CN113604418A - Preparation and application of green south medicinal plant andrographis paniculata protoplast - Google Patents
Preparation and application of green south medicinal plant andrographis paniculata protoplast Download PDFInfo
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Abstract
The invention belongs to the technical field of protoplast preparation, and particularly relates to preparation and application of a protoplast of andrographis paniculata which is a medicinal plant in Lingnan. The invention provides a protoplast enzymolysis liquid, which can remove cell walls better, enable cells to dissociate, maintain good physiological state of the cells, adjust mannitol concentration and maintain cell osmotic pressure when the protoplast is used for transfection, reduce shrinkage or bursting of the protoplast in the transfection process and ensure the cell form of the protoplast. The enzymolysis liquid can shorten the enzymolysis time when the protoplast is prepared, ensures the high activity and the high yield of the protoplast, and can be used for the research of gene transient expression.
Description
Technical Field
The invention belongs to the technical field of protoplast preparation, and particularly relates to preparation and application of a protoplast of andrographis paniculata which is a medicinal plant in Lingnan.
Background
Plant protoplasts are the collective term for various structures within the cell wall, and have the ability to regenerate into whole plants. At present, the main means for preparing plant protoplasts is an enzymolysis method, however, when the plant protoplasts are prepared by the enzymolysis method, such as Huang Hua and the like, the andrographis paniculata protoplasts are prepared by a method of 6 hours of enzymolysis (Huang Hua and the like, establishment and optimization of a preparation method of andrographis paniculata leaf protoplast [ J/OL ]. molecular plant breeding: 1-16.http:// kns. cnki. net/kcms/detail/46.1068.S.20210428.1414.012.html.), the enzymolysis time is long, and the preparation efficiency is not high. How to shorten the time for preparing plant protoplasts is an urgent problem to be solved in the field of protoplast preparation.
Disclosure of Invention
The invention aims to provide a protoplast enzymolysis liquid, which can shorten the enzymolysis time when preparing the protoplast and improve the preparation efficiency of the protoplast.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a protoplast enzymolysis liquid, which comprises 1.5 wt.% of cellulase R-10, 0.75 wt.% of macerozyme R-10, 0.6M mannitol, 20mM MES, 20mM KCl and 10mM CaCl2And 0.1 wt.% BSA.
The invention provides a preparation method of the enzymolysis liquid, which comprises the following steps:
mixing the cellulase R-10, the macerozyme R-10, the mannitol, the MES and the KCl, incubating for 10min at 55 ℃, and cooling to room temperature to obtain a first mixed solution;
mixing the first mixed solution with CaCl2Mixing with BSA, filtering, and sterilizing to obtain enzymatic hydrolysate.
The invention provides a preparation method of andrographis paniculata protoplasts, which comprises the following steps:
cutting hypocotyl and leaf tissue of herba Andrographitis seedling, and regulating osmotic pressure to obtain hypocotyl and leaf fragments of herba Andrographitis seedling;
mixing the fragments with the enzymatic hydrolysate for enzymolysis for 3-4 hours to obtain an andrographis paniculata enzymatic hydrolysate;
mixing the andrographis paniculata enzymolysis liquid with a W5 solution, and filtering to obtain the protoplast.
Preferably, the light-dark time ratio of the culture is 12 h: 12 h; the illumination intensity of the culture is 100 mu mol m-2s-1。
Preferably, the culture time is 20-40 d; the culture temperature is 20-30 ℃.
Preferably, the osmoregulation comprises dark culturing hypocotyl and leaf tissue of the minced plant seedling in an osmoregulation fluid comprising 0.6M mannitol solution for 30 min.
Preferably, the W5 solution comprises 5mM KCl, 154mM NaCl, 125mM CaCl2And 2mM MES.
The invention provides a method for transient transfection of andrographis paniculata protoplasts, which comprises the following steps:
mixing the andrographis paniculata protoplast with exogenous plasmids and a PEG solution, and carrying out dark reaction for 12-15 min to obtain a reaction solution;
the W5 solution was mixed with the reaction solution, and transfected protoplasts were isolated.
Preferably, the volume ratio of the exogenous plasmid, the andrographis paniculata protoplast and the PEG solution is 1: 10: 11.
preferably, the exogenous plasmid contains a marker gene.
The invention provides a protoplast enzymolysis solution, which comprises 1.5 wt.% of cellulase R-10, 0.75 wt.% of macerozyme R-10, 0.6M mannitol, 20mM MES, 20mM KCl, 10mM CaCl2 and 0.1 wt.% BSA. The invention can better remove cell walls by elaborately preparing the protoplast enzymolysis liquid, so that the cells can be dissociated and can maintain a good physiological state.
Furthermore, the invention maintains the osmotic pressure of cells by adjusting the concentration of mannitol, reduces the shrinkage or the expansion of the protoplast in the transfection process and ensures the cell morphology of the protoplast. The enzymolysis liquid can shorten the enzymolysis time when the protoplast is prepared, ensures the high activity and the high yield of the protoplast, and can be used for the research of gene transient expression.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without inventive exercise.
FIG. 1 is a diagram showing the growth state of Andrographis paniculata Nees at the seedling stage in example 1;
FIG. 2 is a schematic diagram of the tissue of Andrographis paniculata seedlings used in the preparation of protoplasts according to example 2, wherein FIG. 2A shows the intact tissue and FIG. 2B shows the minced tissue;
FIG. 3 is a 20-fold microscopic image of the protoplasts prepared in example 2;
FIG. 4 is a 20-fold microscopic image of the protoplast of Andrographis paniculata prepared in example 2 after staining with fluorescein diacetate;
FIG. 5 is an image of pUC-GFP transfected by Andrographis paniculata protoplasts prepared in example 2, wherein FIG. 5A is a GFP fluorescence image, FIG. 5B is a chlorophyll (Chl) autofluorescence image, FIG. 5C is a GFP and Chl integration (Merged), and FIG. 5D is an image of a Bright field channel (Bright).
Detailed Description
The invention provides a protoplast enzymolysis liquid, which comprises 1.5 wt.% of cellulase R-10, 0.75 wt.% of macerozyme R-10, 0.6M mannitol, 20mM MES, 20mM KCl and 10mM CaCl2And 0.1 wt.% BSA.
The protoplast enzymolysis solution provided by the invention comprises 1.5 wt.% of cellulase R-10 and 0.75 wt.% of macerozyme R-10. In the invention, the cellulase R-10 and the macerozyme R-10 are mixed and matched to effectively decompose cell walls. The source of the cellulase R-10 and the macerase R-10 is not critical in the invention, and is preferably purchased from YaKult.
The protoplast enzymolysis solution provided by the invention comprises 0.6M mannitol. The mannitol of the invention maintains the cell osmotic pressure.
The protoplast enzymolysis solution provided by the invention comprises 20mM MES. The MES of the invention acts as a buffer.
The protoplast enzymolysis liquid provided by the invention comprises 20mM KCl and 10mM CaCl2. In the present invention, the KCl and CaCl2The protoplast is not limited by the physiological state of the plant material, the better physiological state of the protoplast is maintained, and the activity and the quality of the protoplast are improved.
The protoplast enzymolysis solution provided by the invention comprises 0.1 wt.% BSA. The BSA of the invention has a protective effect and can reduce or prevent damage to organelles in the enzymolysis process of cell walls.
The protoplast enzymolysis liquid can remove cell walls better through careful preparation, so that cells are dissociated and maintain a good physiological state.
The invention also provides a preparation method of the enzymolysis liquid based on the technical scheme, which comprises the following steps:
mixing the cellulase R-10, the macerozyme R-10, the mannitol, the MES and the KCl, incubating for 10min at 55 ℃, and cooling to room temperature to obtain a first mixed solution;
mixing the first mixed solution with CaCl2Mixing with BSA, filtering, and sterilizing to obtain enzymatic hydrolysate.
The method has no strict requirement on the mode of mixing the cellulase R-10, the macerozyme R-10, the mannitol, the MES and the KCl, and can be realized by adopting a conventional method.
The cellulase R-10, the macerozyme R-10, the mannitol, the MES and the KCl are mixed, incubated for 10min at the temperature of 55 ℃, and cooled to room temperature to obtain a first mixed solution. The incubation according to the invention is capable of inactivating the DNases and proteases present in cellulase R-10 and the macerozyme R-10. The invention has no strict requirement on the cooling mode, and the room temperature is 20-30 ℃.
After the first mixed solution is obtained, the invention mixes the first mixed solution with CaCl2Mixing with BSA, filtering, and sterilizing to obtain enzymatic hydrolysate. When the enzymolysis liquid is prepared, CaCl is added after cooling2And BSA can reduce or prevent the damage to organelles in the enzymolysis process of cell walls, so that the protoplast is not limited by the physiological state of plant materials and maintains a better physiological state.
The invention also provides a method for preparing the andrographis paniculata protoplast based on the enzymolysis liquid, which comprises the following steps:
cutting hypocotyl and leaf tissue of herba Andrographitis seedling, and regulating osmotic pressure to obtain hypocotyl and leaf fragments of herba Andrographitis seedling;
mixing the fragments with the enzymatic hydrolysate for enzymolysis for 3-4 h to obtain an andrographis paniculata enzymatic hydrolysate;
mixing the andrographis paniculata enzymolysis liquid with a W5 solution, and separating to obtain protoplasts.
In the invention, the andrographis paniculata seeds are preferably sown on an MS culture medium for culturing to obtain andrographis paniculata seedlings. According to the invention, the andrographis paniculata seeds are sown on the MS culture medium and cultured, so that the andrographis paniculata seeds can be ensured to germinate well. The invention has no special requirement on the source of the MS culture medium and can be prepared or purchased by oneself.
The andrographis paniculata seeds are preferably selected from andrographis paniculata seeds in south of five Ridges, and further are preferably andrographis paniculata seeds in Guihong City of Guangxi province.
The light-dark time ratio of the culture of the invention is preferably 12 h: 12 h; the light intensity of the culture is preferably 100 μmol m-2s-1(ii) a The culture temperature is preferably 20-30 ℃, more preferably 24-28 ℃, and more preferably 25-26 ℃; the culture time is preferably 20-40 days, more preferably 25-35 days, and even more preferably 28-32 days. The hypocotyl and leaf tissues of the andrographis paniculata seedlings cultured by the method have good growth states and good activity, and the protoplast transfection efficiency prepared from the andrographis paniculata seedlings in the period is higher.
In the invention, before the andrographis paniculata seeds are sowed in the MS culture medium, the andrographis paniculata seeds are preferably soaked in deionized water. The temperature of the deionized water is preferably room temperature, and the soaking time is preferably 12 hours.
According to the invention, 75% ethanol and 3% sodium hypochlorite are preferably used for sequentially disinfecting the soaked andrographis paniculata seeds. The time for sterilization by using 75% ethanol is preferably 15s, and the time for sterilization by using 3% sodium hypochlorite is preferably 20 min.
After obtaining the andrographis paniculata seedlings, the invention preferably cuts off hypocotyl and leaf tissues of the andrographis paniculata seedlings, cuts the hypocotyl and leaf tissues of the andrographis paniculata seedlings and adjusts osmotic pressure to obtain slurry containing the hypocotyl and leaf of the andrographis paniculata seedlings. The mode of regulating osmotic pressure according to the present invention preferably comprises culturing hypocotyl and leaf tissues of the cut plant seedlings in an osmotic pressure regulating solution in the dark for 30 min. When the osmotic pressure is adjusted, the adjustment is preferably performed in a dark environment. The osmotic pressure regulating solution of the present invention is preferably a 0.6M mannitol solution. The invention can maintain the cell osmotic pressure by using 0.6M mannitol solution to adjust the osmotic pressure, reduce shrinkage or expansion of the andrographis paniculata protoplast in the transfection process as much as possible, ensure the cell form of the protoplast and facilitate the later transfection.
After the fragments of hypocotyl and leaves of the andrographis paniculata seedlings are obtained, the fragments and the enzymolysis liquid are mixed and subjected to enzymolysis for 3-4 hours to obtain the andrographis paniculata enzymolysis liquid. The enzymolysis time is preferably 25-30 ℃, and more preferably 26-28 ℃. When the enzymolysis is carried out, the method is preferably carried out in a dark environment, so that the activity of the protoplast is increased. The steps of the preparation of the enzymatic hydrolysate and the enzymatic hydrolysate, and the effect of the enzymatic hydrolysate have been clearly described, and are not described herein again.
After the andrographis paniculata enzymolysis liquid is obtained, the andrographis paniculata enzymolysis liquid is mixed with the W5 solution, and the protoplast is obtained through separation. The volume ratio of the enzymolysis liquid to the W5 solution is preferably 1: 1. The W5 solution of the invention comprises 5mM KCl, 154mM NaCl and 125mM CaCl2And 2mM MES. The preferred mode of mixing the andrographis paniculata enzymolysis liquid with the W5 solution is to shake violently to make the protoplasm body at the cut of the andrographis paniculata leaves drop into the liquid. The time for shaking in the present invention is preferably 10 s. The separation according to the invention is preferably a filtration, preferably using a 40 μm cell filter.
The preparation method of the andrographis paniculata protoplast is simple and efficient, shortens enzymolysis time, can obtain a large amount of andrographis paniculata protoplasts at low cost, and enables the preparation method of the andrographis paniculata protoplasts to be more efficient. The prepared andrographis paniculata protoplast has good cell growth state, is easy to transfect, and is suitable for various research purposes.
The invention also provides a method for transient transfection of the andrographis paniculata protoplast obtained based on the method, which comprises the following steps:
mixing exogenous plasmids, andrographis paniculata protoplasts and PEG solution, and carrying out dark reaction for 12-15 min to obtain reaction liquid;
the reaction solution was diluted with W5 solution, and transfected protoplasts were isolated.
The invention mixes exogenous plasmid, andrographis paniculata protoplast and PEG solution. The andrographis paniculata protoplast of the invention is preferably an MMG solution resuspended protoplast. The concentration of the andrographis paniculata protoplast is preferably 1-2 × 106Individual cells/mL. The MMG solution of the invention comprises 0.6M mannitol, 15mM MgCl2And 4mM MES (pH 5.7). The concentration of the exogenous plasmid is preferably 1-2 mug/muL; the foreign plasmid is preferably an Arabidopsis pUC-GFP plasmid.
The method mixes exogenous plasmid, andrographis paniculata protoplast and PEG solution, and then carries out dark reaction for 12-15 min to obtain reaction liquid. The method has dark reaction for 12-15 min, so that the obtained andrographis paniculata protoplast cells have good activity, and the andrographis paniculata protoplast cells can be used for gene transient expression research.
After the reaction solution is obtained, the invention utilizes the W5 solution to dilute the reaction solution and separates the transfected protoplast. The separation mode of the invention is preferably centrifugation; the time of the centrifugation is preferably 3 min; the rotation speed of the centrifugation is preferably 500 rpm.
The volume ratio of the exogenous plasmid, the andrographis paniculata protoplast and the PEG solution is preferably 1: 10: 11. the exogenous plasmid of the present invention preferably has a marker gene, and more preferably an Arabidopsis plasmid having a marker gene. The invention has no strict requirement on the marker gene and can be a fluorescent marker gene and a resistance gene.
The transfected protoplast can be directly used for subcellular localization, protein interaction and metabolite detection.
The protoplast prepared by the invention successfully transfects exogenous plasmids by adopting a PEG mediated transfection method, establishes an andrographis paniculata protoplast gene transient expression system, and provides technical support for resolving the pharmacological value of andrographis paniculata at a molecular level.
The transfected protoplast obtained by the method is resuspended and cultured for 12-16 hours under the dark condition, and then the protoplast of the transient expression gene can be obtained. The resuspension of the invention is preferably a WI solution, which preferably comprises 0.6M mannitol, 20mM KCl and 4mM MES (pH 5.7).
The MS culture medium needs to be sterilized at the high temperature of 121 ℃ for 20min under high pressure for use; the enzymolysis solution, the W5 solution, the PEG solution, the MMG solution and the WI solution are filtered and sterilized by 0.45 mu m filter membranes.
In order to further illustrate the present invention, the protoplast enzymatic hydrolysate and the preparation method thereof, the preparation of andrographis paniculata protoplast and the transient transfection method and the application thereof provided by the present invention are described in detail below with reference to the drawings and examples, which should not be construed as limiting the scope of the present invention.
The materials and reagents used in the examples of the invention were as follows:
1. seed selection
Selecting seeds of common andrographis herb in Guihong City of Guangxi province.
2. Preparing a reagent:
w5 solution: 5mM KCl, 154mM NaCl, 125mM CaCl22mM MES (pH5.7), 0.45 μm filter sterilized by filtration.
PEG solution: 40% (W/V) PEG4000, 0.3M mannitol, 0.1M CaCl2。
MMG solution: 0.6M mannitol, 15mM MgCl24mM MES (pH5.7), 0.45 μm filter sterilized by filtration.
WI solution: 0.6M mannitol, 20mM KCl, 4mM MES (pH5.7), 0.45 μ M filter sterilized.
MS culture medium: NH (NH)4NO3(825mg/L)，KNO3(950mg/L)，H3BO3(3.1mg/L)，CoCl2·6H2O(0.0125mg/L)，CuSO4·5H2O(0.0125mg/L)，Na2EDTA·2H2O(18.63mg/L)，FeSO4·7H2O(13.9mg/L)，MgSO4(90.35mg/L)，MnSO4·H2O(8.45mg/L)，Na2MoO4·2H2O(0.125mg/L)，KI(0.415mg/L)，KH2PO4(85mg/L)，ZnSO4·7H2O(4.3mg/L)，CaCl2166.1mg/L MES 500mg/L sucrose 30%, KOH to adjust pH to 6.0, and agar powder 7g/LCan be prepared into solid MS culture medium. Sterilizing at 121 deg.C for 20min under high temperature and high pressure.
Example 1
Preparing an enzymolysis solution:
preparing materials according to the following mass and molar concentration: 1.5 wt.% cellulase R-10, 0.75 wt.% macerase R-10, 0.6M mannitol, 20mM MES, 20mM KCl, 10mM CaCl2, and 0.1 wt.% BSA.
Mixing the cellulase R-10, the macerase R-10, the mannitol, the MES and the KCl, incubating at 55 deg.C for 10min, cooling to room temperature, adding 10mM CaCl2And 0.1% BSA, 0.45 μm filter sterilized.
Example 2
1) Soaking herba Andrographitis seed in deionized water at room temperature for 12h, adding 75% ethanol for sterilization for 15s, 3% sodium hypochlorite for sterilization for 20min, and cleaning with deionized water in a super clean bench;
2) placing 80mL MS culture medium in 350mL culture bottles, uniformly sowing the sterilized seeds on the MS culture medium, wherein 5-6 seeds are placed in each culture bottle, and during the culture period, the seeds are irradiated for 12 hours and then treated in darkness for 12 hours, and the seeds are alternately treated and cultured for 40 days in such a way that the light intensity of the irradiation is about 100 mu mol m-2s-1The culture temperature is 26 ℃;
3) shearing 20-30 hypocotyl and leaf tissues of Andrographis paniculata seedlings shown in figure 1 and figure 2A for protoplast preparation, chopping the hypocotyl and leaf tissues with a sharp blade, and dark treating in 0.6M sterile mannitol for 30min to regulate osmotic pressure, as shown in figure 2B;
4) collecting the processed hypocotyl and leaf tissues, putting the enzymolysis liquid into a dark place, and gently shaking (60rpm) for enzymolysis for 3h to enable cells to be free;
5) after enzymolysis, adding a W5 solution with the same volume as the enzyme solution, shaking vigorously by hand for 5-10 s, and collecting in a round-bottom centrifuge tube through a 40-micron cell filter;
6) repeatedly eluting with W5 solution for 3-5 times, centrifuging the protoplast solution collected from the eluent in a horizontal centrifuge at 500rpm for 5min, leaving a small amount of supernatant, and resuspending to obtain herba Andrographitis protoplast, observing the herba Andrographitis protoplast with a fluorescence microscope, wherein the observation result is shown in FIG. 3.
Example 3
The difference from example 2 is that step 4) is carried out for 4 hours.
Example 4
The same as example 2 except that the volume of the MS medium in step 2) was 100 mL.
Example 5
The same as example 2, except that the volume of the MS medium in step 2) was 100mL, and the culture was performed alternately for 20 days.
Test example 1
Detection of Andrographis paniculata protoplast Activity
The andrographis paniculata protoplast prepared in example 2 is stood for 30min in ice bath, supernatant is removed, and the andrographis paniculata protoplast is resuspended in MMG solution and quantified to 1-2 × 106Individual cells/mL. The development of color of the viable cells of the protoplasts of Andrographis paniculata was observed by fluorescence microscopy using 0.01% Fluorescein Diacetate (FDA) staining. The color development results are shown in FIG. 4.
As can be seen from FIG. 3, the viable cells of Andrographis paniculata Nees showed green color, indicating that the obtained protoplast of Andrographis paniculata Nees has physiological activity.
Example 4
Transient transfection of andrographis paniculata protoplasts
1) 10 mu L of 1-2 mu g/mu L arabidopsis 35S, GFP plasmid pUC-GFP (4.5kb), is added into a centrifuge tube;
2) the andrographis paniculata protoplast prepared in example 2 is stood for 30min in ice bath, supernatant is removed, and the andrographis paniculata protoplast is resuspended in MMG solution and quantified to 1-2 × 106Adding 100 mu L of each cell/mL into a centrifuge tube, and gently mixing;
3) adding 110 mu L of PEG solution, turning upside down and fully mixing, and carrying out dark reaction for 12-15 min;
4) the transfection reaction was terminated by diluting the above solution with 440. mu. L W5 solution and mixing well. Horizontally centrifuging at 500rpm for 3min, and removing supernatant;
5) adding 300 mu L-1 mL of WI solution to resuspend the protoplast, transferring the protoplast to 24/12/6 cell culture plates, and culturing for 12-16 hours under the dark condition.
6) After the culture is finished, the WI solution in the culture plate is directly used for blowing and sucking the heavy suspension protoplast, the protoplast is transferred into a centrifugal tube and horizontally centrifuged at 500rpm for 3min, and the supernatant is left for gently flicking the suspended protoplast to obtain the transfected protoplast.
Test example 2
The images of example 4 GFP, chlorophyll autofluorescence (Chl), both integration (merge) and Bright field channel (Bright) were taken and viewed using the THUNDER wide field high resolution imaging system (inverted) and the results are shown in fig. 4.
As can be seen from FIG. 4, the protoplast prepared by the invention successfully transfects pUC-GFP by using the PEG-mediated transfection method, and successfully establishes the transient expression system of the andrographis paniculata protoplast gene.
The above embodiments can be seen that the technical scheme provided by the invention can better remove the cell wall of andrographis paniculata, so that cells can be dissociated, and the cells can maintain a good physiological state. The invention maintains the osmotic pressure of cells by adjusting the concentration of mannitol, reduces the shrinkage or the expansion of the protoplast in the transfection process and ensures the cell form of the protoplast. The enzymolysis liquid can shorten the enzymolysis time when the protoplast is prepared, ensure the high activity and the high yield of the protoplast and successfully establish the gene transient expression system of the andrographis paniculata protoplast.
Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and all of the embodiments belong to the protection scope of the present invention.
Claims (10)
1. A protoplast enzymatic hydrolysate comprising 1.5 wt.% of cellulase R-10, 0.75 wt.% of macerozyme R-10, 0.6M mannitol, 20mM MES, 20mM KCl, 10mM CaCl2And 0.1 wt.% BSA.
2. The method for preparing an enzymatic hydrolysate of claim 1, comprising the steps of:
mixing the cellulase R-10, the macerozyme R-10, the mannitol, the MES and the KCl, incubating for 10min at 55 ℃, and cooling to room temperature to obtain a first mixed solution;
mixing the first mixed solution with CaCl2Mixing with BSA, filtering, and sterilizing to obtain enzymatic hydrolysate.
3. A method for preparing andrographis paniculata protoplasts comprises the following steps:
cutting hypocotyl and leaf tissue of herba Andrographitis seedling, and regulating osmotic pressure to obtain hypocotyl and leaf fragments of herba Andrographitis seedling;
mixing the fragments with the enzymolysis liquid of claim 1 for enzymolysis for 3-4 hours to obtain an andrographis paniculata enzymolysis liquid;
mixing the andrographis paniculata enzymolysis liquid with a W5 solution, and filtering to obtain the protoplast.
4. The method of claim 3, wherein the culturing is performed at a light-to-dark time ratio of 12 h: 12 h; the illumination intensity of the culture is 100 mu mol-2s-1。
5. The method according to claim 3 or 4, wherein the culturing time is 20-40 days; the culture temperature is 20-30 ℃.
6. The method of claim 3, wherein said osmotically conditioning comprises dark culturing hypocotyl and leaf tissue of the minced plant seedling in an osmoconditioning solution comprising 0.6M mannitol solution for 30 min.
7. The method of claim 3, wherein the W5 solution comprises 5mM KCl, 154mM NaCl, 125mM CaCl2And 2mM MES.
8. A method for transient transfection of andrographis paniculata protoplasts comprises the following steps:
mixing the andrographis paniculata protoplast prepared by the method of any one of claims 3 to 7 with exogenous plasmids and a PEG solution, and carrying out dark reaction for 12-15 min to obtain a reaction solution;
the W5 solution was mixed with the reaction solution, and transfected protoplasts were isolated.
9. The method of claim 8, wherein the exogenous plasmid, the andrographis paniculata protoplast, and the PEG solution are present in a volume ratio of 1: 10: 11.
10. the method of claim 8 or 9, wherein said exogenous plasmid comprises a marker gene.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114214305A (en) * | 2022-01-18 | 2022-03-22 | 中国科学院东北地理与农业生态研究所 | Enzymolysis liquid for preparing Lonicera caerulea protoplast and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101402938A (en) * | 2008-11-17 | 2009-04-08 | 中国科学院植物研究所 | Method for producing protoplast |
| CN112680399A (en) * | 2020-12-29 | 2021-04-20 | 广州基迪奥生物科技有限公司 | Preparation method and application of plant protoplast single cell suspension |
-
2021
- 2021-08-30 CN CN202111003791.XA patent/CN113604418A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101402938A (en) * | 2008-11-17 | 2009-04-08 | 中国科学院植物研究所 | Method for producing protoplast |
| CN112680399A (en) * | 2020-12-29 | 2021-04-20 | 广州基迪奥生物科技有限公司 | Preparation method and application of plant protoplast single cell suspension |
Non-Patent Citations (3)
| Title |
|---|
| 刘岭: "中草药植物细胞工程研究进展", 《中草药》 * |
| 王健 等: ""穿心莲转录因子ApNAC1的克隆、亚细胞定位及原核表达"", 《中国中药杂志》 * |
| 黄瑞华 等: ""穿心莲叶片原生质体制备方法的建立与优化"", 《分子植物育种》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114214305A (en) * | 2022-01-18 | 2022-03-22 | 中国科学院东北地理与农业生态研究所 | Enzymolysis liquid for preparing Lonicera caerulea protoplast and preparation method and application thereof |
| CN114214305B (en) * | 2022-01-18 | 2023-05-09 | 中国科学院东北地理与农业生态研究所 | Enzymatic hydrolysate for preparing lonicera caerulea protoplast and preparation method and application thereof |
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