Enzymatic hydrolysate for extracting leymus chinensis protoplast as well as preparation method and application of enzymatic hydrolysate

Abstract

The invention is applicable to the field of protoplast extraction application, and provides an enzymatic hydrolysate for Leymus chinensis protoplast extraction, which comprises the following components: 2.5-5% of cellulase R-10, 0.5-1% of macerozyme R-10, 0.7-1 M of mannitol, 5-9 mM of potassium chloride, 10-20 mM of MES buffer solution, 10-20 mM of calcium chloride, 0.05-0.2% of BSA, and the balance of ultrapure water. The invention also provides a preparation method and an application method of the enzymatic hydrolysate. In conclusion, according to the particularity of the leymus chinensis cell wall, an enzymatic hydrolysate formula is optimized, extraction conditions are adjusted, cell damage is effectively reduced, higher protoplast activity and quantity are obtained in leymus chinensis extraction, the extraction efficiency of the leymus chinensis protoplast is remarkably improved, and further conversion and gene expression research requirements are met.

Description

Technical Field

[0001] The invention relates to the technical field of protoplast extraction, and in particular to an enzymatic hydrolyzate for extracting protoplasts from sheep grasslands, and a preparation method and application thereof. Background Art

[0002] Protoplasts are the remaining parts of plant, bacterial and fungal cells after the cell wall is removed. They contain cell membrane, cytoplasm and nucleus. Protoplast extraction is an important technology in biology, especially in botanical research, and is widely used in genetics, cell biology and biotechnology. The cell wall-free state of protoplasts makes them particularly suitable for experiments such as genetic transformation, cell fusion and protoplast regeneration, and has become an important tool for in-depth research on cell function, gene editing and plant breeding.

[0003] The extraction methods of protoplasts include mechanical separation and enzymatic separation. The mechanical separation method is to break the cell wall by physical means to release the protoplasts. Although the operation is simple, the number of protoplasts obtained is small. The enzymatic separation method uses the action of enzymes to remove the cell wall, and a large number of protoplasts can be obtained. It has a wide range of applications and is a method frequently used in the prior art. In the enzymatic separation method, the enzymatic solution, as a component for removing the cell wall, is critical to the efficient extraction of highly active and complete protoplasts.

[0004] Patent publication number CN117821428A discloses an enzymatic hydrolyzate, a separation method and an application for separating wheat protoplasts; Patent publication number CN117721097A discloses an enzymatic hydrolyzate, a separation method and an application for separating corn protoplasts; Patent publication number CN115873782A discloses an enzymatic hydrolyzate, a kit and a method for preparing protoplasts of cabbage plants.

[0005] In the enzymatic hydrolysate used in the above-mentioned prior art, the added amount of cellulase is generally 1-2%, the pectinase or maceration enzyme is generally 0.5-1.5%, and the potassium chloride concentration is generally 10-20mM; although the protoplasts of wheat, corn, and cabbage plants can be efficiently extracted, and the regeneration rate of the protoplasts is relatively high, due to the difference in the cell wall structure of sheep fescue and other plants, it is impossible to effectively extract highly active and complete sheep fescue bioplasts.

[0006] As a typical grassland plant, the cell wall structure of Leymus chinensis has remarkable characteristics in adapting to harsh environments such as drought and cold. Compared with other plants, the cell wall has some unique biological characteristics:

[0007] 1. Cell wall components

[0008] High lignin content: The cell walls of sheepgrass have a relatively high content of lignin, a hard organic polymer that provides additional mechanical strength and helps the plant maintain structural integrity in arid, cold and windy environments.

[0009] Low pectin content: Pectin contributes to the softness of cell walls and the adhesion between cells. However, unlike some typical dicotyledonous plants (such as legumes and fruit trees), the pectin content in sheep fescue is relatively low in order to enhance the stress resistance of cell walls.

[0010] High cellulose content: The cell wall of sheepgrass is rich in cellulose components, and the cellulose microfibrils are arranged more closely, which can improve the toughness of the cell wall and its ability to resist environmental stress.

[0011] 2. Structure

[0012] Significance of the secondary cell wall: The cell wall of sheepgrass usually has a thicker secondary cell wall, which contains more lignin and other structurally enhanced polymers, making it superior in wind resistance.

[0013] Thicker cuticle: Because sheepgrass often grows in arid environments, it produces a thicker cuticle to help reduce water loss. The cuticle is a waxy substance that can effectively reduce water evaporation.

[0014] 3. Composition of cell wall proteins

[0015] Stress response-related proteins: The cell walls of Leymus chinensis contain some special stress response proteins that can respond quickly when the plant encounters drought or cold, helping the cell wall to adjust its structure to cope with environmental stress. For example, certain antioxidant enzymes and degradation enzymes can reduce damage to the cell wall structure when water is insufficient.

[0016] Presence of hydroxycinnamic acid compounds: The cell walls of Leymus chinensis may contain more hydroxycinnamic acid substances, such as ferulic acid and p-hydroxybenzoic acid. These components can increase the rigidity and toughness of the cell wall by covalently cross-linking cellulose and lignin.

[0017] 4. Adaptive cell wall metabolism

[0018] Flexibility of cell wall reconstruction: The cell wall of Leymus chinensis can be dynamically reconstructed through enzymatic reactions in different seasons and environments. For example, in the dry season, Leymus chinensis can enhance its water retention capacity by reducing the degradation of cell wall polysaccharides.

[0019] Increase β-glucan: β-glucan is commonly found in the cell walls of sheep fescue. These components can increase the elasticity and toughness of the cell walls, making it less likely for sheep fescue to break or fall over under the influence of wind and sand in the grassland environment.

[0020] 5. Regulation of cell wall porosity

[0021] The cell wall porosity of Leymus chinensis is generally low, which helps reduce water loss and improve drought resistance. Compared with other plants, the porosity regulation mechanism of Leymus chinensis cell wall is more flexible, and the opening and closing of its cell wall pores can adapt to environmental changes, thereby maintaining cell function under limited water conditions.

[0022] Due to the above differences in the cell walls of sheepgrass, the common enzymatic hydrolysate often used in the prior art cannot effectively extract highly active and complete sheepgrass bioplastids. The regeneration rate of sheepgrass bioplastids is low, which limits its promotion and use in large-scale breeding applications. Summary of the invention

[0023] In view of this, the present invention proposes an enzymatic hydrolyzate for extracting sheepgrass bioplastids, a preparation method and an application thereof. The enzymatic hydrolyzate formula is optimized according to the particularity of the sheepgrass cell wall, and the extraction conditions are adjusted to effectively reduce cell damage, thereby achieving higher protoplast activity and quantity in the sheepgrass extraction, significantly improving the extraction efficiency of sheepgrass bioplastids, and meeting the needs of further transformation and gene expression research.

[0024] The technical solution of the present invention is achieved in this way:

[0025] The invention provides an enzymolysis solution for extracting bioplastids from sheep grassland. The enzymolysis solution comprises the following components: the mass concentration of cellulase R-10 in the enzymolysis solution is 2.5-5%, the mass concentration of macerozyme R-10 in the enzymolysis solution is 0.5-1%, the molar concentration of mannitol in the enzymolysis solution is 0.7-1M, the molar concentration of potassium chloride in the enzymolysis solution is 5-9mM, the molar concentration of MES buffer in the enzymolysis solution is 10-20mM, the molar concentration of calcium chloride in the enzymolysis solution is 10-20mM, the mass concentration of BSA in the enzymolysis solution is 0.05-0.2%, and the rest is ultrapure water.

[0026] In the present invention, the addition amount of cellulase R-10 is 2.5-5%, and the addition amount is large. This is because the cell wall of Leymus chinensis contains high cellulose and lignin, resulting in the need for more enzymes to effectively lyse its cell wall. The potassium chloride concentration is 5-9mM. The main function of potassium chloride is to maintain the stability of the cell wall. Due to the characteristics of the cell wall composition, structure, protein composition, etc. of Leymus chinensis, the cell wall structure is very stable and can be effectively lysed at a lower potassium chloride concentration. The mannitol concentration is 0.7-1M, which is mainly used for osmotic regulation and protection of cell integrity. High concentrations of mannitol can maintain the permeability of the cell wall, reduce intracellular leakage and damage, and ensure the high activity and integrity of the protoplast.

[0027] The amount of cleavage enzyme R-10 added is basically close to that of the prior art, and is mainly used to decompose pectinase in the cell wall. Calcium chloride is used to stabilize the cell membrane and promote enzyme activity. BSA is mainly used to reduce the adsorption and precipitation of protoplasts on the centrifuge tube and protect the intracellular structure.

[0028] In the present invention, the pH value of the enzymatic hydrolyzate is 5.5-6.

[0029] Cellulase R-10 and cleavage enzyme R-10 are both produced by Japan YAKULT Company, and the product parameters are 10000U / g; mannitol, potassium chloride, MES buffer, calcium chloride, BSA, and ultrapure water are all commercially available reagents. The molar concentration of MES buffer is 0.4-0.6mol / L

[0030] The present invention also provides a method for preparing an enzymatic hydrolysate, comprising the following steps:

[0031] Step 1: Prepare the solution

[0032] Dissolve potassium chloride, calcium chloride and bovine serum albumin (BSA) in ultrapure water to prepare potassium chloride solution, calcium chloride solution and BSA solution respectively;

[0033] Step 2: One-time processing

[0034] Firstly, dissolve mannitol in ultrapure water, then weigh cellulase R-10 and macerate R-10, and continue to dissolve them in the ultrapure water where mannitol is located, and then add the previously prepared potassium chloride solution and MES buffer to obtain a mixed solution 1, and then place the mixed solution 1 in a 50-60°C water bath and heat it for 10-20 minutes.

[0035] The mixed solution is heated in a 50-60°C water bath to thermally inactivate nonspecific enzymes and avoid interference from nonspecific enzymes.

[0036] Step 3: Secondary processing

[0037] The heated mixed solution 1 is shielded from light, allowed to stand at room temperature to 20-25° C., and then calcium chloride solution and BSA solution are added to obtain mixed solution 2; the pH value of the mixed solution 2 is adjusted to 5.5-6 to obtain an enzymatic hydrolyzate.

[0038] The present invention also provides an application method of the enzymatic hydrolysate, comprising the following steps:

[0039] S1: remove the tip of the leymus chinensis leaf blade, wet the leymus chinensis leaf blade, and then add the enzymatic hydrolysate; the ratio of the enzymatic hydrolysate to the leymus chinensis leaf blade is 8-12 ml: 0.1-0.2 g.

[0040] The enzymatic hydrolysis solution configured by the present invention has the best enzymatic hydrolysis effect on the leopard grass leaves when the ratio of the enzymatic hydrolysis solution to the leopard grass leaves is 8-12 ml: 0.1-0.2 g.

[0041] S2 vacuum-treats the sheep fescue leaves added with the enzymatic hydrolysate, and the vacuum degree is reduced to -0.03 to -0.08 MPa. The vacuum treatment time is 0.8 to 1.2 hours.

[0042] The vacuum-treated leaves of Leymus chinensis were mixed by plane oscillation, and enzymolysis was performed on an oscillator at a speed of 28-34 rpm, the enzymolysis time was 2.5-5 h, and the enzymolysis temperature was 25-30°C;

[0043] The leaves of Leymus chinensis are enzymatically lysed under vacuum. This process ensures that the gas in the leaves of Leymus chinensis is fully discharged, thereby helping the enzymatic solution to penetrate into the cells more effectively and achieve a more efficient protoplast separation effect.

[0044] Since the protoplasts of sheep prairie are very fragile, the speed of about 50 rpm often used in the prior art can easily cause mechanical damage and reduce the number and activity of protoplasts.

[0045] The invention performs enzymolysis at a speed of 20-28 rpm, which ensures that the sheepgrass and the enzymolysis solution are evenly mixed, is more conducive to the full contact between the enzyme and the sheepgrass leaf tissue, promotes the gradual lysis of the cell wall, avoids structural damage caused by violent oscillation, and the mild oscillation speed can effectively protect the integrity of the protoplast after the lysis process is completed, providing more ideal conditions for obtaining high-activity and high-quality sheepgrass protoplasts.

[0046] S3: Pass the enzymatically hydrolyzed Leymus chinensis leaves through 100-mesh gauze to filter out unlysed fragments; then filter through a 40 μm cell strainer to remove impurities and collect the crude protoplast mixture.

[0047] After the protoplast hydrolysate decomposes the cell wall, impurities such as larger cell clumps and leaf tissue that has not been completely hydrolyzed may remain in the mixed solution. These residues will not only affect the purity of the protoplasts, but may also hinder subsequent experimental operations.

[0048] S4: centrifuge the crude protoplast mixture at 3-5°C and 100-130g for 1-3 minutes at a low speed, repeat the centrifugation 3-5 times, remove the enzymatic solution, and obtain sheep grassland protoplasts.

[0049] In addition, in the prior art, in order to destroy disulfide bonds or further soften the cell wall, a reducing agent β-mercaptoethanol is generally added, and the structure of the sheep grass cell wall of the present invention is stable, and the main resistance of the sheep grass cell wall comes from its high lignin content. A high concentration of cellulase R-10 is used, and then combined with cleavage enzyme R-10, and a high concentration of mannitol is used at the same time to maintain the permeability of the cell wall, reduce intracellular leakage and damage, and physically soften the cell wall, which is sufficient to meet the cleavage requirements of the sheep grass cell wall; in the prior art, the addition of mercaptoethanol is to cope with its lower degree of lignification, so as to reduce the resistance of polysaccharide bonds on the cell wall and enhance the enzymatic hydrolysis effect, but mercaptoethanol has a strong reducing property and may damage some cell components, thereby affecting the activity of the protoplasts. In the present invention, mercaptoethanol is not added, and the high activity and integrity of the protoplasts can be ensured.

[0050] The enzymatic hydrolysate for extracting bioplastids from sheep grassland of the present invention has the following beneficial effects compared with the prior art:

[0051] 1. Since the cell wall of Leymus chinensis contains high cellulose and lignin, it is difficult to effectively cleave it during the enzymatic hydrolysis process, and a large amount of cellulase R-10 needs to be added. For the 1-2% addition amount in the prior art, if it is applied to the enzymatic hydrolysis of Leymus chinensis of the present invention, the enzymatic hydrolysis is not thorough, the extraction rate is low, and the number of protoplasts obtained is reduced.

[0052] 2. Since the cell wall of Leymus chinensis contains higher cellulose and lignin, thicker secondary cell wall, and special stress response proteins, the strength and resistance of its cell wall are increased, making the cell wall structure very stable. Therefore, the potassium chloride concentration can be reduced to 5-9 mM. The potassium chloride concentration in the prior art is 10-20 mM, which will lead to a waste of potassium chloride when applied to the enzymatic hydrolysis of Leymus chinensis of the present invention.

[0053] 3. Since the cell wall structure of Leymus chinensis is very stable, it is necessary to increase the concentration of mannitol to maintain the permeability of the cell wall and reduce intracellular leakage and damage. The mannitol concentration of the present invention is 0.7-1M. For the 0.2-0.5M mannitol frequently used in the prior art, if it is applied to the enzymatic hydrolysis of Leymus chinensis of the present invention, the permeability of the cell wall cannot be guaranteed, the protoplast damage rate is high, and the activity and integrity of the protoplasts are reduced.

[0054] 4. Mercaptoethanol, a cell protectant often used in the prior art, is used in the enzymatic hydrolysis of Leymus chinensis of the present invention to cope with its lower degree of lignification. Since the cell wall of Leymus chinensis contains higher levels of cellulose and lignin, the activity of the protoplasts will be reduced. Therefore, mercaptoethanol is not used in the enzymatic hydrolyzate formula of the present invention.

[0055] 5. During the preparation of the enzymatic hydrolysate, heat it in a 50-60℃ water bath to thermally inactivate nonspecific enzymes and avoid interference from nonspecific enzymes, making the prepared enzymatic hydrolysate purer and extracting more protoplasts.

[0056] 6. The enzymatic hydrolysis solution configured by the present invention has the best enzymatic hydrolysis effect on the leopard grass leaves when the ratio of the enzymatic hydrolysis solution to the leopard grass leaves is 8-12 ml: 0.1-0.2 g.

[0057] 7. During the application of the enzymatic hydrolyzate, since the protoplasts of sheep grassland are very fragile, the speed of about 50 rpm often used in the prior art can easily cause mechanical damage and reduce the number and activity of the protoplasts; the present invention performs enzymatic hydrolysis at a speed of 20-28 rpm, which can promote the gradual lysis of the cell wall, avoid structural damage caused by violent vibrations, effectively protect the integrity of the protoplasts, and improve the activity of the protoplasts. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0059] Figure 1 The resuspended protoplasts in Example 1 of the present invention;

[0060] Figure 2 This is an image of the sheep grassland bioplastid in Example 2 under an optical microscope;

[0061] Figure 3 This is the image of the sheep grassland bioplastid in comparative example 3 under an optical microscope. DETAILED DESCRIPTION

[0062] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0063] Example 1

[0064] The enzymatic solution for extracting the bioplastid of sheep grassland includes the following components: Cellulase R-10

[0065] (CELLULASE R-10) 2.5%, macerozyme R-10 (MACEROZYME R-10) 0.5%, mannitol 0.7M, potassium chloride 5mM, MES buffer 10mM, calcium chloride 10mM, BSA 0.05%, and the rest are ultrapure water.

[0066] The molar concentration of MES buffer is 0.4 mol / L.

[0067] The preparation method of the enzymatic hydrolysate comprises the following steps:

[0068] Step 1: Prepare the solution

[0069] Dissolve 0.55g potassium chloride in ultrapure water and dilute to 20ml to prepare potassium chloride solution.

[0070] Dissolve 0.88g of calcium chloride in ultrapure water and dilute to 20ml to prepare calcium chloride solution.

[0071] Dissolve 0.1 g of bovine serum albumin (BSA) in ultrapure water and dilute to 1 ml to prepare a BSA solution.

[0072] Step 2: One-time processing

[0073] First, dissolve 4.1g of mannitol in 30ml of ultrapure water, then weigh 0.8g of cellulase R-10 and 0.45g of macerate R-10, and continue to dissolve them in the ultrapure water where the mannitol is located, and then add 1ml of the previously prepared potassium chloride solution and 800ul of 0.4mol / L MES buffer, and make up to 36ml with ultrapure water to obtain a mixed solution 1, and then place the mixed solution 1 in a preheated 51°C water bath and heat it for 10min.

[0074] Step 3: Secondary processing

[0075] The heated mixed solution 1 was shielded from light, allowed to stand at room temperature until it reached 20°C, and then 1 ml of calcium chloride solution and 40 ul of BSA solution were added, and the volume was fixed to 40 ml with ultrapure water to obtain mixed solution 2; the pH value of mixed solution 2 was adjusted to 5.5 to obtain an enzymatic hydrolyzate.

[0076] The application method of the enzymatic hydrolysate comprises the following steps:

[0077] S1 perennial leymus was planted in a growth chamber with a daytime temperature of 25°C, a night temperature of 20°C, and a daily photoperiod of 14 hours. The plants were grown in a substrate of vermiculite, perlite, and peat moss mixed in a ratio of 1:1:3, watered every three days, and fertilized weekly with 1 / 2 Hoagland nutrient solution.

[0078] Select healthy and well-growing plants from 2-week-old cultured sheepgrass, select fresh young leaves from the top, remove the leaf tip and weigh 0.7g, place the leaves in a 9×9cm culture dish, use a disposable syringe to absorb the prepared enzymatic solution, open the 0.45μm filter membrane and install it on the gun tip, add an appropriate amount of enzymatic solution to the pre-cooled culture dish to keep the leaves moist when shredding. Wash the leaves with sterile water and dry the surface moisture with filter paper, put them in the culture dish and cut them into filaments with a width of 0.5mm. After all are completed, add the remaining enzyme solution, and the total amount of enzymatic solution added twice is 56ml. Wrap the culture dish with sealing film and wrap it with tin foil to keep it in a dark environment. S2 slowly moves to the polycarbonate dryer, turns on the AP-9100A vacuum pump, evacuates the vacuum degree to -0.03MPa, and vacuums for 0.8h. Slowly open the vacuum-treated dryer, and then slowly place the tin foil-wrapped culture dish in the oscillator, and keep the enzymatic solution at 28rpm and 25℃ for 2.5h.

[0079] After the S3 enzymatic hydrolysis is completed, take out the W5 solution from the refrigerator. Place a 40μm cell strainer on a sterile-free 50ml centrifuge tube, moisten the filter membrane with the W5 solution, open the tin foil in a dark environment, filter the mixture after enzymatic hydrolysis through a sterilized 100-mesh gauze to remove impurities, use a 5ml pipette tip and use scissors to widen the front end of the tip to minimize mechanical damage to transfer the protoplast enzymatic solution, tilt the test tube to slowly pass the filtered enzymatic solution in the pipette through the 40μm cell strainer, and inject it into the 50ml centrifuge tube, then use 5ml W5 solution to wash the original culture dish and the tissue on the gauze, filter the remaining protoplasts, and repeat adding to the 50ml centrifuge tube containing the 40μm cell strainer.

[0080] S4 Place the 50ml centrifuge tube containing the filtrate in a desktop low-speed refrigerated centrifuge and centrifuge at low speed for 1 minute at 3°C ​​and 100g. Use a 5ml pipette with a widened tip of scissors to slowly probe from the top of the solution and slowly absorb the enzymatic solution. The range of movement should not be too large, and the extraction speed should not be too fast. Discard the supernatant as much as possible. The partial solution of the protoplast precipitation near the bottom of the centrifuge tube can be slowly sucked out with a 1ml pipette with a widened tip of scissors. Resuspend the precipitated protoplasts in 10ml pre-cooled W5 solution, place it in a desktop low-speed refrigerated centrifuge repeatedly, centrifuge it 3 times, and discard all the enzymatic solution. Then resuspend it with 400ul W5 solution and place it on ice to obtain viable and complete sheep grassland protoplasts, see. Figure 1 .

[0081] The preparation steps of W5 solution include: weighing 0.9 g sodium chloride, 1.4 g calcium chloride, 0.4 g potassium chloride and 400 ul of 0.5 mol / L MES solution on a weighing balance, dissolving them in ultrapure water and making the volume to 500 ml, and adjusting the pH value of the solution to 5.5.

[0082] Example 2

[0083] The enzymatic hydrolysate used for extracting the bioplastid of sheep grassland includes the following components: 3% of cellulase R-10, 0.8% of macerozyme R-10, 0.8M of mannitol, 8mM of potassium chloride, 15mM of MES buffer, 18mM of calcium chloride, 0.15% of BSA, and the rest of ultrapure water.

[0084] The molar concentration of MES buffer is 0.5 mol / L.

[0085] The preparation method of the enzymatic hydrolysate comprises the following steps:

[0086] Step 1: Prepare the solution

[0087] Dissolve 0.61g potassium chloride in ultrapure water and make up to 20ml to prepare potassium chloride solution.

[0088] Dissolve 0.90g of calcium chloride in ultrapure water and dilute to 20ml to prepare calcium chloride solution.

[0089] Dissolve 0.1 g of bovine serum albumin (BSA) in ultrapure water and dilute to 1 ml to prepare a BSA solution.

[0090] Step 2: One-time processing

[0091] First, dissolve 4.5g of mannitol in 30ml of ultrapure water, then weigh 0.8g of cellulase R-10 and 0.45g of macerate R-10, and continue to dissolve them in the ultrapure water where the mannitol is located, and then add 1ml of the previously prepared potassium chloride solution and 800ul of 0.5mol / L MES buffer, and make up to 38ml with ultrapure water to obtain a mixed solution 1, and then place the mixed solution 1 in a preheated 57°C water bath and heat it for 16min.

[0092] Step 3: Secondary processing

[0093] The heated mixed solution 1 was shielded from light, allowed to stand at room temperature until it reached 22°C, and then 1 ml of calcium chloride solution and 40 ul of BSA solution were added, and the volume was fixed to 40 ml with ultrapure water to obtain mixed solution 2; the pH value of mixed solution 2 was adjusted to 5.7 to obtain an enzymatic hydrolyzate.

[0094] The application method of the enzymatic hydrolysate comprises the following steps:

[0095] S1 perennial leymus was planted in a growth chamber with a daytime temperature of 25°C, a night temperature of 20°C, and a daily photoperiod of 14 hours. The plants were grown in a substrate of vermiculite, perlite, and peat moss mixed in a ratio of 1:1:3, watered every three days, and fertilized weekly with 1 / 2 Hoagland nutrient solution.

[0096] Select healthy and well-growing plants from 18-day-old cultured Leymus chinensis, select fresh young leaves from the top, remove the leaf tips and weigh 0.8g, place the leaves in a 9×9cm culture dish, use a disposable syringe to absorb the prepared enzymatic solution, open a 0.45μm filter membrane and install it on the gun tip, add an appropriate amount of enzymatic solution to the pre-cooled culture dish to keep the leaves moist when shredding. Wash the leaves with sterile water and use filter paper to absorb the surface moisture, put them in the culture dish and cut them into filaments with a width of 0.5mm. After all are completed, add the remaining enzyme solution, and the total amount of enzymatic solution added twice is 80ml. Wrap the culture dish with sealing film and wrap it with tin foil to keep it in a dark environment.

[0097] S2 was slowly moved to the polycarbonate desiccator, and the AP-9100A vacuum pump was turned on to evacuate the vacuum to -0.05 MPa. The vacuum treatment was performed for 1.0 h. The desiccator after vacuum treatment was slowly opened, and the culture dish wrapped in tin foil was slowly placed in the oscillator. The enzymatic hydrolysis was maintained at 30 rpm and 28 °C for 3.5 h.

[0098] After the S3 enzymatic hydrolysis is completed, take out the W5 solution from the refrigerator. Place a 40μm cell strainer on a sterile-free 50ml centrifuge tube, moisten the filter membrane with the W5 solution, open the tin foil in a dark environment, filter the mixture after enzymatic hydrolysis through a sterilized 100-mesh gauze to remove impurities, use a 5ml pipette tip and use scissors to widen the front end of the tip to minimize mechanical damage to transfer the protoplast enzymatic solution, tilt the test tube to slowly pass the filtered enzymatic solution in the pipette through the 40μm cell strainer, and inject it into the 50ml centrifuge tube, then use 5ml W5 solution to wash the original culture dish and the tissue on the gauze, filter the remaining protoplasts, and repeat adding to the 50ml centrifuge tube containing the 40μm cell strainer.

[0099] S4 Place the 50ml centrifuge tube containing the filtrate in a low-speed refrigerated centrifuge on a tabletop, centrifuge at 4°C, 120g for 2 minutes, and use a 5ml pipette with a widened tip of scissors to slowly probe from the top of the solution and slowly absorb the enzymatic solution. The range of motion should not be too large, and the extraction speed should not be too fast. Discard the supernatant as much as possible. The part of the solution near the bottom of the centrifuge tube where the protoplasts are precipitated can be slowly sucked out with a 1ml pipette with a widened tip of scissors. Resuspend the precipitated protoplasts in 10ml pre-cooled W5 solution, repeatedly place it in a low-speed refrigerated centrifuge on the tabletop, centrifuge it 4 times, and discard all the enzymatic solution. Then resuspend it with 400ul W5 solution and place it on ice to obtain viable and complete sheep grassland bioplasts.

[0100] The preparation steps of W5 solution include: weighing 0.92g sodium chloride, 1.38g calcium chloride, 0.39g potassium chloride and 400ul of 0.5mol / L MES solution on a weighing balance, dissolving them in ultrapure water and making the volume to 500ml, and adjusting the pH value of the solution to 5.7.

[0101] Example 3

[0102] The enzymatic hydrolysate used for extracting the bioplastid of sheep grassland includes the following components: 5% of cellulase R-10, 1% of macerozyme R-10, 1M of mannitol, 9mM of potassium chloride, 20mM of MES buffer, 20mM of calcium chloride, 0.2% of BSA, and the rest is ultrapure water.

[0103] The molar concentration of MES buffer is 0.6 mol / L.

[0104] The preparation method of the enzymatic hydrolysate comprises the following steps:

[0105] Step 1: Prepare the solution

[0106] Dissolve 0.63g potassium chloride in ultrapure water and dilute to 20ml to prepare potassium chloride solution.

[0107] Dissolve 0.94g of calcium chloride in ultrapure water and dilute to 20ml to prepare calcium chloride solution.

[0108] Dissolve 0.1 g of bovine serum albumin (BSA) in ultrapure water and dilute to 1 ml to prepare a BSA solution.

[0109] Step 2: One-time processing

[0110] First, dissolve 4.5g of mannitol in 30ml of ultrapure water, then weigh 0.8g of cellulase R-10 and 0.45g of macerate R-10, and continue to dissolve them in the ultrapure water where the mannitol is located, and then add 1ml of the previously prepared potassium chloride solution and 800ul of 0.6mol / L MES buffer, and make up to 38ml with ultrapure water to obtain a mixed solution 1, and then place the mixed solution 1 in a preheated 60°C water bath and heat it for 20min.

[0111] Step 3: Secondary processing

[0112] The heated mixed solution 1 was shielded from light, allowed to stand at room temperature until it reached 25°C, and then 1 ml of calcium chloride solution and 40 ul of BSA solution were added, and the volume was fixed to 40 ml with ultrapure water to obtain mixed solution 2; the pH value of mixed solution 2 was adjusted to 6 to obtain enzymatic hydrolyzate.

[0113] The application method of the enzymatic hydrolysate comprises the following steps:

[0114] S1 perennial leymus was planted in a growth chamber with a daytime temperature of 25°C, a night temperature of 20°C, and a daily photoperiod of 14 hours. The plants were grown in a substrate of vermiculite, perlite, and peat moss mixed in a ratio of 1:1:3, watered every three days, and fertilized weekly with 1 / 2 Hoagland nutrient solution.

[0115] Select healthy and well-growing plants from 20-day-old cultured Leymus chinensis, select fresh young leaves from the top, remove the leaf tips and weigh 0.5g, place the leaves in a 9×9cm culture dish, use a disposable syringe to absorb the prepared enzymatic solution, open a 0.45μm filter membrane and install it on the gun tip, add an appropriate amount of enzymatic solution to the pre-cooled culture dish to keep the leaves moist when shredding. Wash the leaves with sterile water and use filter paper to absorb the surface moisture, put them in the culture dish and cut them into filaments with a width of 0.5mm. After all are completed, add the remaining enzyme solution. The total amount of enzymatic solution added twice is 60ml. Wrap the culture dish with sealing film and wrap it with tin foil to keep it in a dark environment.

[0116] S2 was slowly moved to the polycarbonate desiccator, and the AP-9100A vacuum pump was turned on to evacuate the vacuum to -0.08 MPa. The vacuum treatment was performed for 1.2 h. The desiccator after vacuum treatment was slowly opened, and the culture dish wrapped in tin foil was slowly placed in the oscillator. The enzymatic hydrolysis was maintained at 34 rpm and 30 °C for 5 h.

[0117] After the S3 enzymatic hydrolysis is completed, take out the W5 solution from the refrigerator. Place a 40μm cell strainer on a sterile-free 50ml centrifuge tube, moisten the filter membrane with the W5 solution, open the tin foil in a dark environment, filter the mixture after enzymatic hydrolysis through a sterilized 100-mesh gauze to remove impurities, use a 5ml pipette tip and use scissors to widen the front end of the tip to minimize mechanical damage to transfer the protoplast enzymatic solution, tilt the test tube to slowly pass the filtered enzymatic solution in the pipette through the 40μm cell strainer, and inject it into the 50ml centrifuge tube, then use 5ml W5 solution to wash the original culture dish and the tissue on the gauze, filter the remaining protoplasts, and repeat adding to the 50ml centrifuge tube containing the 40μm cell strainer.

[0118] S4 Place the 50ml centrifuge tube containing the filtrate in a low-speed refrigerated centrifuge on a tabletop, centrifuge at 5°C, 130g for 3 minutes, and use a 5ml pipette with a widened tip of scissors to slowly probe from the top of the solution and slowly absorb the enzymatic solution. The range of motion should not be too large, and the extraction speed should not be too fast. Discard the supernatant as much as possible. The part of the solution near the bottom of the centrifuge tube where the protoplasts are precipitated can be slowly sucked out with a 1ml pipette with a widened tip of scissors. Resuspend the precipitated protoplasts in 10ml pre-cooled W5 solution, repeatedly place it in a low-speed refrigerated centrifuge on the tabletop, centrifuge it 5 times, and discard all the enzymatic solution. Then resuspend it with 400ul W5 solution and place it on ice to obtain viable and complete sheep grassland bioplasts.

[0119] The preparation steps of W5 solution include: weighing 0.93g sodium chloride, 1.44g calcium chloride, 0.39g potassium chloride and 400ul of 0.5mol / L MES solution on a weighing balance, dissolving them in ultrapure water and making the volume to 500ml, and adjusting the pH value of the solution to 6.

[0120] Comparative Example 1

[0121] On the basis of Example 1, the concentration of mannitol in the enzymatic hydrolysate was adjusted to 0.6 M, and other conditions were the same.

[0122] Comparative Example 2

[0123] On the basis of Example 1, the concentration of mannitol in the enzymatic hydrolysate was adjusted to 0.5 M, and other conditions were the same.

[0124] Comparative Example 3

[0125] On the basis of Example 1, the addition amount of cellulase R-10 in the enzymatic hydrolysate components was adjusted to 2.2%, and other conditions were the same.

[0126] Comparative Example 4

[0127] On the basis of Example 1, the addition amount of cellulase R-10 in the enzymatic hydrolysate components was adjusted to 2%, and other conditions were the same.

[0128] Comparative Example 5

[0129] On the basis of Example 1, the addition amount of cellulase R-10 in the enzymatic hydrolysate components was adjusted to 1.5%, and other conditions were the same.

[0130] Comparative Example 6

[0131] On the basis of Example 2, the potassium chloride concentration in the enzymatic hydrolysate components was adjusted to 10 mM, and other conditions were the same.

[0132] Comparative Example 7

[0133] On the basis of Example 2, the potassium chloride concentration in the enzymatic hydrolysate components was adjusted to 12 mM, and other conditions were the same.

[0134] Comparative Example 8

[0135] On the basis of Example 2, in the first treatment of step 2, the mixed solution 1 is not heated in a water bath, and other conditions are the same.

[0136] Comparative Example 9

[0137] On the basis of Example 2, in the first treatment of step 2, the mixed solution 1 is placed in a 45° C. water bath for heating, and other conditions are the same.

[0138] Comparative Example 10

[0139] On the basis of Example 2, in the first treatment of step 2, the mixed solution was heated for 5 minutes, and the other conditions were the same.

[0140] Comparative Example 11

[0141] On the basis of Example 2, in the first treatment of step 2, the mixed solution was heated for 25 min, and other conditions were the same.

[0142] Comparative Example 12

[0143] On the basis of Example 3, in step S2, the culture dish wrapped in tin foil was slowly placed in an oscillator and enzymatic hydrolysis was performed at a speed of 30 rpm, and other conditions were the same.

[0144] Comparative Example 13

[0145] On the basis of Example 3, in step S2, the culture dish wrapped in tin foil was slowly placed in an oscillator and enzymatic hydrolysis was performed at a speed of 35 rpm, and other conditions were the same.

[0146] Comparative Example 14

[0147] On the basis of Example 3, 0.2% of mercaptoethanol was added to the enzymatic hydrolysate components, and other conditions were the same.

[0148] Comparative Example 15

[0149] On the basis of Example 3, 0.05% of mercaptoethanol was added to the enzymatic hydrolysate components, and other conditions were the same.

[0150] Comparative Example 16

[0151] On the basis of Example 3, the ratio of enzymatic hydrolysate to Leymus chinensis leaf blade was 5 ml:0.1 g, that is, the leaf tip weighed 0.5 g, and the total amount of enzymatic hydrolysate added twice was 25 ml; other conditions were the same.

[0152] Comparative Example 17

[0153] On the basis of Example 3, the ratio of enzymatic hydrolysate to Leymus chinensis leaf blade was 15 ml:0.1 g, that is, the leaf tip weighed 0.5 g, and the total amount of enzymatic hydrolysate added twice was 75 ml; other conditions were the same.

[0154] The protoplasts obtained in Example 1-3 and Comparative Example 1-17 were subjected to protoplast yield statistics and activity detection.

[0155] Protoplast yield statistics

[0156] Based on the selective permeability of the cell membrane, normal cells have a complete cell membrane structure that can control the entry and exit of substances. Therefore, living cells can prevent the trypan blue dye from entering the cell and remain colorless. When the cell dies, the integrity of the cell membrane is lost, and the trypan blue dye can pass through the deformed cell membrane and combine with the disintegrated DNA to color it. The cells that are not stained are living cells, and the cells that are stained blue are dead cells, thus distinguishing dead cells from living cells.

[0157] The protoplast yield statistics include the following steps:

[0158] a. Prepare the protoplast heavy suspensions in Examples and Comparative Examples respectively.

[0159] b. Take out a clean cell counting plate, cover the counting area with a coverslip, and slowly add 8ul of protoplast resuspension from the sample ports on both sides of the counting pool. Be careful not to have bubbles. Four samples can be counted at the same time. Then add 1ul of trypan blue (0.4%) dye to one side and let it slowly penetrate into the sample.

[0160] c. Place the counting plate on the microscope, find the counting area, and let it stand for 2 minutes. Only when the cells settle on the counting plate and no longer drift with the liquid can they be counted. Only the unstained spherical green cells without damage are selected for counting.

[0161] d. Count cells within the grid. When counting cells close to the grid border (double lines), follow the principle of "counting the top but not the bottom, and counting the left but not the right". If the number of cells in each grid differs by more than 20, it indicates that the cells are unevenly distributed. Recount until the cells are evenly distributed and the number of cells is calculated. Count the 16 small squares in each grid in the counting area in turn, and follow the S-shaped counting principle.

[0162] (See Figure 2 and Figure 3 , since there are many pictures obtained in the activity detection experiment of the present invention, the present invention only shows two pictures).

[0163] Protoplast Viability Assay

[0164] The present invention measures the activity of protoplasts by the FDA method, which is a prior art and the specific detection steps will not be described in detail.

[0165] The yield statistics and vitality test results of protoplasts are shown in Table 1.

[0166]

[0167]

[0168] Generally, the protoplast concentration used in transient gene expression and transformation experiments needs to reach 10 5 -106 cells / mL. This concentration range can usually ensure transformation efficiency and experimental reproducibility. If a larger-scale transformation or a more complex experiment is performed, such as protein-protein interaction or subcellular localization experiments, the protoplast concentration needs to be increased to 5×10 6 cells / mL or above.

[0169] It can be seen from Examples 1-3 that the present invention can separate 5.6×10 7 -9.2×10 7 The number of protoplasts is large, and the activity can reach more than 95%, which shows that the number and activity of the extracted protoplasts are very good and can meet the needs of large-scale or high-complexity experiments.

[0170] In Comparative Examples 1-17, the number or activity of protoplasts was affected due to changes in the components or the addition amounts of the components in the enzymatic hydrolysate formula, or changes in the preparation method or use method of the enzymatic hydrolysate.

[0171] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An enzymatic hydrolysate for extracting bioplastids from sheep grassland, characterized in that: The invention comprises the following components: 2.5-5% of cellulase R-10, 0.5-1% of macerate R-10, 0.7-1M of mannitol, 5-9mM of potassium chloride, 10-20mM of MES buffer, 10-20mM of calcium chloride, 0.05-0.2% of BSA, and the rest is ultrapure water.

2. The enzymatic hydrolyzate for extracting bioplastids from sheep grassland as claimed in claim 1, characterized in that: The pH value of the enzymatic hydrolysate is 5.5-6.

3. The enzymatic hydrolyzate for extracting bioplastids from sheep grassland according to claim 1, characterized in that: The molar concentration of the MES buffer is 0.4-0.6 mol / L.

4. A method for preparing the enzymatic hydrolyzate according to claim 1, characterized in that: The steps include: Step 1: Prepare the solution Dissolve potassium chloride, calcium chloride and BSA in ultrapure water to prepare potassium chloride solution, calcium chloride solution and BSA solution respectively; Step 2: One-time processing Firstly, dissolve mannitol in ultrapure water, then weigh cellulase R-10 and macerate R-10, and continue to dissolve them in the ultrapure water where mannitol is located, and then add the potassium chloride solution and MES buffer to obtain a mixed solution 1; then, place the mixed solution 1 in a 50-60° C. water bath and heat it for 10-20 minutes; Step 3: Secondary processing The heated mixed solution 1 is shielded from light, allowed to stand at room temperature to 20-25° C., and then the calcium chloride solution and the BSA solution are added to obtain a mixed solution 2; the pH value of the mixed solution 2 is adjusted to 5.5-6 to obtain an enzymatic hydrolyzate.

5. A method for using the enzymatic hydrolysis solution according to claim 4, characterized in that: The steps include: S1: removing the tip of the chinensis leaf blade, wetting the chinensis leaf blade, and then adding the enzymolysis solution; S2 vacuum-treats the chinensis leaves added with the enzymolysis solution, and then enzymolyzes the vacuum-treated chinensis leaves on an oscillator at a speed of 28-34 rpm, the enzymolysis time is 2.5-5 hours, and the enzymolysis temperature is 25-30° C.; S3: filtering the enzymatically hydrolyzed chinensis leaves to collect the crude protoplast mixture; S4: centrifuging the crude protoplast mixture at a low speed for 1-3 min at 3-5° C. and 100-130 g, repeating the centrifugation 3-5 times, removing the enzymatic solution, and obtaining sheep grassland protoplasts.

6. The method for using the enzymolysis solution according to claim 5, characterized in that: In the step S1, the ratio of the enzymatic hydrolysate to the chinensis leaves is 8-12 ml: 0.1-0.2 g.

7. The method for using the enzymolysis solution according to claim 5, characterized in that: In the step S2, during the vacuum treatment, the vacuum degree is evacuated to -0.03 to -0.08 MPa, and the vacuum treatment time is 0.8 to 1.2 h.

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