an embodiment of the present invention. DETAILED DESCRIPTION
[0017] The present invention will be further described in detail below with reference to the embodiments shown in the accompanying drawings.
[0018] The present invention is divided into the following parts from the perspective of experimental method and data processing:
[0019] (1) Soil total protein extraction
[0020] Prepare a protein extract prepared by dissolving 1 mM phenylmethylsulfonyl fluoride, 150 mM sodium chloride, 0.1 mM ethylenediaminetetraacetic acid, 1 mM magnesium chloride, 50 mM dithiothiol, and 5% (w / v) sodium dodecyl sulfate in Tris-HCl buffer. Adjust the pH to 8.5 with concentrated hydrochloric acid and store at 4°C. When using, ultrasonically reconstitute and return to room temperature to reduce the impact on the protein.
[0021] Weigh an appropriate amount of revived soil into a centrifuge tube, add an appropriate amount (v:w = 2-4) of protein extract, and place in a vortexer at maximum speed (3200 rpm) for 5 minutes to thoroughly mix the soil and extract. Transfer the mixture to a glass serum bottle, seal it with a rubber stopper and aluminum cap, and heat it in a water bath in boiling water for 1 hour. The glass bottle should not directly contact the high-temperature heating plate to ensure uniform heating of the mixture, terminate the soil reaction, and fully denature and precipitate the soil protein. Heat-resistant glass bottles are necessary during this process, and the mixture should be shaken during heating to prevent precipitation.
[0022] Remove the glass bottle, let it cool, and then transfer it to a centrifuge tube. Vortex at maximum speed for 5 minutes and centrifuge at 2100g for 10 minutes to precipitate the sand and gravel particles in the soil. Take the supernatant and distribute it in a 2ml centrifuge tube. Take 100% (m / V) TCA solution and mix it with the supernatant in a ratio of 1:3. Shake to mix thoroughly and place it in a -20℃ static incubation for 8-10 hours. After incubation, thaw the supernatant in an ice-water mixture, balance it, and centrifuge it at 21000g and 4℃ for 20 minutes to precipitate impurities in the mixture. Gently pour off the supernatant, making sure to retain the black and transparent precipitates at the bottom of the centrifuge tube.
[0023] Take 1 ml of chilled acetone (stored at -20°C) and add it to the centrifuge tube containing the precipitate. Repeatedly aspirate the suspension to thoroughly wash it with the acetone. Vortex the suspension intermittently for 1 minute and centrifuge at 21,000 g at 4°C for 10 minutes. Repeat this step at least three times to wash away impurities and humic substances in the precipitate, leaving a clear precipitate.
[0024] Discard the upper acetone solution, leaving the transparent precipitate. Air-dry the precipitate and completely evaporate the acetone in a sterile, ventilated, clean hood at 4°C. Prepare guanidine buffer: 6 M guanidine hydrochloride, 10 mM dithiothreitol, 50 mM tris(hydroxymethyl)aminomethane, and 10 mM calcium chloride. Adjust the pH to 7.6 with NaOH. Enrich the protein pellets in the precipitate based on the estimated total protein content in the soil. This can be achieved by increasing the soil mass and the number of centrifuge tubes used. Repeatedly dissolve the protein pellet in multiple 2 ml centrifuge tubes using 1 ml of guanidine buffer to increase the total protein concentration in the soil. Proteins should ideally be stored as pellets or in guanidine buffer at -80°C. Avoid repeated freeze-thaw cycles. More than three freeze-thaw cycles may reduce protein quality and lead to denaturation and enzymatic degradation. Determine total soil protein concentration using the BCA assay.
[0025] (2) Protein desalting, purification and mass spectrometry analysis
[0026] The supernatant was desalted using a Sep-Pak C18 column: 5 ml of acidified acetonitrile was rinsed through the Sep-Pak column, followed by 5 ml of acidified water via syringe. The sample was then injected via syringe onto the C18 resin to capture the peptides. The cartridge was washed with 5 ml of acidified water to remove the buffer and salts. The captured peptides were eluted with 5 ml of acidified acetonitrile into a fresh Eppendorf tube. The initial solution was dried using a vacuum centrifuge to obtain a lyophilized protein pellet.
[0027] Before mass spectrometry analysis, the lyophilized particles were redissolved in 0.1% formic acid in water (phase A) and 200 ng of sample was loaded (AUR3-15075C18 analytical column: 15 cm × 75 μm i.d, 1.7 μm particle size, pore size, IonOpticks), 60min gradient separation of samples, column temperature was 50℃. The column flow rate was controlled at 400nl / min, the gradient started from 2.2% of 80% acetonitrile, 0.1% formic acid (phase B), increased to 28% in 46min, increased to 38% in 8min, and reached 90% in 3min, and maintained for 3min. DIA data acquisition was performed in diaPASEF mode, with a scan range from 349-1229m / z and an isolation window width set to 40Da. During the PASEF MSMS scan, the collision energy increased linearly with the ion mobility from 59eV (1 / K0=1.6Vs / cm 2 ) rises to 20eV (1 / K0=0.6Vs / cm 2 ).
[0028] (3) Establishment of nitrogen cycle protein database
[0029] Download protein sequence files from the Ncyc database. Use Entrez Direct in a Linux operating system to download the first 1 million sequences of all target proteins related to nitrogen-cycling microorganisms from NCBI (amoA, amoB, HAO, nirS, etc.) and merge them into a single faa protein sequence file. These sequences were then compiled to create a nitrogen-cycling protease database.
[0030] (4) Identification and quantification of nitrogen cycle microbial proteases
[0031] DIA data were analyzed using Spectronaut 19 default parameters (BGS Factory Settings (default)). The sequence database was the nitrogen cycle protein database mentioned above, and Trypsin enzymatic digestion was set. The iRT peptide software can automatically correct the retention time and mass window and automatically determine the ideal extraction window. Protein qualitative standards: PrecursorThreshold 1.0% FDR, Protein Threshold 1.0% FDR. The Decoy database was generated using a mutated strategy, which is similar to scrambling a random number of amino acid sequences (minimum 2 amino acids, maximum half of the total length of the peptide segment). Spectronaut performed automatic correction and used a local normalization strategy for data normalization. Peptides with an FDR of less than 1.0% were quantified using MaxLFQ for protein groups. The library search was completed to obtain qualitative and relative quantitative results of nitrogen cycle microbial proteases, and the corresponding sequence and description information of the protease were output at the same time.
[0032] (5) Identification and quantification of nitrogen-cycling microorganisms
[0033] The bacterial and fungal protein sequence libraries were downloaded from NCBI, and the bacterial and fungal databases were constructed respectively using the makeblastdb command. The protease sequences detected by the above DIA method were aligned with the database using the blastp command. The alignment parameters were specified as follows: significance threshold (evalue) -1e-5, minimum alignment score (threshold) = 11, sequence coverage percentage (qcov_hsp_perc) = 70, and output format (outfmt) 6qseqid sseqid pident length mismatch evaluebitscore. The significant results evalue and comparison score (bitscore) were filtered in the output results, and sequences with bitscore>30 were filtered out to obtain the microbial species corresponding to the proteases in the nitrogen cycle process.
[0034] Experimental Example 1
[0035] like Figure 1 As shown, Figure 1 The following is a flow chart of a method for accurately quantifying soil nitrogen cycle microbial enzyme activity provided by an embodiment of the present invention. The method comprises the following steps:
[0036] Three groups of 30g dryland crops (corn, barley, lettuce, peanut) were mixed with fresh soil. Group A was added with (NH4)2SO4, Group B was added with 92mgN / kg soil (NH4)2SO4 and 100mg / kg soil nitrification inhibitor syringic acid, and Group C was added with 92mgN / kg soil (NH4 +)2SO4 and 100mg / kg soil nitrification inhibitor dicyandiamide, cultured at room temperature for 3 days, and according to the soil total protein extraction method in the above specific embodiment, 60ml of soil protein extract was added to each group, and after high-temperature cooking, cooled and centrifuged at 2100g, the supernatant was dispersed into 2ml centrifuge tubes, each group had about 40 centrifuge tubes, and three groups had a total of 120 centrifuge tubes. After centrifugation at 21000g, the precipitate was washed with acetone until transparent, the acetone was air-dried, and guanidine buffer was added. The BCA method (Pierce TM BCA protein detection kit) were used to measure the total protein concentration of soil in groups A, B, and C. Figure 2 The figure shows the standard concentration of total soil protein in a method for accurately quantifying soil nitrogen cycle microbial enzyme activity provided by an embodiment of the present invention.
[0037] The remaining protein solution was desalted by column, and 100 ng of protein was loaded into each group. The mass spectrometer was used to output the DIA data-dependent peak results. The above-mentioned nitrogen cycle microbial enzyme activity reference database was input into Spectronaut 19 to analyze the protease activity and relative abundance of the nitrogen cycle process detected in each group. The corresponding microorganisms were annotated by blastp, and different proteases were compared between groups to obtain the changes in the enzyme activity of the nitrogen cycle process in the soil under different treatment conditions, such as Figure 3 The embodiment of the present invention provides a method for accurately quantifying the activity of soil nitrogen cycle microbial enzymes, which is related to the changes in AMO, nirS, gdhA and other proteases involved in the ammonia oxidation, denitrification and nitrogen assimilation processes of soil microorganisms.
[0038] References
[0039] Chen Lx, Hu M, Huang Ln, et al. Comparative metagenomic and metatranscriptomic analyzes of microbial communities in acid mine drainage[J]. The ISME Journal, 2015, 9(7): 1579-1592.
[0040] Urich T, Lanzén A, Qi J, et al. Simultaneous assessment of soil microbialcommunity structure and function through analysis of the meta-transcriptome[Z]. PloS one.2008:e2527.10.1371 / journal.pone.0002527.
Claims
1. A method for accurately quantifying soil nitrogen cycle microbial enzyme activity, comprising the following steps: A method for accurately quantifying soil nitrogen cycle microbial enzyme activity comprises the following steps: first, preparing an appropriate soil protein extract, adding the protein extract to soil from which total protein extraction is required at an appropriate extract / soil ratio, terminating the reaction by high-temperature steaming, and extracting and enriching the soil total protein according to experimental procedures; then, quantifying the extracted total protein, performing electrophoresis, enzymatic hydrolysis, and desalting, and then analyzing the extracted total protein using a timsTOF Pro2 mass spectrometer via LC-MS / MS and DIA data acquisition using diaPASEF mode to calculate peak areas, establish a database of protein sequences and microbial sequences related to the microbial nitrogen cycle, perform database search and analysis on the DIA data using Spectronaut 19, and output the identified protease species and abundance; The protein extraction reagents involve drugs: sodium dodecyl sulfate, Tris-HCl, sodium chloride, magnesium chloride, dithiothioate, EDTA, phenylmethylsulfonyl fluoride; The sample is any soil sample involving microorganisms, such as crop soil, wetland soil, sediment, etc.; The protein was quantified using Pierce TM BCA protein detection kit was used for BCA method quantification; The protein electrophoresis adopts SDS-PAGE electrophoresis method; The microbial nitrogen cycle protein database is established based on NCBI, Ncyc, and Uniprot; The mass spectrometer model is: timsTOF Pro2 mass spectrometer (Bruker Daltonik, Bremen, Germany) connected in series with an UltiMate 3000 system (Thermo Fisher Scientific, MA, USA).
2. The method for accurately quantifying soil nitrogen cycle microbial enzyme activity according to claim 1, characterized in that: The soil protein extraction reagent prepared is: 1 mM phenylmethylsulfonyl fluoride, 150 mM sodium chloride, 0.1 mM ethylenediaminetetraacetic acid, 1 mM magnesium chloride, 50 mM dithiothiol, 5% (w / v) sodium dodecyl sulfate dissolved in Tris-HCl buffer solution, and the pH is adjusted to 8.5 using concentrated hydrochloric acid.
3. The method for accurately quantifying soil nitrogen cycle microbial enzyme activity according to claim 1, characterized in that: When the soil sample is soybean, peanut, or barley soil, the incubation time is 72 hours and the incubation time is 60 minutes.
4. The method for accurately quantifying soil nitrogen cycle microbial enzyme activity according to claim 1, characterized in that: To construct a nitrogen cycle protein database, we downloaded protein sequence files from the Ncyc database, downloaded the first 1 million sequence information of all target proteins related to nitrogen cycle microorganisms from NCBI and merged them into a faa protein sequence file, and organized the above sequences to synthesize a nitrogen cycle protease database.
5. The method for accurately quantifying soil nitrogen cycle microbial enzyme activity according to claim 1, characterized in that: The microbial species corresponding to the proteases in the soil nitrogen cycle were annotated. The protease sequences detected by the DIA method were compared with the database using the blastp command with strict comparison parameters.