CN111004766B - Effective method for extracting and purifying endoplasmic reticulum and protein thereof from plant leaves - Google Patents
Effective method for extracting and purifying endoplasmic reticulum and protein thereof from plant leaves Download PDFInfo
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- CN111004766B CN111004766B CN201911405514.4A CN201911405514A CN111004766B CN 111004766 B CN111004766 B CN 111004766B CN 201911405514 A CN201911405514 A CN 201911405514A CN 111004766 B CN111004766 B CN 111004766B
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Abstract
The invention discloses an effective method for extracting and purifying endoplasmic reticulum and protein thereof from plant leaves. The ultrasonic extraction method is replaced by a mode of directly grinding the leaves and fresh leaves of plants with proper age for about 15 days by adding a buffer solution, a fixed-angle rotor is replaced by a horizontal rotor during the centrifugation at the rotating speed of 12000Xg, the endoplasmic reticulum protein positive markers GRP78 and CNX are respectively increased by about 3.3 times and 6.1 times after the purification, and the chloroplast positive protein RCA and the cell nucleus positive protein Histone H3 are respectively reduced by about 84.1 percent and 74.4 percent. The invention establishes an effective system suitable for extracting and purifying endoplasmic reticulum protein of plant leaves for the first time, and has reference significance for constructing the endoplasmic reticulum protein extracting and purifying system by other plant leaves.
Description
Technical Field
The invention relates to an effective method for extracting and purifying endoplasmic reticulum and protein thereof of plant leaves, which is suitable for separating and purifying endoplasmic reticulum protein of the plant leaves and belongs to the technical field of plant biology.
Background
The endoplasmic reticulum is a continuous membrane system consisting of a smooth endoplasmic reticulum and a rough endoplasmic reticulum with attached ribosomes (Wang et al, 2016). Its main function is to participate in the biosynthesis of proteins and lipids in cells, and also to be the center of quality control of proteins (Borgese et al, 2006). Modern studies have shown that in plants such as soybean (Wang et al, 2016), spinach (Anderson et al, 1994), arabidopsis thaliana (Liu et al, 2007), the endoplasmic reticulum has an important function, and can respond to abiotic stresses such as drought, flooding, cold, salt and the like, and alleviate the damage to the plants.
At present, the extraction of endoplasmic reticulum from plant leaves is not reported. Early studies show that the subcellular structure of mulberry leaves is remarkably changed after ultraviolet B (UVB) stress, and proteomics studies show that both the primary metabolic system and the secondary metabolic system are affected (corn poppy, etc., 2016). Therefore, the extraction of high-quality mulberry leaf endoplasmic reticulum for endoplasmic reticulum proteomics analysis becomes a key for further developing research works such as plant leaf UVB stress resistance mechanism and the like.
Disclosure of Invention
The invention aims to provide an effective method for extracting and purifying endoplasmic reticulum and protein thereof of plant leaves, which is used for separating and purifying the endoplasmic reticulum protein of the plant leaves.
The invention is realized by adopting the following technical scheme:
an effective method for extracting and purifying endoplasmic reticulum and protein thereof from plant leaves specifically comprises the following steps:
1. tissue disruption of plant leaves
(1) Weighing a proper amount of fresh plant leaves, and cleaning and wiping the fresh plant leaves with clear water to obtain clean plant leaves which can be used for subsequent experiments;
(2) taking the tissue leaves, cutting the tissue leaves into pieces of 0.2-2.0 cm, placing the tissue leaves in a precooled mortar, adding 1-5mL of precooled grinding buffer and 1-10 mu L of 100x protease inhibitor into each 1g of the tissue leaves, and grinding the tissue leaves on ice to form homogenate.
2. Centrifuging the homogenate of the plant leaf to separate endoplasmic reticulum
(1) Homogenizing the plant leaves, subpackaging the homogenate in a 15mL centrifuge tube, centrifuging the homogenate for 10 to 30 minutes at the temperature of between 0 and 10 ℃ at the rotating speed of 800-;
(2) subpackaging the supernatant into a thick-wall ultracentrifuge tube (or equivalent centrifuge tube) made of clean and precooled polycarbonate, centrifuging the tube in an ultracentrifuge at the temperature of 0-10 ℃ and the rotating speed of 10000-;
(3) subpackaging the supernatant into a clean and precooled polycarbonate thick-wall ultracentrifuge tube (or equivalent centrifuge tube), centrifuging for 30-120 minutes at the rotating speed of 60000-.
3. Cracking the extracted endoplasmic reticulum to extract protein
(1) The lysate is prepared at present, and 50 mu L of 100xPMSF is added into 5mL of RIPAlysis to obtain the lysate;
(2) adding 200-500 μ L of lysis solution into the precipitate, grinding and resuspending the precipitate in a centrifuge tube with a cleaned and precooled homogenate pestle for lysis, and sucking out the resuspension solution into a clean and precooled 1.5 or 2mL centrifuge tube;
(3) adding 30-100 μ L of lysis solution to wash the centrifuge tube and homogenate pestle, collecting into resuspension to obtain endoplasmic reticulum protein of plant leaf, and storing at-80 deg.C or immediately detecting.
4. Detecting the purity of the endoplasmic reticulum protein
Purity assays were performed using a comparison of the relative intensities of the bands in a Western blot (Western blot) of endoplasmic reticulum protein and total protein.
(1) Total protein extraction from plant leaves before the experiment: weighing a proper amount of fresh plant leaves, adding 3mL of lysate into every 0.5g of fresh plant leaves, grinding the mixture on ice into uniform slurry, filtering the slurry by using a 100-micron cell sieve, and collecting filtrate, namely the total protein of the plant leaves;
(2) protein quantification and sample preparation: protein concentration is measured by using a BCA kit method, each protein is diluted to the same concentration by using lysate and 5x loading buffer, the solution is heated for 15 minutes at 100 ℃, and supernatant is taken after short-time centrifugation to obtain a sample solution;
(3) SDS-PAGE electrophoresis was used as loading control: referring to an SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoresis method, the loading amount of ER and TP proteins is 20ug, electrophoresis is carried out under the constant voltage of 80-100V until the dye reaches the bottom, silver staining and imaging are carried out on the gel, and the loading amount is determined to be close.
(4) The relative intensity of the bands is checked by a Western blot experiment method: western blot experiments were carried out in the amounts as described in (3) above, using GRP78 and CNX as the endoplasmic reticulum protein positive marker, RCA as the chloroplast protein positive marker, and Histone H3 as the nuclear protein positive marker. Scanning to obtain an electronic plate strip, performing gray value analysis by using Image J software, and performing statistical analysis by using SPSS software.
In the invention, the plant leaves are leaves of mulberry leaves, anemone indica leaves or Chinese mahonia.
The invention has the beneficial effects that:
the method for extracting endoplasmic reticulum and protein thereof from plant leaves provided by the invention has the following advantages:
(1) in order to avoid that homogenate is sticky in texture and is difficult to grind uniformly, and meanwhile, nuclear protein pollution can be removed, plant leaves with proper age about 15 days from germination are selected as experimental materials, so that the integrity of endoplasmic reticulum is effectively guaranteed;
(2) the method of grinding fresh leaves by directly adding buffer solution replaces the method of ultrasonic grinding on ice after liquid nitrogen grinding, thereby greatly improving the content of the extracted product;
(3) selecting a horizontal rotor instead of a fixed-angle rotor during the rotation speed centrifugation at 12000Xg, thereby effectively removing the pollution of chloroplast protein;
(4) the method of directly cracking the precipitate in the centrifugal tube and then sucking out the resuspension by using the glass homogenate pestle solves the problem that the precipitate in the ultracentrifuge tube is difficult to transfer, accelerates the protein extraction efficiency and reduces the protein degradation.
The invention establishes an effective system suitable for extracting and purifying endoplasmic reticulum protein of plant leaves (including leaves of mulberry leaves, anemone leaves or Chinese mahonia leaves) for the first time, and has reference significance for constructing the endoplasmic reticulum protein extracting and purifying system by other plant leaves. The invention can solve the problem of the research technology of endoplasmic reticulum of plant leaves, and effectively extract high-quality endoplasmic reticulum protein from the plant leaves. Compared with the total protein, the endoplasmic reticulum protein positive markers GRP78 and CNX of the purified plant leaf are obviously higher than the total protein and respectively increase by about 3.3 times and 6.1 times, which indicates that the endoplasmic reticulum protein is obviously enriched; both the chloroplast-positive protein RCA and the nucleus-positive protein Histone H3 were significantly lower in endoplasmic reticulum protein than in total protein, by about 6.3 times and 3.9 times, respectively, indicating that the contaminated organelles were effectively removed. The endoplasmic reticulum protein of the plant leaves extracted by the method can be used for further research.
Drawings
FIG. 1: endoplasmic reticulum protein and total protein loading control;
FIG. 2: western blot histograms of endoplasmic reticulum and total protein;
FIG. 3: statistical analysis of grey values of western blot bands (. p <0.05,. p <0.01, n ═ 3).
Detailed Description
The invention is further illustrated by the following examples in combination with corresponding and specific embodiments.
The method is further described by taking mulberry leaves as an example in the embodiment of the invention. The potted seedling of folium Mori (Morus alba L.) used in this study was purchased from Hangzhou Gesang Yuan technology Co., Ltd, and cultivated in a greenhouse (air temperature 28-30 deg.C; relative humidity 70-80%; white light irradiance 160 μmolm) at the Harbour school district of purple Jiang university-2s-1) Medium growth. Common reagents and kits used in the invention, such as PMSF, BCA protein concentration determination kit, silver staining kit and the like, are purchased from Hangzhou Tangpu science and technology Limited; mulberry leaf grinding buffer and protease inhibitor were purchased from NOVUS, USA; antibodies GRP78, RCA, Histone H3 were purchased from Hangzhou Huanan Biotechnology, Inc., and CNX was purchased from Agrisera, Sweden. The preparation method comprises the following steps:
1. grinding and centrifuging reagents
Grinding buffer solution: 5x Isoplastic localization Buffer (KC-414), consisting of HEPES, Sucrose, KCl.
Protease inhibitors: 100X Protease Inhibitor Cocktail (PIC, KC-121).
Preparation of reagents relevant to SDS-PAGE electrophoresis and preparation of gel
SDS-PAGE buffer a (1M Tris-HCl, PH 6.8): weighing Tris 12.1g, adding 80mL of ultrapure water for dissolving, adjusting the pH value to 6.8 by concentrated hydrochloric acid, and adding ultrapure water for constant volume of 100 mL.
SDS-PAGE buffer B (5M Tris-HCl, PH 8.8): weighing 18.17g of Tris, adding 80mL of ultrapure water for dissolving, adjusting the pH value to 8.8 by concentrated hydrochloric acid, and adding ultrapure water for constant volume of 100 mL.
10% ammonium persulfate: 0.1g of ammonium persulfate was weighed and dissolved in 1mL of ultrapure water. The reagent is ready for use.
5 × Tris-glycine electrophoresis buffer: 94g of glycine, 15.1g of Tris and 5g of SDS are weighed, dissolved in 800mL of ultrapure water and then added to 1000mL of solution. It is diluted five times when used.
The reagent formulation for a 1.0mm thick SDS-PAGE electrophoresis gel is shown in Table 1:
TABLE 1 SDS-PAGE electrophoretic separation gel and concentrated gel solution formulation
| Name of reagent | 15% separation gel | 5% concentrated gum |
| H2O(mL) | 2.3 | 2.7 |
| 30% acrylamide (mL) | 5.0 | 0.67 |
| Buffer A (mL) | / | 0.5 |
| Buffer B (mL) | 2.5 | / |
| 10％SDS(μL) | 0.1 | 0.04 |
| 10% ammonium persulfate (mL) | 0.1 | 0.04 |
| TEMED(μL) | 0.004 | 0.004 |
Preparation of main reagent, antibody and gel for Western blot experiment
10 × anode electrophoresis buffer: weighing 24.2g of Tris base, and adding double distilled water to reach the constant volume of 1000 mL. Before use, dilute to 1x with double distilled water and add concentrated hydrochloric acid to pH 8.9.
Cathode electrophoresis buffer: weighing 12.1g of Tris base and 1g of SDS, adding 800mL of double distilled water for dissolution, dissolving 12.3g of tricine powder, adjusting the pH value to 8.25, and adding the double distilled water for constant volume to 1000 mL. The buffer solution can be recycled for multiple times.
Gel buffer (gel buffer): weighing 18.15g of Tris base and 0.3g of SDS, adding 80mL of double distilled water for dissolution, adding concentrated hydrochloric acid until the pH value is 8.45, and adding the double distilled water for constant volume until the volume is 100 mL.
Gel buffer (stacking gel buffer): weighing 3.63g Tris base and 0.4g SDS, adding 80mL double distilled water for dissolution, adding concentrated hydrochloric acid until the pH value is 7.8, and adding the double distilled water for constant volume until the volume is 100 mL.
1.0M Tris-HCl (pH 8.3): weighing 60.57g of Tris, adding 400mL of double distilled water for dissolution, adding concentrated hydrochloric acid until the pH value is 8.3, and adding the double distilled water for constant volume until the volume is 500 mL.
And (3) membrane transfer buffer solution: 14.413g of glycine was weighed and dissolved in an appropriate amount of double distilled water, 25mL of 1M Tris-HCl (pH 8.3) and 200mL of methanol were added, and double distilled water was added to the solution to a volume of 1000 mL. Precooling for more than half an hour before use, repeatedly using for 2-3 times, and according to the voltage condition, if the voltage is lower, using the fresh preparation.
10 × TBS: weighing 80g of NaCl and 24g of Tris, adding 800mL of double distilled water for dissolution, adding concentrated hydrochloric acid until the pH value is 7.6, and adding the double distilled water for constant volume until the volume is 1000 mL.
TBST: 10 × TBS 100mL and Tween 20(Tween-20)1mL are measured, and the volume is adjusted to 1000mL by double distilled water.
Confining liquid (5% skim milk): weighing 5g of skimmed milk powder, and making the volume of the skimmed milk powder to be 100mL by TBST. It is prepared according to the actual use amount and can not be repeatedly used.
The antibodies used were: all are diluted to corresponding concentrations by TBST, stored in a refrigerator at-20 ℃ and can be used repeatedly.
The reagent preparation of 1 Western blot electrophoresis gel with a thickness of 1.5mm is shown in Table 2
TABLE 2 Western blot electrophoretic separation gel and concentrated gel solution preparation
| Name of reagent | 7.5% separation gel | Concentrated glue |
| 30% acrylamide (mL) | 2.25 | 0.85 |
| gel buffer(mL) | 3 | / |
| stacking buffer(mL) | / | 1.5 |
| Glycerol (g) | 2 | / |
| H2O(mL) | 2.55 | 3.65 |
| 10% ammonium persulfate (mL) | 0.05 | 0.05 |
| TEMED(μL) | 5 | 7.5 |
The operation process is as follows:
1. crushing tissue of mulberry leaf
(1) Weighing 1.25g of fresh mulberry leaves, and cleaning and wiping the fresh mulberry leaves with clear water to obtain clean plant leaves which can be used for subsequent experiments;
(2) taking the tissue leaves, shearing the tissue leaves into 0.2-2.0 cm, adding a precooled grinding buffer solution into a precooled mortar, adding 5mL of grinding buffer solution and 50 mu L of 100x protease inhibitor, and grinding the mixture on ice to form homogenate.
2. Centrifuging the homogenate of the mulberry leaf tissue to separate endoplasmic reticulum
(1) Taking the folium Mori homogenate, transferring to a 15mL centrifuge tube, centrifuging in a freezing type low speed centrifuge (H1850R, Xiang instrumental centrifuge Co., Ltd.) at 4 deg.C and 1000Xg for 10 minutes, precipitating cell debris and cell nucleus, and collecting supernatant;
(2) transferring the supernatant to a clean and precooled 3.5mL thick-walled ultracentrifuge tube (349622, Beckman Coulter, USA) made of polycarbonate, selecting a horizontal rotor (MLS50, Beckman Coulter, USA), centrifuging for 15 minutes at 12000Xg at 4 ℃ in a desktop ultrarefrigerated centrifuge (MAX-XP, Beckman Coulter, USA), and collecting the supernatant after mitochondria and chloroplast organelles are precipitated;
(3) transferring the supernatant to a clean and precooled thick-wall ultracentrifuge tube made of 3.5mL polycarbonate, centrifuging the tube for 60 minutes at the rotating speed of 90000Xg at the temperature of 4 ℃ in a rotor and a centrifuge which are the same as those in the step (2), and removing the supernatant, wherein the precipitate is the endoplasmic reticulum.
3. Cracking the extracted endoplasmic reticulum to extract protein
(1) Adding 10 μ L of 100x PMSF into 1mL RIPA lysine (Biyunstian Biotechnology Co., Ltd.) to prepare the lysate on site;
(2) adding 300 mu L of lysate into the precipitate, grinding and cracking the precipitate in a centrifuge tube by using a clean and precooled glass homogenizer accessory homogenizing pestle, and sucking out the heavy suspension into a clean and precooled 1.5mL centrifuge tube;
(3) and adding about 50 mu L of lysis solution to wash the centrifuge tube and the homogenate pestle, collecting the lysate into the resuspension solution to obtain the mulberry leaf endoplasmic reticulum protein, and immediately detecting the mulberry leaf endoplasmic reticulum protein.
4. Detecting the purity of the endoplasmic reticulum protein
Purity assays were performed using a comparison of the relative intensities of the bands in a Western blot (Western blot) of endoplasmic reticulum protein and total protein.
(1) Total protein extraction from mulberry leaves was performed before the experiment: weighing 0.5g of fresh mulberry leaves, adding 3mL of lysate, grinding on ice into slurry, filtering the slurry by using a 100 mu m cell sieve, and collecting filtrate, namely the total protein of the mulberry leaves;
(2) protein quantification and sample preparation: protein concentration is measured by using a BCA kit method, each protein is diluted to the same concentration by using lysate and 5x loading buffer, the solution is heated for 15 minutes at 100 ℃, and supernatant is taken after short-time centrifugation to obtain a sample solution;
(3) SDS-PAGE electrophoresis was used as loading control: referring to SDS-PAGE electrophoresis, the loading of ER and TP proteins was 20ug, electrophoresis was performed at a constant voltage of 80V until the dye reached the bottom, and the gel was silver stained and imaged, as shown in FIG. 1, indicating that the loading of the endoplasmic reticulum protein sample and the total protein sample were close.
(4) The relative intensity of the bands is checked by a Western blot experiment method: western blot experiments were carried out in the amounts as described in (3) above, using GRP78 and CNX as the endoplasmic reticulum protein positive marker, RCA as the chloroplast protein positive marker, and Histone H3 as the nuclear protein positive marker. Scanning to obtain an electronic plate strip, performing gray value analysis by using Image J software, and performing statistical analysis by using SPSS software.
The data show that after purification, the endoplasmic reticulum protein positive markers GRP78 and CNX are increased by 3.3(p <0.05, n ═ 3) times and 6.1(p <0.01, n ═ 3) times, respectively, compared with the total protein, and the chloroplast-positive protein RCA and the cell nucleus-positive protein Histone H3 are reduced by about 84.1% (p <0.05, n ═ 3) and 74.4% (p <0.05, n ═ 3), respectively, indicating that the purity of the isolated and purified endoplasmic reticulum protein is higher and the significant enrichment is obtained. See fig. 2, 3.
Claims (3)
1. An effective method for extracting and purifying endoplasmic reticulum and protein thereof from plant leaves is characterized by comprising the following steps:
1) crushing the tissues of the plant leaves, and grinding the plant leaves into homogenate; the plant leaves are leaves of mulberry leaves, anemone indica leaves or Chinese mahonia;
2) carrying out centrifugal separation on the plant leaf tissue homogenate to obtain an endoplasmic reticulum;
3) cracking endoplasmic reticulum to extract protein;
the step 2) is specifically as follows:
(1) the plant leaf homogenate obtained in the step 1) is subpackaged in a centrifuge tube, centrifuged for 10-30 minutes at the rotation speed of 800-;
(2) subpackaging the supernatant into clean and precooled ultracentrifuge tubes, centrifuging the ultracentrifuge tubes for 10 to 30 minutes at 10000-;
(3) subpackaging the supernatant into clean and precooled ultracentrifuge tubes, centrifuging at the temperature of 0-10 ℃ at the rotating speed of 60000-;
the step 3) is specifically as follows:
(1) preparing the lysate in situ, and adding 50 μ L of 100 × PMSF into 5mL of RIPA lysine;
(2) adding 500 mu L of 200-fold lysis solution into the endoplasmic reticulum sediment obtained in the step 2), grinding and resuspending the sediment in a centrifuge tube by using a cleaned and precooled homogenate pestle for lysis, and sucking out the resuspension solution into a clean and precooled centrifuge tube;
(3) adding 30-100 μ L lysate to wash the centrifuge tube and homogenate pestle, collecting into resuspension to obtain endoplasmic reticulum protein of plant leaf, and storing at-80 deg.C or immediately detecting.
2. The method as claimed in claim 1, wherein the plant leaves are leaves of mulberry, neya, or mahonia.
3. The effective method for extracting and purifying endoplasmic reticulum and proteins thereof from plant leaves as claimed in claim 1, wherein said step 1) is specifically:
(1) taking fresh plant leaves, cleaning and wiping the fresh plant leaves with clear water to obtain clean plant leaves for subsequent experiments;
(2) cutting the tissue leaves into pieces of 0.2-2.0 cm, placing the pieces in a precooled mortar, adding 1-5mL of precooled grinding buffer and 1-10 muL of 100 Xprotease inhibitor into each 1g of leaves, and grinding the mixture on ice to form a homogenate.
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