Method for finely extracting nuclear protein and cytoplasm protein from small-volume plant sample

Abstract

The invention belongs to the technical field of plant protein separation, and relates to a method for finely extracting nuclear protein and cytoplasm protein from a small-volume plant sample. The invention provides a method for synchronously and finely extracting nucleoprotein and cytoplasm protein from a small-volume plant sample, which comprises the following steps: taking leaves of the small-volume plant sample, grinding, adding a first nucleoprotein extraction reagent for extraction, and centrifugally collecting supernate and precipitate; extracting the obtained precipitate by using a second nucleoprotein extraction reagent and a third nucleoprotein extraction reagent, and centrifuging to obtain supernate, namely a cell nucleoprotein extract; and extracting the obtained supernate by using an RIPA extracting solution, and centrifuging to obtain a precipitate dissolving solution, namely the cytoplasm protein extract. The method aims at small-size crops, and synchronous and high-purity extraction of the nuclear protein and the cytoplasm protein can be achieved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant protein separation, and relates to a method for simultaneously extracting nuclear proteins and cytoplasmic proteins from a small-volume plant sample. BACKGROUND

[0002] The accurate separation of nuclear and cytoplasmic proteins is a core technical basis for analyzing plant gene expression regulation and signal transduction mechanisms. The nuclear-cytoplasmic distribution of nuclear-cytoplasmic proteins, as a key mechanism for plant signal regulation, is involved in cell proliferation, stress response and many other physiological processes. High-quality nuclear-cytoplasmic protein samples are a prerequisite for identifying nuclear transport receptor substrates and analyzing protein functions. With the widespread use of model plants such as Arabidopsis thaliana in molecular biology research, the demand for nuclear-cytoplasmic protein extraction techniques for small-volume crop tissues is increasingly urgent.

[0003] Existing plant nuclear-cytoplasmic protein extraction methods are mainly based on sucrose gradient centrifugation or differential centrifugation techniques. Although these methods have been applied to plant tissue separation, they have significant drawbacks. Traditional methods are designed for large amounts of plant tissue or animal cells, and when directly applied to small-volume samples, the protein yield is extremely low due to insufficient starting material. Additionally, the starch granules, chloroplast fragments and cell nuclei in plant tissues have similar densities or sizes, which can contaminate the nuclear protein sample. Moreover, the process is complex, requires an ultracentrifuge, and uses multiple types of reagents, which are costly and can cause cross-linking and degradation of secondary metabolites with proteins, severely restricting the development of small-volume crop-related research.

[0004] Although some improved protein extraction methods have been reported, most of them are not specifically designed for the simultaneous extraction of nuclear and cytoplasmic proteins from small-volume crops. For example, the method for extracting high-purity nuclear and membrane proteins disclosed in CN109942666A, or focuses on a single component, or does not systematically optimize the lysis and separation steps under micro-sample conditions, making it difficult to effectively solve the cross-contamination problem of nuclear and cytoplasmic protein components while ensuring high yield. Therefore, there is an urgent need to develop a method that is specifically for small-volume crops, simplifies the process, controls costs, and can simultaneously obtain high-purity nuclear and cytoplasmic proteins. SUMMARY

[0005] The present application proposes a standardized method for simultaneously extracting nuclear and cytoplasmic proteins from small-volume plant samples, which is of great significance for promoting plant molecular biology research.

[0006] To achieve the above-mentioned purpose, the technical solutions of the present application are as follows:

[0007] On the one hand, the present application provides a method for simultaneously extracting nuclear and cytoplasmic proteins from a small-volume plant sample, comprising the following steps: Preliminary separation of nuclear protein and cytoplasmic protein: take small volume plant sample leaves, grind in liquid nitrogen, add pre-cooled first nuclear protein extraction reagent, mix and place on ice, mix again, filter, centrifuge to collect supernatant and precipitate; Extraction of nuclear protein: resuspend the obtained precipitate with second nuclear protein extraction reagent and centrifuge until the supernatant is green-free, resuspend the precipitate after centrifugation with third nuclear protein extraction reagent; add the resuspension to the upper layer of a centrifuge tube containing third nuclear protein extraction reagent, centrifuge to collect the precipitate, lyse the precipitate with nuclear lysis buffer, and the supernatant after centrifugation again is the nuclear protein extract; Extraction of cytoplasmic protein: centrifuge the obtained supernatant again, add RIPA extraction solution to the supernatant after centrifugation, mix and place on ice, add pre-cooled acetone to the supernatant after standing to precipitate overnight, dissolve the precipitate after centrifugation with HEPES buffer to obtain the cytoplasmic protein extract.

[0008] In the present application, small volume plant sample refers to whole or partial tissue with small size, low biomass, easy to miniaturize operation and high throughput processing of living or in vitro plant material; its core features include miniaturization, low biomass requirement and physiological integrity. For example, small volume plant sample can be potato tissue culture seedlings, tomato tissue culture seedlings, Arabidopsis, miniature rice varieties and aquatic ferns.

[0009] Preferably, the first nuclear protein extraction reagent, the second nuclear protein extraction reagent and the third nuclear protein extraction reagent each comprise sucrose, Tris-HCl, MgCl2, β-mercaptoethanol, phenylmethylsulfonyl fluoride and Triton X-100; The first nuclear protein extraction reagent, the second nuclear protein extraction reagent and the third nuclear protein extraction reagent each need to add protease inhibitor cocktail when used.

[0010] Preferably, the nuclear lysis buffer comprises Tris-HCl, ethylenediaminetetraacetic acid, sodium dodecyl sulfate and phenylmethylsulfonyl fluoride; The nuclear lysis buffer needs to add protease inhibitor cocktail when used.

[0011] Preferably, the RIPA extraction solution comprises Tris-HCl, NaCl, Triton X-100, deoxycholic acid sodium, sodium dodecyl sulfate, NaF, sodium orthovanadate, ethylenediaminetetraacetic acid and PVPP.

[0012] More preferably, 250 mL of the first nuclear protein extraction reagent is prepared according to the following formula: sucrose 34.23 g, 1 mol / L Tris-HCl (pH = 8.0) 2.5 mL, MgCl20.51 g, β-mercaptoethanol 1.25 mL, 0.1 mol / L benzyl sulfonyl fluoride 250 μL, and Triton X-100 2.5 mL; 50 mL of the second nuclear protein extraction reagent is prepared according to the following formula: sucrose 4.279 g, 1 mol / L Tris-HCl (pH = 8.0) 500 μL, MgCl20.218 g, β-mercaptoethanol 0.25 mL, 0.1 mol / L benzyl sulfonyl fluoride 50 μL, and Triton X-100 0.5 mL; 50 mL of the third nuclear protein extraction reagent is prepared according to the following formula: sucrose 29.09 g, 1 mol / L Tris-HCl (pH = 8.0) 500 μL, MgCl20.02 g, β-mercaptoethanol 250 μL, 0.1 mol / L benzyl sulfonyl fluoride 50 μL, and Triton X-100 75 μL.

[0013] More preferably, 50 mL of the nuclear lysis buffer is prepared according to the following formula: 1 mol / L Tris-HCl (pH = 8.0) 2.5 mL, 0.5 mol / L ethylenediaminetetraacetic acid 0.5 mL, sodium dodecyl sulfate 0.5 g, and benzyl sulfonyl fluoride 1 mL.

[0014] More preferably, 50 mL of the RIPA extraction solution is prepared according to the following formula: 1 mol / L Tris-HCl 2.5 mL, 1.5 mol / L NaCl 5 mL, 1% Triton X-100 0.5 mL, sodium deoxycholate 0.5 g, sodium dodecyl sulfate 0.05 g, 100 mmol / L NaF 2.5 mL, 100 mmol / L sodium orthovanadate 0.5 mL, 50 mmol / L ethylenediaminetetraacetic acid 0.1 mL, 2% (m / v) PVPP 1 g.

[0015] In another aspect, the present application provides a kit for simultaneously extracting nuclear proteins and cytoplasmic proteins from a small-volume plant sample, the kit comprising a first nuclear protein extraction reagent, a second nuclear protein extraction reagent, a third nuclear protein extraction reagent, a nuclear lysis buffer, and a RIPA extraction solution; The first nuclear protein extraction reagent, the second nuclear protein extraction reagent, and the third nuclear protein extraction reagent each comprises sucrose, Tris-HCl, MgCl2, β-mercaptoethanol, benzyl sulfonyl fluoride, and Triton X-100; The nucleus lysis buffer comprises Tris-HCl, ethylenediaminetetraacetic acid, sodium dodecyl sulfate and benzylsulfonyl fluoride; The RIPA extraction solution comprises Tris-HCl, NaCl, Triton X-100, sodium deoxycholate, sodium dodecyl sulfate, NaF, sodium orthovanadate, ethylenediaminetetraacetic acid and PVPP.

[0016] The application also provides the use of the method or the kit in the preparation of a sample for studying small-volume plant signal transduction, gene expression regulation and protein nucleocytoplasmic transport.

[0017] The application also provides the use of the method or the kit in the preparation of a sample for studying small-volume plant signal transduction, gene expression regulation and protein nucleocytoplasmic transport.

[0018] It should be noted that a variety of different plant cell nucleus separation techniques have been developed, and most separation methods consist of similar sequential steps, including disruption, filtration, centrifugation, dissolution and separation (all on ice). Due to the differences in size and density of different organelles in cells, density gradient centrifugation and differential centrifugation are widely used for the purification of different subcellular organelles.

[0019] The complete plant cell nucleus is denser than most organelles (such as chloroplasts, mitochondria) and cell debris. After filtration, the homogenate is placed on a 2.0 mol / L sucrose pad containing the same buffer, and under the action of centrifugal force, the cell nucleus is deposited at the bottom of the tube or enriched at the interface, and the chloroplasts, mitochondria and cell debris are left in the upper layer or supernatant. Density gradient centrifugation is essential for removing contaminated organelles, especially chloroplasts. However, excessive grinding and high viscosity of the homogenate can cause the cell nucleus to rupture or be lost, resulting in reduced ChIP efficiency.

[0020] In the differential centrifugation process, the homogenate is centrifuged in a stepwise increasing centrifugal force manner. Larger organelles (such as cell nuclei, chloroplasts, starch granules) can be quickly precipitated under lower centrifugal force, while smaller organelles (such as mitochondria, peroxisomes, microsomes) need higher centrifugal force to precipitate within a reasonable time. Differential centrifugation is one of the most direct and fastest methods for separating organelles, and only needs to adjust the centrifugal force and time parameters to obtain the precipitates of different components, can handle larger volumes of homogenate, and is suitable as a preliminary enrichment step before subsequent fine purification (such as density gradient centrifugation), effectively concentrating the target organelles. However, differential centrifugation can only be used for preliminary enrichment with low purity requirements.

[0021] It should be noted that the traditional method of nuclear separation mainly has the following technical problems: ① easy to be contaminated: the density of starch granules is close to that of the nucleus, and it is difficult to separate from the nucleus in the density gradient. At the same time, incomplete chloroplast fragmentation will produce fragments similar in size to the nucleus, contaminating the nuclear preparation. ② Not friendly to rare cells or trace samples: high loss rate. ③ Interference of secondary metabolites: plant tissues are rich in polyphenols, tannins and other substances, which are easy to oxidize and cross-link with proteins / nucleic acids during homogenization, resulting in nuclear aggregation, precipitation or degradation of biomolecules. ④ Complex process: requires an ultracentrifuge, and gradient preparation and nuclear collection require skilled operation. ⑤ High cost: the process of nuclear separation is complicated, and many reagents are required, which is costly. The reasons for the problems are: first, the integrity of the nuclear membrane is difficult to maintain absolutely, and mechanical shear force or the action of a detergent can easily cause it to be damaged, resulting in leakage of DNA, histone and regulatory proteins in the nucleus into the cytoplasmic component. Second, the physical and biochemical connections between subcellular components are close, the outer nuclear membrane is connected to the endoplasmic reticulum membrane system, making it easy for membrane-bound ribosomes and other components to co-precipitate with the nucleus; at the same time, the dynamic interaction between chromatin and nuclear skeleton, nucleolus and specific cytoplasmic mRNP particles also makes it difficult to completely dissociate some components during separation. In addition, the resolution of traditional centrifugal separation methods based on size and density is limited, and some organelles with similar size or density to the nucleus (such as mitochondria, peroxisomes, lysosomes, and stubborn chloroplasts and starch granules in plant cells) cannot be effectively removed, resulting in co-precipitation and contamination.

[0022] It should be noted that the existing general method is usually designed for animal cells or large amounts of plant tissue, and direct application to small volume plant samples will result in insufficient starting material, resulting in low protein yield or experimental failure. The present application optimizes the process and reagent system, so that it can still work effectively in trace samples (such as tens of milligrams of fresh weight). The high sample utilization rate of the method of the present application enables researchers to complete experiments with less material, especially suitable for mutant, valuable transgenic materials or high-throughput screening scenarios. At the same time, since the present application is aimed at the common problems of small volume crops, its principles and technical solutions can be extended to other similar crops (such as potato tissue culture seedlings, tomato tissue culture seedlings, Arabidopsis, miniature rice varieties, aquatic ferns, etc.), showing good universality.

[0023] Existing technologies tend to focus on membrane proteins or single components, or lack finely optimized cytoplasmic-nuclear separation steps, easily leading to cross-contamination. This invention, through a key design of fractional lysis and differential centrifugation, achieves physical separation of cytoplasmic and nuclear proteins in the early stages of separation. This logically reduces the mixing of organelle debris and protein components, thus ensuring minimal cytoplasmic protein residue in the nuclear protein preparation, and vice versa. Simultaneously, the optimization of the activity-protecting components in the buffer system theoretically guarantees the native conformation and biological activity of the extracted proteins (especially transcription factors and kinases), providing a reliable foundation for subsequent functional studies (such as enzyme activity detection and protein-protein interactions).

[0024] Traditional methods often rely on researchers' experience, leading to subjective differences in various steps and inconsistent results. This invention standardizes and quantifies all key variables such as lysis conditions, centrifugation parameters, and buffer formulations, forming a clear workflow. This significantly reduces technical differences and human error between operators, ensuring high reproducibility and comparability of results from different laboratories and batches. The integrated and optimized workflow also avoids lengthy trial-and-error steps and condition optimization, allowing researchers, even novices, to obtain reliable results in a shorter time, significantly improving experimental efficiency.

[0025] This invention goes beyond mere methodological improvement; its ultimate goal is to solve the problem of obtaining high-quality functional protein components from limited materials. Therefore, this method directly serves cutting-edge scientific questions, such as plant immunity and the crucial nucleocytoplasmic shuttle process in stress responses. Its technological advantages can be translated into more reliable and sensitive experimental data. Furthermore, because the method system of this invention is complete, the steps are clear, and the reagent formulation is well-defined, it is very easy to convert into standardized pre-packaged reagent kits, providing the life science research market with a much-needed specialized tool, possessing clear commercial prospects and industrial value.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention proposes a complete method for the simultaneous, efficient, and high-purity separation and extraction of cytoplasmic and nuclear proteins from small-volume plant samples. This method aims to solve the key problems of insufficient handling of trace samples, cross-contamination of cytoplasmic and nuclear protein components, and low yield in existing technologies. Its direct application value lies in providing high-quality protein samples for in-depth analysis of mechanisms such as signal transduction and gene expression regulation within plant cells, and laying the technical foundation for the development of specialized plant subcellular protein extraction kits. Specifically, this invention achieves the following improvements by optimizing the extraction reagents: ① Achieving efficient cytoplasmic-nuclear protein separation from trace samples: This invention enables high-quality cytoplasmic-nuclear protein separation from small-volume plant samples by adjusting the amount of reagents used in the reaction system. ② Reducing reagent usage: By optimizing the preparation of the cytoplasmic-nuclear protein separation solution, the types and amounts of reagents required are greatly reduced, lowering costs. ③ Improving the extraction rate of cytoplasmic and nuclear proteins: By adjusting the centrifugation speed, time, and solution preparation, the separation efficiency is improved. ④ By optimizing the solution preparation for sucrose density gradient centrifugation, not only were problems such as easy contamination and complex process in traditional nucleus-mass separation methods solved, but experimental costs were also significantly reduced and the efficiency of nucleus-mass separation was improved.

[0027] (2) First, the method of this invention establishes a mild yet efficient graded lysis strategy for small-volume plant tissues. By precisely controlling the lysis conditions and centrifugal force parameters, cytoplasmic proteins are preferentially released while maintaining the integrity of the cell nucleus, thereby effectively separating the two types of protein components at the physical level. Second, through systematic optimization of the first, second, and third nuclear protein extraction reagents, nuclear lysis buffer, and RIPA extraction solution, the natural activity and integrity of the extracted proteins are ensured, minimizing degradation and artificial modification. Finally, the method of this invention seamlessly integrates the extraction process with downstream trace protein detection technology, forming a reproducible and standardized solution that fundamentally overcomes the technical defects of traditional methods when applied to small-volume crops, such as cumbersome procedures, poor purity, and poor reproducibility. Attached Figure Description

[0028] Figure 1 Figure A shows the results of the protein subcellular localization verification experiment; A is immunofluorescence imaging; B is immunoblotting analysis; Total represents total protein extract, Cytosol represents cytoplasmic protein extract, Nucleus represents nuclear protein extract, and α-Histone H3 represents anti-histone H3 antibody.

[0029] Figure 2This is an immunoblot verification image for the purity of separation between nuclear and cytoplasmic proteins in Arabidopsis thaliana cells; Cytosol represents cytoplasmic protein extract, Nucleus represents nuclear protein extract, Anti-GFP represents anti-GFP tag antibody, Anti-Histone H3 represents anti-histone H3 antibody, Anti-Actin represents anti-actin antibody, Anti-Rubisco represents anti-chloroplast photosynthetic carbon fixation enzyme antibody, and Ponceau S represents Ponceau S staining. Detailed Implementation

[0030] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention clearer. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0031] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods and detection methods described in each embodiment are conventional methods; unless otherwise specified, the reagents and materials are all commercially available.

[0032] In this embodiment of the invention, 250 mL of the first nucleoprotein extraction reagent was prepared according to the following formula: 34.23 g sucrose, 2.5 mL 1 mol / L Tris-HCl (pH=8.0), 0.51 g MgCl2, 1.25 mL β-mercaptoethanol, 250 μL 0.1 mol / L benzyl sulfonyl fluoride, and 2.5 mL Triton X-100.

[0033] 50 mL of the second nucleoprotein extraction reagent was prepared according to the following formula: 4.279 g sucrose, 500 μ L 1 mol / L Tris-HCl (pH=8.0), 0.218 g MgCl2, 0.25 mL β-mercaptoethanol, 50 μ L 0.1 mol / L benzyl sulfonyl fluoride, and 0.5 mL Triton X-100.

[0034] 50 mL of the third nucleoprotein extraction reagent was prepared according to the following formula: 29.09 g sucrose, 500 μ L 1 mol / L Tris-HCl (pH=8.0), 0.02 g MgCl2, 250 μ L β-mercaptoethanol, 50 μ L 0.1 mol / L benzyl sulfonyl fluoride, and 75 μ L Triton X-100.

[0035] Prepare 50 mL of nuclear lysis buffer according to the following formula: 2.5 mL of 1 mol / L Tris-HCl (pH=8.0), 0.5 mL of 0.5 mol / L ethylenediaminetetraacetic acid, 0.5 g of sodium dodecyl sulfate, and 1 mL of benzyl sulfonyl fluoride.

[0036] 50 mL of RIPA extract was prepared according to the following formula: 2.5 mL of 1 mol / L Tris-HCl, 5 mL of 1.5 mol / L NaCl, 0.5 mL of 1% Triton X-100, 0.5 g of sodium deoxycholate, 0.05 g of sodium dodecyl sulfate, 2.5 mL of 100 mmol / L NaF, 0.5 mL of 100 mmol / L sodium orthovanadate, 0.1 mL of 50 mmol / L ethylenediaminetetraacetic acid, and 1 g of 2% (m / v) PVPP.

[0037] It should be noted that the first nuclear protein extraction reagent, the second nuclear protein extraction reagent, the third nuclear protein extraction reagent, and the nuclear lysis buffer all require the addition of a protease inhibitor mixture (B14001 protease inhibitor Cocktail, without EDTA and 100X DMSO stock solution) during use.

[0038] Example This embodiment provides a method for simultaneously extracting nuclear proteins and cytoplasmic proteins from small-volume plant samples, including the following steps: Preliminary separation of nuclear proteins from cytoplasmic proteins: Take 1g of Arabidopsis thaliana leaves, grind them with liquid nitrogen, add 4mL of pre-cooled first nuclear protein extraction reagent, vortex to mix, and place on ice; mix by rotating at 4℃ in the dark for 30min; filter the solution through two layers of gauze into a new 50mL centrifuge tube; centrifuge at 6000rpm and 4℃ for 20min, and collect the supernatant and precipitate; Extraction of nuclear proteins: Gently pour off the supernatant, resuspend the resulting precipitate in 1 mL of second nuclear protein extraction reagent, and transfer it to a new 2 mL centrifuge tube; centrifuge at 12000 rpm and 4℃ for 10 min; remove the supernatant, resuspend in 1 mL of second nuclear protein extraction reagent, and centrifuge at 12000 rpm and 4℃ for 10 min until the supernatant of the extract no longer appears green; remove the supernatant, resuspend in 500 μL of third nuclear protein extraction reagent, avoiding foaming; in a new 1.5 mL centrifuge tube, first add 500 μL of third nuclear protein extraction reagent; then take 500 μL of the solution resuspended in 500 μL of third nuclear protein extraction reagent and carefully cover it on the clean 500 μL of third nuclear protein extraction reagent; centrifuge at 14000 rpm and 4℃ for 60 min; remove the supernatant, resuspend in 200 μL of nuclear lysis buffer; centrifuge at 14000 rpm and 4℃ for 10 min, and store the supernatant at -80℃ to obtain the nuclear protein extract; Extraction of cytoplasmic proteins: Centrifuge the obtained supernatant again at 4℃ and 6000rpm for 20min; take 2mL of supernatant, add 500μL of RIPA extraction buffer, vortex for 30s, and let stand on ice for 20min; take 2.5mL of supernatant, add 2.5mL of acetone pre-chilled at -20℃, and precipitate overnight at -20℃; centrifuge at 4℃ and 12000rpm for 15min; discard the supernatant, and dry the acetone in a fume hood; dissolve the precipitate with 500μL of 30mmol / L HEPES buffer (pH=7.5) to obtain the cytoplasmic protein extract.

[0039] 100 μL of cytoplasmic protein extract, 72 μL of nuclear protein extract, and 172 μL of total protein extract (100 μL of cytoplasmic protein extract and 72 μL of nuclear protein extract) obtained in the examples were respectively used for protein subcellular localization experiments. Figure 1 ).Depend on Figure 1 As can be seen, the white fluorescent spots observed in the immunofluorescence experiment are concentrated in the cell nucleus region. The white fluorescent spots represent the distribution of histone H3, and the subcellular localization of the protein within the cell can be directly observed. Figure 1 (A). This invention successfully separates nuclear proteins (histone H3) from cytoplasmic proteins, and the separated nuclear protein components have high purity. Histone H3 is only present in the nuclear protein extract, while no histone H3 signal is found in the cytoplasmic protein extract. Effective separation of nuclear and cytoplasmic components is achieved without cross-contamination. Figure 1 (B)

[0040] 500 μL of cytoplasmic protein extract and 200 μL of nuclear protein extract obtained in the examples were respectively used for immunoblotting verification of the purity of nuclear protein and cytoplasmic protein separation. Figure 2 ).Depend on Figure 2It can be seen that the purity of nuclear proteins separated meets the standard, with no cross-contamination; the purity of cytoplasmic proteins separated meets the standard, with no nuclear proteins mixed in; the experimental results of this invention are not affected by sample loading differences, and the data are reliable. Specifically, the GFP fusion protein only showed a band in the Cytosol lane, and no signal in the Nucleus lane; indicating that the GFP fusion protein is only located in the cytoplasm. The Anti-Histone H3 detection results showed that histone H3 only showed a clear band in the Nucleus lane, and no signal in the Cytosol lane; indicating that the purity of the nuclear protein extract is extremely high, with no cytoplasmic contamination; and there is no nuclear leakage in the cytoplasmic protein extract. The Anti-Actin detection results showed that Actin only showed a band in the Cytosol lane, and no signal in the Nucleus lane; indicating that the purity of the cytoplasmic protein extract is high, with no nuclear contamination; and there is no cytoplasmic contamination in the nuclear protein extract. Anti-Rubisco detection results showed that Rubisco bands appeared in the Cytosol lane, while no signal was detected in the Nucleus lane; this indicates that the nuclear protein extraction process was not contaminated by chloroplast debris, and this invention solves the technical problem of starch / chloroplast co-precipitation in traditional methods. Ponceau S staining results showed that the total protein content in all lanes was approximately equal, indicating that the experimental procedure was standardized and the results were reliable.

[0041] It should be understood that the disclosed invention is not limited to the specific methods, schemes, and substances described, as these are all subject to variation. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims.

Claims

1. A method for simultaneous extraction of nuclear and cytoplasmic proteins from small volume plant samples, characterized in that, The method comprises the following steps: Preliminary separation of nuclear proteins and cytoplasmic proteins: a small volume of plant sample leaves is taken, ground in liquid nitrogen, and then added to pre-cooled first nuclear protein extraction reagent, mixed and placed on ice, mixed again, filtered, and the supernatant and precipitate are collected by centrifugation; Extraction of nuclear proteins: the obtained precipitate is resuspended with second nuclear protein extraction reagent and centrifuged until the supernatant is green-free, and the precipitate after centrifugation is resuspended with third nuclear protein extraction reagent; the resuspension liquid is added to the upper layer of a centrifuge tube containing third nuclear protein extraction reagent, and the precipitate is collected by centrifugation, and the precipitate is lysed with nuclear lysis buffer, and the supernatant after centrifugation is the nuclear protein extract; Extraction of cytoplasmic proteins: the obtained supernatant is centrifuged again, RIPA extraction solution is added to the supernatant after centrifugation, mixed and placed on ice, pre-cooled acetone is added to the supernatant after standing to precipitate overnight, and the precipitate after centrifugation is dissolved with HEPES buffer to obtain the cytoplasmic protein extract.

2. The method of claim 1, wherein The first nuclear protein extraction reagent, the second nuclear protein extraction reagent and the third nuclear protein extraction reagent each comprise sucrose, Tris-HCl, MgCl2, β-mercaptoethanol, phenylmethylsulfonyl fluoride and Triton X-100; The first nuclear protein extraction reagent, the second nuclear protein extraction reagent and the third nuclear protein extraction reagent each need to add a protease inhibitor mixture when used.

3. The method of claim 1, wherein The nuclear lysis buffer comprises Tris-HCl, ethylenediaminetetraacetic acid, sodium dodecyl sulfate and phenylmethylsulfonyl fluoride; The nuclear lysis buffer needs to add a protease inhibitor mixture when used.

4. The method of claim 1, wherein, The RIPA extraction solution comprises Tris-HCl, NaCl, Triton X-100, sodium deoxycholate, sodium dodecyl sulfate, NaF, sodium orthovanadate, ethylenediaminetetraacetic acid and PVPP.

5. The method of claim 2, wherein 250mL of the first nuclear protein extraction reagent is prepared according to the following formula: sucrose 34.23g, 1mol / L Tris-HCl 2.5mL, MgCl2 0.51g, β-mercaptoethanol 1.25mL, 0.1mol / L phenylmethylsulfonyl fluoride 250μL and Triton X-100 2.5mL; 50mL of the second nuclear protein extraction reagent is prepared according to the following formula: sucrose 4.279g, 1mol / L Tris-HCl 500μL, MgCl2 0.218g, β-mercaptoethanol 0.25mL, 0.1mol / L phenylmethylsulfonyl fluoride 50μL and Triton X-100 0.5mL; 50 mL of the third nuclear protein extraction reagent is prepared according to the following formula: sucrose 29.09 g, 1 mol / L Tris-HCl 500 μL, MgCl2 0.02 g, β-mercaptoethanol 250 μL, 0.1 mol / L phenylmethylsulfonyl fluoride 50 μL and Triton X-100 75 μL.

6. The method of claim 3, wherein, 50 mL of the nuclear lysis buffer is prepared according to the following formula: 1 mol / L Tris-HCl 2.5 mL, 0.5 mol / L ethylenediaminetetraacetic acid 0.5 mL, sodium dodecyl sulfate 0.5 g and phenylmethylsulfonyl fluoride 1 mL.

7. The method of claim 4, wherein, 50 mL of the RIPA extraction solution is prepared according to the following formula: 1 mol / L Tris-HCl 2.5 mL, 1.5 mol / L NaCl 5 mL, 1% Triton X-100 0.5 mL, sodium deoxycholate 0.5 g, sodium dodecyl sulfate 0.05 g, 100 mmol / L NaF 2.5 mL, 100 mmol / L sodium orthovanadate 0.5 mL, 50 mmol / L ethylenediaminetetraacetic acid 0.1 mL, 2% PVPP 1 g.

8. A kit for simultaneous extraction of nuclear and cytoplasmic proteins from small volume plant samples, comprising, The kit comprises a first nuclear protein extraction reagent, a second nuclear protein extraction reagent, a third nuclear protein extraction reagent, a nuclear lysis buffer and a RIPA extraction solution; The first nuclear protein extraction reagent, the second nuclear protein extraction reagent and the third nuclear protein extraction reagent each comprises sucrose, Tris-HCl, MgCl2, β-mercaptoethanol, phenylmethylsulfonyl fluoride and Triton X-100; The nuclear lysis buffer comprises Tris-HCl, ethylenediaminetetraacetic acid, sodium dodecyl sulfate and phenylmethylsulfonyl fluoride; The RIPA extraction solution comprises Tris-HCl, NaCl, Triton X-100, sodium deoxycholate, sodium dodecyl sulfate, NaF, sodium orthovanadate, ethylenediaminetetraacetic acid and PVPP.

9. Use of the method of any one of claims 1-7 or the kit of claim 8 in simultaneous extraction of nuclear proteins and cytoplasmic proteins from a small volume of plant sample.

10. Use of the method of any one of claims 1-7 or the kit of claim 8 in preparation of a sample for studying signal transduction, gene expression regulation and protein nucleocytoplasmic shuttling in a small volume of plant.

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