Extraction method and application of moso bamboo mitochondrial protein PeUCP5

Abstract

The invention discloses a moso bamboo mitochondrial protein PeUCP5 extraction method and application thereof, a specific buffer solution is prepared, then a moso bamboo tissue block is subjected to differential centrifugation and density gradient centrifugation, and sediment of a mitochondrial zone is collected to obtain moso bamboo mitochondria; and then extracting mitochondrial protein from moso bamboo mitochondria by adopting a protein extracting solution, and analyzing through LC-MS / MS mass spectrometry to complete separation, extraction and identification of the moso bamboo mitochondrial protein PeUCP5. Pollutions of cell nucleuses, chloroplasts and the like are removed through differential centrifugation and Percoll density gradient centrifugation, interference of non-mitochondrial proteins (such as cytoplasm or cell nucleus proteins) is avoided, it is ensured that a mass spectrometry analysis result only reflects mitochondrial proteomes, and high expression of the proteins in rapidly-elongated sections of moso bamboos is disclosed for the first time.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a method for extracting bamboo mitochondrial protein PeUCP5 and application thereof. Background Art

[0002] Mitochondria are crucial organelles within cells. They are semi-autonomous, double-membrane organelles that contain their own DNA, RNA, and ribosomes and are capable of independently synthesizing some proteins. Their primary function is to convert chemical energy from nutrients into a form of energy—adenosine triphosphate (ATP)—through oxidative phosphorylation, providing energy for various cellular activities. Mitochondria also participate in many other important physiological processes within cells, such as the regulation of apoptosis, the storage and regulation of intracellular calcium, and the generation and clearance of reactive oxygen species (ROS).

[0003] Mitochondrial proteomic analysis requires the isolation of highly pure and intact mitochondria from cells to ensure accurate and reliable results. Although mitochondrial affinity purification technology has been developed in model plants, centrifugation-based methods are still the main means of mitochondrial isolation due to the difficulty in ensuring stable transformation in crops and woody plants. Centrifugation separation methods (including differential centrifugation and density gradient centrifugation) can be used to separate crude mitochondria from many fruit species, such as avocados and bananas. Due to the low purity of mitochondria separated by differential centrifugation, subsequent optimization has improved the purity of mitochondria isolated from fruits using Sucrose or Percoll density gradient centrifugation, enabling the characterization of the mitochondrial proteomes of various fruit crops. This method relies on the extraction solution formulation and centrifugation settings, and optimizing these conditions is crucial to obtaining highly pure and active mitochondria.

[0004] However, isolating high-purity, intact mitochondria from bamboo shoots remains challenging due to the large amounts of primary and secondary metabolites and lignin present in bamboo shoot tissue. Furthermore, the difficulty in obtaining stably transformed bamboo shoots and the lack of available endogenous antibodies hinder the application of affinity purification techniques in mitochondrial isolation.

[0005] Bamboos are known for their rapid internode elongation, reaching growth rates of up to one meter per day. However, the energy metabolism mechanisms underlying this cell elongation remain largely unknown. Existing research has primarily focused on genes involved in cell wall relaxation (such as expansins) or hormone signaling (such as gibberellins), while research on the functions of mitochondria and the proteins within them remains underdeveloped.

[0006] Therefore, it is crucial to establish a simple and rapid protocol to obtain highly pure mitochondria, which will facilitate high-throughput characterization of the mitochondrial proteome and provide new insights into the relationship between mitochondrial metabolism and rapid growth of bamboo. Summary of the Invention

[0007] The purpose of the present invention is to provide a method for extracting PeUCP5, a mitochondrial protein from bamboo, and its application in view of the deficiencies in the prior art.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a method for extracting the mitochondrial protein PeUCP5 from bamboo, comprising the following steps:

[0010] Step (1), sampling bamboo, and cutting into pieces to obtain bamboo tissue blocks;

[0011] Step (2), mixing bamboo tissue blocks with buffer A to squeeze out juice, and filtering to obtain a filtrate; the buffer A is an aqueous solution comprising the following components: 0.33M sorbitol, 0.2M 3-morpholinepropanesulfonic acid (MOPS, pH 7.8), 7.5mM ethylenediaminetetraacetic acid (EDTA), 0.1% bovine serum albumin (BSA) by mass, 1.5% polyvinylpyrrolidone (PVP-40) by mass, and 2mM dithiothreitol (DTT);

[0012] Step (3), adding buffer B to the filtrate and performing differential centrifugation to obtain a crude mitochondrial extract; the buffer B is an aqueous solution comprising the following components: 50 mM 3-morpholinepropanesulfonic acid (MOPS) and 0.33 M sorbitol, with a pH of 7.5;

[0013] Step (4) adding different volume fractions of Percoll gradient solution to the ultracentrifuge tube, wherein the volume fractions of the Percoll gradient solution are as follows: 33% Percoll 6 mL, 24% Percoll 18 mL, and 20% Percoll 8 mL, respectively; layering the crude mitochondrial extract (about 6 mL) on the top layer of the Percoll gradient solution for density gradient centrifugation, and collecting the precipitate of the mitochondrial zone;

[0014] Step (5), mixing the precipitate with buffer B and centrifuging, collecting the precipitate to obtain bamboo mitochondria;

[0015] Step (6), using a protein extract to extract the mitochondrial protein PeUCP5 from the mitochondria of the bamboo, and analyzing it by LC-MS / MS mass spectrometry; the protein extract is an aqueous solution comprising the following components: 8M urea, 50mM tris (hydroxymethyl)aminomethane (Tris), 4% sodium dodecyl sulfate (SDS) by mass fraction, 10mM EDTA, 5mM DTT and 1% protease inhibitor by mass fraction.

[0016] Furthermore, the pH value of the buffer solution A and the buffer solution B is 7.5.

[0017] Furthermore, the differential centrifugation comprises the following steps: centrifuging the filtrate at 1500 g centrifugal force for 5 min, collecting the supernatant and centrifuging at 3000 g centrifugal force for 10 min, collecting the supernatant and centrifuging at 12000 g centrifugal force for 15 min, and collecting the precipitate.

[0018] Furthermore, the differential centrifugation is performed twice to remove cell debris, cell nuclei and other components as much as possible to obtain a high-quality crude mitochondrial extract.

[0019] Furthermore, the density gradient centrifugation adopts ultracentrifugation, the centrifugal force is 50000g, and the ultracentrifugation time is 60min.

[0020] Furthermore, the mitochondrial zone was collected by piercing the side wall of the centrifuge tube with a syringe (25G) to avoid contact with other components; the mitochondrial zone was located between 24% and 33% Percoll gradients.

[0021] Furthermore, the pH value of the protein extract is 8.0.

[0022] Furthermore, the mitochondrial protein extraction is specifically as follows: adding protein extract to the bamboo mitochondria, ultrasonically treating on ice, and centrifuging at 20,000 g for 10 minutes at 4°C to obtain a supernatant containing mitochondrial component proteins.

[0023] In a second aspect, the present invention provides the use of the bamboo PeUCP5 protein extracted by the above method in accelerating the internode growth of bamboo. The PeUCP5 protein is located in the mitochondria of bamboo and is upregulated in the rapidly elongating nodes of bamboo, thereby participating in the rapid elongation process of bamboo internodes.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention provides a method for extracting and identifying mitochondrial proteins from bamboo (Moso bamboo) by combining mitochondrial separation with mass spectrometry. Considering that bamboo contains relatively few acidic substances such as organic acids and has a high cellular pH, the present invention optimizes the pH of the bamboo mitochondrial extract to prevent mitochondrial stress caused by excessively high pH. Furthermore, the present invention combines differential centrifugation with density gradient centrifugation to enrich and collect daughter mitochondrial bands. The Percoll concentration of the density gradient is optimized, resulting in clear mitochondrial stratification at Percoll concentrations of 20%, 24%, and 33%.

[0026] In addition, the present invention adopts a large-volume (38.6 mL centrifuge tube) separation system during density gradient centrifugation, which can effectively precipitate mitochondria in the crude mitochondrial fluid.

[0027] The present invention overcomes the difficulty of isolating and purifying bamboo mitochondria, providing a stable research foundation for subsequent studies. Contamination such as the nucleus and chloroplasts is removed through differential centrifugation and Percoll density gradient centrifugation, avoiding interference from non-mitochondrial proteins (such as cytoplasmic or nuclear proteins), ensuring that mass spectrometry analysis results only reflect the mitochondrial proteome. After purity verification using fluorescent dyes and transmission electron microscopy, proteins identified by mass spectrometry are more likely to be derived from mitochondria rather than other cellular components.

[0028] The present invention reveals the mitochondrial function and growth regulation mechanism of UCP5 of moso bamboo. Mitochondrial proteome data extracted by optimized method reveal that the expression level of UCP5 (PH02Gene28514.t1) in rapidly elongating nodes is significantly higher than that in apical division nodes, suggesting that it specifically regulates internode elongation. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a flow chart of the present invention.

[0030] Figure 2 The figure shows the verification of mitochondrial enrichment bands after differential centrifugation. A shows the verification of sampling by dividing the sample into six parts after differential centrifugation, and B shows the mitochondrial enrichment results of the six parts of the sample after confocal microscopy with mitochondrial dye staining. The scale bar in the figure is 25 μm.

[0031] Figure 3 These are the results of Mitotracker observation using fluorescent dye and transmission electron microscopy observation of mitochondrial organelles after mitochondrial purification, where A is a confocal observation photo after Mitotracker staining; B is a transmission electron microscopy photo.

[0032] Figure 4 This figure shows the subcellular localization of UCP5 protein in bamboo protoplasts. UCP5-GFP is the fluorescence observed by confocal microscopy after transient transformation of bamboo protoplasts with the pbi221-UCP5-GFP vector. MitoTracker is a mitochondrial-specific dye and serves as a positive control. Chlorophyll represents chloroplast autoluminescence. Merged is a merged image. Yellow areas indicate colocalization. DETAILED DESCRIPTION

[0033] The present invention will be further described below with reference to the embodiments and accompanying drawings.

[0034] like Figure 1 As shown, the present invention provides a method for extracting and identifying bamboo mitochondrial protein, wherein all buffers need to be pre-cooled at 4°C, and all operations are performed on ice or at 4°C; specifically, the method comprises the following steps:

[0035] (1) Bamboo sampling: The internodes of bamboo shoots (about 3 meters) in the middle and above the bamboo shoots at the high growth stage were collected as bamboo tissue. After sampling in the bamboo forest, the bamboo shoots were quickly returned to the laboratory, the bamboo shoots were removed, and the bamboo tissue was cut into small pieces (about 2 cm in size). The pieces were first placed on ice in Extraction buffer (i.e., buffer A) for 10 minutes. The formula of Extraction buffer is shown in Table 1:

[0036] Table 1 Mitochondrial Extaction Buffer Recipe

[0037]

[0038] (2) Juice was extracted from 300 g of bamboo tissue blocks and a mixture of buffer A (600 mL) at a low speed, and filtered through Millipore (Miracloth 22-25 μm) to obtain a filtrate;

[0039] (3) Add washing buffer (i.e., buffer B) to the filtrate and centrifuge at 1500 g for 5 min at 4°C.

[0040] (4) Collect the supernatant and centrifuge at 3000 g for 10 min at 4°C.

[0041] (5) Collect the supernatant and centrifuge at 12000g for 15 min at 4°C.

[0042] The formula of Washingbuffer is shown in Table 2:

[0043] Table 2 Mitochondrial Washing Buffer Formula

[0044]

[0045] (6) Discard the supernatant and immediately add a small amount of Washing Buffer (i.e., Buffer B). Use a Pasteur pipette to gently pipette back and forth to resuspend the precipitate, which is the crude mitochondrial precipitate.

[0046] (7) Add different volume fractions of Percoll gradient solution to ultracentrifuge tubes (Beckman catalog number: 344058). The volume fractions of the Percoll gradient solution are as follows: 33% Percoll 6 mL, 24% Percoll 18 mL, and 20% Percoll 8 mL from bottom to top. Slowly layer the crude mitochondrial pellet (about 6 mL) on the top layer of the Percoll gradient solution, with about 6-7 mL per tube. Use an ultracentrifuge (Beckman) and ultracentrifuge at 4°C and 50,000 g for 60 min.

[0047] The formulas of Percoll gradient solutions with different concentrations are shown in Table 3:

[0048] Table 3 Percoll gradient solution formula

[0049]

[0050] 2×Washing buffer is twice the concentration of Washing buffer.

[0051] In order to determine the enrichment bands after density gradient centrifugation, the crude mitochondrial fluid after centrifugation was divided into six parts: Fraction-Ⅰ, Fraction-Ⅱ, Fraction-Ⅲ, Fraction-Ⅳ, Fraction-Ⅴ, Fraction-Ⅵ (such as Figure 2 The mitochondrial fluorescent dye Mito-Tracker was used to stain the cells to determine the enrichment status of mitochondria in each part.

[0052] The verification results showed that there were almost no mitochondria in Fraction-Ⅰ and Fraction-Ⅱ, and most of them were other components of the cell. There were very few mitochondria in Fraction-Ⅲ, Fraction-Ⅳ, and Fraction-VI, which was caused by the different sizes of mitochondria and the damage of mitochondria. A large number of mitochondria were enriched in Fraction-Ⅴ, and there was almost no contamination from other components (such as Figure 2 The above results indicate that mitochondria are enriched between 24% Percoll and 33% Percoll (Fraction-V).

[0053] (9) Use a 5 mL syringe to pierce the tube wall and carefully aspirate the mitochondrial zone between the 24% and 33% Percoll gradients, approximately 3 mL.

[0054] (10) Add 30 mL of 10-fold volume of washing buffer, dilute thoroughly, and centrifuge at 15,000 g for 5 min at 4°C.

[0055] (11) Add a small amount of washing buffer to the precipitate obtained above, transfer to a 1.5 mL centrifuge tube, and centrifuge at 15,000 g for 5 min at 4°C.

[0056] (12) Aspirate part of the supernatant, add protease inhibitors, and store at -80°C to obtain bamboo mitochondria.

[0057] The results showed that the present invention adopted two rounds of differential centrifugation and discontinuous Percoll density gradient centrifugation to separate the mitochondria of bamboo, and the mitochondria were clearly layered when the Percoll concentration was 20%, 24%, and 33%. Taking into account that bamboo tissue has less water content than citrus pulp and less acidic substances such as organic acids, the molar concentrations of Sorbitol and MOPS in the extraction buffer were optimized, and the pH of the buffer was adjusted to 7.5 to prevent mitochondrial stress caused by excessively high pH. In addition, in order to reduce contamination during the mitochondrial collection process, the side wall syringe tube tying method was used for collection to avoid contact with other components. The present invention uses fluorescent dye Mitotracker confocal microscopy observation and transmission electron microscopy (TEM) to evaluate the purity of mitochondria, such as Figure 3 As shown, the results suggest that the method of the present invention can separate a large number of mitochondria with complete mitochondrial morphology.

[0058] (13) Mitochondrial proteins were extracted from bamboo mitochondria using a protein extract solution, wherein the protein extract solution was an aqueous solution comprising the following components: 8 M urea, 50 mM tris(hydroxymethyl)aminomethane, 4% by mass of sodium dodecylsulfonate, 10 mM ethylenediaminetetraacetic acid, 5 mM dithiothreitol, and 1% by mass of a protease inhibitor.

[0059] Mitochondria were isolated and proteins were extracted from spring bamboo shoots (20 cm above the ground), 15 nodes (middle of bamboo, with long internodes, representing rapid elongation) of high-growing bamboo shoots (3 m), and the top (40-42 nodes, the apical splitting node) of high-growing bamboo shoots (3 m) according to the method described in steps (1)-(13). The extracted proteins were subjected to LC-MS / MS analysis. 100 μg of protein was enzymatically digested using filter-assisted sample processing (FASP). The resulting peptides were dissolved in solvent A (0.1% formic acid) and loaded onto an Acclaim PepMap 100C18 trapping column (Dionex, 75 μm × 2 cm) using an Ultimate 3000 nanoliter ultra-high performance liquid chromatography system (Dionex). The peptides were then eluted onto an Acclaim PepMap RSLC C18 analytical column (Dionex, 75 μm × 25 cm). Nanoelectrospray ionization (NSI) mass spectrometry analysis was performed using a Q Exactive HFx mass spectrometer (Thermo Scientific) coupled to an online ultra-high performance liquid chromatography system. The raw data were uploaded to ProteomeXchange with ID number PXD055053. https: / / www.proteomexchange.org / ),

[0060] Tandem mass spectrometry data were analyzed for peptide identification and protein quantification using MaxQuant software (version 1.5.2.8). Raw mass spectrometry data were compared against a non-redundant protein database from bamboo (Moso bamboo). The mass error tolerances for precursor ions and fragment ions were set to 10 ppm and 0.02 Da, respectively. Trypsin was used as the digestion enzyme, and a maximum of two missed cleavage sites was tolerated. Protein intensities were calculated using the label-free quantification (LFQ) algorithm. A protein was considered successfully identified when at least two unique peptides matched.

[0061] In the database, annotation analysis found that the expression level of PH02Gene28514.t1 protein (PeUCP5) in the rapidly elongating nodes was significantly higher than that in the apical division nodes, and it was involved in the rapid elongation process of internodes of bamboo. That is, this protein regulates the rapid growth process of bamboo and is upregulated in the rapidly elongating nodes of bamboo.

[0062] Table 1. Tissue differential expression analysis results of PeUCP5 in bamboo

[0063]

[0064] Note: IG is spring bamboo shoots, RE is rapid elongation node, SD is apical division node, FC is differential expression fold, the threshold value is FC>1.50or<0.66, and P-value<0.05, indicating that there is a difference.

[0065] The amino acid sequence of PH02Gene28514.t1 is:

[0066] >PH02Gene28514.t1[mRNA]locus=hic_scaffold_13:58138210:58139181:+[translate_table:standard]

[0067]

[0068] like Figure 4 As shown in the figure, subcellular localization analysis demonstrated that UCP5 of Moso bamboo is a mitochondrial protein and can co-localize with mitochondria-specific dyes.

[0069] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by persons of ordinary skill in the art within the spirit and scope of the present invention shall also fall within the scope of protection of the present invention.

Claims

1. A method for extracting PeUCP5, a mitochondrial protein from bamboo, characterized in that: The following steps are involved: Step (1), sampling bamboo, and cutting into pieces to obtain bamboo tissue blocks; Step (2), mixing the bamboo tissue block with buffer A to extract juice, and filtering to obtain a filtrate; Step (3), adding buffer B to the filtrate and performing differential centrifugation to obtain a crude mitochondrial extract; Step (4), adding different volume fractions of Percoll gradient solution to the ultracentrifuge tube, layering the crude mitochondrial extract on the top layer of Percoll gradient solution for density gradient centrifugation, and collecting the precipitate of the mitochondrial zone; Step (5), mixing the precipitate with buffer B and centrifuging, collecting the precipitate to obtain bamboo mitochondria; Step (6): extracting the bamboo mitochondrial protein PeUCP5 from the bamboo mitochondria using a protein extract.

2. The extraction method according to claim 1, wherein The buffer solution A is an aqueous solution comprising the following components: 0.33M sorbitol, 0.2M 3-morpholinepropanesulfonic acid, 7.5mM ethylenediaminetetraacetic acid, 0.1% bovine serum albumin by mass, 1.5% polyvinylpyrrolidone by mass, and 2mM dithiothreitol; the pH value of the buffer solution A is 7.

5.

3. The extraction method according to claim 1, wherein The buffer solution B is an aqueous solution comprising the following components: 50 mM 3-morpholinepropanesulfonic acid and 0.33 M sorbitol; the pH value of the buffer solution B is 7.

5.

4. The extraction method according to claim 1, wherein The volume fractions of the Percoll gradient liquid are as follows from bottom to top: 33% Percoll 6 mL, 24% Percoll 18 mL, 20% Percoll 8 mL, and the mitochondrial zone is located between the 24% and 33% Percoll gradients.

5. The extraction method according to claim 1, wherein The protein extract is an aqueous solution comprising the following components: 8M urea, 50mM tris(hydroxymethylaminomethane), 4% by mass of sodium dodecylsulfonate, 10mM ethylenediaminetetraacetic acid, 5mM dithiothreitol and 1% by mass of a protease inhibitor; the pH value of the protein extract is 8.

0.

6. The extraction method according to claim 1, characterized in that The differential centrifugation comprises the following steps: centrifuging the filtrate at 1500 g centrifugal force for 5 minutes, collecting the supernatant and centrifuging it at 3000 g centrifugal force for 10 minutes, collecting the supernatant and centrifuging it at 12000 g centrifugal force for 15 minutes, and collecting the precipitate.

7. The extraction method according to claim 1, characterized in that The density gradient centrifugation adopts ultracentrifugation with a centrifugal force of 50,000 g and an ultracentrifugation time of 60 min.

8. The extraction method according to claim 1, characterized in that The Percoll gradient solution was diluted with buffer C, which was an aqueous solution comprising the following components: 0.4 M 3-morpholinepropanesulfonic acid and 0.66 M sorbitol.

9. The extraction method according to claim 1, characterized in that The H value of the buffer C is 7.

5.

10. Use of the bamboo mitochondrial protein PeUCP5 extracted according to any one of claims 1 to 9 in accelerating the internode growth of bamboo, characterized in that: The PeUCP5 protein is up-regulated in the rapidly elongating nodes of bamboo.

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