CN115340972A - Preparation method and application of banana protoplast - Google Patents
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Abstract
The invention discloses a preparation method and application of banana protoplasts, and belongs to the technical field of plant protoplasts. The invention adopts the inner layer tissue of banana bud absorption, improves the formula of an enzymolysis solution, and obtains the high-purity banana protoplast by a method of multiple low-speed centrifugation and heavy suspension, wherein the purity of the high-purity banana protoplast reaches 1.22 multiplied by 10 per gram of tissue 7 And (4) protoplasts. The method has high reproducibility, can stably obtain high-quality banana protoplasts, and achieves the effect that the transformation efficiency reaches more than 75%. The invention provides an effective research technology for molecular biology research such as banana protein subcellular localization, promoter function verification and the like, and has wide application value.
Description
Technical Field
The invention belongs to the technical field of plant protoplasts, and particularly relates to a preparation method and application of a banana protoplast.
Background
Plant protoplasts are an ideal genetic transformation receptor, and transient expression by using the plant protoplasts is an important technical means for analyzing analysis such as gene expression, subcellular localization, protein interaction, promoter activity and the like in molecular genetic research. Successful protoplast transformation systems are currently reported in a wide variety of species, including model species. For bananas, embryogenic cell suspension lines and callus are considered as good protoplast separation materials, but the direct separation of viable protoplasts from banana plant tissues is rarely reported.
Bananas, as the second largest fruit in the world, are second only to citrus in planting area and yield, and are one of the important economic and food crops in tropical and subtropical regions. At present, the common cultivated varieties of bananas have poor resistance and are often seriously damaged by soil-borne diseases, particularly banana vascular wilt. Most banana cultivars belong to vegetative propagation, and genetic diversity of the cultivars is lost due to polyploidy and high sterility of the cultivars, so that the traditional breeding method is difficult to implement. Therefore, a new technical system is urgently needed to breed a high-quality new banana variety, so that the healthy and continuous development of the banana industry is the current important task. By utilizing the banana protoplast separation and transformation system and the protoplast fusion technology, the problem of difficult traditional breeding of bananas can be solved, and the acquisition of new varieties is accelerated. The instant expression system of the banana protoplast is perfected, a good research technical means can be provided for the subsequent research of the functional genome of the banana, and the foundation is further laid for the research of the disease resistance breeding of the banana. Therefore, the construction of the efficient and stable protoplast preparation and transient transformation method has important significance for banana genetic research and breeding utilization.
Disclosure of Invention
Aiming at the technical problems, the invention provides a preparation method and application of banana protoplast, solves the problems that the viable protoplast is directly separated from banana plant tissue and genetic transformation is difficult to carry out, and provides a stable and efficient technical method for molecular genetic researches such as banana protein subcellular localization, promoter function verification and the like.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a preparation method of banana protoplast comprises the following steps:
(1) Collecting banana suckers, peeling off layer by layer to obtain inner-layer tender parts, and cutting the tender parts into suckers with the diameter of 0.5-1 mm;
(2) Soaking the bud sucking segment in a culture dish containing 0.6M mannitol, sealing the culture dish with tinfoil paper, and standing for 5 min;
(3) After completely sucking mannitol by a liquid transfer gun, transferring the bud suction small segment to a 50 mL beaker, adding 5mL of enzymolysis liquid, and vacuumizing in a vacuum pump for 2-3 times, 5 min each time;
(4) Sealing the beaker by using tin foil paper, horizontally oscillating at 50 rpm, and performing enzymolysis at room temperature (26 ℃) until the protoplast is dissociated from the bud-sucking small segment;
(5) After the enzymolysis is finished, filtering the enzymolysis liquid into a 50 mL centrifuge tube by using a 200-mesh cell sieve, washing the small beaker for multiple times by using a W5 solution precooled on ice, and filtering into the 50 mL centrifuge tube;
(6) Centrifuging the solution filtered in the step (5) for 3 min at the rotating speed of 400 g (slowly increasing and decreasing), and removing the supernatant; slowly adding 15mL of precooled W5 solution, gently resuspending the cells, centrifuging again for 3 min at the rotating speed of 400 g, and then sucking out the redundant supernatant;
(7) Repeating the step (6) for 2 times, re-suspending the protoplast obtained by the last centrifugation in 15mL of precooled W5 solution, placing the solution in a refrigerator for precooling for 30 min at 4 ℃, centrifuging for 3 min at the rotating speed of 400 g, and removing the supernatant;
(8) And adding an appropriate amount of MMG solution, and fully suspending the cell precipitate for later use.
Preferably, the enzymolysis time in the step (4) is 6 hours.
Preferably, the components and contents of the enzymolysis liquid are as follows: 10 mM MES pH7.5, 0.6M D-Mannitol, 2.5% (w/v) Cellulase (Cellulase R-10), 1% (w/v) Segrelase (Macerozyme R-10), 10 mM CaCl 2 0.1% (w/v) BSA 0.35% (v/v) 2-mercaptoethanol (v/v)β-Mercaptoethanol）。
Preferably, the components and the contents thereof in the W5 solution are as follows: 1.5 mM MES pH7.5, 155 mM NaCl, 125 mM CaCl 2 、5 mM KCl、5 mM D-(+)-Glucose。
Preferably, the components and contents of the MMG solution are as follows: 4mM MES pH7.5, 0.6M D-Mannitol, 10 mM MgCl 2 ·6H 2 O。
The banana protoplast prepared by the invention can be used for genetic transformation, banana protein subcellular localization and banana breeding.
Has the advantages that:
(1) The invention can obtain 1.22 multiplied by 10 per gram of tissue 7 The protoplast effectively solves the problem of directly separating the viable protoplast from the banana plant tissue. Meanwhile, a matched conversion system is optimized, so that the conversion efficiency reaches over 75 percent.
(2) The invention provides an effective research technical means for molecular genetics research such as banana protein subcellular localization, promoter function verification and the like. From the technical point of view, the invention is realized by improving a plurality of simple and innovative technical processes, and the operation difficulty is reduced, so that the popularization and application value and the commercial development potential of the invention are more prominent.
Drawings
FIG. 1 is a schematic diagram of expanding propagation seedlings (A), rooting seedlings (C) and suckers (E) of bananas, wherein the left diagram of the diagram E is the complete banana suckers, and the right diagram is tender parts of the banana suckers after the outer layer is peeled off; bar =1 cm; b, D and F are diagrams of protoplasm yield of banana propagated seedlings, rooted seedlings and suckers respectively after enzymolysis for 8 hours under different enzymolysis liquid conditions;
FIG. 2 is a graph of protoplast yield of banana suckers isolated at different enzymatic hydrolysis times;
FIG. 3 is a diagram showing the expression effect of YFP in banana protoplast after transformation, wherein the left diagram is a GFP field, the middle diagram is a bright field, and the right diagram is a merged field; bar =50 μm.
Detailed Description
The reagent consumables used in the invention are as follows:
MES (Sigma，V900336-500G)
KCl (Xilongsu science corporation)
MgCl 2 •6H 2 O (Sigma，M2393-500G)
CaCl 2 (Sigma，C4901-500G)
PEG4000 (Sigma，81240-1KG)
NaCl (Xilongsu science Co., ltd.)
D-Mannitol (Sigma，M1902-1kg)
Cellulase R-10 (Yakult，L0012)
Macerozyme R-10 (Yakult，JM6033)
Pectinase Y-23 (Yakult, P8220-100)
D-(+)-Glucose（Sigma，G8270-1KG）
BSA (Lamborlidide Biotechnology Co., ltd.)
β-ME (Sigma，M3148)
The invention is further described below with reference to the accompanying drawings and specific embodiments.
The following examples were prepared with the following solutions:
(1) The preparation method of the enzymolysis solution comprises the following steps:
dissolving 39.048 g MES in 1L distilled water, adjusting pH to 7.5 with KOH to obtain 0.2M MES mother liquor with pH of 7.5, and storing in dark at 4 deg.C; dissolving 146.536 g of D-Mannitol into 1L of distilled water to obtain 0.8M of D-Mannitol mother liquor, and storing at room temperature; dissolving 149.1 g of KCl in 1L of distilled water to obtain 2M KCl mother liquor, and storing at room temperature; 110.98 g of CaCl 2 Dissolving in 1L distilled water to obtain 1M CaCl 2 Mother liquor is stored at room temperature; 203.3 g of MgCl 2 ·6H 2 O dissolved in 1LDistilled water to give 1M MgCl 2 ·6H 2 And (4) storing the mother liquor at room temperature.
1 mL of 0.2M MES (final concentration 10 mM), 15mL of 0.8M D-Mannitol (final concentration 0.6M), 200. Mu.L of 0.2M KCl (final concentration 20 mM), 0.5 g of Cellulase Cellulase R-10 (final concentration 2.5%), 0.2 g of the eductase Macerozyme R-10 (final concentration 1%), (each 20 mL of the enzymatic hydrolysate). Placing the enzymolysis buffer solution in 55 deg.C water bath for 10 min, mixing by reversing every 2-3 min, cooling to room temperature on ice, and adding 200 μ L1M CaCl2 (final concentration 10 mM), 0.02 g BSA (final concentration 10%) and 7 μ L BSA respectivelyβ-ME (final concentration 0.35%), and the prepared enzymolysis liquid is finally obtained and can be used as it is and stored at 4 ℃ in a short time.
(2) The preparation method of the PEG4000 solution comprises the following steps:
per 2.5 mL of PEG4000 solution are added with 1.125 g of PEG4000 (final concentration 45%), 625. Mu.L of 0.8M D-Mannitol (final concentration 0.2M), 250. Mu.L of 1M CaCl 2 (final concentration 0.1M), finally obtaining the prepared 45% PEG solution which is prepared just before use.
(3) The preparation method of the W5 solution comprises the following steps:
10 mL of 0.2M MES (final concentration 2 mM), 90 g NaCl (final concentration 154 mM), 125 mL of 1M CaCl were added to 1L of the W5 solution 2 (final concentration 125 mM), 2.5 mL of 2M KCl (final concentration 5 mM), and 0.9 g of D- (+) -Glucose (final concentration 5 mM) to obtain a W5 solution, adjusting the volume to 1L with distilled water, adjusting the pH to 5.7 with 1M KOH, and sterilizing at high temperature and high pressure, and storing at 4 ℃.
(4) The preparation method of the MMG solution comprises the following steps:
to each 10 mL of MMG solution were added 0.2 mL of 0.2M MES (final concentration 4 mM), 7.5 mL of 0.8M D-Mannitol (final concentration 0.6M), and 150. Mu.L of 1M MgCl 2 ·6H 2 O (final concentration 15 mM), constant volume with distilled water, and storage at 4 deg.C.
Example 1
Preparation of banana protoplasts, the preparation method comprising the steps of:
(1) Material culture: collecting banana suckers, peeling layer by layer, only remaining soft white tender inner layer parts, and cutting the obtained suckers into small sections of 0.5-1mm by using a surgical scalpel, as shown in FIG. 1E;
(2) Enzymolysis: soaking the bud sucking segment in a culture dish containing 0.6M mannitol, sealing the culture dish with tinfoil paper, and standing for 5 min; after completely sucking mannitol by a liquid transfer gun, transferring the bud suction small segment to a 50 mL beaker, adding 5mL of enzymolysis liquid, and vacuumizing in a vacuum pump for 2-3 times, 5 min each time; sealing the beaker by using tin foil paper, horizontally oscillating at 50 rpm, and performing enzymolysis at room temperature (26 ℃) until the protoplast is dissociated from the bud-sucking small segment; microscopic examination of the enzymolysis result;
(3) And (3) terminating enzymolysis: filtering the enzymolysis liquid into a 50 mL centrifuge tube by using a 200-mesh cell sieve, washing the small beaker with a W5 solution precooled on ice for multiple times, and filtering into the 50 mL centrifuge tube;
(4) Collecting and rinsing protoplasts: centrifuging the solution filtered in the step (3) for 3 min at the rotating speed of 400 g (slowly increasing and decreasing), and removing the supernatant; slowly adding 15mL of precooled W5 solution, gently resuspending the cells, centrifuging again for 3 min at the rotating speed of 400 g, and then sucking off the redundant supernatant;
(5) Ice-bath protoplast: repeating the step (4) for 2 times, re-suspending the protoplast obtained by the last centrifugation in 15mL of precooled W5 solution, placing the solution in a refrigerator for precooling for 30 min at 4 ℃, centrifuging for 3 min at the rotating speed of 400 g, and removing the supernatant;
(6) Resuspending protoplasts in MMG solution: an appropriate amount of MMG solution was added and the cell pellet was resuspended thoroughly for future use.
Example 2
Banana protoplasts were prepared according to the method of example 1, and control groups of different tissues (propagating seedling, rooting seedling and suckling) and different enzymatic hydrolysates were set.
The results are shown in FIG. 1, and the number of banana protoplasts obtained from the expanded propagation seedlings and the rooted seedlings in all combinations of the enzymolysis solutions (FIG. 1B, D) is significantly less than that obtained from the young suckers (FIG. 1F) under the condition of 8h enzymolysis time. For young suckers, the number of protoplasts prepared by the enzymolysis groups 1-8 and 10 was significantly less than the number of protoplasts prepared in example 1 (enzymolysis group 9).
Example 3
Banana protoplasts were prepared according to the method of example 1 and control groups consisting of different enzymatic hydrolysis times were set.
The results are shown in fig. 2, the banana protoplasts were obtained at the enzymolysis time (1 h, 2h, 3h, 4h, 5h, 6h, 7h, 8 h), and the highest value of the number of the protoplasts was reached at the enzymolysis time of 6h (fig. 2).
Example 4
Use of transient expression of banana protoplasts, said use comprising the steps of:
(1) Adding 10 μ L of 35S YFP plasmid (concentration greater than 1 μ g/μ L) and 200 μ L of protoplast into 2 mL centrifuge tube, mixing, adding 220 μ L of PEG4000 solution, mixing immediately, standing in dark at room temperature for 15 min;
(2) Terminating the conversion reaction: adding 1 mL of W5 solution, reversing, mixing uniformly, horizontally centrifuging (4 ℃,400 g) for 3 min, discarding the supernatant, collecting protoplasts, and repeating the step for 2 times;
(3) Culturing for 12-16 h under weak light;
(4) The dark cultured protoplasts were taken and observed by microscopy for YFP expression (see FIG. 3).
According to the green fluorescent protein gene transformation signal diagram adopted by the protoplast prepared by banana bud picking in the figure 3, the protoplast prepared by the invention can successfully carry out genetic transformation.
It will be understood that the invention is not limited to the examples described above, but that modifications and variations will occur to those skilled in the art in light of the above teachings, and that all such modifications and variations are considered to be within the scope of the invention as defined by the appended claims.
Claims (5)
1. A preparation method of banana protoplasts is characterized by comprising the following steps:
(1) Collecting banana suckers, peeling layer by layer to obtain inner-layer tender parts, and cutting the inner-layer tender parts into small sections of 0.5-1 mm;
(2) Soaking the bud sucking segment in a culture dish containing 0.6M mannitol, sealing the culture dish with tinfoil paper, and standing for 5 min;
(3) After completely sucking mannitol by a liquid transfer gun, transferring the bud suction small segment to a beaker, adding 5mL of enzymolysis liquid, and vacuumizing for 5 min each time for 2-3 times in a vacuum pump;
(4) Sealing the beaker by using tin foil paper, horizontally oscillating at 50 rpm, and performing enzymolysis for 6 hours at room temperature until the protoplast is dissociated from the bud-sucking small section;
(5) After the enzymolysis is finished, filtering the enzymolysis liquid into a centrifuge tube by using a 200-mesh cell sieve, washing the beaker with a W5 solution precooled on ice for multiple times, and filtering into the centrifuge tube;
(6) Centrifuging the solution filtered in the step (5) for 3 min, and removing the supernatant; slowly adding 15mL of precooled W5 solution, gently resuspending the cells, centrifuging for 3 min again, and then sucking out the redundant supernatant;
(7) Repeating the step (6) for 2 times, re-suspending the protoplast obtained by the last centrifugation in 15mL of precooled W5 solution, placing the solution in a refrigerator for precooling for 30 min at 4 ℃, centrifuging for 3 min, and removing the supernatant;
(8) The MMG solution is added again, and the cell pellet is fully resuspended for later use.
2. The method for preparing banana protoplasts according to claim 1, wherein the enzymatic hydrolysate comprises the following components in percentage by weight: 10 mM MES pH7.5, 0.6M D-Mannitol, 2.5% (w/v) cellulase, 1% (w/v) Segrenase, 10 mM CaCl 2 0.1% (w/v) BSA, 0.35% (v/v) 2-mercaptoethanol.
3. The method for preparing banana protoplasts according to claim 1, wherein the W5 solution comprises the following components: 1.5 mM MES pH7.5, 155 mM NaCl, 125 mM CaCl 2 、5 mM KCl、5 mM D-(+)-Glucose。
4. The method for preparing banana protoplasts according to claim 1, wherein the MMG solution comprises the following components: 4mM MES pH7.5, 0.6M D-Mannitol, 10 mM MgCl 2 ·6H 2 O。
5. Use of banana protoplasts prepared by the method according to any one of claims 1 to 4 for genetic transformation, subcellular localization of banana proteins, and banana breeding.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115820532A (en) * | 2022-12-09 | 2023-03-21 | 广州元信生物科技有限公司 | Dissociation method of banana radicle protoplast |
| CN115820532B (en) * | 2022-12-09 | 2023-10-31 | 广州元信生物科技有限公司 | Dissociation method of banana young root protoplast |
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