CN113025556A - Separation method of wheat protoplast, cytoplasm and chloroplast - Google Patents
Separation method of wheat protoplast, cytoplasm and chloroplast Download PDFInfo
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Abstract
The invention discloses a separation method of wheat protoplast, cytoplasm and chloroplast. Firstly, extracting protoplasts from wheat leaves, and then further purifying the protoplasts by using a sucrose density gradient centrifugation method to obtain nearly complete protoplasts; the protoplasts are then disrupted, the chloroplasts are left intact, and the intact chloroplasts and cytoplasts are separated by centrifugation. The invention can effectively separate chloroplast and cytoplasm, has less cross contamination, and can further carry out deeper research on different components of cells. The invention has important application value.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a separation method of wheat protoplast, cytoplasm and chloroplast.
Background
Studies have demonstrated that hybridization is one of the important factors in producing reproductive isolation and is the major driving force for speciation and differentiation. Both crossing and polyploidization result in the fusion of nuclear genomes of different parents, but cytoplasm is only from one parent. Thus, hybrid and polyploid species must address not only the conflict between the two sets of genomic fusions, but also compatibility issues between nuclear and cytoplasmic organelles. For example, polyploid crops such as wheat, cotton and peanut are broken through the original mode, a new mode of coexistence of cell nucleus and organelle is re-established, and the male sterile line in rice and soybean is caused by incompatibility of cell nucleus and mitochondria. Separating organelles (such as chloroplasts) and cytoplasm and researching corresponding mechanisms can provide new ideas and methods for crop improvement.
The separation of plant cytoplasm and chloroplast is mainly divided into two parts of high-quality protoplast extraction and cytoplasm and chloroplast separation. At present, the methods for extracting protoplasts of higher plants are mature, but certain problems still exist, such as the required initial amount is large, the obtained protoplasts are fragile, and the like, which causes great difficulty for the next work. There are few techniques for separating the cytoplasm from the chloroplasts, and there are only a few chloroplast-associated extraction methods, but these methods generally do not take into account the cytoplasmic components. Therefore, the technology for separating cytoplasm and chloroplast in the wheat leaf is provided, a new angle is provided for the research of origin and evolution of wheat, and a new technology is provided for germplasm innovation of wheat.
Disclosure of Invention
The object of the invention is to isolate wheat cytoplasm, chloroplast and protoplast.
The invention firstly protects a method for separating wheat cytoplasm and/or chloroplast, which comprises the following steps:
(a1) placing chopped wheat leaves in 0.3-0.5M mannitol solution (such as 0.3-0.4M mannitol solution, 0.4-0.5M mannitol solution, 0.3M mannitol solution, 0.4M mannitol solution, 0.5M mannitol solution), and standing in dark for 5-15min (such as 5-10min, 10-15min, 5min, 10min or 15 min);
(a2) after completion of step (a1), filtering and collecting the precipitate; adding enzymolysis liquid, keeping out of the sun, and performing enzymolysis;
the enzymolysis solution has a solute concentration of 15-25mM (such as 15-20mM, 20-25mM, 15mM, 20mM or 25mM) MES, 0.2-0.6M (such as 0.2-0.4M, 0.4-0.6M, 0.2M, 0.4M or 0.6M) mannitol, 10-30mM (such as 10-20mM, 20-30mM, 10mM, 20mM or 30mM) KCl, 1-2% (such as 1-1.5%, 1.5% -2%, 1%, 1.5% or 2%) cellulase, 0.5-1% (such as 0.5% -0.75%, 0.75% -1%, 0.5%, 0.75% or 1%) enzyme, 5-15mM (such as 5-10mM, 10-15mM, 5mM, 10mM or 15mM) CaCl, 5-25mM, 0.2-0.6M (such as 1-20 mM), 1-2% or 1% of the enzyme20.1% -0.5% (such as 0.1% -0.3%, 0.3% -0.5%, 0.1%, 0.3% or 0.5%) BSA, 3-8mM (such as 3-5mM, 5-8mM, 3mM, 5mM or 8mM) beta-mercaptoethanol, water as solvent, and natural pH;
(a3) after the step (a2) is completed, adding W5 washing solution, and processing in dark at 26-30 deg.C (such as 26-28 deg.C, 28-30 deg.C, 26 deg.C, 28 deg.C or 30 deg.C), 60-80rpm (such as 60-70rpm, 70-80rpm, 60rpm, 70rpm or 80rpm) for 5-15min (such as 5-10min, 10-15min, 5min, 10min or 15 min);
the W5 lotion has a solute concentration of 100-200mM (e.g., 100-154mM, 154-200mM, 100mM, 154mM or 200mM) NaCl, 100-150mM (e.g., 100-125mM, 125-150mM, 100mM, 125mM or 150mM) CaCl22-8mM (e.g., 2-5mM, 5-8mM, 2mM, 5mM, or 8mM) KCl, 1-4mM (e.g., 1-2mM, 2-4mM, 1mM, 2mM, or 4mM) MES in water at a pH of 5.7-6.0 (e.g., 5.7, 5.8, 5.9, or 6.0);
(a4) after step (a3) is completed, filtering and collecting filtrate; centrifuging and collecting precipitate; washing the precipitate with the W5 washing solution for more than 3 times;
(a5) after the step (a4) is completed, adding FMA solution, FMB solution and FMC solution, centrifuging, and collecting liquid between the FMB solution and the FMC solution interface, namely wheat protoplast;
the FMA solution has a solute concentration of 0.3-0.6M (such as 0.3-0.5M, 0.5-0.6M, 0.3M, 0.5M or 0.6M) sucrose, 0.8-1.2mM (such as 0.8-1.0mM, 1.0-1.2mM, 0.8mM, 1.0mM or 1.2mM) MgCl2HEPES (pH7.0, 3-7mM (such as 3-5mM, 5-7mM, 3mM, 5mM or 7mM), in water at a natural pH;
the FMB solution has a solute concentration of 0.2-0.5M (e.g., 0.2-0.4M, 0.4-0.5M, 0.2M, 0.4M, or 0.5M) sucrose, 0.8-1.2mM (e.g., 0.8-1.0mM, 1.0-1.2mM, 0.8mM, 1.0mM, or 1.2mM) MgCl2HEPES (pH7.0), HEPES (pH 3-5mM, 5-7mM, 3mM, 5mM or 7mM), sorbitol (0.05-0.15M (0.05-0.10M, 0.10-0.15M, 0.05M, 0.10M or 0.15M), with water as solvent and natural pH;
the FMC solution has a solute concentration of 0.8-1.2mM (e.g., 0.8-1.0mM, 1.0-1.2mM, 0.8mM, 1.0mM, or 1.2mM) MgCl2pH7.0, 3-7mM (e.g., 3-5mM, 5-7mM, 3mM, 5mM, or 7mM) HEPES, 0.3-0.8M (e.g., 0.3-0.5M, 0.5-0.8M, 0.3M, 0.5M, or 0.8M) sorbitol, water as a solvent, and a natural pH;
(a6) after step (a5) is completed, adding WH solution into wheat protoplast, keeping away from light, and standing overnight at 3-6 deg.C (such as 3-4 deg.C, 4-6 deg.C, 3 deg.C, 4 deg.C or 6 deg.C);
the WH solution has a solute and a concentration of 0.3-0.8M (e.g., 0.3-0.5M, 0.5-0.8M, 0.3M, 0.5M, or 0.8M) mannitol, 10-30mM (e.g., 10-20mM, 20-30mM, 10mM, 20mM, or 30mM) KCL, 2-6mM (e.g., 2-4mM, 4-6mM, 2mM, 4mM, or 6mM) MES, a solvent of water, and a pH of 5.7;
(a7) after step (a6) is completed, collecting the precipitate, adding a localization buffer solution, and mixing uniformly;
the solute of the localization buffer and its concentration are 0.3-0.5M (such as 0.3-0.4M, 0.4-0.5M, 0.3M, 0.4M or 0.5M) sucrose, 2-4mM (such as 2-3mM, 3-4mM, 2mM, 3mM or 4mM) EDTA, 40-60mM (such as 40-50mM, 50-60mM, 40mM, 50mM or 60mM) Tris-HCL, 1.5-2.5M (such as 1.5-2.0M, 2.0-2.5M, 1.5M, 2.0M or 2.5M) DTT, the solvent is water, pH value is natural;
(a8) after the step (a7) is completed, crushing, centrifuging and collecting the precipitate; the precipitate is the wheat chloroplast;
(a9) after the step (a7) is completed, crushing, centrifuging and collecting supernatant; the supernatant is the wheat cytoplasm.
In the step (a1), the wheat leaves may be wheat leaves grown to 3-leaf stage (e.g., wheat leaves grown to day 16).
In the step (a1), the cutting may be performed by placing the wheat leaves in a culture dish on ice and cutting into filaments to obtain cut leaves.
In the step (a2) or the step (a4), the filtration may be performed by miracle filter cloth filtration.
In the step (a4), the centrifugation parameters may be 80g centrifugation at room temperature for 5 min.
In the step (a6), the volume ratio of the wheat protoplast to the WH solution can be 1:3-5 (e.g., 1:3-4, 1:4-5, 1:3, 1:4, or 1: 5).
In the step (a7), the ratio of the precipitation buffer to the homogenesis buffer may be 2.0g of the precipitate obtained from wheat leaves: 1-3ml (e.g., 1ml, 2ml, or 3ml) of the homogenesis buffer.
In the step (a8), the crushing may be performed 50 to 60 times (e.g., 50 to 55 times, 55 to 60 times, 50 times, 55 times, or 60 times) of grinding. The centrifugation is performed at 3-5 deg.C (such as 3-4 deg.C, 4-5 deg.C, 3 deg.C, 4 deg.C or 5 deg.C), 600-1000g (such as 600-800g, 800-1000g, 600g, 800g or 1000g), and 10-20min (such as 10-15min, 15-20min, 10min, 15min or 20 min).
The step (a8) or the step (a9) may be performed by crushing on ice using pre-cooled Glass/Teflon Potter Elvehjem homogenerizers.
In the step (a9), the crushing may be performed 4 to 10 times (e.g., 4 to 5 times, 5 to 10 times, 4 times, 5 times, or 10 times) grinding. The centrifugation can be performed more than 2 times. The 1 st centrifugation can be performed at 3-5 deg.C (such as 3-4 deg.C, 4-5 deg.C, 3 deg.C, 4 deg.C or 5 deg.C), 600-1000g (such as 600-800g, 800-1000g, 600g, 800g or 1000g) for 10-20min (such as 10-15min, 15-20min, 10min, 15min or 20min), and collecting the supernatant. Centrifuging for more than 2 times at 3-5 deg.C (such as 3-4 deg.C, 4-5 deg.C, 3 deg.C, 4 deg.C or 5 deg.C), 8000-.
The invention also provides a method for separating the wheat protoplast, which comprises the following steps:
(b1) placing chopped wheat leaves in the above mannitol solution, and standing in dark for 5-15min (such as 5-10min, 10-15min, 5min, 10min or 15 min);
(b2) after completion of step (b1), filtering and collecting the precipitate; then adding any one of the enzymolysis solutions, keeping out of the sun, and carrying out enzymolysis;
(b3) after the step (b2) is completed, adding any one of the W5 washing solutions, and performing light-shielding treatment at 26-30 ℃ (such as 26-28 ℃, 28-30 ℃, 26 ℃, 28 ℃ or 30 ℃), 60-80rpm (such as 60-70rpm, 70-80rpm, 60rpm, 70rpm or 80rpm) for 5-15min (such as 5-10min, 10-15min, 5min, 10min or 15 min);
(b4) after completion of step (b3), filtering and collecting the filtrate; centrifuging and collecting precipitate; washing the precipitate with any one of the W5 washes for more than 3 times;
(b5) after step (b4) is completed, adding any one of the FMA solutions, any one of the FMB solutions and any one of the FMC solutions, centrifuging, and collecting the liquid at the interface between the FMB solution and the FMC solution, i.e., wheat protoplasts.
In the step (a2) or the step (b2), the ratio of the precipitate to the enzymolysis solution may be 2.0g of the precipitate obtained from wheat leaves: 30-50ml (such as 30-40ml, 40-50ml, 30ml, 40ml or 50ml) of enzymolysis solution. The enzymolysis parameter can be 26-30 deg.C (such as 26-28 deg.C, 28-30 deg.C, 26 deg.C, 28 deg.C or 30 deg.C), 60-80rpm (such as 60-70rpm, 70-80rpm, 60rpm, 70rpm or 80rpm), and 3-5 hr (such as 3-4 hr, 4-5 hr, 3 hr, 4 hr or 5 hr).
In the step (a3) or the step (b3), the volume ratio of the W5 washing solution to the enzymolysis solution may be 1:0.5 to 1.5 (e.g., 1:0.5 to 1.0, 1:1.0 to 1.5, 1:0.5, 1:1.0, 1: 1.5).
In the step (a5) or the step (b5), the ratio of the precipitate, the FMA solution, the FMB solution and the FMC solution may be specifically 2.0g of the precipitate obtained from wheat leaves: 15ml FMA solution: 7.5ml FMB solution: 3ml FMC solution.
Any of the above mannitol solutions may be an aqueous mannitol solution.
Any of the above waters may be RO water.
The invention also protects a kit A or a kit B.
The kit A comprises any one of the enzymolysis solutions, any one of the W5 lotion, any one of the FMA solutions, any one of the FMB solutions, any one of the FMC solutions, any one of the WH solutions and any one of the Homogenization buffer solutions.
The kit A may specifically comprise any one of the above-mentioned enzymatic hydrolysates, any one of the above-mentioned W5 lotions, any one of the above-mentioned FMA solutions, any one of the above-mentioned FMB solutions, any one of the above-mentioned FMC solutions, any one of the above-mentioned WH solutions and any one of the above-mentioned Homogenization buffers.
The kit B comprises any one of the enzymolysis liquid, any one of the W5 lotion, any one of the FMA solution, any one of the FMB solution and any one of the FMC solution.
The kit B can specifically comprise any one of the enzymolysis solutions, any one of the W5 washing solutions, any one of the FMA solutions, any one of the FMB solutions and any one of the FMC solutions.
The invention also protects at least one of A1) -A6).
A1) Use of any of the methods described above for isolating wheat cytoplasm and/or chloroplast.
A2) Use of any of the above methods in wheat cytoplasm and/or chloroplast genome or protein extraction.
A3) Use of any of the above methods for isolating wheat protoplasts.
A4) The use of any of the above methods in wheat protoplast genome or protein extraction.
A5) The use of any one of the kits a in the isolation of wheat cytoplasm and/or chloroplast.
A6) The application of any one of the kit B in separating wheat protoplasts.
The invention has the following advantages:
1) the existing method for extracting the protoplast is easy to cause the protoplast to be crushed and the consumption of the required raw materials is larger, but the method provided by the invention can further purify the crushed protoplast, thereby obtaining a large amount of complete protoplast;
2) the existing chloroplast extraction technology only extracts the chloroplast which is an organelle, and does not consider the cytoplasm, but the method provided by the invention can separate the chloroplast and the cytoplasm, and the cytoplasm is rarely polluted by the chloroplast.
3) The invention can effectively obtain high-quality chloroplast and cytoplasm, and the extracted chloroplast and cytoplasm are suitable for tests such as protein extraction, protein mass spectrometry, protein quantification and the like.
The invention has important application value.
Drawings
FIG. 1 shows the process of extracting wheat protoplasts.
FIG. 2 shows the purification of wheat protoplasts by sucrose density gradient centrifugation.
FIG. 3 shows the state of wheat protoplasts before and after purification under a microscope.
FIG. 4 shows the Western blot detection results of chloroplast, cytoplasmic, and protoplast concentrated proteins.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The cellulase R-10 (hereinafter referred to as cellulase) is a product of Yakult Honsha company, and the catalog number of the product is L0012-10 g. The specific activity of the cellulase is more than 10000U/g.
Cellulase enzyme activity is defined as: 1g enzyme powder (or 1ml enzyme solution) at 50 deg.C and pH4.8, the enzyme amount for hydrolyzing substrate (filter paper, CMC, absorbent cotton or saligenin) for 1min to generate 1 μ g glucose is 1 enzyme activity unit.
The isolation enzyme R-10 (hereinafter referred to as isolation enzyme) is a product of Yakult Honsha company, and the catalog number of the product is L0021-10 g. The specific activity of the eductase is more than 3000U/g.
The enzyme activity of the eductase is defined as: 1g enzyme powder (or 1ml enzyme solution) decomposes pectin at 50 deg.C and pH 3.5 for 1 hr to generate 1mg galacturonic acid as 1 enzyme activity unit.
Triticum aestivum TAA10, a natural hexaploid TAA10, is described in the following documents: the evolution research of transcriptome of the Bibo.triticum aestivum BBAA subgenome in the allophexaploid track [ D ].2014, the doctrine of doctorals at northeast university, hereinafter, triticum aestivum TAA10 is abbreviated as wheat.
The following examples relate to the following compositions of solutions:
the enzymolysis solution contains 20mM MES (morpholine ethanesulfonic acid), 0.4M mannitol, 20mM KCl, 1.5% cellulase, 0.75% eductase, and 10mM CaCl20.1% BSA (bovine serum albumin), 5mM beta-mercaptoethanol, water as solvent, and natural pH.
The solute of the W5 lotion and its concentration were 154mM NaCl and 125mM CaCl25mM KCl, 2mM MES, water as solvent, and pH value of 5.7-6.0.
The solute of FMA solution is 0.5M sucrose, 1.0mM MgCl25mM HEPES (pH7.0), water as a solvent, and a natural pH value.
The solute of FMB solution is 0.4M sucrose, 1.0mM MgCl25mM HEPES (pH7.0), 0.1M sorbitol, water as solvent, pH natural.
Solute of FMC solution and its concentration is 1.0mM MgCl25.0mM HEPES (pH7.0), 0.5M sorbitol, water as solvent, pH fromHowever.
The WH solution had a solute concentration of 0.5M mannitol, 20mM KCL, 4mM MES, water as solvent, and a pH of 5.7 (adjusted with NAOH).
The solute and concentration of the localization buffer solution are 0.4M sucrose, 3mM EDTA, 50mM Tris-HCL and 2mM DTT, the solvent is water, and the pH value is natural. It should be noted that DTT is added before use.
The solute and concentration of the IP lysis buffer are 100mM Tris-HCl, 150mM NaCl, 5mM EGTA, 5mM EDTA, 2mM DTT, 0.5% Triton X-100 and 10% Cocktail, the solvent is water, and the pH value is natural.
Example 1 establishment of a method for isolation of wheat protoplasts, cytoplasm and chloroplasts
The inventor establishes a separation method of wheat protoplast, cytoplasm and chloroplast through a large number of experiments. The method comprises the following specific steps:
the extraction process of wheat protoplasts is shown in FIG. 1.
1. 2.0g of wheat leaves which normally grow to day 16 were taken, and placed in a petri dish on ice and minced into a filamentous form to obtain crushed leaves.
2. After the completion of step 1, the crushed leaves were placed in a 0.4M mannitol aqueous solution and allowed to stand for 10min in the dark.
3. After the completion of the step 2, the crushed leaves are filtered by miraculous filter cloth, then put into a triangular flask filled with 40ml of enzymolysis liquid, the triangular flask is wrapped by tinfoil, and put into a shaking table with the temperature of 28 ℃ and the rpm of 70 to slowly shake for 4 hours (for enzymolysis).
4. After completing step 3, the lotion W5 with the same volume was added to the flask, wrapped with tinfoil and placed back on the shaker at 28 deg.C and 70rpm for 10min to obtain a mixed solution.
5. After the step 4 is finished, filtering the mixed solution obtained in the step (4) by miraculous filter cloth, collecting filtrate, transferring the filtrate into a 50ml centrifuge tube wrapped by tinfoil, centrifuging for 5min at 80g under the condition of room temperature, collecting precipitate, and re-suspending by using 15ml W5 lotion to obtain a re-suspension 1; centrifuging 80g of the heavy suspension 1 for 5min at room temperature, collecting precipitates, and carrying out heavy suspension by using 15ml of W5 washing liquor to obtain a heavy suspension 2; the resuspension 2 was centrifuged at 80g for 5min at room temperature, and the pellet was collected. The precipitate was wheat protoplasts before purification.
6. After completion of step 5, 15ml of FMA solution was slowly added to the pellet followed by 7.5ml of FMB solution and 3ml of FMC solution in sequence with a syringe (no needle required); centrifuging at 250g for 5min at 4 deg.C (see FIG. 2); the liquid (dark band at the interface between FMB and FMC solutions) was collected using a syringe (stainless steel plain needle) into a new 15ml centrifuge tube, which was the purified wheat protoplast.
And observing the wheat protoplast before purification and the wheat protoplast after purification under a microscope.
The results are shown in FIG. 3.
7. After the step 6 is completed, 4 times of the volume of WH solution is slowly added into the centrifuge tube (containing the wheat protoplast), and the protoplast is naturally settled at 4 ℃ overnight after being wrapped by tinfoil.
8. After completion of step 7, the supernatant was aspirated, the pellet was retained, 2ml of Homogenization buffer was added, gently shaken and then randomly divided into two parts, designated as suspension A (for cytoplasmic extraction) and suspension B (for chloroplast extraction).
9. After completion of step 8, the suspension A was crushed on ice using pre-cooled Glass/Teflon Potter Elvehjem homogenerizers (the number of grinding times was set to 55) to give a mixed solution; transferring the mixed solution to a new 2ml centrifuge tube, centrifuging for 15min at the temperature of 4 ℃ at 800g, and collecting precipitate, wherein the precipitate is the wheat chloroplast.
10. After completion of step 8, the suspension B was crushed (5 times of grinding) on ice using pre-cooled Glass/Teflon Potter Elvehjem homogenerizers to obtain a mixed solution; the mixed solution was transferred to a new 2ml centrifuge tube, centrifuged at 800g at 4 ℃ for 15min, and the supernatant 1 was collected.
11. After the step 10 is finished, transferring the supernatant 1 into an ultracentrifuge tube, putting the ultracentrifuge tube into an ultracentrifuge, centrifuging the ultracentrifuge tube at 10000g for 15min at 4 ℃, and collecting a supernatant 2; transferring the supernatant 2 to a new ultracentrifuge tube, placing the ultracentrifuge tube into an ultracentrifuge, centrifuging the ultracentrifuge tube at 10000g for 30min at 4 ℃, and collecting a supernatant 3. The supernatant 3 is the wheat cytoplasm.
Example 2, verification of wheat chloroplast and wheat cytoplasm extracted in example 1
1. And (3) adding 200ul of IP lysis buffer into the wheat chloroplast extracted in the step 9 in the example 1, fully lysing, centrifuging at 12000rpm at 4 ℃ for 15min, and collecting supernatant, namely chloroplast protein supernatant.
2. And transferring the chloroplast protein supernatant to a 3kDa Ultrafree centrifugal filtration and concentration device, and centrifuging at 4000g for 30min at 4 ℃ to obtain the chloroplast protein concentrate.
3. The cytoplasm of wheat extracted in step 11 of example 1 was added with 200ul of IP lysis buffer, and after sufficient lysis, it was centrifuged at 12000rpm at 4 ℃ for 15min, and the supernatant, i.e., the supernatant of cytoplasmic protein, was collected.
4. Transferring the supernatant of the cytoplasmic protein to a 3kDa Ultrafree centrifugal filtration and concentration device, and centrifuging at 4000g for 30min at 4 ℃ to obtain the cytoplasmic concentrated protein.
5. The purified wheat protoplast extracted in step 6 of example 1 was added with 200ul of IP lysis buffer, and after sufficient lysis, centrifuged at 12000rpm at 4 ℃ for 15min, and the supernatant, i.e., the supernatant of the protoplast protein, was collected.
6. Transferring the supernatant of the protoplast protein to a 3kDa Ultrafree centrifugal filtration and concentration device, and centrifuging at 4000g for 30min at 4 ℃ to obtain the protoplast concentrated protein.
7. Western Blot was performed on a protein (chloroplast, cytoplasmic or protoplast concentrated protein), and chloroplast was detected using a specific antibody cFBPase (product of Agriera, catalog No. AS04043) AS a primary antibody and RbcL (product of Agriera, catalog No. AS03037A) AS a primary antibody.
The results are shown in FIG. 4. The result shows that the chloroplast concentrated protein and the cytoplasm concentrated protein have higher purity and have no obvious mutual pollution; namely, the wheat chloroplast and the wheat cytoplasm extracted in the example 1 are pure and have no pollution.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Claims (10)
1. A method of isolating wheat cytoplasm and/or chloroplast comprising the steps of:
(a1) placing chopped wheat leaves in 0.3-0.5M mannitol solution, and standing in dark for 5-15 min;
(a2) after completion of step (a1), filtering and collecting the precipitate; adding enzymolysis liquid, keeping out of the sun, and performing enzymolysis;
the solute and the concentration of the enzymolysis liquid are 15-25mM MES, 0.2-0.6M mannitol, 10-30mM KCl, 1-2% cellulase, 0.5-1% eductase and 5-15mM CaCl20.1 to 0.5 percent BSA, 3 to 8mM beta-mercaptoethanol, water as a solvent and natural pH value;
(a3) after the step (a2) is finished, adding W5 washing liquid, and carrying out light-shielding treatment for 5-15min at 26-30 ℃ and 60-80 rpm;
the solute of the W5 lotion and the concentration thereof are 200mM NaCl and 150mM CaCl, respectively22-8mM KCl, 1-4mM MES, water as solvent, and pH value of 5.7-6.0;
(a4) after step (a3) is completed, filtering and collecting filtrate; centrifuging and collecting precipitate; washing the precipitate with the W5 washing solution for more than 3 times;
(a5) after the step (a4) is completed, adding FMA solution, FMB solution and FMC solution, centrifuging, and collecting liquid between the FMB solution and the FMC solution interface, namely wheat protoplast;
the solute of FMA solution and its concentration are 0.3-0.6M sucrose and 0.8-1.2mM MgCl2pH7.0, 3-7mM HEPES, water as solvent, and natural pH value;
the solute of the FMB solution and the concentration thereof are 0.2-0.5M sucrose and 0.8-1.2mM MgCl2pH7.0, 3-7mM HEPES, 0.05-0.15M sorbitol, water as solvent, and natural pH value;
the solute of the FMC solution and the concentration thereof are 0.8-1.2mM MgCl2pH7.0, 3-7mM HEPES, 0.3-0.8M sorbitol, water as solvent, and natural pH value;
(a6) after the step (a5) is completed, adding WH solution into the wheat protoplast, keeping out of the sun, and standing overnight at 3-6 ℃;
the solute and the concentration of the WH solution are 0.3-0.8M mannitol, 10-30mM KCL and 2-6mM MES, the solvent is water, and the pH value is 5.7;
(a7) after step (a6) is completed, collecting the precipitate, adding a localization buffer solution, and mixing uniformly;
the solute and the concentration of the localization buffer solution are 0.3-0.5M sucrose, 2-4mM EDTA, 40-60mM Tris-HCL and 1.5-2.5M DTT, the solvent is water, and the pH value is natural;
(a8) after the step (a7) is completed, crushing, centrifuging and collecting the precipitate; the precipitate is the wheat chloroplast;
(a9) after the step (a7) is completed, crushing, centrifuging and collecting supernatant; the supernatant is the wheat cytoplasm.
2. The method of claim 1, wherein: in the step (a6), the volume ratio of the wheat protoplast to the WH solution is 1: 3-5.
3. The method of claim 1, wherein: in said step (a7), the ratio of the precipitation to the homogenesis buffer was 2.0g of the precipitate obtained from wheat leaves: 1-3ml of the homogenization buffer.
4. The method of claim 1, wherein: in the step (a8), crushing is carried out for 50-60 times of grinding; the centrifugation is carried out at the temperature of 3-5 ℃ and the temperature of 600-.
5. The method of claim 1, wherein: in the step (a9), crushing for 4-10 times of grinding; centrifuging for more than 2 times; centrifuging for 10-20min at the temperature of 3-5 ℃ and the temperature of 600-; centrifuging at 3-5 deg.C and 8000-.
6. A method of isolating wheat protoplasts comprising the steps of:
(b1) placing chopped wheat leaves in 0.3-0.5M mannitol solution, and standing in dark for 5-15 min;
(b2) after completion of step (b1), filtering and collecting the precipitate; adding enzymolysis liquid, keeping out of the sun, and performing enzymolysis;
the solute and the concentration of the enzymolysis liquid are 15-25mM MES, 0.2-0.6M mannitol, 10-30mM KCl, 1-2% cellulase, 0.5-1% eductase and 5-15mM CaCl20.1 to 0.5 percent BSA, 3 to 8mM beta-mercaptoethanol, water as a solvent and natural pH value;
(b3) after the step (b2) is finished, adding W5 washing liquid, and carrying out light-shielding treatment for 5-15min at 26-30 ℃ and 60-80 rpm;
the solute of the W5 lotion and the concentration thereof are 200mM NaCl and 150mM CaCl, respectively22-8mM KCl, 1-4mM MES, water as solvent, and pH value of 5.7-6.0;
(b4) after completion of step (b3), filtering and collecting the filtrate; centrifuging and collecting precipitate; washing the precipitate with the W5 washing solution for more than 3 times;
(b5) after the step (b4) is completed, adding FMA solution, FMB solution and FMC solution, centrifuging, and collecting liquid between the FMB solution and the FMC solution interface, namely wheat protoplast;
the solute of FMA solution and its concentration are 0.3-0.6M sucrose, 0.8-1.2mM MgCl2pH7.0, 3-7mM HEPES, water as solvent, and natural pH value;
the solute of the FMB solution and the concentration thereof are 0.2-0.5M sucrose and 0.8-1.2mM MgCl2pH7.0, 3-7mM HEPES, 0.05-0.15M sorbitol, water as solvent, and natural pH value;
the FMC solution has a solute concentration of 0.8-1.2mM MgCl2pH7.0, 3-7mM HEPES, 0.3-0.8M sorbitol, water as solvent, and natural pH.
7. The method of claim 1 or 6, wherein: in the step (a2) or the step (b2), the proportion of the precipitate to the enzymolysis solution is 2.0g of the precipitate obtained from wheat leaves: 30-50ml of enzymolysis liquid; the enzymolysis parameter is 26-30 deg.C, and the treatment is carried out at 60-80rpm for 3-5 h.
8. The method of claim 1 or 6, wherein: in the step (a3) or the step (b3), the volume ratio of the W5 washing liquid to the enzymolysis liquid is 1: 0.5-1.5.
9. Kit A or kit B;
the kit A, comprising the enzymatic hydrolysate, the W5 lotion, the FMA solution, the FMB solution, the FMC solution, the WH solution and the localization buffer of claim 1;
the kit B comprising the enzymatic hydrolysate, the W5 lotion, the FMA solution, the FMB solution and the FMC solution of claim 1.
10, a1) -a 6):
A1) use of the method of any one of claims 1, 2, 3, 4, 5, 7 or 8 for isolating wheat cytoplasm and/or chloroplast;
A2) use of the method of any one of claims 1, 2, 3, 4, 5, 7 or 8 for cytoplasmic and/or chloroplast genome or protein extraction of wheat;
A3) use of the method of any one of claims 6 to 8 for the isolation of wheat protoplasts;
A4) use of the method of any one of claims 6-8 for wheat protoplast genome or protein extraction;
A5) the use of the kit A as claimed in claim 9 for isolating wheat cytoplasm and/or chloroplast;
A6) use of the kit of claim 9 for the isolation of wheat protoplasts.
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