CN113897330A - Enzymolysis method for quickly removing cell walls of poplar or eucalyptus and application - Google Patents
Enzymolysis method for quickly removing cell walls of poplar or eucalyptus and application Download PDFInfo
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Abstract
The invention relates to an enzymolysis method for quickly removing cell walls of poplar or eucalyptus, which is characterized in that poplar or eucalyptus leaves pretreated by plasmolysis are placed into an enzymolysis solution by utilizing a specially prepared enzymolysis solution and treated at room temperature, so that a poplar or eucalyptus protoplast with the integrity of 90% can be obtained. The method has great application value for cultivating excellent new varieties of poplar and eucalyptus through the field of genetic engineering by quickly removing the cell walls of the poplar and the eucalyptus.
Description
Technical Field
The invention relates to the field of plant genetic engineering, in particular to an enzymolysis method for quickly removing cell walls of poplars and eucalyptuses.
Technical Field
The poplar is a main artificial forest planting tree species in the yellow river basin of China, is widely planted in northern areas of China due to rapid growth, drought resistance, barren resistance and beautiful tree shape, is a main tree species for landscape greening of urban streets and expressways, is also an important tree species for maintaining and controlling wind prevention, sand fixation and returning to farming in ecological environment, and plays an important role in forestry industries such as wood processing, paper making and the like. At present, the total area of poplar artificial forests in China is 850 million hectares, which exceeds 1 hundred million acres (2018 data), and the poplar artificial forests are the first place in the world. However, the traditional poplar seedling breeding has the defects of long breeding period and difficulty in performing diversified integration on excellent characters, and simultaneously has the defect of relatively single high-quality variety, so that the diversified requirements of our country on ecological construction, vegetation recovery and forest carbon sink capacity increase are difficult to meet.
Eucalyptus is an economic tree species widely planted in Guangxi, Guangdong, Fujian and other places in south China, contributes to more than one third of wood yield in China every year, and plays a key supporting role in maintaining wood safety in China and building strategic stock bases of wood. Only Guangxi province of China, the planting area reaches more than 3000 mu and ten thousand mu, the wood yield is more than 2500 million cubic meters, and the economic benefit scale reaches more than 4700 hundred million. Meanwhile, the eucalyptus absorbs more than 8.5 hundred million tons of carbon dioxide every year in the aspects of emission reduction and carbon absorption and environmental protection, and also makes great contribution to the environmental protection industry of China. However, eucalyptus is covered by the external numbers of a water pump and a fertilizer extractor in the development process, because eucalyptus grows rapidly, and the demand for water and fertilizer is great. How to cultivate new eucalyptus species by means of genetic engineering and efficiently utilizing 'moisture' and 'fertilizer' has important significance for winning 'turning over in the breeding industry' and solving the difficult points in the forestry seedling industry.
In order to better solve the breeding problem of populus and eucalyptus, plant protoplasts need to be separated, the problem of wall breaking of cell walls needs to be solved, and the cell walls consisting of pectin, cellulose, hemicellulose, lignin and the like need to be removed. Forest plants are more complex in their cell wall structure due to their generally higher degree of lignification than herbaceous plants. In the early stage of this century, a mechanical separation method was used, in which mesophyll cells, callus and liquid suspension culture cells were placed in a hypertonic sugar solution to separate the walls and the plastids, and the protoplasts were contracted into spheres. The tissue is then cut with scissors, and the cell wall can be dissected to obtain a small number of intact protoplasts. However, this method separates too few protoplasts and is only suitable for a part of the tissue. The enzymatic hydrolysis method invented in the later stage opens up a new technology for separating a large amount of protoplasts, but how to rapidly remove the cell walls of the poplar and the eucalyptus and obtain the protoplasts of the poplar and the eucalyptus with the integrity of 90% or more is not reported so far.
Disclosure of Invention
In order to solve the technical problems, the invention provides an enzymolysis method for quickly removing the cell walls of poplar or eucalyptus, which comprises the steps of carrying out enzymolysis on the leaves of the poplar or eucalyptus by using a prepared enzymolysis solution to remove the cell walls, and carrying out enzymolysis incubation to obtain the protoplast of the poplar or eucalyptus. The method can rapidly and completely dissociate the cell walls of the poplar and the eucalyptus, the dissociation degree can reach 90% or above, and the method has great application potential.
The invention also provides a protoplast prepared by an enzymolysis method for quickly removing the cell wall of the poplar or the eucalyptus.
The invention also provides the application of the protoplast in the transformation of exogenous DNA plasmids.
The invention is realized by the following technical scheme:
an enzymolysis method for quickly removing the cell wall of poplar or eucalyptus at least comprises the following steps: and performing enzymolysis on the leaves of the poplar or the eucalyptus by using the prepared enzymolysis solution to remove cell walls, and performing enzymolysis incubation.
Further preferably, the enzymatic hydrolysis method for rapidly removing the cell wall of the poplar or the eucalyptus comprises the following steps: soaking leaves of poplar and eucalyptus in the solution I; and (3) placing the poplar or eucalyptus leaves soaked by the solvent I into an enzymolysis solution for enzymolysis to obtain an enzymolysis solution, and filtering and purifying after the enzymolysis is finished.
In one embodiment, the composition of the enzymatic hydrolysis solution of the present invention includes the following components by mass percentage or concentration: 1-2% (w/v) cellulase R-10, 0.4-0.8% (w/v) macerozyme R-10, 0.2-1.0% (w/v) pectinase Y-23,0.4-0.8mol/L mannitol, 18-22mmol/L potassium chloride KCl, 18-22mmol/L2- (N-morpholinyl) ethanesulfonic acid (MES), 8-12mmol/L calcium chloride, 0.03-0.08mmol/L beta-mercaptoethanol, 0.08-0.12% (w/v) Bovine Serum Albumin (BSA).
The enzymolysis solution is suitable for preparing the poplar or eucalyptus protoplast, and the specific type and proportion of the enzymolysis solution can be subjected to enzymolysis quickly without damaging the poplar or eucalyptus protoplast. The pectinase can degrade pectin in the cell walls of populus and eucalyptus to separate the cells, and then cellulase R-10 is used for treating and degrading the cell walls. Pectase is added to degrade pectic substance, so that cell wall can be rapidly eliminated, and protoplast can be obtained. The addition of bovine serum albumin reduces or prevents damage to organelles during wall degradation.
In one embodiment, the composition of the enzymolysis solution of the present invention, the cellulase R-10 may be 1.3-1.4% (w/v), 1.4-1.5% (w/v), 1.5-1.8% (w/v), or 1.8-2.0% (w/v) by mass.
In one embodiment, the composition of the enzymolysis solution of the present invention, the mass percentage of the pectinase Y-23 may be 0.3-0.4% (w/v), 0.4-0.5% (w/v), 0.5-0.6% (w/v), 0.6-0.7% (w/v), or 0.7-0.8% (w/v).
In an embodiment, the composition of the enzymatic hydrolysis solution of the present invention, the Bovine Serum Albumin (BSA) may be 0.08-0.09% (w/v), 0.09-0.10% (w/v), 0.10-0.11% (w/v), or 0.11-0.12% (w/v), by mass.
In one embodiment, the composition of the enzymatic hydrolysis solution of the present invention includes the following components by mass percentage or concentration: 1.5% (w/v) cellulase R-10, 0.4% (w/v) macerozyme R-10, 0.5% (w/v) pectinase Y-23,0.6mol/L mannitol, 20mmol/L potassium chloride KCl, 20mmol/L2- (N-morpholinyl) ethanesulfonic acid (MES), 10mmol/L calcium chloride, 0.05mmol/L beta-mercaptoethanol, 0.1% (w/v) Bovine Serum Albumin (BSA).
The invention relates to an enzymolysis method for quickly removing cell walls of poplar or eucalyptus, wherein a solvent I is a mannitol solution, and the preferable concentration is 0.4-1.2 mol/L. The aim of soaking the poplar or eucalyptus leaves in the mannitol solution can be to primarily separate protoplasts from cell walls, and the cell walls can be rapidly removed by enzymolysis. The preferred concentration may be from 0.4 to 0.5mol/L, from 0.5 to 0.6mol/L, from 0.7 to 0.8mol/L, from 0.8 to 0.9mol/L or from 0.9 to 1.0 mol/L.
The purpose of the mannitol soaking is to separate the cell membrane and the cell wall for plasmolysis and facilitate the next enzymolysis and dissociation. Too high a concentration will result in too high an osmotic pressure for cell death, and too low a concentration will prevent plasmolysis of the cells.
In one embodiment, the mannitol pretreated aspen or eucalyptus leaves are drained and then placed in an enzymatic solution to avoid carrying the mannitol solution as much as possible, and the mannitol soaking is performed to separate the cell membrane and the cell wall for the purpose of plasmolysis and dissociation.
The enzymolysis condition is in dark condition, the mixture is placed on a horizontal shaking bed and is subjected to enzymolysis for 3 to 5 hours at the temperature of between 20 and 24 ℃. Under the condition, the enzyme activity can be improved, and the enzymolysis can be carried out quickly, so that the cell wall can be separated quickly; the complete dissociation degree can reach 90% or more within 5 hours.
In one embodiment, the enzymolysis conditions of the present invention may be carried out at 20-21 deg.C, 21-22 deg.C, 22-23 deg.C or 23-24 deg.C.
The method for terminating the enzymolysis comprises the step of adding a W5 solution into the enzymolysis liquid.
The filtering of the invention adopts a 200-400-mesh filter screen for filtering. Can be 200-300 mesh or 300-400 mesh.
The purification of the invention adopts multiple times of centrifugation and washing for purification.
In one embodiment, the washing comprises adding a W5 solution to the filtered and centrifuged enzymatic solution and centrifuging to remove the supernatant, and is performed a plurality of times.
The invention relates to an enzymolysis method for rapidly removing the cell wall of poplar or eucalyptus, wherein the poplar or eucalyptus leaves are obtained by aseptic subculture and culture of tissue culture seedlings of poplar or eucalyptus through a culture medium.
The invention provides a protoplast of poplar or eucalyptus, which is prepared by an enzymolysis method for quickly removing the cell wall of the poplar or eucalyptus.
The invention also providesApplication of poplar or eucalyptus protoplast in exogenous DNA plasmid transformation, and protoplast, exogenous DNA plasmid and PEG/Ca prepared by the method2+And MMg solution for incubation transformation.
Advantageous effects
The invention provides a specific enzymolysis solution formula and a series of implementation means, which are used for rapidly dissociating the cell walls of the poplar and the eucalyptus, wherein the complete dissociation degree can reach 90 percent or more within 5 hours, and the industrial problem that the cell walls of trees are difficult to remove due to complex cell wall components is solved. The method can be used for obtaining the protoplast with high integrity and activity, is used for subsequent regeneration and other ways, can accelerate the genetic engineering breeding of the poplar in the northern area and the eucalyptus in the southern area of China, and has important application value for cultivating new varieties of excellent poplar and eucalyptus and ensuring the wood safety in China. The method can be suitable for removing and dissociating the cell walls of populus tomentosa, populus alba, populus 84K, populus deltoides, populus Sinkiangensis and populus tomentosa in the populus tremuloides pie, populus tremuloides and populus tremuloides in the populus tremuloides pie, and eucalyptus grandis, eucalyptus grandis and eucalyptus grandis in the eucalyptus, and has important significance for promoting genetic engineering breeding of improved varieties of forest trees.
The invention finds that the protoplast enzymolysis method only uses the eductase and the pectinase, is difficult to degrade the woody plants such as poplar and eucalyptus, which have complex cell wall components, and has extremely low efficiency. On the basis of cellulase and eductase, pectinase is additionally added, and an optimal concentration is set for carrying out an enzymolysis experiment, so that a large amount of pectin in the cell walls of the poplar and the eucalyptus can be removed by enzymolysis, and a protoplast is dissociated.
Drawings
FIG. 1 is a schematic flow chart of an enzymatic hydrolysis method for rapidly removing cell walls of populus alba and eucalyptus.
FIG. 2 "poplar" protoplasts after removal of the cell wall.
FIG. 3 "Eucalyptus" protoplasts after removal of the cell wall.
Detailed Description
The invention will be described in detail hereinafter with reference to the accompanying drawings in conjunction with embodiments. It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict. The experimental procedures used in the following examples are conventional ones unless otherwise specified, and the reagents and materials used therein are commercially available ones unless otherwise specified. Wherein cellulase R-10, macerase R-10 and pectinase Y-23 are available from Yakult, Inc., and various pharmaceutical reagents, such as Sigma, are available without specific reference.
The first embodiment is as follows: culture of tissue culture seedling aseptic seedling of poplar and eucalyptus
Carrying out vegetative propagation on the tissue culture seedlings of the populus tomentosa through the following culture media respectively:
the formula of the culture medium is as follows:
callus induction medium: MS + KT 0.5mg/L +0.1 mg/L2, 4-D +6.5g/L agar
Differentiation and budding culture medium: MS + TDZ 0.05mg/L +6-BA0.5mg/L +6.5g/L agar
Shoot elongation medium: MS +6-BA0.5mg/L +6.5g/L agar
Rooting culture medium: 1/2MS + NAA0.5mg/L +6g/L agar
(1) The method comprises cutting stem of wild grown Chinese white poplar, brushing gently, washing with running water for 12 hr, air drying, making into stem of 3cm length, soaking in 75% alcohol for 30s on a superclean bench, sterilizing with sterile water for 3 times (1 min each) with 2% sodium hypochlorite (NaClO) water solution for 10min, washing with sterile water for 3 times (1 min each), sucking surface water with sterile filter paper, culturing in "callus induction medium" for 30 days in dark, wherein the medium is changed every 10 days until white transparent callus tissue grows on the edge of leaf;
(2) callus induction, cutting off callus carefully, placing on "differentiation bud culture medium", placing under light for culturing for 30d, wherein culture medium is changed every 10 days until callus grows bud;
(3) and (3) bud induction: placing the callus and the buds on a bud elongation culture medium, growing for about 20 days, and growing obvious stems from the straight buds;
(4) rooting culture: cutting off the stem segment, inserting the stem segment into a rooting culture medium, culturing for 30d under the light, and growing a complete plant for later use.
Carrying out vegetative propagation on the eucalyptus tissue culture seedlings by respectively passing through the following culture media:
the formula of the culture medium is as follows:
callus induction and differentiation germination culture medium: MS + TDZ0.5mg/L + NAA0.1mg/L +6.5g/L agar subculture medium: MS + KT0.2mg/L +6-BA0.2mg/L + NAA0.1mg/L +6.5g/L agar rooting medium: 1/2MS + NAA0.5mg/L + IBA0.5mg/L +6.0g/L agar
(5) Firstly, stem segments of field-grown eucalyptus grandis are treated according to a method for disinfecting an external cultured body of the populus tomentosa, then the stem segments are placed on a eucalyptus callus induction and differentiation bud culture medium and are placed in the dark for culturing for 30 days, wherein the culture medium is replaced every 10 days, and then the stem segments are transferred to the light for bud induction until young and tender buds grow.
(6) Placing the stem segments with the buds and the callus on a subculture medium, and growing for about 30 days until obvious stems grow from the straight buds;
(7) the stem segments are cut off, inserted into a 'rooting medium' and cultured under the light for 30d until a complete plant is grown for standby.
Example two: mass-wall separation pretreatment
(8) Preparing 0.8M mannitol solution by preparing a disposable culture dish with a diameter of 9cm, weighing 2.912g mannitol, and dissolving in 20ml ddH2And O, fully dissolving and uniformly mixing mannitol.
(9) Cutting the leaves of Populus alba and Eucalyptus globulus into strips with width of about 1mm with scissors or a blade, and soaking in the mannitol solution prepared in the previous step to ensure that the strips are all soaked in the solution.
(10) Standing at room temperature for 20 min.
Example three: enzymatic cell wall removal
10ml of enzymolysis solution with the following formula is prepared by a 10ml centrifuge tube:
0.15g cellulase R-10
0.04g of the eductase R-10
0.05g of pectinase Y-23
1.092g mannitol
1ml 200mM KCl
1ml 200mM MES
Add 5ml of ddH2Incubating at 55 deg.C for 10min, cooling to room temperature, adding
100μl 1M CaCl2
66.67. mu.l of 7.5mM beta-mercaptoethanol
1ml 1％BSA
The volume is adjusted to 10ml for standby.
(11) Taking out the fine strips of the leaves of the poplar and the eucalyptus which are treated by the mannitol, draining, putting into the enzymolysis liquid (the mannitol solution is not brought out as much as possible), and loosely and uniformly soaking in a 10ml centrifuge tube containing 10ml of the enzymolysis liquid.
(12) And (3) placing the mixture on a horizontal shaker under a dark condition, and carrying out enzymolysis for 3-5 hours at 23 ℃ and 10 rpm.
(13) After 3h of enzymolysis, absorbing the enzymolysis liquid by a gun head with a cut tip every 1h, and observing and analyzing under a microscope.
(14) Filtering with 400 mesh filter screen, sucking the filtrate into 10ml plastic centrifuge tube, centrifuging at room temperature for 2min at 100g, and discarding the supernatant.
Example four: observation and analysis
(15) 2ml of a pre-chilled W5 solution (154mM NaCl,125mM CaCl) were added (gently, not too quickly) to a 10ml centrifuge tube25mM KCl,2mM MES,5mM glucose), the protoplasts were washed and mixed by gentle inversion.
(16) Centrifuge at 100g, 4 deg.C for 1min, and discard the supernatant.
(17) An additional 2ml of pre-cooled W5 solution was added and the pellet suspended with gentle shaking.
(18) Observation statistics of protoplast integrity were performed under an optical microscope.
(19) 1ml of protoplast solution is sucked, 50 mul of Fluorescein Diacetate (FDA) solution with the concentration of 5mg/ml is added, the solution is placed under a fluorescence microscope for observation after being dyed for 2min, and the activity of the protoplast is analyzed.
The above results indicate that protoplasts were observed under a fluorescence microscope, and that the yellow-green cells were viable cells, and the viable cells accounted for more than 90% of the total cells.
A schematic diagram of the detection of poplar protoplast viability on a scale of 200 μm with a fluorescent channel is shown in FIG. 2.
A schematic diagram of the detection of the activity of eucalyptus protoplasts on a scale of 200 μm in a fluorescence channel is shown in FIG. 3.
Examples comparative data for pentamannitol pretreatment and no pretreatment
The subsequent dissociation effect is greatly influenced by respectively using the pretreatment with mannitol and the pretreatment without, the yield of the protoplast obtained by the subsequent dissociation is very low and is about 2x10 without the pretreatment3One/ml, and the yield of the subsequent protoplast is 1-5x10 after the plasmolysis is carried out firstly by using mannitol5One per ml.
Example screening test for formulation of six enzymatic solutions
The base plate formulation was 1% cellulase R-10, 0.3% Segrelase, 0.4M mannitol, 20mmol/L KCl, 20mmol/L MES and 10mmol/L CaCl2. Degrading main components of cell wall with cellulase and isolation enzyme, maintaining osmotic pressure with mannitol, KCl, MES, CaCl2Has effect of maintaining the activity of the cells. Setting 1%, 1.5%, 2% cellulase, 0.4%, 0.6%, 0.8% macerozyme R-10, and adding the concentration gradient of 0.2%, 0.5%, 1.0% pectase, totally nine enzymolysis liquid experimental schemes, the other components are the same as the third embodiment, finally determining the best enzymolysis liquid concentration of the invention is 1.5% cellulase R-10, 0.4% macerozyme and 0.5% pectase, the highest yield of the obtained protoplast can be obtained, the dissociation degree can reach 90% and above.
Claims (10)
1. An enzymolysis method for quickly removing the cell wall of poplar or eucalyptus is characterized by at least comprising the following steps: performing enzymolysis on the leaves of the poplar or the eucalyptus by using the prepared enzymolysis solution to remove cell walls, and performing enzymolysis incubation; preferably, the method comprises the following steps: soaking leaves of poplar and eucalyptus in the solution I; placing the poplar or eucalyptus leaves soaked by the solvent I into an enzymolysis solution for enzymolysis to obtain an enzymolysis solution; after the enzymolysis is finished, filtration and purification are carried out.
2. The enzymatic hydrolysis method according to claim 1, wherein the composition of the enzymatic hydrolysis solution comprises the following components in percentage by mass or concentration: 1-2% (w/v) cellulase R-10, 0.4-0.8% (w/v) macerozyme R-10, 0.2-1.0% (w/v) pectinase Y-23,0.4-0.8mol/L mannitol, 18-22mmol/L potassium chloride KCl, 18-22mmol/L2- (N-morpholinyl) ethanesulfonic acid (MES), 8-12mmol/L calcium chloride, 0.03-0.08mmol/L beta-mercaptoethanol, 0.08-0.12% (w/v) Bovine Serum Albumin (BSA); preferably, the composition of the enzymolysis solution comprises the following components in percentage by mass or concentration: 1.5% (w/v) cellulase R-10, 0.4% (w/v) macerozyme R-10, 0.5% (w/v) pectinase Y-23,0.6mol/L mannitol, 20mmol/L potassium chloride KCl, 20mmol/L2- (N-morpholinyl) ethanesulfonic acid (MES), 10mmol/L calcium chloride, 0.05mmol/L beta-mercaptoethanol, 0.1% (w/v) Bovine Serum Albumin (BSA).
3. The method of claim 1 or 2, wherein: the solvent I is mannitol solution, and the preferable concentration is 0.4-1.2 mol/L.
4. The method of claim 1 or 2, wherein: the enzymolysis condition is dark condition, placing on a horizontal shaking table, and carrying out enzymolysis for 3-5 hours at 20-24 ℃.
5. The method of claim 1 or 2, wherein: the method for terminating the enzymolysis is to add a W5 solution into the enzymolysis liquid.
6. The method of claim 1 or 2, wherein: the filtration is carried out by adopting a 200-mesh and 400-mesh filter screen, and the purification is carried out by adopting multiple times of centrifugation and cleaning; preferably, the washing includes adding a W5 solution to the filtered and centrifuged enzymolysis solution and centrifuging to remove a supernatant, and is performed a plurality of times.
7. The method of claim 6, wherein: the formula of the W5 solution comprises the following components by mass percent, 154mmol/L sodium chloride, 125mmol/L calcium chloride, 5mmol/L potassium chloride,
2mmol/L2- (N-morpholinyl) ethanesulfonic acid, 5mmol/L glucose.
8. The method of claim 1 or 2, wherein: wherein the poplar or eucalyptus leaves are obtained by aseptic subculture and culture of the poplar or eucalyptus tissue culture seedlings by using the culture medium.
9. Protoplasts of aspen or eucalyptus obtained by the method of claims 1 to 8.
10. The application of the poplar or eucalyptus protoplast in the transformation of exogenous DNA plasmids is characterized in that: protoplast, foreign DNA plasmid, PEG/Ca prepared according to claim 92+And MMg solution for incubation transformation.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114591882A (en) * | 2022-03-03 | 2022-06-07 | 东北林业大学 | Method for transient transformation of acanthopanax cells |
| CN115428734A (en) * | 2022-09-22 | 2022-12-06 | 北京林业大学 | Method for inducing massive multiplication of poplar leaf callus |
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| AU2020101602A4 (en) * | 2020-07-31 | 2020-09-17 | Anhui Science And Technology University | A Method for Rapidly Preparing Stevia Leaf Protoplasts |
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