CN109706113B - Method for extracting soybean mitochondria by using eutectic solvent - Google Patents
Method for extracting soybean mitochondria by using eutectic solvent Download PDFInfo
- Publication number
- CN109706113B CN109706113B CN201910151077.1A CN201910151077A CN109706113B CN 109706113 B CN109706113 B CN 109706113B CN 201910151077 A CN201910151077 A CN 201910151077A CN 109706113 B CN109706113 B CN 109706113B
- Authority
- CN
- China
- Prior art keywords
- mitochondria
- soybean
- eutectic solvent
- extraction
- extracting
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Abstract
The invention discloses a method for extracting soybean mitochondria by using a eutectic solvent, which comprises the following steps: a) cutting soybean roots, adding liquid nitrogen, and grinding to obtain powder; b) transferring the powder into an extraction buffer solution to prepare a suspension; c) adding a eutectic solvent into the suspension, uniformly mixing the eutectic solvent and the suspension, wherein the eutectic solvent comprises choline chloride and polyethylene glycol in a molar ratio of 1: 1-3, and filtering to obtain a filtrate; d) centrifuging 3000-5000 g of the filtrate for 8-12 min to obtain a supernatant; e) centrifuging 11000-13000 g of the supernatant for 18-22 min, and taking a precipitate to obtain mitochondria. The method utilizes the eutectic solvent as the solvent part of the extracting solution, has simple and efficient extraction process and few steps, and can extract mitochondria obtained by extraction at 4 ℃ in large quantity and high activity.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for extracting soybean mitochondria by using a eutectic solvent.
Background
Mitochondria is an important organelle in plant tissue cells, is present in most living cells, is functionally an important and unique organelle in eukaryotic cells, is widely present in aerobic biological cells, converts a proton concentration gradient into ATP through oxidative phosphorylation, provides most of energy required for various vital activities of cells, and is a "kinetic factory" for cells. Therefore, the study of the structure, function and physicochemical properties of components such as the inner and outer membranes of mitochondria, respiratory chain enzymes and mitochondria has become an important subject in biological studies. In addition, the soybeans are important agricultural crops, are important food sources and have wide application value. Mitochondria are increasingly used as important organelles for researching plant variety resources, cytoplasmic male sterility, plant stress resistance and apoptosis, and extraction of active mitochondria is the basis of mitochondrial research. Since mitochondria are present in large amounts in cells with vigorous metabolism, such as plant seeds or roots, the preparation of mitochondria in large amounts is carried out from these tissue cells.
At present, mitochondria are extracted by a plurality of methods, common methods comprise a conventional mitochondria kit extraction method, a density gradient centrifugation method and a differential centrifugation method, and different laboratories often adopt different methods according to actual experimental conditions. Factors considered when selecting the mitochondrial extraction method mainly include whether the experimental method can reduce the pollution of cytoplasm in the extraction process, whether the activity of mitochondria obtained by extraction is strong, whether the extraction method is convenient and fast, and the like. The conventional mitochondrial extraction kit method and the sucrose density gradient centrifugation method belong to the traditional methods, have high cost, need an ultra-low temperature centrifuge, have high requirements on instrument and equipment conditions, have low efficiency and low activity rate of extracted mitochondria, waste time and labor, and have limited number of extracted mitochondria.
Disclosure of Invention
The invention provides a method for extracting soybean mitochondria by using a eutectic solvent, which is used for solving the problems that the method for extracting the soybean mitochondria in the prior art is time-consuming and labor-consuming and the number of the extracted mitochondria is limited.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a method for extracting soybean mitochondria using a eutectic solvent, comprising:
a) cutting soybean roots, adding liquid nitrogen, and grinding to obtain powder;
b) transferring the powder into an extraction buffer solution to prepare a suspension;
c) adding a eutectic solvent into the suspension, uniformly mixing the eutectic solvent and the suspension, wherein the eutectic solvent comprises choline chloride and polyethylene glycol in a molar ratio of 1: 1-3, and filtering to obtain a filtrate;
d) centrifuging 3000-5000 g of the filtrate for 8-12 min to obtain a supernatant;
e) centrifuging 11000-13000 g of the supernatant for 18-22 min, and taking a precipitate to obtain mitochondria.
In one embodiment: in the step a), the soybean roots are cleaned and disinfected before grinding.
In one embodiment: in said step a), the grinding is carried out at room temperature.
In one embodiment: in said step a), grinding is carried out at least three times to sufficiently break the cells.
In one embodiment: in step a), the time for separating the soybean roots from the body is not more than 2 hours, preferably not more than 0.5 hour.
In one embodiment: in the step b), the extraction buffer solution comprises 0.4-0.6 mol/L sucrose and 0.9-1.1 mmol/L MgCl 2 0.9-1.1 mmol/L EGTA, 4.5-5.5 g/L PVP, 0.9-1.1 g/L cys, 1.3-1.5 mL/L mercaptoethanol0.9-1.1 g/L BSA and 48-52 mmol/L Tris-HCl, pH 7.4-7.8.
In one embodiment: in the step b), the extraction buffer solution is sterilized before use and is stored at 0-4 ℃ for at least 30 min.
In one embodiment: in the step c), the eutectic solvent comprises 0.8-1.2 mol/L choline chloride and 1.8-2.2 mol/L polyethylene glycol.
In one embodiment: in said step c), the eutectic solvent is sterilized before use.
In one embodiment: in the step c), the mass volume ratio of the soybean root to the eutectic solvent is 1: 1-7, preferably 1: 6.
In one embodiment: in the step c), the extraction temperature of the eutectic solvent is 4-25 ℃, and preferably 4 ℃.
In one embodiment: in the step c), 6 layers of gauze are adopted for filtering.
Compared with the background technology, the technical scheme has the following advantages:
according to the method for extracting mitochondria, the soybean root powder ground by liquid nitrogen is subjected to two-step centrifugation, the eutectic solvent is used as the solvent part of the extracting solution, the extraction process is simple and efficient, the steps are few, the extracted mitochondria are large in quantity, and the activity of the extracted mitochondria can be maintained.
Detailed Description
The present invention is specifically illustrated by the following examples:
example 1 extraction of Soybean mitochondria Using different volumes of eutectic solvents
a) Selecting soybean roots of 5g, culturing for 14 days at 28 ℃ in the dark, washing the soybean roots with distilled water and carrying out 75% alcohol disinfection treatment within 30min, drying soybean root tissues with absorbent paper, cutting the soybean roots into 0.2cm small sections with a scalpel, transferring the small sections into a mortar, and grinding the small sections into powder with liquid nitrogen at room temperature; the powder is ground more than three times to fully break the cells;
b) transferring the powder into a beaker containing 30mL of extraction buffer solution to prepare suspension; four groups are repeated in parallel; the preparation method of the extraction buffer solution comprises the following steps: 500mL (0.5 mo)L/L sucrose, 1mmol/L MgCl 2 1mmol/L EGTA (ethylene glycol bis (2-aminoethyl ether) tetraacetic acid), 2.5g PVP (polyvinylpyrrolidone), 0.5g cys (cysteine), 0.7mL mercaptoethanol, 0.5g BSA (bovine serum albumin), 50mmol/L Tris-HCl and pH 7.4-7.8, and are prepared by distilled water, the extraction buffer solution is sterilized before use, and the extraction buffer solution is stored in an ice bath for at least 30min for reuse after sterilization;
c) one of the four repeated groups is used as a control, 10mL, 25mL and 30mL of eutectic solvents with different volumes are respectively added into the suspension according to the mass-to-volume ratios of 1:2, 1:5 and 1:6 (when the mass-to-volume ratios are 1:5 and 1:6, the extraction effect is remarkably increased, and when the mass-to-volume ratios are 1:5 and 1:6, the mitochondrial loss rate is low), the mixture is uniformly mixed and stirred for 30s, and the mixture is filtered to a beaker by 6 layers of gauze; the preparation method of the eutectic solvent comprises the following steps: 500mL (0.5mol choline chloride, 1mol polyethylene glycol 2000, prepared with distilled water), sterilized before use;
d) adding the filtrate into a 2mL centrifuge tube, and centrifuging for 10 minutes by using 4000 g;
e) adding the supernatant obtained by centrifugation into a 2mL centrifuge tube, centrifuging at 12000g for 20 minutes (the centrifugation operation is carried out at 4 ℃), and discarding the supernatant; the pellet in the precipitate is the mitochondria.
Example 2 extraction of soybean mitochondria by conventional method
a) Selecting soybean roots of 5g, culturing for 14 days at 28 ℃ in the dark, washing the soybean roots with distilled water and carrying out 75% alcohol disinfection treatment within 30min, drying soybean root tissues with absorbent paper, cutting the soybean roots into 0.2cm small sections with a scalpel, transferring the small sections into a mortar, and grinding the small sections into powder with liquid nitrogen at room temperature; the powder is ground more than three times to fully break the cells;
b) transferring the powder to a beaker filled with 30mL of extraction buffer solution, uniformly mixing and stirring for 30s, and filtering the mixture to the beaker by 6 layers of gauze; the preparation method of the extraction buffer solution is the same as that of example 1, namely: 500mL (0.5mol/L sucrose, 1mmol/L MgCl 2 1mmol/L EGTA, 2.5g PVP, 0.5g cys, 0.7mL mercaptoethanol, 0.5g BSA and 50mmol/L Tris-HCl, pH 7.4-7.8, prepared with distilled water), sterilizing the extraction buffer before use, and sterilizingStoring in ice bath for at least 30min for reuse;
c) the filtrate was added to a 2mL centrifuge tube and centrifuged at 4000g for 10 minutes
d) Adding the supernatant obtained by centrifugation into a 2mL centrifuge tube, centrifuging at 12000g for 20 minutes (the centrifugation operation is carried out at 4 ℃), and discarding the supernatant; the pellet in the precipitate is the mitochondria.
Example 3 measurement of Soybean mitochondrial cytochrome C oxidase Activity
In four cuvettes, 0.1mL of 0.01M phosphate buffer, 0.07mL of 1% ferrous cytochrome C (cytochrome C dissolved in 0.01M potassium phosphate buffer pH 7.0), and 0.83mL of distilled water were added, respectively. One of them was added with 0.01mol of 0.1M potassium ferrocyanide as a control, and the other three were added with 10. mu.l of different groups of mitochondrial preparations, respectively. At 550nm, absorbance was measured using a spectrophotometer of Shimadzuuv-120-02 type, and absorbance at the beginning was read as a0 every 15s interval at and after 15s, as a 1. K is obtained by plotting the slope of the line obtained by plotting ln (A1-A1+ Delta) against t.
The experimental results are shown in table 1, the first reaction rate constant k is used as the final basis for data comparison in the experiment, and the first reaction rate k value calculated according to the formula is shown in the table, so that the group with the larger reaction rate constant A has the better relative effect in the mitochondrial extraction experiment. The comparison shows that the activity of the mitochondria extracted by the eutectic solvent is higher than that of the mitochondria extracted by the traditional method, and the extraction activity is best when the volume of the eutectic solvent is 30 mL. The mitochondrial cytochrome C oxidase extracted by the extraction method has good activity (P is less than 0.05).
TABLE 1 results of mitochondrial cytochrome C oxidase activity of mitochondria obtained by different extraction methods
| Group of | k value |
| Control group | 17.26 |
| 17.26 | |
| Mass to volume ratio of 1:2 | 26.52 |
| 19.14 | |
| Mass to volume ratio of 1:5 | 24.32 |
| 41.58 | |
| Mass to volume ratio of 1:6 | 30.65 |
| 41.58 |
Example 4 extraction of Soybean mitochondria Using eutectic solvent at various temperatures
a) Selecting soybean roots of 5g, culturing for 14 days at 28 ℃ in the dark, washing the soybean roots with distilled water and disinfecting with 75% alcohol within 30min, drying the soybean root tissues with absorbent paper, cutting the soybean roots into 0.2cm small sections with a scalpel, transferring the small sections into a mortar, and grinding the small sections into powder with liquid nitrogen at room temperature; the powder is ground more than three times to fully break up the cells;
b) transferring the powder into a beaker containing 30mL of extraction buffer solution to prepare suspension; four groups are repeated in parallel; the preparation method of the extraction buffer solution is the same as that of example 1, namely: 500mL (0.5mol/L sucrose, 1mmol/L MgCl 2 1mmol/L EGTA, 2.5g PVP, 0.5g cys, 0.7mL mercaptoethanol, 0.5g BSA and 50mmol/L Tris-HCl, pH 7.4-7.8, and are prepared by distilled water), the extraction buffer solution is sterilized before use, and the extraction buffer solution is stored in an ice bath for at least 30min for reuse after sterilization;
c) one of the four repeated groups is used as a control, 30mL of eutectic solvents with different volumes are added into the suspension according to the mass volume ratio of 1:6 for the rest three groups, the mixture is respectively placed in different extraction temperature environments at 4 ℃, 25 ℃ and 50 ℃, and is uniformly mixed and stirred for 30s, and 6 layers of gauze are used for filtering the mixture to a beaker; the preparation method of the eutectic solvent is the same as that of example 1, namely: 500mL (0.5mol choline chloride, 1mol polyethylene glycol 2000, prepared with distilled water), sterilized before use;
d) adding the filtrate into a 2mL centrifuge tube, and centrifuging for 10 minutes by using 4000 g;
e) adding the supernatant obtained by centrifugation into a 2mL centrifuge tube, centrifuging at 12000g for 20 minutes (the centrifugation operation is carried out at 4 ℃), and discarding the supernatant; the pellet in the precipitate is the mitochondria.
Example 5 quantitative detection of Soybean mitochondrial protein
BCA (bicinchoninic acid) protein quantification kit (san gon Biotech, Inc. of Industrial science) was used. The procedure was followed as described in the protocol for the protein quantification kit. Protein standards were prepared and a standard curve plot was plotted. Uniformly mixing the solution A and the solution B in the kit according to the proportion of 50:1, adding 100 mu L of a sample to be detected into a centrifuge tube, adding 1mL of BCA working solution, uniformly mixing, and carrying out water bath at 37 ℃ for 30 min. The assay procedure was performed at a spectrophotometer wavelength of 562nm, following the procedure of the BCA protein quantification kit instructions. Protein standards were prepared and standard curve plots were plotted. And (4) taking the light absorption value of each centrifugal tube, and calculating the protein concentration of the sample. The result of quantifying the mitochondrial protein by the BCA method is shown in Table 2, and the result shows that under the same root mass, 30mL (mass-to-volume ratio of 1:6) of eutectic solvent is used for the best extraction effect at 4 ℃, the concentration of the soybean mitochondrial protein extracted by the extraction method is higher than that extracted by the traditional method, namely the number of the soybean mitochondria extracted by the extraction method is larger than that extracted by the traditional method, and the difference has statistical significance (P is less than 0.05).
TABLE 2 results of mitochondrial concentration obtained by different extraction methods
The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims and their equivalents.
Claims (6)
1. A method for extracting soybean mitochondria by using a eutectic solvent is characterized in that: the method comprises the following steps:
a) cutting soybean roots, adding liquid nitrogen, and grinding to obtain powder;
b) transferring the powder into an extraction buffer solution to prepare a suspension; the extraction buffer solution comprises 0.4-0.6 mol/L sucrose and 0.9-1.1 mmol/L MgCl 2 0.9-1.1 mmol/L EGTA, 4.5-5.5 g/L PVP, 0.9-1.1 g/L cys, 1.3-1.5 mL/L mercaptoethanol, 0.9-1.1 g/L BSA and 48-52 mmol/L Tris-HCl, and the pH value is 7.4-7.8;
c) adding a eutectic solvent into the suspension, wherein the extraction temperature of the eutectic solvent is 4 ℃; the mass volume ratio of the soybean root to the eutectic solvent is 1: 1-7; the eutectic solvent comprises choline chloride and polyethylene glycol in a molar ratio of 1: 1-3, the concentration of the choline chloride is 0.8-1.2 mol/L, the concentration of the polyethylene glycol is 1.8-2.2 mol/L, the mixture is uniformly mixed, and a filtrate is obtained after filtration;
d) centrifuging 3000-5000 g of the filtrate for 8-12 min to obtain a supernatant;
e) centrifuging 11000-13000 g of the supernatant for 18-22 min, and taking a precipitate to obtain mitochondria.
2. The method for extracting soybean mitochondria according to claim 1, wherein: in the step a), the root of the soybean is cleaned and disinfected before grinding; grinding is carried out at room temperature; grind at least three times to break up the cells sufficiently.
3. The method for extracting soybean mitochondria according to claim 1, wherein: in the step a), the time for separating the soybean roots from the body does not exceed 2 hours.
4. The method for extracting soybean mitochondria according to claim 1, wherein: in the step b), the extraction buffer solution is sterilized before use and is stored at 0-4 ℃ for at least 30 min.
5. The method for extracting soybean mitochondria according to claim 1, wherein: in said step c), the eutectic solvent is sterilized before use.
6. The method for extracting soybean mitochondria according to claim 1, wherein: in the step c), 6 layers of gauze are adopted for filtering.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910151077.1A CN109706113B (en) | 2019-02-28 | 2019-02-28 | Method for extracting soybean mitochondria by using eutectic solvent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910151077.1A CN109706113B (en) | 2019-02-28 | 2019-02-28 | Method for extracting soybean mitochondria by using eutectic solvent |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109706113A CN109706113A (en) | 2019-05-03 |
| CN109706113B true CN109706113B (en) | 2022-08-26 |
Family
ID=66265908
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910151077.1A Active CN109706113B (en) | 2019-02-28 | 2019-02-28 | Method for extracting soybean mitochondria by using eutectic solvent |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109706113B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117143796B (en) * | 2023-09-06 | 2024-02-13 | 广东龄值生物科技有限公司 | Method for extracting plant tissue mitochondria |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB201303666D0 (en) * | 2013-03-01 | 2013-04-17 | Goldsborough Andrew S | Sample fixation and stabilisation |
| CN106518590B (en) * | 2016-09-18 | 2018-11-23 | 曲阜师范大学 | The method that polyphenols in granatum is extracted using eutectic solvent |
| CN108143836B (en) * | 2018-01-18 | 2021-03-23 | 华侨大学 | Method for extracting different-form phenolic compounds from tea seed oil by using eutectic solvent |
| CN108553937B (en) * | 2018-05-15 | 2020-12-25 | 广州科帆生物科技有限公司 | Natural eutectic solvent and application thereof in plant extraction |
-
2019
- 2019-02-28 CN CN201910151077.1A patent/CN109706113B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN109706113A (en) | 2019-05-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102639694B (en) | Methods of preparing mesenchymal stem cells, compositions and kit thereof | |
| CN104651524A (en) | Method for storing biological samples and kit | |
| CN111088319B (en) | Inactivated virus sample RNA preservation solution and preparation method thereof | |
| CN110583619A (en) | Colorectal cancer solid tumor tissue sample preservation solution | |
| CN109706113B (en) | Method for extracting soybean mitochondria by using eutectic solvent | |
| CN104839146A (en) | Composition and application thereof, placenta preservative and preparation method of placenta preservative | |
| CN101629167B (en) | Method for extracting superoxide dismutase | |
| Keck | Culturing and experimental manipulation of Acetabularia | |
| CN116773306B (en) | Vaginal secretion fluorescent staining solution and preparation method thereof | |
| CN106399235A (en) | Method for isolating human umbilical cord mesenchymal stem cells | |
| CN107904196B (en) | Streptomyces yanshi and application thereof | |
| CN116814520A (en) | Method for improving fucoxanthin content in Isochrysis galbana by using spermidine | |
| Leal et al. | Lack of pectic enzyme production by non-pathogenic species of Verticillium | |
| CN104212820A (en) | Enzyme with catalase activity and encoding gene of enzyme | |
| Rushizky et al. | Ribonuclease-sensitive infectious units from tomato bushy stunt virus | |
| CN105767016B (en) | A kind of suppression food Chroococcus algae-inhibiting agent and preparation method thereof | |
| CN111394301B (en) | Application of piceatannol in increasing number of secreted exosomes of pluripotent stem cells and bioactivity | |
| CN109988251B (en) | Preparation method of needle mushroom acidic polysaccharide with antioxidant activity | |
| CN103421764A (en) | Kit for quickly extracting double-stranded RNA of mycovirus and application of kit | |
| CN105532581A (en) | In-vitro study method of insect symbiotic bacterium transovarial vertical transmission | |
| Kung et al. | Studies with deoxyribonucleic acid from blue-green algae | |
| CN106434545B (en) | The separation method and kit of high-purity cord blood stem cell | |
| CN110982783A (en) | Method for culturing spermatogonial stem cells and application thereof | |
| CN105316283A (en) | Clinical-grade placenta mesenchymal stem cell preparation method | |
| CN109182255A (en) | A kind of non-animal source tree shrew sperm microcytotoxicity liquid and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |