Method for screening interaction protein for obtaining bait protein in plant based on AP-SWATH technology

Abstract

The invention discloses a method for screening interaction protein of bait protein from plants based on an AP-SWATH technology, which comprises the following steps: after obtaining a transgenic plant of an overexpression bait gene fused with an affinity tag, extracting total protein of the plant, and capturing a protein compound by an immunoprecipitation method so as to detect and obtain the interaction protein of the bait protein. According to the invention, the interaction protein is screened in the plant body through the AP-SWATH technology for the first time, the specific interaction protein which is high in specificity and accurately enriched to the target protein is stably and efficiently searched in the plant, and accurate identification and quantification of the compound protein are realized; the efficiency of verifying the protein interaction process is high, the interaction protein of a certain determined gene in a plant body can be captured, and the method has universality. The obtained sample is high in recovery rate, the operation speed of capturing transient interaction is high, and a high-purity protein compound is easier to obtain.

Description

Technical Field

[0001] The invention relates to a method for screening and obtaining interacting proteins of bait proteins in plants based on AP-SWATH technology, belonging to the technical field of protein interaction omics. Background Art

[0002] Protein-protein interactions are the main contributors to various basic functions of cells and participate in almost all important life activities. Therefore, the study of protein-protein interactions and the establishment of a network diagram of interactions are of great significance and are also a hot topic in the current field of protein research. Affinity purification combined with SWATH (AP-SWATH) technology is an important technology for studying protein interactions developed in recent years. This technology can accurately enrich the specific interacting proteins of the target protein with high specificity and realize the accurate identification and quantification of complex proteins. AP-SWATH technology is a proteomics analysis method that combines affinity purification (Affinity Purification) and SWATH-MS (Sequential Window Acquisition of All Theoretical Mass Spectra), which is often used to study the detailed composition of protein complexes and their interactions. AP-SWATH combines the specificity of affinity purification with the comprehensiveness and quantitative ability of SWATH-MS, and can accurately quantify the captured proteins and their interacting proteins, while ensuring that the mass spectrometry data of all peptides are acquired unbiasedly. AP-SWATH technology is currently widely used in biomedical research, especially in the process of cell signaling, and can study key proteins in signaling pathways and their interacting partners. In addition, in protein interaction networks, complex protein interaction networks can be drawn to reveal the regulatory mechanisms of biological processes. Importantly, AP-SWATH also plays a huge role in disease research. In the study of diseases such as cancer and neurodegenerative diseases, changes in protein interactions can be analyzed to find potential therapeutic targets. In general, AP-SWATH technology has been fully applied in animal research, but its application in plant research has not yet been explored.

[0003] Due to their fixed growth mode, plants are exposed to various stresses in the natural environment for a long time. To cope with these challenges, plants have evolved complex mechanisms to perceive external stresses and trigger multi-level responses from physiological to molecular and cellular levels. The construction of such a complex and sophisticated signal regulation network depends on the synergistic action of thousands of molecules. In early studies, scientists tried to reveal protein regulation mechanisms by identifying and quantifying proteins. However, the evidence provided by simple protein abundance change information is limited, and it can only preliminarily predict the potential association between proteins. It is difficult to use it as direct evidence to prove their relationship, and it is impossible to determine whether these relationships are direct or indirect. In addition, the function of many proteins is not necessarily accompanied by changes in abundance. Because the physical interaction between proteins is the basis of signal transduction and protein regulation, proteins can respond quickly to external stimuli and promote cell regulation processes through interactions with other proteins, thereby maintaining the homeostasis of cells and the entire organism. Protein interactomics technology has the advantages of globality and high throughput, and has played an important role in protein function research. However, the application of this technology in plant systems still faces many challenges. Plants have diverse metabolic pathways, and the genomes of many plant species are complex and severely under-annotated. In addition, interactome analysis methods rely on genetic manipulation, which is difficult in plant systems, especially polyploid plants.

[0004] AP-SWATH is a rapid, highly selective and sensitive tool for detecting the interaction between a protein of interest (bait protein) and its interacting proteins (prey protein) under near-physiological conditions. The technology uses antibodies to target bait proteins, thereby capturing protein complexes from cell lysates under native conditions. However, due to the limited number of antibodies targeting plant proteins and the potential shielding of the interaction sites of the bait protein, there are no reports on the screening of interacting proteins in plants using the AP-SWATH technology. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for exploring the interacting proteins of bait proteins in plants based on AP-SWATH technology.

[0006] Technical solution: To solve the above technical problems, the present invention provides a method for exploring the interacting proteins of bait proteins in plants based on AP-SWATH technology, comprising the following steps:

[0007] Step S1: constructing an overexpression vector fused with an affinity tag to obtain a corresponding transgenic plant;

[0008] Step S2: taking transgenic plant tissues for cross-linking reaction and plant total protein extraction;

[0009] Step S3: immunoprecipitation of the extracted plant total protein and identification of protein complexes;

[0010] Step S4: Mass spectrometry data analysis and result verification.

[0011] As a further illustration of the present invention, the plant materials are selected from Arabidopsis, poplar, soybean, wheat, corn and the like.

[0012] As a further illustration of the present invention, the method for exploring the interacting proteins of bait proteins in plants based on AP-SWATH technology specifically comprises the following steps:

[0013] Step S1: Use pMDC32 vector or other overexpression vector, connect affinity tag, connect target gene corresponding to bait protein, construct empty vector with affinity tag, transfer into Agrobacterium, infect plant, and obtain transgenic plant;

[0014] Step S2: Take 0.2g of plant tissue from each sample, wash it with ddH2O, wipe it dry, soak it in 1% formaldehyde solution and vacuum it for 15min, leave it at room temperature for 10min, add glycine to a final concentration of 125mM, vacuum it again for 15min, leave it at room temperature for 5min, take it out and wash it 5 times with ddH2O, wipe it dry with dust-free paper, and then quickly freeze it in liquid nitrogen and grind it. Add protease inhibitors to the 4℃ pre-cooled PBS lysate, use a handheld homogenizer to grind it at low temperature, shake it at low temperature for 30min, ultrasonically break it for 5min, and then centrifuge it at 4℃, 13000rpm, 30min, and take the supernatant to a new EP tube, which is the total plant protein;

[0015] Step S3: Take 10 μl of agarose beads or magnetic beads (beads) with affinity-tagged antibodies, add 1 ml IP buffer to wash the beads, gently invert to mix, and centrifuge, wash twice in total. Add 1 ml of total plant protein to the washed beads, gently invert to mix, and incubate at 4°C for 4-6 hours, or overnight. Centrifuge the incubated proteins and beads, remove the supernatant, and repeat the wash 5 times with pre-cooled IP buffer, gently invert to mix for 3 minutes each time, remove as much liquid as possible, then wash 3 times with pre-cooled PBS, and store at -20°C;

[0016] Step S4: The protein complex attached to the beads was subjected to SWATH mass spectrometry detection, and the DIA raw data file was analyzed by DIA-NN software (v 1.8) to obtain protein quantitative information. The final result was screened for parent ions and protein levels at 1% FDR. The quantitative information of the protein group after screening was used for subsequent analysis.

[0017] More preferably, in step S1, the vector construction includes the following steps: using the pMDC32 vector, connecting the 3HA affinity tag (common tags include FLAG, Myc, hemagglutinin, GFP, etc., and in some cases, multiple tags are fused together), designing primers for specific amplification of the target gene (using NCBI blast for primer design), using a high-fidelity enzyme (NoviZan, p520-01) to amplify the target fragment (98°C 30sec, 98°C 10sec, 55°C 10sec, 72°C, 5-10sec / kb, 35 cycles starting from the second step), and then using the homologous recombination method to connect the target fragment to the enzyme-cut vector (enzyme cutting sites AscⅠ, SpeⅠ; 40 μl system, 37°C 20min, 85°C 20min).

[0018] More preferably, in step S1, the constructed recombinant plasmid and the empty vector are respectively transferred into Agrobacterium GV3101 or EHA105, etc., and infected using a transgenic method for a certain plant. The transgenic method for a certain plant includes: Arabidopsis using the inflorescence infection method; soybean using the Agrobacterium Ti or Ri plasmid-mediated method; poplar using the leaf disc method; wheat, corn, etc. can also use the gene gun method.

[0019] More preferably, in step S2, plant tissue is preferably selected from areas such as leaves where protein is easily available.

[0020] More preferably, in step S2, the PBS lysate comprises 200 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, 25 mM Sodium pyrophosphate, 10 mM β-glycerophosphate, 10 mM EDTA, and 10 mM Na3VO4.

[0021] More preferably, in step S2, the IP buffer comprises 0.485 g of 20 mM Tris, 150 mM NaCl, and 1 mM EDTA, which is adjusted to pH 7.4, fixed to volume, and then sterilized at high temperature.

[0022] More preferably, in step S2, a small amount of plant total protein and protein loading buffer are taken for SDS-PAGE electrophoresis and western blot verification.

[0023] More preferably, in step S3, the above centrifugation conditions are used when agarose beads with affinity tags corresponding to antibodies are used, and a magnetic stand is used instead when magnetic beads are used. Further, step S3 comprises:

[0024] Take 10 μl of agarose beads with affinity-tagged antibodies, add 1 ml IP buffer to wash the beads, gently invert and mix for 3 minutes, and then centrifuge at 1000 rcf, 4°C, 1 min, and wash twice in total; add 1 ml of plant total protein to the washed beads, gently invert and mix, and then incubate at 4°C for 4-6 hours or overnight; centrifuge the incubated proteins and beads at 1000 rcf, 4°C, 1 min, remove the supernatant, and repeat the washing 5 times with pre-cooled IP buffer, gently invert and mix for 3 minutes each time, remove as much liquid as possible, then wash 3 times with pre-cooled PBS, and finally store with PBS buffer at -20°C;

[0025] Alternatively, take 10 μl of magnetic beads with the antibody Anti-HA corresponding to the affinity tag, add 1 ml IP buffer to wash the magnetic beads, gently blow with a pipette and fully resuspend the Anti-HA magnetic beads; place on a magnetic stand for separation for 10 seconds, and wash twice in total. Add 1 ml of the above-mentioned total plant protein to the washed magnetic beads, gently invert to mix, and place on a side-swing shaker or rotary mixer to incubate at 4°C for 4-6 hours or overnight; after the protein and magnetic beads are incubated, place on a magnetic stand for separation for 10 seconds, remove the supernatant, and repeat the washing 5 times with pre-cooled IP buffer to remove as much liquid as possible, and finally wash 3 times with pre-cooled PBS, and finally store the magnetic beads with PBS buffer at -20°C;

[0026] More preferably, in step S4, a relatively high difference fold is selected, and a log2 foldchange ratio is greater than 4.

[0027] As a further illustration of the present invention, the present invention also provides the application of the method described in the rapid screening of bait protein interacting proteins in plants.

[0028] The affinity-based AP-SWATH technology of the present invention usually fuses the bait protein with an affinity tag. The bait protein and its interacting complex are co-purified by affinity enrichment method, and non-specifically bound proteins are removed by washing. Finally, the eluted protein complex is analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0029] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0030] 1) The present invention is the first to screen interacting proteins in plants using AP-SWATH technology, and to determine the interactions between proteins using highly sensitive analytical techniques. By analyzing the mass spectrometry data of protein complexes, the existence of protein interactions can be identified and proteins can be quantified. At the same time, affinity purification is used to identify synergistic prey proteins using affinity tags and capture probes on bait proteins, and proteins that interact directly or indirectly can be identified.

[0031] 2) Common methods for screening interacting proteins include yeast two-hybrid, subcellular localization, co-immunoprecipitation, chemical cross-linking and surface, etc. However, the problems with these methods are: ① The number of interacting proteins found is limited; ② There may be no interaction inside the actual cell; ③ Indirect protein interactions cannot be found; This method can make up for the shortcomings of the above point-to-point screening and verification of protein interactions, and ensure that the captured protein interactions occur in the specific plant tissue cells under study, preserving the endogenous state of the interacting proteins and complexes.

[0032] 3) The present invention verifies that the protein interaction process is highly efficient and can capture the interacting proteins of a certain gene in the plant, which is more extensive. The obtained sample recovery rate is high and the operation speed of capturing transient interactions is fast, making it easier to obtain high-purity protein complexes. These interactions help describe the functions of proteins in plants, provide a detailed catalog of protein complexes and proteins involved in biological processes, and provide a basis for biological process networks across the entire protein range. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a WB verification result diagram of the transgenic plant protein in Example 1;

[0034] Figure 2 This is a WB verification result diagram of the magnetic beads bound to the bait protein in Example 1;

[0035] Figure 3 Figure 1. Y2H validation results of bait proteins of interest screened by mass spectrometry results;

[0036] Figure 4 BiFC validation results of the bait proteins of interest screened by mass spectrometry results: Interaction validation results of U1-70k and d01~d05 respectively;

[0037] Figure 5 BiFC validation results of the bait proteins of interest screened by mass spectrometry results: Interaction validation results of U1-70k and d06-d07 respectively;

[0038] Figure 6 Figure 2 shows the results of BiFC validation of the bait protein of interest in the control group: d01 to d05;

[0039] Figure 7 Figure 3 is the BiFC validation result of the bait protein of interest in the control group: d06~d07. DETAILED DESCRIPTION

[0040] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0041] Example 1

[0042] The model plant Arabidopsis thaliana was selected as the plant material, and the PtU1-70k-OE-3HA vector was constructed (using the pMDC32 vector, connected to the 3HA affinity tag, and then the primers for specific amplification of the target gene were designed (F:cgggccccccctcgaggcgcgccAGTGAATTCTGTTTAAGATGTTGAACTCT, R:ggcggccgct ctagaactagtAGAAACTTTATTGCCAAATGTTTGAA using NCBI blast for primer design;), use high-fidelity enzyme (NoviZan, p520-01) to amplify the target fragment (amplification program: 98℃30sec, 98℃10sec, 55℃10sec, 72℃, 5-10sec / kb, 35 cycles from the second step), then use homologous recombination to connect the target fragment to the enzyme-cut vector (enzyme cutting site AscⅠ, SpeⅠ; 40 microliter system, 37℃20min, 85℃20min) and empty vector, and transfer into Agrobacterium GV3101, and inflorescence infection method to infect Arabidopsis, Obtain a transgenic Arabidopsis strain with a stable HA affinity tag. Select three different transgenic strains for sampling, take 0.2g of transgenic Arabidopsis and empty Arabidopsis leaf tissue, wash with ddH2O, wipe dry, soak in 1% formaldehyde solution and vacuum for 15 minutes, leave at room temperature for 10 minutes, add glycine to a final concentration of 125mM, vacuum again for 15 minutes, leave at room temperature for 5 minutes, take out and wash with ddH2O 5 times, wipe dry with dust-free paper, and quickly freeze and grind in liquid nitrogen. Add PBS lysis buffer pre-cooled at 4°C. Among them, the composition of PBS lysis buffer is 200mM Tris (pH 7.5), 150mM NaCl, 1% Triton X-100, 25mM Sodium pyrophosphate, 10mMβ-glycerophosphate, 10mM EDTA, 10mMNa3VO4. Use a handheld homogenizer to grind at low temperature, shake at low temperature for 30min, ultrasonically break for 5min, then centrifuge at 4℃, 13000rpm, 30min, take the supernatant to a new EP tube, and obtain plant total protein. Take 50μl of total protein, add 5μl protein loading buffer (5×), heat at 95℃ for 10 minutes, perform SDS-PAGE electrophoresis and western blot verification, the results are detailed in the attached Figure 1 , Figure 1This is a graph showing the WB verification results of the transgenic plant protein in Example 1. Column M in the figure represents a protein marker, and columns 1, 2, and 3 in the figure represent three biological replicates of the sample, respectively. The results show that after the protein extracted from the plant material was verified by the WB experiment, there was a band of the same size, which was 58 kda, proving that the transgenic plant material had a 3HA affinity tag and was fused with the target protein.

[0043] Take 10μl Anti-HA magnetic beads (Biyuntian, p2121), add 1ml IP buffer (ingredients are 20mMTris0.485g, 150mM NaCl, 1mM EDTA, adjusted to pH=7.4, fixed volume, and sterilized at high temperature) to wash the magnetic beads, gently blow with a pipette and fully resuspend the Anti-HA magnetic beads. Place on a magnetic stand for separation for 10 seconds, and wash twice in total. Add 1ml of the above-mentioned total plant protein to the washed magnetic beads, gently invert and mix, and place on a side-swing shaker or a rotating mixer to incubate overnight at 4°C. After the protein and magnetic beads are incubated, place on a magnetic stand for separation for 10 seconds, remove the supernatant, and repeat the washing 5 times with pre-cooled IP buffer to remove as much liquid as possible, and finally wash 3 times with pre-cooled PBS, and finally store the magnetic beads with PBS buffer. Take 30 μl of the above PBS and magnetic beads mixture and 6 μl of protein loading buffer, mix them, heat at 95℃ for 10 minutes, perform SDS-PAGE electrophoresis and western blot verification. The results are detailed in the attached Figure 2 , Figure 2 This is a WB verification result diagram of the magnetic beads bound to the bait protein in Example 1. Column M in the figure represents the protein marker, and columns 1, 2, and 3 in the figure represent three biological replicates of the sample, respectively. The results show that after immunoprecipitation, the magnetic beads bind to the bait protein with a 3HA affinity tag.

[0044] Add reaction solution (1% sodium deoxycholate (SDC), 100mM Tris-HCL pH=8.5, 10mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP), 40mM chloroacetamide (CAA)) to the above-mentioned immunoprecipitated magnetic beads, and incubate at 60°C for 1h to perform protein denaturation, reduction and alkylation in one step. After adding an equal volume of ultrapure water for dilution, add trypsin at a mass ratio of 1:50 between enzyme and protein, and incubate at 37°C with shaking overnight for enzyme digestion (enzymatic digestion system is to add 1ug trypsin (1ug / uL) to the diluted 200uL reaction solution (final concentration: 0.5% SDC, 50mM Tris-HCL, 5mM TCEP, 20mM CAA), and the reaction temperature is 37°C overnight (about 16h)). On the second day, trifluoroacetic acid (TFA) was added to terminate the enzyme cleavage, and the supernatant was centrifuged at 16000g and desalted using a homemade SDB desalting column (the homemade desalting column was prepared by filling a 200uL pipette tip with ReproSil-Pur 120C18-AQ, 1.9μm filler). After drying, the supernatant was frozen at -20°C for later use.

[0045] After the desalted peptides were re-dissolved with 0.1% formic acid, 200 ng was taken for injection and subsequent mass spectrometry detection through an autosampler. Mass spectrometry analysis was performed using Bruker's ion mobility-quadrupole-time of flight mass spectrometer timsTOF Pro. Sample injection and separation were performed using an UltiMate 3000RSLCnano liquid chromatograph connected online to the mass spectrometer. The peptide samples were inhaled through the autosampler and bound to a Trap column (75μm×20mm, 2μm particle size, pore size, Thermo), and then eluted to an analytical column (75 μm × 250 mm, 1.6 μm particle size, pore size, ionoptics) for separation. Two mobile phases (mobile phase A: 0.1% formic acid in H2O and mobile phase B: 0.1% formic acid in ACN) were used to establish an analytical gradient of 60 min (the gradient was: 0 min, the volume fraction of mobile phase A was 94%, and the volume fraction of mobile phase B was 6%; 0-5 min, the volume change fraction of mobile phase A was 94→89%, and the volume change fraction of mobile phase B was 6→11%; 5-40 min, the volume change fraction of mobile phase A was 89→75%, and the volume change fraction of mobile phase B was 11→25%; 40-55.5 min, the volume change fraction of mobile phase A was 75→50%, and the volume change fraction of mobile phase B was 25→50%; 55.5-56 min, the volume change fraction of mobile phase A was 50→10%, and the volume change fraction of mobile phase B was 10→25%. The volume change fraction was 50→90%; from 56 to 58.5 min, the volume change fraction of mobile phase A was 10→6%, and the volume change fraction of mobile phase B was 90→94%; from 58.5 to 60 min, the volume fraction of mobile phase A was 6%, and the volume fraction of mobile phase B was 94%. The flow rate of the liquid phase was set to 300 nL / min. The peptide segment entered the mass spectrometer for scanning through the CaptiveSpray nanoliter ion source, the TIMS function was turned on, and the diaPASEF scanning mode was used. The DIA window was defined in the timsControl software. Each scan cycle consisted of 1 MS1 scan and a defined diaPASEF scan.

[0046] The DIA raw data files were analyzed using the DIA-NN software (v 1.8). The database used for the search was the Uniprot database ( https: / / www.uniprot.org / ) Arabidopsis_thaliana proteome reference database. A spectrum library was predicted by the deep learning algorithm in DIA-NN, and the predicted spectrum library and the spectrum library obtained by the MBR function were used to extract the DIA raw data to obtain protein quantitative information. The final result was screened at 1% FDR for the parent ion and protein levels. The screened proteome quantitative information was used for subsequent analysis. When performing functional annotation and enrichment analysis, based on the principle that the functions of proteins are similar if the sequences are identical or similar, the Diamond program of eggNOG-mapper software was used to obtain the GO term (the second, third, and fourth level annotation information of the three aspects of Biological Process, Cellular Component, and Molecular Function), KEGG pathway, and COG (Cluster of Orthologous Groups of proteins) categories corresponding to the submitted protein sequence for annotation. After obtaining the annotation information of all proteins identified by mass spectrometry, the relevant information of the differentially expressed proteins was extracted, the categories and numbers were counted, and the functional enrichment analysis was performed by hypergeometric test to screen out the significant functional categories related to the experiment.

[0047] According to the mass spectrometry analysis, some genes (see Table 1 for details) were selected for interaction verification, and the interaction success rate was high, which verified the accuracy and feasibility of this method.

[0048] Table 1: Bait proteins of interest screened by mass spectrometry results

[0049]

[0050] Yeast two-hybrid experiment verifies whether the target protein interacts with the bait protein of interest screened by mass spectrometry results. Use pGBKT7 vector (restriction sites BamHⅠ, EcoRⅠ), and connect AtU1-70k (AT3G50670) through homologous recombination (Novizen, c112-01), and connect the bait protein with pGADT7 (restriction sites BamHⅠ, EcoRⅠ). Add 300ng of the constructed recombinant plasmids and 5μL denatured carrier DNA (salmon sperm DNA) to 100μL Y2H competent medium (Y2Hgold), mix gently, and then add 600μL PEG / LiAc (Coolaber, YT0006) for transformation experiment, mix gently; incubate in a 30℃ water bath for 30min, mix gently every 10min; gently add 70μL DMSO and mix, heat shock in a 42℃ water bath for 20min, gently mix once every 5min (slowly); centrifuge at 12000rpm for 15-30s, remove the supernatant; add 150μL YPDA medium, 30℃, 250rpm, 30min; 12000rpm, 15-30s, discard the supernatant; keep 50μL to apply to the second-deficient plate SD / -Trp-Leu plate, and culture at 30℃ for 2-3d. Transfer the second-deficient plate monoclonal to the fourth-deficient plate SD / -Ade-His-Trp-Leu. Figure 3 The Y2H verification results of the bait protein of interest screened by the mass spectrometry results. For example, AD-U1 vs. BD-d01 in the figure represents the interaction verification results of U1-70k and d01. The results show that each experimental group grows normally on the four-deficient plate and the control group does not grow, indicating that the bait protein of interest screened from the mass spectrometry results interacts with the U1-70k protein, thereby proving the feasibility and high success rate of this method.

[0051] BiFC experiment verifies whether the target protein interacts with the bait protein of interest screened by mass spectrometry results. The pEGP2×35S-cYFP-NOS vector (restriction site NcoⅠ) and the AtU1-70k (AT3G50670) fragment are connected by homologous recombination (NoviZan, c112-01). The bait protein is connected to pEGP2×35S-nYFP-NOS (restriction site NcoⅠ). The recombinant plasmid is transferred into Agrobacterium GV3101. The Agrobacterium monoclonal clone containing the target vector is picked and placed in 5ml LB medium containing the corresponding antibiotics (Kana, Rif, working concentration 50μM). It is cultured at 28℃ and 200rpm for 2d; 1ml of the cultured Agrobacterium culture liquid is transferred to 20ml LB medium containing the corresponding antibiotics (Kana, Rif, working concentration 50μM) for expansion culture. Cultivate at 28℃ and 200rpm until the growth logarithmic phase of Agrobacterium (OD 600=0.5-0.6); centrifuge at 5000 rpm for 10 min at room temperature to collect the cells, and suspend the Agrobacterium cells in an infiltration solution (containing 10 mM MgCl2, 10 mM MES, 150 μM acetosyringone, pH = 5.6) until OD 600 = 1.0. Mix two bacteria containing different plasmids in equal volumes and let stand at room temperature (at least 0.5h, no more than 3h); use a 1ml needle to gently make a small hole on the back of the tobacco leaf (be careful not to puncture it), then use a needle without the needle to absorb the bacterial solution and inject it into the leaf from the wound; after the injected plants are cultured at about 21℃ for 2 days, observe whether the area of ​​tobacco injected with Agrobacterium has fluorescence. Tear off the injection area of ​​the tobacco leaf (you don’t have to tear it, just use a knife to dig out the part of the leaf close to the wound) and use a laser confocal microscope to perform fluorescence imaging (the epidermal cell layer on the back of the leaf is better observed). Figures 4 to 7 The BiFC verification results of the bait protein of interest screened by mass spectrometry results. For example, nYFP-U1vs.cYFP-d01 in the figure represents the interaction verification results of U1-70k and d01. The results show that fluorescence was observed in tobacco in each experimental group, but not in the control group, indicating that the bait protein of interest screened from the mass spectrometry results interacts with the U1-70k protein, thereby proving the feasibility and high success rate of this method.

[0052] The present invention focuses on using AP-SWATH technology to stably and efficiently search for specific interacting proteins that are accurately enriched with target proteins with high specificity in plants, and to achieve accurate identification and quantification of complex proteins.

[0053] The above description is only an example of the implementation of the present invention and does not limit the present invention in any form. The protection scope of the present invention shall be based on the claims and is not limited by the above specific embodiments. Any simple modification or equivalent changes and modifications made to the above implementation according to the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for screening and obtaining interacting proteins of bait proteins in plants based on AP-SWATH technology, characterized in that: The specific steps include: Step S1: using an overexpression vector, connecting an affinity tag, connecting a target gene corresponding to the bait protein, and constructing an empty vector with an affinity tag, transferring it into Agrobacterium, infecting plants, and obtaining transgenic plants; Step S2: obtaining the total protein of the transgenic plant described in step S1; adding agarose beads with antibodies corresponding to the affinity tags to the total protein of the plant, mixing and incubating, centrifuging, removing the supernatant, and washing to remove the liquid; Step S3: The protein complex attached to the beads was subjected to SWATH mass spectrometry detection, and the DIA raw data file was analyzed by DIA-NN software to obtain protein quantitative information; the final result was screened for parent ion and protein levels at 1% FDR; the quantitative information of the screened proteome was analyzed, and the interacting proteins were obtained when the log2 foldchange ratio was greater than 4.

2. The method for screening and obtaining interacting proteins of bait proteins in plants based on AP-SWATH technology according to claim 1, characterized in that: In step S1, the vector construction includes the following steps: using the pMDC32 vector, connecting the 3HA affinity tag, designing primers for specific amplification of the target gene, using a high-fidelity enzyme to amplify the target fragment, and then connecting the target fragment to the enzyme-cut vector by homologous recombination.

3. The method for screening and obtaining interacting proteins of bait proteins in plants based on AP-SWATH technology according to claim 1, characterized in that: In step S1, the constructed recombinant plasmid and the empty vector are respectively transferred into Agrobacterium GV3101 or EHA105, and the transgenic method for a certain plant is used for infection; the transgenic method for a certain plant includes: inflorescence infection method for Arabidopsis; Agrobacterium Ti or Ri plasmid-mediated method for rapeseed, soybean, cabbage, cucumber and indica rice; leaf disc method for poplar; gene gun method for indica rice, wheat and corn.

4. The method for screening and obtaining interacting proteins of bait proteins in plants based on AP-SWATH technology according to claim 1, characterized in that: In the step S2, the total protein of the plant is obtained from plant tissues from which protein is easily available.

5. The method for screening and obtaining interacting proteins of bait proteins in plants based on AP-SWATH technology according to claim 1, characterized in that: In step S2, the plant total protein and protein loading buffer are taken for SDS-PAGE electrophoresis and western blot verification.

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