Efficient plant DNA binding protein extraction method
Through liquid nitrogen grinding and vortex shock, plant DNA-binding proteins are directly extracted, solving the problems of complex extraction process and low purity in the existing methods, achieving efficient and simple DNA-binding protein extraction, significantly improving histone content and Extraction rate.
Patent Information
- Application Number
- CN202411663516.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-20
- Publication Date
- 2025-01-17
AI Technical Summary
Existing methods cannot directly extract DNA-binding proteins, resulting in complex extraction process and low purity. Classic methods require isolation of cell nuclei, increasing the operation steps and the presence of contaminants.
The plant material was ground into powder with liquid nitrogen, filtered after vortex and shock, and suspended and sonicated with NaCl solution. The NaCl concentration was diluted to separate DNA-binding proteins, reducing dependence on the cell nucleus and improving extraction efficiency and purity.
It significantly improves the purity and yield of DNA-binding proteins, reduces the contamination of cytoplasmic proteins and chloroplasts, simplifies the operation process, and makes extraction more efficient and simple.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of protein extraction methods, and in particular relates to an efficient method for extracting plant DNA binding proteins. Background Art
[0002] DNA binding proteins include histone modifiers, chromatin remodeling factors, and non-histone proteins. DNA binding proteins play a key role in regulating gene expression, epigenetic regulation, maintaining chromatin structure, and maintaining genome integrity. They also provide a scaffold for DNA packaging and form the basis for epigenetic regulation and genome maintenance. For example, histone modifiers play an important role in post-translational modification (PTM) of histones and are essential for epigenetic modification. Recent studies have shown that histone acetyltransferases such as GCN5 are involved in regulating plant stress response genes, thereby enhancing plant adaptability to environmental stress. Chromatin remodeling complexes, such as the SWI / SNF complex, use ATP hydrolysis to reposition or reorganize nucleosomes, thereby increasing the accessibility of DNA transcription. In Arabidopsis, the SWI / SNF complex is involved in the regulation of flowering time and response to environmental stimuli. Non-histone DNA binding proteins, such as transcription factors and structural proteins, also play an important role in chromatin dynamics. For example, the MYB transcription factor family has been shown to interact with chromatin and regulate the expression of flavonoid biosynthesis-related genes under ultraviolet light. In plants, these proteins are involved in a variety of biological processes, including development, stress responses, and epigenetic regulation. DNA-binding proteins are crucial in various developmental processes in plants. For example, Polycomb group (PcG) proteins are involved in repressing the expression of specific developmental genes. Recent studies have shown that the PcG complex regulates gene expression patterns through epigenetic silencing, thereby affecting the development of Arabidopsis stems and roots. In addition to developmental roles, DNA-binding proteins also mediate plant responses to biotic and abiotic stresses. The histone variant H2A.Z is associated with driving the expression of stress response genes under drought conditions, indicating that it plays a key role in plant stress resistance.
[0003] Identifying a complete list of DNA-binding proteins will help deepen our understanding of gene regulation and other chromatin-related processes. The study of DNA-binding proteins has driven the development of a variety of techniques, such as chromatin immunoprecipitation (ChIP), which provides researchers with the ability to study the binding patterns of DNA-binding proteins on the genome. This provides new ideas for understanding the regulatory networks of plant gene expression. For example, ChIP-seq analysis revealed the relationship between certain histone modifications and gene expression profiles during rice seed development. In addition, the integration of multi-omics approaches, including genomics, transcriptomics, and epigenomics, has begun to elucidate the complex interplay between chromatin dynamics and gene regulation in plants.
[0004] Therefore, the separation of DNA-binding proteins is very important in life science research, but the current methods for separating DNA-binding proteins are not ideal. The classic method is to separate the nucleus first and then extract the DNA-binding protein. However, the classic method not only has a complicated extraction process, but also has a high background, especially the high content of cytoplasmic proteins. Generally, nuclear proteins only account for about 25% of the proteins obtained. In addition, during the liquid nitrogen grinding process, a large number of cell nuclei will be ground and broken, resulting in a decrease in the yield of cell nuclei, thereby reducing the yield of nuclear proteins. The most important problem is that the classic method is to extract nuclear proteins, not directly extract DNA-binding proteins, and there are a large number of non-DNA-binding proteins in nuclear proteins, which will affect subsequent analysis. Therefore, it is urgent to establish a method that can directly extract DNA-binding proteins. Summary of the invention
[0005] In order to solve the problem that the existing method can only extract nuclear protein but cannot directly extract DNA binding protein, the present invention provides an efficient method for extracting plant DNA binding protein.
[0006] The technical solution of the present invention:
[0007] An efficient method for extracting plant DNA binding protein comprises the following steps:
[0008] Step 1: Grind the plant material into powder using liquid nitrogen;
[0009] Step 2: Add the plant powder obtained in step 1 to solution 1 and vortex shake the mixture, filter the mixture after sufficient shaking to collect the filtrate, and centrifuge the filtrate to collect the precipitate;
[0010] Step 3, resuspending the precipitate obtained in step 2 with NaCl solution, vortexing and ultrasonicating;
[0011] Step 4: Add ultrapure water to the system after ultrasonic treatment in step 3 to dilute the NaCl concentration to 0.14 mol / L, collect the precipitate by centrifugation, and obtain a DNA-protein complex;
[0012] Step 5: extract and purify the protein from the DNA-protein complex obtained in step 4, thereby obtaining the plant DNA binding protein.
[0013] Furthermore, step one also includes the step of immersing the plant material in a formaldehyde solution to perform DNA and protein cross-linking, and then grinding the obtained DNA protein cross-linked material into powder using liquid nitrogen.
[0014] Furthermore, the solution 1 in step 2 is a 10 mM Tris-HCl buffer solution with a pH of 8.0 containing 0.4 M sucrose, 1.5 mM MgCl2 and 1 mM DTT.
[0015] Furthermore, in step 2, the mass volume ratio of the plant powder to the solution 1 is 1 g:10 mL, and the vortex oscillation time is 10 to 20 min.
[0016] Furthermore, the filtration in step 2 is performed by filtering with 2 layers of 300-mesh nylon cloth or 2-4 layers of Miracloth filter cloth.
[0017] Furthermore, the specific conditions of the centrifugation in step 2 are 4° C., 1000-1200 rpm, 10-20 min.
[0018] Furthermore, the concentration of the NaCl solution in step three is 1 mol / L.
[0019] Furthermore, the ultrasonic treatment in step 3 is carried out in an ice bath, with an ultrasonic output power of 100 to 200 W, a working time of 3 s, an interval time of 12 s, and a total operation time of 20 to 30 min.
[0020] Furthermore, the specific conditions of the centrifugation in step 4 are 4° C., 10,000-25,000 rpm, 5-30 min.
[0021] Furthermore, when step five needs to obtain active plant DNA binding protein, the DNA-protein complex is treated with micrococcal nuclease and the plant DNA binding protein is extracted and purified; when it is not necessary to obtain active plant DNA binding protein, conventional protein extraction methods are used to extract and purify the plant DNA binding protein from the DNA-protein complex.
[0022] Furthermore, when the DNA-protein complex is treated with micrococcal nuclease, the amount of micrococcal nuclease used is 2 μL, and the activity of the micrococcal nuclease is 2000 gel units / μL.
[0023] Beneficial effects of the present invention:
[0024] The present invention provides an efficient plant DNA binding protein extraction method, which utilizes the different solubility of DNA binding protein in NaCl solution of different concentrations to separate DNA binding protein and other pollutants, and can directly extract DNA binding protein. The purity of DNA binding protein obtained by the present invention is significantly improved compared with the classical method, the histone content is high, the contamination of cytoplasmic protein and chloroplast is significantly reduced, and the yield of DNA binding protein is 2 to 3 times higher than that of the classical method.
[0025] The DNA binding protein extraction method provided by the present invention does not need to separate the cell nucleus, thus reducing the operation steps, making the DNA binding protein extraction experiment simpler, time-saving and efficient. The DNA binding protein extracted by the present invention can release DNA by cross-linking under the action of proteinase K, which is convenient for the operation of subsequent experiments.
[0026] The present invention can be used for the extraction of normal DNA-binding proteins, is also suitable for the extraction of DNA and protein cross-linked materials, can be used to study protein-DNA interactions, and can also be used to separate proteins that are directly or indirectly bound to chromatin without the need for other nuclear proteins, thereby providing more convenience for the study of transcriptional regulation, epigenetics and chromatin structure. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 This is a comparison chart of the amount of DNA separated in Example 2 and Comparative Example 1;
[0028] Figure 2 This is a comparison chart of the amount of DNA separated in Example 3 and Comparative Example 2;
[0029] Figure 3 This is a comparison chart of the number of chloroplast residues in chromatin DNA extracted from Example 2 and Comparative Example 1;
[0030] Figure 4 This is a comparison chart of the number of chloroplast residues in chromatin DNA extracted from Example 3 and Comparative Example 2;
[0031] Figure 5 The SDS-PAGE results of the separated nucleoproteins obtained in Example 2 and Comparative Example 1 and in Example 3 and Comparative Example 2 are compared;
[0032] Figure 6 It is a comparison diagram of the protein immunoblotting results of the nuclear proteins separated from Example 2 and Comparative Example 1, and from Example 3 and Comparative Example 2;
[0033] Figure 7 It is a comparison chart of the protein immunoblotting results of nuclear proteins of different concentrations separated from Example 2 and Comparative Example 1, and from Example 3 and Comparative Example 2. DETAILED DESCRIPTION
[0034] The technical solution of the present invention is further described below in conjunction with the embodiments, but it is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be included in the protection scope of the present invention. The process equipment or devices not specifically noted in the following embodiments are all conventional equipment or devices in the art. If not specifically specified, the raw materials used in the embodiments of the present invention can be obtained commercially; if not specifically specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.
[0035] Example 1
[0036] This embodiment provides an efficient method for extracting plant DNA binding proteins, comprising the following steps:
[0037] Step 1: Grind the plant material into powder using liquid nitrogen;
[0038] Step 2: Add the plant powder obtained in step 1 to solution 1 at a mass volume ratio of 1 g: 10 mL, and vortex for 10 minutes. Filter the mixed system after sufficient shaking through 2 layers of 300-mesh nylon cloth, collect the filtrate, and centrifuge the obtained filtrate at 4°C, 1000 rpm for 15 minutes to collect the precipitate; solution 1 is a 10mM Tris-HCl buffer solution with a pH of 8.0 containing 0.4M sucrose, 1.5mM MgCl2 and 1mM DTT (dithiothreitol).
[0039] Step 3: The precipitate obtained in step 2 was resuspended in a 1 mol / L NaCl solution, vortexed and ultrasonically treated in an ice bath with an ultrasonic output power of 100 W, a working time of 3 s, an interval time of 12 s, and a total operation time of 24 min.
[0040] Step 4: Add ultrapure water to the system after ultrasonic treatment in step 3 to dilute the NaCl concentration to 0.14 mol / L, collect the precipitate by centrifugation, and obtain a DNA-protein complex;
[0041] Step 5: extracting and purifying protein from the DNA-protein complex obtained in step 4, i.e., obtaining plant DNA binding protein; when it is necessary to obtain active plant DNA binding protein, treating the DNA-protein complex with micrococcal nuclease and extracting and purifying the plant DNA binding protein; when it is not necessary to obtain active plant DNA binding protein, extracting and purifying the plant DNA binding protein from the DNA-protein complex using conventional protein extraction methods.
[0042] Example 2
[0043] This embodiment provides an efficient method for extracting plant DNA binding proteins.
[0044] 1. The plant materials and culture conditions of this example are as follows:
[0045] Populus davidianus is cultured on the growth medium for 20-30 days.
[0046] Growth medium: 1 / 2MS + 0.05mg·L -1 6-BA+0.5mg·L -1 NAA.
[0047] 2. The PdbSCL1 gene was transiently transformed into Populus davidianus. The transient genetic transformation method of this embodiment is as follows:
[0048] 1. Agrobacterium tumefaciens EHA105 strain was cultured in a liquid medium containing LB, kanamycin and rifampicin in a culture volume of 10 ml. The culture was incubated overnight at 28°C until it reached the early logarithmic phase, i.e., the OD600 value was between 0.6 and 0.8;
[0049] 2. Centrifuge at 3000g and 4°C for 10 minutes to collect bacterial cells. Dilute the collected cells with transformation solution containing 1 / 2MS, 2.5% sucrose, 100mM acetosyringone and Tween20, and adjust OD600 to 0.8;
[0050] 3. Soak the plant tissue of Populus shanxin in the transformation solution and culture it in a 250mL flask at 90 rpm at 25°C for 2.5 hours. Rinse the transformed plant samples thoroughly with distilled water. After absorbing excess water with filter paper, transfer the plants to a solid culture medium containing 1 / 2MS nutrient solution.
[0051] 3. The method for extracting DNA binding protein from transformed Populus sphenanthera in this example is as follows:
[0052] Step 1: Soak the Populus dahliae material in a formaldehyde solution to crosslink DNA and protein, and grind the plant material into powder with liquid nitrogen after the crosslinking is completed;
[0053] Step 2: Add the plant powder obtained in step 1 to solution 1 at a mass volume ratio of 1 g: 10 mL, and vortex for 10 minutes. Filter the mixed system after sufficient shaking through 2 layers of 300-mesh nylon cloth, collect the filtrate, and centrifuge the obtained filtrate at 4°C, 1000 rpm for 15 minutes to collect the precipitate; solution 1 is a 10mM Tris-HCl buffer solution with a pH of 8.0 containing 0.4M sucrose, 1.5mM MgCl2 and 1mM DTT (dithiothreitol).
[0054] Step 3: The precipitate obtained in step 2 was resuspended in a 1 mol / L NaCl solution, vortexed and ultrasonically treated in an ice bath with an ultrasonic output power of 100 W, a working time of 3 s, an interval time of 12 s, and a total operation time of 24 min.
[0055] Step 4: Add ultrapure water to the system after ultrasonic treatment in step 3, dilute the NaCl concentration to 0.14 mol / L, centrifuge at 10000 rpm at 4°C for 20 min, collect the precipitate, and obtain a DNA-protein complex;
[0056] Step 5: Use the phenol-chloroform method (refer to Experiment 1 of the Principles of Molecular Biology Experimental Techniques) to extract and purify the protein from the DNA-protein complex obtained in Step 4, thereby obtaining the plant DNA binding protein.
[0057] Example 3
[0058] This embodiment provides an efficient method for extracting plant DNA binding proteins.
[0059] 1. The plant materials and culture conditions of this example are as follows:
[0060] Populus davidianus is cultured on the growth medium for 20-30 days.
[0061] Growth medium: 1 / 2MS + 0.05mg·L -1 6-BA+0.5mg·L -1 NAA.
[0062] 2. The PdbSCL1 gene was transiently transformed into Populus davidianus, and the ATHSfA gene was transiently transformed into Arabidopsis thaliana. The transient genetic transformation method of this embodiment is as follows:
[0063] 1. Agrobacterium tumefaciens EHA105 strain was cultured in a liquid medium containing LB, kanamycin and rifampicin in a culture volume of 10 ml. The culture was incubated overnight at 28°C until it reached the early logarithmic phase, i.e., the OD600 value was between 0.6 and 0.8;
[0064] 2. Centrifuge at 3000g and 4°C for 10 minutes to collect bacterial cells. Dilute the collected cells with transformation solution containing 1 / 2MS, 2.5% sucrose, 100mM acetosyringone and Tween20, and adjust OD600 to 0.8;
[0065] 3. Soak the plant tissues of Populus shanxin and Arabidopsis thaliana in the transformation solution and culture them at 25°C for 2.5 hours at 90 rpm in a 250 mL flask. Rinse the transformed plant samples thoroughly with distilled water. After absorbing excess water with filter paper, transfer the plants to a solid culture medium containing 1 / 2MS nutrient solution.
[0066] 3. The method for extracting DNA binding proteins from transformed Populus davidianus and Arabidopsis thaliana in this embodiment is as follows:
[0067] Step 1: Soak the Populus davidianus and Arabidopsis thaliana materials in a formaldehyde solution to perform DNA and protein cross-linking, and grind the plant materials into powder using liquid nitrogen after the cross-linking is completed;
[0068] Step 2: Add the plant powder obtained in step 1 to solution 1 at a mass volume ratio of 1 g: 10 mL, and vortex for 10 minutes. Filter the mixed system after sufficient shaking through 2 layers of 300-mesh nylon cloth, collect the filtrate, and centrifuge the obtained filtrate at 4°C, 1000 rpm for 15 minutes to collect the precipitate; solution 1 is a 10mM Tris-HCl buffer solution with a pH of 8.0 containing 0.4M sucrose, 1.5mM MgCl2 and 1mM DTT (dithiothreitol).
[0069] Step 3: The precipitate obtained in step 2 was resuspended in a 1 mol / L NaCl solution, vortexed and ultrasonically treated in an ice bath with an ultrasonic output power of 100 W, a working time of 3 s, an interval time of 12 s, and a total operation time of 24 min.
[0070] Step 4: Add ultrapure water to the system after ultrasonic treatment in step 3, dilute the NaCl concentration to 0.14 mol / L, centrifuge at 10000 rpm at 4°C for 20 min, collect the precipitate, and obtain a DNA-protein complex;
[0071] Step 5: Use the phenol-chloroform method (refer to Experiment 1 of the Principles of Molecular Biology Experimental Techniques) to extract and purify the protein from the DNA-protein complex obtained in Step 4, thereby obtaining the plant DNA binding protein.
[0072] Comparative Example 1
[0073] The difference between this comparative example and Example 2 is that the method for extracting plant DNA binding protein using the classical method in this comparative example is:
[0074] After isolating the nuclei by the classical method, the nuclei were dissolved in SDS solution, DNA-binding proteins were extracted by the phenol method, and DNA was obtained by extraction with trisphenol and chloroform.
[0075] Comparative Example 2
[0076] The difference between this comparative example and Example 3 is that the method for extracting plant DNA binding protein in this comparative example is:
[0077] After isolating the nuclei by the classical method, the nuclei were dissolved in SDS solution, DNA-binding proteins were extracted by the phenol method, and DNA was obtained by extraction with trisphenol and chloroform.
[0078] Experimental Example 1
[0079] This experiment indirectly reflects the efficiency of separating DNA binding proteins by comparing the plant DNA binding protein extraction method in Example 2 and Example 3 with the efficiency of separating chromatin by the classical method in Comparative Example 1 and Comparative Example 2.
[0080] The two extraction methods were compared using the same number of samples. The DNA-protein complexes obtained in step 4 of Example 2 and Example 3 were respectively extracted using the phenol-chloroform method (refer to Experiment 1 of the Principles of Molecular Biology Experimental Techniques).
[0081] Comparative Examples 1 and 2 After the cell nuclei were separated by the classical method, the cell nuclei were dissolved in SDS solution, and the DNA was extracted by the phenol-chloroform method (refer to Experiment 1 of Principles of Molecular Biology Experimental Technology).
[0082] In this experiment, 11 transcription factor genes were selected from Populus dahliae transiently transformed with PdbSCL1 gene and 15 transcription genes were selected from Arabidopsis thaliana transiently transformed with ATHSfA for qRT-PCR to compare the efficiency of chromatin separation of the two methods.
[0083] The specific method of qRT-PCR (fluorescence quantitative PCR) is as follows:
[0084] Quantitative PCR was performed using a qTower 2.2 quantitative PCR instrument (Analytik Jena). The PCR reaction system included 10 μL SYBR Green Real-time PCR Master Mix (Toyobo), 0.5 mM forward and reverse primers, 0.5 mM each, and 2 μL PCR template, with a total volume of 20 μL. The parameters of the PCR reaction were set as follows: initial denaturation at 94°C for 1 minute; followed by denaturation at 94°C for 12 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 45 seconds, completing 40 cycles. Eleven transcription factor genes were selected from Populus davidianus transiently transformed with the PdbSCL1 gene, and 15 transcription factor genes were selected from Arabidopsis thaliana transiently transformed with ATHSfA, and primers were designed at their conserved coding sequences (CDS). Populus davidianus actin (PdbActin) was selected as an internal reference gene, and the endogenous actin gene (ATActin) of Arabidopsis thaliana was selected as an internal reference gene. The experiment was repeated three times, using 2 -ΔΔCt Methods The experimental results were analyzed and the data were expressed as average expression values.
[0085] like Figure 1 and Figure 2As shown, the results show that the amount of DNA separated by the present invention is 3.4 times and 2.4 times that of the classical method, respectively, indicating that the present invention is more effective in separating chromatin and should have higher efficiency in extracting DNA-binding proteins than the classical method.
[0086] This experiment further compared the purity of chromatin extracted by the two methods.
[0087] Chloroplast contamination is one of the main contaminations when isolating cell nuclei. Therefore, qPCR was used to compare the chloroplasts remaining in the cell nuclei isolated by the present invention with those remaining in the cell nuclei isolated by the classical method. Figure 3 and Figure 4 As shown, the results show that the chloroplast residues of chromatin DNA isolated by the classical method using Populus davidianus as the material are 3.6 times that of the chromatin DNA isolated by the present invention, and the chloroplast residues of chromatin DNA isolated by the classical method using Arabidopsis thaliana as the material are 2.6 times that of the chromatin DNA isolated by the present invention, indicating that the present invention can isolate chromatin with higher purity than the classical method.
[0088] Experimental Example 2
[0089] In order to compare the quality of the nuclear proteins separated by the present invention and the classical method, the nuclear proteins of Populus dahliae and Arabidopsis thaliana were separated by the present invention and the classical method, respectively, and firstly analyzed by SDS-PAGE.
[0090] After the plant DNA binding protein obtained in Example 2, Example 3, Comparative Example 1 and Comparative Example 2 was combined with the sample solution, the mixture was separated by SDS-PAGE gel electrophoresis, and the electrophoresis conditions were 90V for the concentrated gel and 120V for the separation gel, and the electrophoresis time was 40 minutes. After the electrophoresis, the protein was transferred to a polyvinylidene fluoride membrane (Millipore, Massachusetts, USA). In order to reduce nonspecific binding, a Quick Block TM Western ELISA kit (Beyotime, Shanghai, China) was used for blocking. Subsequently, the membrane was incubated with histone H3.1 polyclonal antibody (Abmart, P30266M) and PCK1 antibody (Abmart, TD6770S) at 4°C overnight. Secondary antibodies and enhanced chemiluminescence (ECL) solution were used for development (Beyotime, Shanghai, China).
[0091] like Figure 5 As shown, the results show that compared with the classical method, the protein separated by the present invention contains more 20KD protein. Since the molecular weight of histones is between 10-20kd, this shows that the present invention can harvest more histones than the classical method.
[0092] In order to compare the efficiency of the present invention and the classical method, we used protein immunoblotting technology to study the quantity and purity of the nuclear proteins of Populus dahliae and Arabidopsis thaliana separated by the present invention and the classical method.
[0093] The nuclear proteins in the same amount of samples were extracted using Example 2, Example 3, Comparative Example 1 and Comparative Example 2, respectively, dissolved with the same amount of buffer, and subjected to protein immunoblotting. The amount of nuclear proteins and cytoplasmic proteins was detected using anti-histone H3 antibody and anti-PEPC antibody (phosphoenolpyruvate carboxylase, PEPC), respectively, and the total protein was used as a control.
[0094] like Figure 6 As shown, when using H3 antibody, the results show that both methods can separate nuclear proteins; however, the present invention produces more nuclear proteins than the classical method. When using anti-pck1 antibody for protein immunoblotting, no hybridization signal is observed for the protein separated by the present invention, while the classical method has hybridization signals, indicating that the classical method has contamination on cytoplasmic proteins, while the protein obtained by the present invention has no obvious contamination on cytoplasmic proteins.
[0095] In order to further compare the extraction amount of the present invention and the classical method, the nuclear proteins in the samples extracted in equal amounts in Example 2, Example 3, Comparative Example 1 and Comparative Example 2 were diluted 0.5 times, 0.25 times and 0.125 times respectively, and protein immunoblotting was performed using anti-histone H3 antibody as the primary antibody.
[0096] like Figure 7 As shown, the results show that when the protein obtained by the present invention is diluted to 0.125 times, its hybridization signal is the same as the hybridization signal when it is diluted 0.5 times by the classical method, indicating that the yield of the protein obtained by the present invention is 4 times that of the classical method.
[0097] Experimental Example 3
[0098] In this experiment, mass spectrometry was used to compare the plant DNA binding protein extracted in Example 2 with the protein obtained by the classical method in Comparative Example 1. At the same time, total protein was separated by mass spectrometry as a control.
[0099] The specific method of proteomic analysis in this experiment is as follows:
[0100] Pre-cooled acetone was added to precipitate plant DNA binding proteins, and then vortexed thoroughly to mix. Next, 4 volumes of pre-cooled acetone were added and precipitated at -20°C for 2 hours. The resulting precipitate was washed 2-3 times with pre-cooled acetone. The protein sample was then redissolved in 200mM TEAB and dispersed by ultrasound. For the initial digestion, trypsin was added at a mass to protein ratio of 1:50 for overnight digestion. The sample was reduced with 5mM dithiothreitol at 56°C for 30 minutes and then alkylated with 11mM iodoacetamide at room temperature for 15 minutes in the dark. Finally, the peptides were desalted using a Strata X solid phase extraction column. The peptides were analyzed using a timsTOF Pro 2 mass spectrometer after a capillary source. Precursors and fragments were analyzed on the TOF detector. The timsTOFPro was operated in data-independent parallel accumulation serial fragmentation (DIA-PASEF) mode. The full MS scan was set to 300–1500 (MS / MS scan range) and 20 PASEF (MS / MS mode) scans were acquired per cycle. The MS / MS scan range was set to 400–850 and the isolation window was set to 7 m / z.
[0101] The statistics of nuclear protein quantity are shown in Table 1.
[0102] Table 1
[0103]
[0104] The results are shown in Table 1. The control sample (separated total protein) identified 904 nucleoproteins, accounting for 17% of the total protein. The classical method identified 942 nucleoproteins, accounting for 26.8% of the total number of identified proteins, indicating that compared with the total protein, nucleoproteins have been enriched in large quantities. However, the protein obtained by the present invention identified 1477 nucleoproteins, accounting for 45% of the total number of identified proteins. These results show that the present invention can separate nucleoproteins more effectively than the traditional method, and is a powerful method for separating nucleoproteins.
Claims
1. An efficient method for extracting plant DNA binding protein, characterized in that: The steps include: Step 1: Grind the plant material into powder using liquid nitrogen; Step 2: Add the plant powder obtained in step 1 to solution 1 and vortex shake the mixture, filter the mixture after sufficient shaking to collect the filtrate, and centrifuge the filtrate to collect the precipitate; Step 3, resuspending the precipitate obtained in step 2 with NaCl solution, vortexing and ultrasonicating; Step 4: Add ultrapure water to the system after ultrasonic treatment in step 3, dilute the NaCl concentration to 0.14 mol / L, collect the precipitate by centrifugation, and obtain a DNA-protein complex; Step 5: extract and purify the protein from the DNA-protein complex obtained in step 4, thereby obtaining the plant DNA binding protein.
2. An efficient method for extracting plant DNA binding proteins according to claim 1, characterized in that: Step one also includes soaking the plant material in a formaldehyde solution to perform DNA-protein cross-linking, and then grinding the obtained DNA-protein cross-linked material into powder using liquid nitrogen.
3. An efficient method for extracting plant DNA binding protein according to claim 1 or 2, characterized in that, Step 2: Solution 1 is a 10 mM Tris-HCl buffer solution with a pH of 8.0 containing 0.4 M sucrose, 1.5 mM MgCl2 and 1 mM DTT.
4. An efficient method for extracting plant DNA binding protein according to claim 3, characterized in that: In step 2, the mass volume ratio of the plant powder to solution 1 is 1 g:10 mL, and the vortex oscillation time is 10 to 20 min.
5. An efficient method for extracting plant DNA binding protein according to claim 4, characterized in that: The filtration in step 2 is performed using 2 layers of 300-mesh nylon cloth or 2-4 layers of Miracloth filter cloth.
6. An efficient method for extracting plant DNA binding protein according to claim 5, characterized in that: The specific conditions of the centrifugation in step 2 are 4° C., 1000-1200 rpm, 10-20 min.
7. An efficient method for extracting plant DNA binding protein according to claim 6, characterized in that: The concentration of the NaCl solution in step 3 is 1 mol / L.
8. An efficient method for extracting plant DNA binding protein according to claim 7, characterized in that: The ultrasonic treatment in step 3 is carried out in an ice bath, with an ultrasonic output power of 100 to 200 W, a working time of 3 s, an interval time of 12 s, and a total operation time of 20 to 30 min.
9. An efficient method for extracting plant DNA binding protein according to claim 8, characterized in that: The specific conditions of the centrifugation in step 4 are 4° C., 10,000-25,000 rpm, and 5-30 min.
10. An efficient method for extracting plant DNA binding protein according to claim 9, characterized in that: In step 5, when it is necessary to obtain active plant DNA binding protein, the DNA-protein complex is treated with micrococcal nuclease and the plant DNA binding protein is extracted and purified; when it is not necessary to obtain active plant DNA binding protein, the conventional protein extraction method is used to extract and purify the plant DNA binding protein from the DNA-protein complex.