Method for searching differential expression protein in corn wild type and Zmpt7 mutant thereof

Abstract

The invention provides a method for searching differential protein expression in a corn wild type and a Zmpt7 mutant thereof. Protein data of corn under the low-nitrogen condition is enriched by analyzing differential protein expression plasmic omics of the corn wild type and the Zmpt7 mutant of the corn wild type in low-nitrogen and normal nitrogen, and GO enrichment analysis and protein pathway enrichment analysis are adopted on the basis of proteomics data to play a role in further research on the biological function of Zmpt7.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically a method for finding wild-type maize and its... Zmpt7 Methods for differentially expressed proteins in mutants. Background Technology

[0002] Phosphorus deficiency leads to stunted plant growth, reduced branching, smaller and darker leaves, and inhibits nitrogen fixation. Due to the generally low bioavailability of inorganic phosphate in soil, plants have evolved a sophisticated phosphate transport system to acquire phosphorus from the soil and maintain phosphorus homeostasis. ZmPT7, an important member of the maize PHT1 gene family, plays a crucial role in phosphorus absorption and redistribution. Previous studies have shown extensive cross-interactions of nitrogen and phosphorus nutrient signals within plants: in Arabidopsis thaliana, nitrate... The expression of SPX family proteins (AtSPX1 / 2 / 4) can be downregulated by the transcriptional repressor AtNIGT1, thereby activating the phosphorus starvation response signaling pathway. Under nitrogen-deficient conditions, the transcriptional level of the phosphate export protein AtPHO2 is significantly increased, and this process is co-regulated by AtNIGT1 and the nitrate transporter AtNRT1.1. Further studies in rice have revealed that under low nitrogen conditions, OsSPX4 can form a complex in the cytoplasm with the core phosphorus signaling transcription factor OsPHR2 and the nitrogen signaling regulatory protein OsNLP3, preventing their entry into the nucleus and thus synergistically inhibiting the expression of downstream nitrogen and phosphorus response genes. However, these nitrogen-phosphorus interaction regulatory networks established in model plants still lack systematic validation in crops such as maize.

[0003] To elucidate the molecular mechanism of nitrogen and phosphorus co-utilization in maize, this study used wild-type and... Zmpt7 Using mutants as materials, proteomics analysis was conducted under both normal nitrogen supply and low nitrogen conditions. Differentially expressed proteins were screened using high-precision mass spectrometry, aiming to reveal… ZmPT7 The key components of the nitrogen-phosphorus interaction pathway mediated by the study provide a theoretical basis for the efficient utilization of crop nutrients. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a method for identifying wild-type maize and its... Zmpt7 Methods for differential protein expression in mutants.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: (1) Cultivation of wild-type maize ND101 and its offspring under normal nitrogen (4 mM) and low nitrogen (0.05 mM) conditions Zmpt7 The mutant, specifically: Corn seedlings germinated in a mixture of vermiculite and sand under controlled conditions of 24°C–27°C and 55% humidity. After 7 days of growth, the seedlings were carefully removed from the pots, their roots were thoroughly rinsed with distilled water, and then transferred to culture solutions of different nutrient concentrations for treatment: normal... Concentration 4 mM (labeled as NN), low The concentration was 0.05 mM (LN), and the maize seedlings were then cultured for another 14 days. A portion of the seedlings were harvested for physiological parameter determination, while the remaining samples were rapidly frozen in liquid nitrogen and stored at -80°C for subsequent proteomics analysis.

[0006] (2) Extraction of wild-type and Zmpt7 Total maize protein and membrane proteins in mutants, including the following steps: Take 0.3 g of frozen maize root sample, and grind it together with 0.2 g of alumina powder and 500 μL of buffer A in a centrifuge tube for 2 min. Then transfer the mixture to the built-in filter of the kit to separate and enrich membrane proteins. Finally, collect the membrane proteins by centrifugation precipitation. The obtained membrane protein precipitate is dissolved with the dissolving reagent provided with the kit, and the protein concentration is determined by BCA method. The separation of whole maize root protein is based on a modified phenol extraction method (this method is described in Qi, J., X. Zhao, and Z. Li. 2020. “iTRAQ‐Based Quantitative Proteomic Analysis of the Arabidopsis Mutant opr3‐1 in Response to Exogenous MeJA.” International Journal of Molecular Sciences 21: 571). The extraction of maize root membrane proteins is carried out using a micro plasma membrane protein separation kit designed specifically for plants (Invent Biotechnologies, Eden Prairie, Minn., USA).

[0007] (3) Digesting the protein, including the following steps: Whole protein samples were digested using a modified filter-assisted sample preparation method (FASP, described in Zhao, X., X. Bai, C. Jiang, and Z. Li. 2019. “Phosphoproteomic Analysis of Two Contrasting Maize Inbred Lines Provides Insights into the Mechanism of Salt‐Stress Tolerance.” International Journal of Molecular Sciences 20: 1886.). Membrane protein digestion was performed using a modified SP3 workflow (described in Hughes, CS, S. Foehr, DA Garfield, EE Furlong, LM Steinmetz, and J. Krijgsveld. 2014. “Ultrasensitive Proteome Analysis Using Paramagnetic Bead Technology.” Molecular Systems Biology 10: 757). At the start of the process, the pH of the membrane protein solution was adjusted to 8.5 using 50 mM NH4HCO3. The protein was then reduced with 100 mM DTT at 56°C and alkylated with 500 mM IAM for 30 min at room temperature in the dark, maintaining a total solution volume of less than 35 μL. Subsequently, 15 μL of hydrophilic / hydrophobic paramagnetic beads (Cytiva, 1:1 ratio) were added, and the mixture was shaken at 1000 rpm for 10 min. The beads were washed twice with 100 μL of 80% ethanol, followed by one wash with acetonitrile. The protein adsorbed on the beads was digested overnight with trypsin at 37°C. The resulting trypsin-digested peptides were quantified using a peptide quantification kit (Thermo Scientific), desalted using a self-made C18 stage pipette tip (comprising multiple tiny C18 solid-phase extraction packing materials housed in a standard 200 µL pipette tip), and then vacuum-dried.

[0008] (4) The sample was analyzed by nano-liquid chromatography-mass spectrometry (nanoLC-MS), including the following steps: Protein identification and quantification were performed using an Orbitrap Fusion Lumos ultra-high resolution mass spectrometer coupled with an EASY-nLC1200 nanoliter high-performance liquid chromatography system. Peptides were resuspended in 0.1% formic acid solution and an iRT internal standard peptide (Biognosys) was added. The peptide mixture was then loaded onto an Acclaim PepMap trapping column (75 µm × 2 cm, 3 µm, C18, 100 Å, ThermoScientific). Separation was then performed at a flow rate of 400 nL / min through a custom analytical column (100 µm inner diameter) packed with a 20 cm length of C18 stationary phase (Aqua C18, 1.8 µm, 125 Å, Phenomenex). Mobile phases A and B were an aqueous solution containing 0.1% formic acid and a mixed solvent of acetonitrile, water, and formic acid in a 79.9:20:0.1 (v / v) ratio, respectively. Peptides were separated by a 90-minute gradient elution program, and the samples were analyzed using dynamic ion focusing (DIA) mode.

[0009] (5) Perform corn database search and annotation analysis on the results, including the following steps: DIA data were analyzed using Spectronaut 15.0 software compared with a maize database. Parameters were set as follows: trypsin was specified as the specific enzyme, allowing a maximum of two uncut sites. Oxidative modifications (M) and acetylation modifications (N-terminus of proteins) were set as variable modification sites, and peptides were filtered with a 1% false discovery rate (FDR).

[0010] The obtained protein library was subjected to bioinformatics analysis, which included protein annotation, protein function enrichment, cluster analysis of protein function enrichment, and protein interaction network analysis. The protein annotation included pathway annotation, protein domain annotation, subcellular localization, and gene ontology analysis. The protein function enrichment included GO enrichment analysis, pathway enrichment analysis, and protein domain enrichment analysis.

[0011] The present invention describes a method for finding wild-type maize and its Zmpt7 The beneficial effects of differentially expressed proteins in mutants are as follows: This invention analyzes wild-type and Zmpt7 The differential protein expression proteomics of mutants under low and normal nitrogen conditions enriched the protein data of maize under low nitrogen conditions. This invention, based on proteomics data, employed GO enrichment analysis and protein pathway enrichment analysis to provide a basis for further research. ZmPT7 Its biological functions come into play. Attached Figure Description

[0012] The present invention includes the following figures: Figure 1Displaying Venn diagrams Zmpt7 Membrane proteins identified in three biological replicates from the root.

[0013] Figure 2 GO enrichment analysis revealed differentially expressed membrane proteins between wild-type-LN and Zmpt7-LN.

[0014] Figure 3 To downregulate the protein-protein interaction network of membrane proteins. Detailed Implementation

[0015] The present invention will be further described in detail below with reference to the accompanying drawings.

[0016] The following embodiments are provided to better understand the present invention, but are not limited thereto. These embodiments are for illustrative purposes only and do not limit the scope of protection of the present invention in any way.

[0017] Unless otherwise specified, the equipment and reagents used in each embodiment are commercially available.

[0018] (1) Extraction of wild type and Zmpt7 Total protein and membrane protein in corn InventBio's plasma membrane protein isolation kit can extract over 100 µg of membrane proteins from just 0.3 g of plant material, but requires a dissolving solvent containing strong surfactants to dissolve the extracted membrane proteins. The SP3 method was then used to remove the detergent, achieving efficient enzymatic digestion of the membrane proteins. Simultaneously, a modified phenol extraction technique was used to analyze the whole proteome of these seedlings. Over 8,000 proteomes were successfully identified for each sample. Over 93% of the proteins detected in all three biological replicates were co-occurring, and a strong correlation was observed between two replicates (R² = 0.81). Over 50% of the identified proteins were membrane proteins. Figure 1 These results demonstrate the efficiency and reproducibility of our proteomics workflow. The screening criteria for differentially expressed proteins (DRPs) were: a fold change in expression level between different genotypes or treatment groups >2 and a Q value <0.05.

[0019] (2) GO enrichment analysis Gene Ontology (GO) enrichment analysis was performed using the Omicsolution platform (https: / / www.omicsolution.com / wkomics / passwd / GOEnrich / ), a bioinformatics analysis platform.

[0020] Gene Ontology (GO) enrichment analysis revealed that these proteins are involved in nitrogen metabolism, development, transport, and protein synthesis. Proteins involved in the metabolism of nitrogen-related compounds... Zmpt7-LN The level was adjusted upwards. Meanwhile, compared to wild-type -LN, Zmpt7-LN Downregulated proteins are enriched in transport processes, post-translational modifications, and transcription initiation. Figure 2 This indicates that in NO3 - In the absence of conditions, ZmPT7 Mutations can disrupt nitrogen transport and the synthesis of nitrogen-related compounds, thereby affecting the growth and development of maize plants.

[0021] (3) Protein pathway enrichment analysis Protein-protein interaction analysis was performed using the STRING database (https: / / cn.string-db.org / ). The String 11.5 database was used for further analysis. Zmpt7-LN In-depth analysis of differentially expressed membrane proteins between wild-type and wild-type LN revealed the interaction mechanism of nitrogen and phosphorus signaling in maize roots. These proteins can be classified into three major categories ( Figure 3 ): Single-cell localization (Category 1), carbohydrate synthesis (Category 2), and transport activity (Category 3).

[0022] The contents not described in detail in this specification are existing technologies known to those skilled in the art.

Claims

1. A method for identifying wild-type maize and... Zmpt7 A method for differentially expressing proteins in mutants, characterized in that, Includes the following steps: Step 1: Cultivate wild-type maize and its Zmpt7 mutant under normal nitrogen (4 mM) and low nitrogen (0.05 mM) conditions; Step 2, regarding the above Wild type and Zmpt7 Total protein and membrane protein were extracted from mutant corn. Step 3: Digest the protein obtained in Step 2; Step 4: Perform nano-liquid chromatography-mass spectrometry analysis on the sample obtained in step 3; Step 5: Perform corn database search and annotation analysis on the analysis results of Step 4.

2. The method as described in claim 1, characterized in that, Step 2 specifically involves: Take 0.3 g of frozen corn root sample, and grind it together with 0.2 g of alumina powder and 500 μL of buffer A in a centrifuge tube for 2 min; The mixture is transferred to the built-in filter cartridge of the kit to achieve the separation and enrichment of membrane proteins; Membrane proteins were collected by centrifugation precipitation. The resulting membrane protein precipitate was dissolved using the dissolving reagent provided with the kit, and the protein concentration was determined by the BCA method. The total protein from maize roots was separated using a modified phenol extraction method. The above kit is a micro-volume plasma membrane protein separation kit.

3. The method as described in claim 1, characterized in that, Step 3 specifically involves: A modified membrane-assisted sample preparation method was used to digest whole protein samples; a modified SP3 workflow was used to digest membrane proteins. The pH of the membrane protein solution was adjusted to 8.5 with 50 mM NH4HCO3; the protein was then reduced with 100 mM DTT at 56°C and alkylated with 500 mM IAM for 30 min at room temperature in the dark, while keeping the total solution volume below 35 μL; then 15 μL of hydrophilic / hydrophobic paramagnetic beads were added and the solution was shaken at 1000 rpm for 10 min. The magnetic beads were washed twice with 100 μL of 80% ethanol, and then washed once with acetonitrile. Proteins adsorbed onto magnetic beads were digested overnight with trypsin at 37°C; the resulting trypsin-digested peptides were quantified using a peptide quantification kit, desalted using a C18 pipette tip, and then vacuum dried.

4. The method as described in claim 1, characterized in that, Step 4 specifically involves: Proteins were identified and quantified using an ultra-high resolution mass spectrometer combined with a high performance liquid chromatography system. The peptides were resuspended in 0.1% formic acid solution and an iRT internal standard peptide was added. The peptide mixture was loaded onto a trapping column and then separated by passing it through an analytical column at a flow rate of 400 nL / min. The stationary phase in the analytical column was C18, and the mobile phases A and B were an aqueous solution containing 0.1% formic acid and a mixed solvent of acetonitrile, water, and formic acid in a volume ratio of 79.9:20:0.1, respectively. The peptides were separated by a gradient elution program of 90 min, and the samples were analyzed using dynamic ion focusing mode.

5. The method as described in claim 1, characterized in that, Step 5 specifically involves: DIA data were analyzed by comparing them against a maize database using Spectronaut 15.0 software; parameters were set as follows: trypsin was specified as the specific enzyme, allowing a maximum of two uncut sites; oxidative and acetylation modifications were set as variable modification sites, and peptides were filtered with a 1% false discovery rate. The obtained protein library was subjected to bioinformatics analysis, which included protein annotation, protein function enrichment, cluster analysis of protein function enrichment, and protein interaction network analysis. The protein annotation included pathway annotation, protein domain annotation, subcellular localization, and gene ontology analysis. The protein function enrichment included GO enrichment analysis, pathway enrichment analysis, and protein domain enrichment analysis.

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