A method and kit for extracting heat shock nucleoprotein from protoplasts of leafy vegetables

Abstract

This invention belongs to the field of vegetable molecular biology technology, specifically relating to a method and kit for extracting heat shock nucleoproteins from protoplasts of leafy vegetables. The kit provided by this invention includes reagents A1, A2, B1, B2, B3, B4, and C; reagent A1 contains dissociative enzymes, cellulase, etc.; reagent A2 is a cell preservation and cell processing buffer; reagent B1 contains ethylenediaminetetraacetic acid, benzyl sulfonyl fluoride, etc.; reagent B2 contains Triton X-100, etc.; reagent B3 contains Glycerol, etc.; reagent B4 is a cell nucleus preservation solution; and reagent C is a cell nucleus lysis buffer. This kit enables precise extraction of heat shock nucleoproteins at the cellular level, yielding high-quality heat shock nucleoproteins and providing technical support for studying the expression and regulation of nuclear proteins in leafy vegetable cells.

Description

Technical Field

[0001] This invention belongs to the field of vegetable molecular biology technology, specifically relating to a method for extracting heat shock nucleoproteins from protoplasts of leafy vegetables and its reagent kit. Background Technology

[0002] Studying the response mechanisms of plants to high-temperature stress is of great significance for the sustainable development of agricultural production and food security. Among these, heat shock nucleoproteins are a diverse class of proteins found in the cell nucleus, playing a crucial role in the response to high-temperature stress. Therefore, extracting high-purity and high-concentration nucleoproteins is essential for studying chromatin organization and regulation, gene expression regulation, and DNA repair and recombination. Furthermore, the extraction of heat shock nucleoproteins is fundamental to studying the transcriptional regulation and signal transduction of functional genes under high-temperature stress. Currently, methods for extracting nucleoproteins from some plants have been reported. For example, Chinese invention patent CN117866871A discloses a method for extracting cell nuclei from plant leaves suitable for ATAC-seq, including the following steps: S1. Grinding plant leaf tissue into powder using liquid nitrogen; S2. Adding a composite cell wall degrading enzyme reagent to enzymatically hydrolyze the cell wall; S3. Preparing N types of cell nucleus extraction buffer reagents (N>1), extracting cell nuclei using differential centrifugation, and resuspending the precipitate with any one of the N cell nucleus extraction buffer reagents after each centrifugation; S4. Removing impurities by Percoll density gradient centrifugation. This method employs a composite cell wall degrading enzyme reagent (disintegrating enzyme and cellulase) to achieve efficient enzymatic hydrolysis of plant cell wall structural components. It also utilizes components of a nuclear extraction buffer reagent more suitable for differential centrifugation. The NPB reagent uses CHAPS, which does not alter protein activity but can lyse the cell membrane; HEPES maintains the stability of the nuclear structure, helping to preserve the integrity of the nucleus and reducing the impact on subsequent library construction and sequencing analysis results. However, current methods mainly extract nuclear proteins by grinding large pieces of plant tissue, such as leaves and stems, and have not been refined to the cellular level. Furthermore, there are few reports on the extraction of heat-shock nuclear proteins. Moreover, the composition of plant cell walls varies considerably among different species, making these methods unsuitable for extracting nuclear proteins from leafy vegetables. While Chinese invention patent CN112779210B discloses a kit for extracting cytoplasmic and nuclear proteins, this kit was initially designed for animal cells and has only been validated on HeLa, SW480, and HCT116 cell lines. Due to the numerous differences in structure, composition, and physiological characteristics between plant and animal cells, these differences may affect the applicability of the kit. Furthermore, there are currently few methods for extracting heat shock nucleoproteins from protoplasts by lysing the cell wall to release them, and even fewer reports on using protoplast-extracted nucleoproteins in conjunction with DNA pull-down techniques to screen upstream regulatory factors. The lack of refinement in cell preservation, processing efficiency, and extraction techniques makes it difficult to study heat shock transcription factors in the nuclei of leafy vegetable cells using protoplasts. Therefore, it is necessary to develop a method suitable for extracting heat shock nucleoproteins from protoplasts in vegetables. Summary of the Invention

[0003] To overcome the shortcomings of the prior art, this invention provides a method and kit for extracting nuclear proteins from leafy vegetable cells, enabling precise extraction of heat shock nuclear proteins at the cellular level. This method can extract high-quality heat shock nuclear proteins, providing technical support for the study of the expression and regulation of nuclear proteins in leafy vegetable cells.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0005] The first aspect of the present invention provides a kit for extracting heat shock nucleoproteins from protoplasts of leafy vegetables, the kit comprising reagent A1, reagent A2, reagent B1, reagent B2, reagent B3, reagent B4, and reagent C;

[0006] Reagent A1 contains Macerozyme-R10, Cellulase R-10, Mannitol, MES, and KCl; Reagent A2 contains NaCl, CaCl2, KCl, MES, and Glucose; Reagent B1 contains Tris-HCl (pH 7.4), Glycerol, KCl, EDTA, MgCl2, Sucrose, Beta-mercaptoethanol, PMSF, Leupeptin, NaF, Na3VO4, and Beta-glycerophosphate; Reagent B2 contains Tris-HCl (pH 7.4), Glycerol, MgCl2, and Triton X-100; Reagent B3 contains pH 7.4. The reagents are: Tris-HCl, Glycerol, and MgCl2; reagent B4 contains Tris-HCl, Glycerol, MgCl2, Sucrose, NaF, Na3VO4, Beta-glycerophosphate, and PMSF at pH 7.4; and reagent C contains Tris-HCl, NaCl, Glycerol, EDTA, SDC, Tritonx-100, CHAPS, HEPES, NP-40, CAPSO, Sucrose, PMSF, Leupeptin, NaF, Na3VO4, and Beta-glycerophosphate at pH 8.0.

[0007] The kit provided by this invention is suitable for the precise extraction of heat shock nuclear proteins from leafy vegetables at the cellular level. Reagent A1 is used to extract intact protoplasts from leafy vegetables. The cell wall is broken down using Macerozyme-R10 (a dissociative enzyme) and Cellulase R-10 (a cellulase), Mannitol provides osmotic pressure support, and MES and KCl adjust the solution pH and ionic strength to ensure stable protoplast extraction. Reagent A2 serves as a cell culture and processing buffer for leafy vegetables, containing inorganic salts (NaCl, CaCl2, KCl), the buffer MES, and the energy source glucose to maintain cell viability. The B series of reagents is specifically designed for the extraction of nuclear proteins from leafy vegetable protoplast cells. B1 contains enzyme inhibitors and stabilizers (such as PMSF and Leupeptin) to prevent protein degradation; B2 and B3 further process the cell nucleus; and B4 serves as a cell nucleus preservation solution to ensure the integrity of the cell nucleus. Reagent C is a nuclear lysis buffer containing detergents (Tritonx-100, NP-40) and buffers (HEPES), which disrupt the nuclear membrane, releasing and maintaining the activity of nucleoproteins. pH and ionic strength / osmotic pressure adjustments ensure that enzyme and protein activity are not affected during extraction. Therefore, this invention provides a kit for extracting heat shock nucleoproteins from protoplasts of leafy vegetables, enabling precise extraction of heat shock nucleoproteins at the cellular level. This kit can extract high-quality heat shock nucleoproteins, providing technical support for studying the expression and regulation of nucleoproteins in leafy vegetable cells.

[0008] Preferably, based on a 10 mL system, reagent A1 comprises 0.05 g of Macerozyme-R10, 0.10-0.40 g of Cellulase R-10, 2 mL of 0.1 mol MES, 1 mL of 0.2 mol KCl, 5 mL of 0.8 mol Mannitol, and 1.9 mL of ddH2O; based on a 50 mL system, reagent A2 comprises 1.5 mL of 0.1 mol MES, 2.8 mL of 3 mol NaCl, 3 mL of 0.1 mol Glucose, 6 mL of 1 mol CaCl2, 1.5 mL of 0.2 mol KCl, and 35.2 mL of ddH2O; based on a 10 mL volume, reagent B1 comprises 200 μL of 1M Tris-HCl (pH 7.4), 5000 μL of 50 v / v% Glycerol, 1000 μL of 0.2M KCl, 40 μL of 0.5M EDTA, and 1M The reagent B2 consists of 25 μL MgCl2, 0.855 g Sucrose, 22 μL 100 v / v% Beta-mercaptoethanol, 100 μL 0.1 M PMSF, 100 μL 1 g / mL Leupeptin, 100 μL 0.1 M NaF, 100 μL 0.1 M Na3VO4, 100 μL 0.1 M Beta-glycerophosphate, and 10 μL ddH2O. In a 10 mL system, reagent B2 comprises 200 μL 1 M Tris-HCl (pH 7.4), 5 mL 50 v / v% Glycerol, 25 μL 1 M MgCl2, 20 μL 100x Triton X-100, and ddH2O added to 10 mL. In a 20 mL system, reagent B3 comprises 400 μL 1 M Tris-HCl (pH 7.4), 50 v / v% Glycerol... 10 mL, 1M MgCl2 50 μL, ddH2O added to 20 mL; Based on a 6 mL system, reagent B4 comprises 120 μL of 1M Tris-HCl (pH 7.4), 3 mL of 50 v / v % Glycerol, 15 μL of 1M MgCl2, 0.906 g of Sucrose, 60 μL of 0.1M NaF, 60 μL of 0.1M Na3VO4, 60 μL of 0.1M Beta-glycerophosphate, 60 μL of 0.1M PMSF, ddH2O added to 6 mL; Based on a 100 mL system, reagent C comprises 2.5 mL of 2M Tris-HCl (pH 8.0), 2 mL of 5M NaCl, 10 mL of 100 v / v % Glycerol, 0.1M EDTA 10mL, SDC 1g, 100x Tritonx-1000.5mL, CHAPS 0.5g, HEPES0.5g, 100x NP-400.5mL, CAPSO 1g, Sucrose 34.23g, 0.1M PMSF 1mL, 1g / mL Leupeptin 1mL, 0.1M NaF 1mL, 0.1M Na3VO41mL, 0.1M Beta-glycerophosphate 1mL, ddH2OAdd to 100mL. .

[0009] More preferably, the pH value of reagent A1 is 6.8, and the final concentration of Cellulase R-10 is 0.25 g / 10 mL.

[0010] The second aspect of this invention provides a method for extracting heat shock nucleoproteins from protoplasts of leafy vegetables. The method utilizes the kit described in the first aspect to extract heat shock nucleoproteins from protoplasts of leafy vegetables, and specifically includes the following steps:

[0011] S1. Obtain mesophyll protoplast cells and subject them to heat shock treatment:

[0012] S11. Take tender true leaves, cut the leaves into thin strips, and put them into reagent A1 that has been pre-treated in a 50-60℃ water bath and cooled for enzymatic hydrolysis to obtain the enzymatic hydrolysis product.

[0013] S12. After filtration, the enzymatic hydrolysis products are washed with A2 reagent and the protoplasts are cultured with A2 reagent.

[0014] S13. After culturing, filter to remove dead cells, collect the filtrate to obtain intact protoplasts, and then subject the obtained protoplasts to heat shock treatment.

[0015] S2. Extraction of cell nuclei from protoplast precipitation:

[0016] S21. After removing reagent A2, add reagent B1 to the protoplast precipitate to fully lyse the protoplasts, then wash repeatedly with reagent B2 to remove impurities, discard the supernatant, and retain the precipitate until the precipitate turns off-white or pure white.

[0017] S22. Add reagent B3 to wash the precipitate to remove excess Triton X-100 contained in reagent B2. Collect the precipitate to obtain the cell nucleus.

[0018] S3. Lyse the cell nucleus and extract heat shock nucleoprotein: Add reagent C to the cell nucleus sample, mix well, and let stand in an ice bath. During the standing period, vortex for 7-15 seconds every 4-7 minutes, and then sonicate in an ice bath to break up the nucleus. Finally, centrifuge and collect the supernatant to obtain the heat shock nucleoprotein.

[0019] The principle of the kit provided in this application for extracting heat shock nucleoproteins from protoplasts of leafy vegetables is as follows: First, intact protoplasts of leafy vegetables are extracted: leaf cells are treated with reagent A1 to release the protoplasts, which are then stored in reagent A2. Next, heat shock treatment is performed: using a yellow fluorescent protein driven by a heat-related promoter in leafy vegetables as a reporter gene, the heat shock time required for fluorescent cell luminescence is determined by transforming leafy vegetable protoplasts; the protoplasts are then subjected to heat shock treatment for the same duration to induce heat shock nucleoprotein expression. Then, nuclear proteins are extracted: cell nuclei are separated using reagent series B, and then the nuclei are lysed using reagent C to release the nucleoproteins. Finally, protein detection and analysis are performed: the previously obtained HSP promoter is biotin-labeled and incubated with heat shock nucleoproteins for DNA pull-down and mass spectrometry detection. By combining the above methods with the kit provided in this application, precise extraction of heat shock nucleoproteins at the cellular level is achieved, resulting in high-quality heat shock nucleoproteins and providing technical support for the study of the expression and regulation of nuclear proteins in leafy vegetable cells.

[0020] Preferably, the temperature of the heat shock treatment in S13 is 30-40°C and the time is 0.5-2h.

[0021] Preferably, the enzymatic hydrolysis in S11 is performed at room temperature and in the dark on a shaker at 70-90 rpm for 2-4 hours.

[0022] Preferably, S12 uses reagent A2 to culture protoplast cells overnight.

[0023] Preferably, B4 reagent is added to the cell nuclei obtained in S22, the precipitate is resuspended, and the sample is rapidly frozen with liquid nitrogen and then placed at -80°C for storage.

[0024] Preferably, the ice bath is left to stand for 25-40 minutes.

[0025] Preferably, the ice bath ultrasonic breaking time is 1-3 minutes, the power is 15-25%, the working time is 3 seconds, and the interval is 3 seconds.

[0026] Compared with the prior art, the beneficial effects of the present invention are:

[0027] (1) Currently, there are no methods or kits for extracting heat shock nucleoproteins from leafy vegetable cells. Almost all methods extract nucleoproteins by grinding large pieces of plant tissue, such as seeds, leaves, stems, etc., without refining the extraction to the cellular level (e.g., Chinese invention patent CN202311700710.0). Therefore, this invention provides a method and kit suitable for extracting nuclear proteins from leafy vegetables. The kit includes reagents A1, A2, B1, B2, B3, B4, and C. Reagent A1 contains Macerozyme-R10, Cellulase R-10, Mannitol, MES, and KCl. Reagent A2 contains NaCl, CaCl2, KCl, MES, and Glucose. Reagent B1 contains Tris-HCl (pH 7.4), Glycerol, KCl, EDTA, MgCl2, Sucrose, Beta-mercaptoethanol, PMSF, Leupeptin, NaF, Na3VO4, and Beta-glycerophosphate. Reagent B2 contains Tris-HCl (pH 7.4), Glycerol, MgCl2, and Triton... X-100; Reagent B3 contains Tris-HCl (pH 7.4), Glycerol, and MgCl2; Reagent B4 contains Tris-HCl (pH 7.4), Glycerol, MgCl2, Sucrose, NaF, Na3VO4, Beta-glycerophosphate, and PMSF; Reagent C contains Tris-HCl (pH 8.0), NaCl, Glycerol, EDTA, SDC, Tritonx-100, CHAPS, HEPES, NP-40, CAPSO, Sucrose, PMSF, Leupeptin, NaF, Na3VO4, and Beta-glycerophosphate. Using the kit of this invention, precise extraction of heat-shock nuclear proteins at the cellular level is achieved. This method first extracts intact leaf protoplasts for culture, then performs heat shock treatment before extracting nuclear proteins, resulting in more accurate heat-shock expressed nuclear proteins.

[0028] (2) To date, there have been no reports on screening upstream regulatory factors using nuclear proteins extracted from protoplasts combined with DNA pull-down technology. In existing reports, most plant nuclear proteins are extracted directly from plant tissues and used to study upstream regulatory genes (e.g., Li X, He L, An X, Yu K, Meng N, Duan CQ, PanQH. VviWRKY40, a WRKY Transcription Factor, Regulates Glycosylated Monoterpenoid Production by VviGT14 in Grape Berry. Genes (Basel). 2020 Apr 29; 11(5):485. doi:10.3390 / genes11050485.PMID:32365554; PMCID:PMC7290806). This invention also provides a method and kit for extracting heat shock nuclear proteins from protoplasts of leafy vegetables that are suitable for DNA pull-down.

[0029] (3) Existing nuclear protein extraction kits are relatively expensive (e.g., Thermo Fisher Scientific's NE-PER). TM The present invention provides a cost-effective and efficient method and kit for extracting plant nuclear proteins, including nuclear and cytoplasmic extraction reagents (catalog number: 78833). This method can extract high-quality heat-shock nuclear proteins, providing technical support for the study of the expression and regulation of nuclear proteins in leafy vegetable cells. Attached Figure Description

[0030] Figure 1 Coomassie brilliant blue staining results for heat shock nucleoproteins in Chinese kale protoplasts;

[0031] Figure 2 The image shows the cellular fluorescence of a yellow fluorescent protein driven by a heat shock promoter under high temperature conditions.

[0032] Figure 3 This is the secondary spectrum of the BoHSF-3721 protein. Detailed Implementation

[0033] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the experimental materials used in the following examples are all commercially available. Cellulase R10 (model L0012) and analyte R10 (model L0021) were purchased from Guangzhou Zuoke Biotechnology Development Co., Ltd., and other conventional biochemical reagents were purchased from Guangzhou Dingguo Biotechnology Co., Ltd. and Sangon Biotech (Shanghai) Co., Ltd. Centrifuge tubes and other general consumables were purchased from Guangzhou Newprobio Biotechnology Co., Ltd.

[0035] Example 1: A kit for extracting heat shock nucleoproteins from leafy vegetable cells

[0036] The kit includes reagent A1, reagent A2, reagent B1, reagent B2, reagent B3, reagent B4, and reagent C.

[0037] Reagent A1 contains Macerozyme-R10 (analyte), Cellulase R-10 (cellulase), Mannitol, MES (morpholinoethanesulfonic acid), and KCl, with a pH of 6.8. The final concentration of Cellulase R-10 is 0.10-0.40 g / 10 mL (preferably 0.25 g / 10 mL), and the specific component configuration is shown in Table 1 below:

[0038] Table 1 Content of the combined formulation in reagent A1

[0039]

[0040] The reagent A2 is a cell preservation and cell processing buffer, which contains NaCl, CaCl2, KCl, MES (morpholinoethanesulfonic acid) and glucose. The specific components are shown in Table 2.

[0041] Table 2 Content of the combined formulation in reagent A2

[0042]

[0043] The reagent B1 contains Tris-HCl (pH 7.4), Glycerol, KCl, EDTA (ethylenediaminetetraacetic acid), MgCl2, Sucrose, Beta-mercaptoethanol, PMSF (phenylmethylsulfonyl fluoride), Leupeptin, NaF, Na3VO4, and Beta-glycerophosphate (sodium β-glycerophosphate, pentahydrate), and the specific composition is shown in Table 3.

[0044] Table 3 Content of the combined formulation in reagent B1

[0045]

[0046]

[0047] The reagent B2 contains Tris-HCl (pH 7.4), Glycerol, MgCl2, and Triton X-100, and the specific composition is shown in Table 4.

[0048] Table 4 Content of the combined formulation in reagent B2

[0049]

[0050] The reagent B3 contains Tris-HCl (pH 7.4), Glycerol, and MgCl2, and the specific composition is shown in Table 5.

[0051] Table 5 Content of the combined formulation in reagent B3

[0052]

[0053] Reagent B4 is a cell nucleus preservation solution containing Tris-HCl (pH 7.4), Glycerol, MgCl2, Sucrose, NaF, Na3VO4, Beta-glycerophosphate, and PMSF (phenylmethylsulfonyl fluoride). The specific component configuration is shown in Table 6.

[0054] Table 6 Content of the combined formulation in reagent B4

[0055]

[0056] Reagent C is a cell nuclear lysis buffer, containing Tris-HCl (pH 8.0), NaCl, Glycerol, EDTA (ethylenediaminetetraacetic acid), SDC (sodium deoxycholate), Tritonx-100, CHAPS (3-[3-(cholamidopropyl)dimethylamino]-1-propanesulfonic acid), HEPES (N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)), NP-40 (nonylphenol polyoxyethylene ether, detergent), CAPSO (3-(cyclohexylamine)-2-hydroxy-1-propanesulfonic acid, free acid), Sucrose, PMSF, Leupeptin, NaF, Na3VO4, and Beta-glycerophosphate. The specific component configuration is shown in Table 7.

[0057] Table 7 Content of the combined formulation in reagent C

[0058]

[0059]

[0060] Example 2: A method for extracting heat shock nucleoproteins from leafy vegetables based on protoplasts

[0061] This invention details the method for extracting heat shock nucleoprotein from protoplasts of Chinese kale, using the extraction of heat shock nucleoprotein from Chinese kale protoplasts as an example. Because the inventors previously cloned the HSP (heat shock protein gene) promoter of a Chinese kale inbred line “JL5” (Guangdong Provincial Crop Germplasm Resource Conservation Bank GDI4E00049), it was necessary to extract heat shock nucleoprotein from protoplasts under heat treatment conditions in order to study its upstream heat shock regulatory genes. The study focused on "JL5" as the target gene and used the HSP promoter as the target gene. Mass spectrometry detection was performed using DNA pull-down (DNA pull-down is an existing technique: Wang, Y.-C., Wei, Y., Li, X.-Y., Zhang, H.-M., Meng, X., Duan, C.-Q., & Pan, Q.-H. (2024). Ethylene-responsive VviERF003 modulates glycosylated monoterpenoid synthesis by upregulating VviGT14 in grapes. Horticulture Research, 11(4). https: / / doi.org / 10.1093 / hr / uhae065.). The procedure for extracting heat shock nucleoprotein from Chinese kale protoplasts was as follows: obtaining Chinese kale leaf mesophyll protoplasts and performing heat shock treatment—extracting the cell nuclei from the protoplasts—nucleus lysis to release heat shock nucleoprotein. Subsequent DNA pull-down and mass spectrometry analysis were performed by Wuhan Jinkairui Biotechnology Co., Ltd.

[0062] The full-length DNA sequence (SEQ ID NO:1) of the cloned HSP (heat shock protein gene) promoter is as follows:

[0063]

[0064] I. The process of extracting heat shock nucleoprotein from Chinese kale protoplasts using the kit of Example 1 specifically includes the following steps;

[0065] (1) Obtain protoplasts of Chinese kale leaf mesophyll and subject them to heat shock treatment

[0066] 1) Take the 2nd-3rd true leaves from the base of Chinese kale seedlings that are 18-20 days old, excluding the cotyledons; the leaves at this time are in the optimal state for extracting Chinese kale protoplasts, which is beneficial for subsequent cell processing.

[0067] 2) Extraction of leaf protoplasts: Pre-treat reagent A1 in a 55℃ water bath for 10 min and cool to room temperature. Then, cut true leaves of Chinese kale into thin, non-repetitive strips approximately 0.8 cm long and add them to 15 mL of cooled reagent A1. Incubate at 85 rpm for 2.5 h (room temperature, protected from light). After hydrolysis, filter the hydrolysate through a 100-mesh nylon mesh (discard the filtrate). Add an equal volume of reagent A2 to the filtered hydrolysate and centrifuge at 60 rcf for 5 min at 4℃, repeating twice to wash away the hydrolysate. Finally, add 5 mL of reagent A2 and incubate overnight in the dark for 12 h. After incubation, filter through a 100-mesh nylon mesh to remove dead cells and collect the filtrate to obtain intact Chinese kale protoplasts. Aliquot the protoplasts into centrifuge tubes and heat-shock them in a 35℃ water bath for 1.5 h before further processing.

[0068] 2. Extracting cell nuclei from protoplasts

[0069] 1) Centrifuge to remove reagent A2. Add 1 mL of reagent B1 to approximately 250 mg of protoplast precipitate, vortex several times to ensure complete cell lysis, and take 10 μL of the lysate containing protoplasts to observe under a microscope to see if the cells are completely broken. (If lysis is insufficient, the extracted nucleus precipitate will contain more other impurities, affecting the extraction of nuclei). Then add 1 mL of reagent B2 and wash repeatedly, centrifuging at 1500 g for 10 min each time to wash away impurities. Discard the supernatant and retain the precipitate until the green precipitate turns off-white or pure white. This precipitate is the cell nucleus.

[0070] 2) Because reagent B2 contains Triton X-100, which has a certain lytic effect on the nuclear membrane, after cleaning the cell nuclei, reagent B3 is added to wash the precipitate to remove residual Triton X-100. During the washing process, 1.5 mL of reagent B3 is added to the centrifuge tube to resuspend the cell nuclei, and then all the liquid is transferred to a new centrifuge tube. The tube is then centrifuged at 1500g for 10 min, and this process is repeated twice. The supernatant is removed, and the precipitate is retained.

[0071] 3) Preservation of cell nuclei (this step is suitable for cases where cell nuclei cannot be processed immediately): Add 500 μL of B4 reagent to the precipitate sample from step 2), resuspend the precipitate, rapidly freeze the sample using liquid nitrogen, and then store it in an ultra-low temperature freezer at -80°C.

[0072] 3. Nucleus lysis releases heat shock nucleoproteins

[0073] Before nuclear lysis, the sample was centrifuged at 1500g for 10 min to remove the supernatant, retaining the precipitate. Then, 500 μL of reagent C was added to the nuclear sample, and the mixture was gently mixed with a pipette and incubated on ice for 30 min. During the 30 min incubation period, the sample was vortexed for 10 s every 5 minutes, followed by sonication on ice using an ultrasonic cell disruptor for 2 min at 20% power, with a 3 s working interval followed by a 3 s pause. Finally, the sample was centrifuged at 12000 rpm for 10 min at 4°C. The resulting supernatant was the heat shock nucleoprotein. The Coomassie brilliant blue staining results for the nucleoprotein are shown below. Figure 1 As shown.

[0074] If the nucleoproteins frozen and preserved in step 3) of stage 2 above are to be extracted, the preserved samples must first be thawed at low temperature, and then centrifuged at 1500g for 10 minutes. The supernatant should be removed and the precipitate retained. Then, the heat-shocked nucleoproteins should be extracted according to the method of stage 3.

[0075] II. Detection of heat shock nucleoproteins

[0076] Using a yellow fluorescent protein driven by the HSP promoter as a reporter gene, the heat shock time for fluorescence in fluorescent cells was determined by transforming kale protoplasts. Figure 2 As shown, the proHSP-YFP plasmid was transformed into Chinese kale protoplast cells. Laser confocal microscopy revealed fluorescence in the cells 1.5 hours after heat shock treatment, indicating the presence of upstream heat-related factors within the cells. Therefore, further investigation of the heat shock nucleoprotein at this time point is necessary. The previously obtained HSP promoter was biotinylated and incubated with the heat shock nucleoprotein for DNA pull-down and mass spectrometry detection. Comparison with the UniProt protein database revealed that BoHSF-3721 (SEQ ID NO:2) is a heat shock transcription factor detected by mass spectrometry (transcription factors typically bind to DNA in the cell nucleus to initiate or regulate gene transcription. Therefore, transcription factors can be considered proteins that perform specific functions in the cell nucleus, i.e., a type of nuclear protein). Figure 3 This is a secondary spectrum of the BoHSF-3721 protein. This demonstrates that this method can effectively extract heat shock nucleoprotein from Chinese kale protoplasts, and can be used for further research.

[0077] The amino acid sequence (SEQ ID NO:2) of the transcription factor BoHSF-3721 in the heat shock nucleoprotein obtained from the Uniprot database is as follows:

[0078] MDESNHGGSLPPFLTKTYEMVDDSSSDSIVSWSQSNKSFIVWNPPEFSRDLLPKFFKHNNFASFIRQLNTYGFRKADPEQWEFANDDFVRGQPHLMKNIHRRKPVHSHSSPSLQPHPLTDSERQRMNDQIERLTKEKEVLLQELHKQEKEREMFQQQVKELKDQLQHMEKRQKTMVSFVSQ VLEKPELALNLSPCLLETNERKRRFPRIGLEGSTSPSSQARELQVEQLESSIAVWENLVSEDSSESLGQEKRSMMTLDVDESSTCPESPPLPCIQLSIDTCPNCPTSPRTIDMNSEPDTSKEPPPAAGVNDVFWQQLLTENPGSTEQKEVQAERKDDKAEECWWDLRNVNTLTEQLGHLTS.

[0079] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A kit for extracting heat shock nucleoproteins from protoplasts of leafy vegetables, characterized in that, The kit includes reagent A1, reagent A2, reagent B1, reagent B2, reagent B3, reagent B4, and reagent C; Reagent A1 contains Macerozyme-R10, Cellulase R-10, Mannitol, MES, and KCl; Reagent A2 contains NaCl, CaCl2, KCl, MES, and Glucose; Reagent B1 contains Tris-HCl (pH 7.4), Glycerol, KCl, EDTA, MgCl2, Sucrose, β-mercaptoethanol, PMSF, Leupeptin, NaF, Na3VO4, and sodium β-glycerophosphate; Reagent B2 contains Tris-HCl (pH 7.4), Glycerol, MgCl2, and Triton... X-100; Reagent B3 contains Tris-HCl, Glycerol, and MgCl2 at pH 7.4; Reagent B4 contains Tris-HCl, Glycerol, MgCl2, Sucrose, NaF, Na3VO4, sodium β-glycerophosphate, and PMSF at pH 7.4; Reagent C contains Tris-HCl, NaCl, Glycerol, EDTA, sodium deoxycholate, Triton X-100, CHAPS, HEPES, NP-40, CAPSO, Sucrose, PMSF, Leupeptin, NaF, Na3VO4, and sodium β-glycerophosphate at pH 8.

0.

2. The kit for extracting heat shock nucleoproteins from protoplasts of leafy vegetables according to claim 1, characterized in that, The pH value of reagent A1 is 6.8, and the final concentration of Cellulase R-10 is 0.25 g / 10 mL.

3. A method for extracting heat shock nucleoproteins from protoplasts of leafy vegetables, characterized in that, The extraction of heat shock nucleoproteins from protoplasts of leafy vegetables using the kit described in claim 1 or 2 specifically includes the following steps: S1. Obtain mesophyll protoplast cells and subject them to heat shock treatment: S11. Take tender true leaves, cut the leaves into thin strips, and put them into reagent A1 that has been pre-treated in a 50-60℃ water bath and cooled for enzymatic hydrolysis to obtain the enzymatic hydrolysis product. S12. After filtration, the enzymatic hydrolysis products are washed with A2 reagent and the protoplasts are cultured with A2 reagent. S13. After culturing, filter to remove dead cells, collect the filtrate to obtain intact protoplasts, and then subject the obtained protoplasts to heat shock treatment at a temperature of 30-40℃ for 0.5-2h. S2. Extraction of cell nuclei from protoplast precipitation: S21. After removing reagent A2, add reagent B1 to the protoplast precipitate to fully lyse the protoplasts, then wash repeatedly with reagent B2 to remove impurities, discard the supernatant, and retain the precipitate until the precipitate turns off-white or pure white. S22. Add reagent B3 to wash the precipitate to remove excess Triton X-100 contained in reagent B2. Collect the precipitate to obtain the cell nucleus. S3. Lyse the cell nucleus and extract heat shock nucleoprotein: Add reagent C to the cell nucleus sample, mix well, and let stand in an ice bath. During the standing period, vortex for 7-15 seconds every 4-7 minutes, and then sonicate in an ice bath to break up the nucleus. Finally, centrifuge and collect the supernatant to obtain the heat shock nucleoprotein.

4. The method for extracting heat shock nucleoprotein from protoplasts of leafy vegetables according to claim 3, characterized in that, The enzymatic hydrolysis described in S11 is performed at room temperature and in the dark on a shaker at 70-90 rpm for 2-4 hours.

5. The method for extracting heat shock nucleoprotein from protoplasts of leafy vegetables according to claim 3, characterized in that, S12 used A2 reagent to culture protoplast cells overnight.

6. The method for extracting heat shock nucleoprotein from protoplasts of leafy vegetables according to claim 3, characterized in that, Add B4 reagent to the cell nuclei obtained from S22, resuspend the precipitate, and then rapidly freeze the sample with liquid nitrogen and store it at -80°C for sample storage.

7. The method for extracting heat shock nucleoprotein from protoplasts of leafy vegetables according to claim 3, characterized in that, The ice bath should be left to stand for 25-40 minutes.

8. The method for extracting heat shock nucleoprotein from protoplasts of leafy vegetables according to claim 3, characterized in that, The ice bath ultrasonic fragmentation time is 1-3 minutes, the power is 15%-25%, the working time is 3 seconds, and the interval is 3 seconds.

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