CN111548984A - Method for preparing and transforming wheat endosperm cell protoplast - Google Patents
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Abstract
The invention discloses a method for preparing and transforming wheat endosperm cell protoplast. The invention provides a method for preparing wheat endosperm cell protoplast, which comprises the following steps: taking fresh wheat seeds 8-11 days after flowering, splitting along abdominal seams to obtain endosperm tissues, soaking and washing the endosperm tissues in a buffer solution 1, then placing the endosperm tissues in a buffer solution 2 for enzymolysis, and performing ice bath standing for gradient sedimentation to obtain purified wheat endosperm cell protoplasts. The invention provides a method for preparing and transforming protoplast with high efficiency, economy and rapidness aiming at wheat endosperm cells containing a large amount of storage starch granules, which can rapidly and effectively separate the protoplast of the endosperm cells, can be used for subcellular localization, transcription factor activity analysis, protein interaction identification and enzyme activity analysis of wheat endosperm expression factors, creates conditions for the research of molecular biology in vivo of the factors and effectively avoids experimental deviation caused by the error expression of specific expression genes of the endosperm in other cells.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for preparing and transforming wheat endosperm cell protoplasts.
Background
Wheat endosperm is a vegetative storage organ, containing about 75% storage starch, 15% storage protein and 1-2% fat. During endosperm development, a large number of genes are involved in the synthesis, transport and storage of nutrients. Understanding the regulation and function of these endosperm expression genes is a prerequisite for improving wheat yield and quality by genetic means. Because of long growth period of wheat and high difficulty in stable genetic transformation, large-scale development of biological function research of endosperm expression genes in wheat bodies is difficult. Therefore, development of alternative technologies is urgently needed.
Transient protoplast expression is a conventional technology for researching protein subcellular localization and biological functions, and at present, mature Arabidopsis thaliana mesophyll cell protoplast expression systems (Chenglu, Jiayan chrysanthemum, Zhang cui ru. Arabidopsis thaliana mesophyll cell protoplast separation and influencing factors. Hibei university report (Nature science edition), 2008,28(4): 423-. However, endosperm cells are not structurally and functionally identical to mesophyllic cells. Structurally, mesophyllic cells have chloroplasts, while endosperm cells have subcellular organelles such as amyloplasts and proteosomes of single membrane structure. Functionally, mesophyllic cells utilize the assimilation forces formed in the photoreaction to transport CO2Conversion to carbohydrates; the endosperm cells take sucrose and free amino acids output by mesophyll cells as raw materials to synthesize storage starch and storage protein. Therefore, it is not clearly appropriate to study the biological function of endosperm-expressed genes, in particular endosperm-specific expressed genes, in mesophyllic cell protoplasts.
The development of the cereal kernel-type endosperm is divided into a free nucleus phase, a cellularization phase, a growth differentiation phase and a maturation phase (OlsenOA. nucleic end period degradation in cereals and Arabidopsis thaliana. plant cell,2004,16: 214-222.). 4 days after wheat blossoms, endosperm is cellularized (Jingyanping, Liuda Tong, Lidong Beam, Lixiaogang, Xian, Gushijie, Wangzhuang. development of wheat endosperm cells and starch body. journal of wheat crops, 2013,33(4):818 and 824.); then entering a cell rapid growth stage until the number of days after the flower is basically stable (Ulmus pumila, Guo Wen, squaring, Zhang Chaien, Zhu Xin, Pengyoxin, wheat grain endosperm cell proliferation and substance enrichment dynamic. plant bulletin, 2003,29(5): 779-784.). Two kinds of starch grains are synthesized by the powder-making body of the wheat endosperm cells: the A-type starch grains have large diameter (10-40 μm) and small number (about 10% of total starch grains), and are synthesized from 4 days after flowering; type B starch granules are small (<10 μm) in diameter and abundant in number (about 90% of total starch granules), and are synthesized starting from 12 days after flowering. Under the influence of the quantity and volume of starch granules, the cell nucleus in endosperm cell begins to deform from 16 days after flowering, inducing programmed cell death (Jingyanping, Liuda Tong, Lidong, Libang, Hawai Xiao, Xian, Huizhi, Wangzhi, wheat endosperm cell and its starch body development, wheat crops journal, 2013,33(4): 818-. Besides starch granules, the endosperm cells contain a certain number of proteosome. Small proteosome begins to appear 8 days after blooming, and is fused with the development of endosperm to form large proteosome which is filled in the gap of starch granules (Jingyanping, Liuda Tong, Lidong beam, Libanggang, unfortunately, Zhaochen, Wangzhi, wheat endosperm cells and the development of starch body. the journal of wheat crops, 2013,33(4): 818-.
The large-ball endosperm cells of the wheat are adhered together and are more difficult to dissociate than the mesophyll cells; the presence of type a starch granules makes the dissociated endosperm cells more susceptible to mechanical damage leading to cell disruption; starch granules inside and outside cells adsorb a large amount of vectors in the protoplast transformation process, which is not beneficial to the expression of target genes. Obviously, the methods for the preparation and transformation of mesophyllic cell protoplasts are not applicable to wheat endosperm cell protoplasts.
Disclosure of Invention
The present invention aims to solve at least one of the above technical problems to a certain extent. Therefore, the invention provides a method for preparing and transforming the protoplast of the wheat endosperm cell, which can effectively, economically and rapidly extract the protoplast of the wheat endosperm cell.
In a first aspect, the invention claims a method of preparing wheat endosperm cell protoplasts.
The method for preparing the wheat endosperm cell protoplast claimed by the invention can comprise the following steps:
s1: taking fresh wheat seeds 8-11 days after flowering, splitting along abdominal seams to obtain endosperm tissues;
s2: soaking and washing endosperm tissues in a buffer solution 1;
s3: placing the washed endosperm tissues in a buffer solution 2 for enzymolysis to obtain crude wheat endosperm cell protoplasts;
s4: and (3) treating the crude wheat endosperm cell protoplast (aiming at removing starch granules) by adopting an ice bath standing time gradient sedimentation method to obtain the purified wheat endosperm cell protoplast.
In step S1, fresh wheat grain 8-11 days after flowering was selected as the test material, because the present invention found that the material can provide endosperm cells with large amount, well-developed and small content of starch granules, which is important for the separation of endosperm protoplasts.
In step S2, the osmotic pressure stabilizer in the buffer solution 1 is sucrose and mannitol, wherein the concentration of sucrose is 20g/L, and the concentration of mannitol is 130 g/L.
In step S2, the solvent of buffer 1 is water, and the solutes and concentrations are as follows: 130g/L of D-mannitol, 20g/L of sucrose, 0.2mM of dipotassium hydrogen phosphate (KH)2PO4) 1.0mM potassium nitrate (KNO)3) 10.0mM calcium chloride dihydrate (CaCl)2·2H2O), 1.0mM magnesium sulfate heptahydrate (MgSO)4·7H2O), 1.0 μ M potassium iodide (KI), 0.1 μ M copper sulfate pentahydrate (CuSO)4·5H2O) and 2.0mM MES (2- (N-morpholino) ethanesulfonic acid) pH 5.7. Buffer 1 was filter sterilized and ready for use.
Wherein the amount of hydrate can be converted to the amount of corresponding non-hydrate substance to be replaced by non-hydrate.
In step S2, the endosperm tissue is cut into thin strips (e.g., strips with a width of 0.5 mm), soaked in the buffer 1 for 30-60min (e.g., 45min), mixed by gently inverting, and then washed 3-5 times (e.g., 3 times) with the buffer 1 until the buffer 1 becomes clear.
In step S3, the buffer solution 2 contains Cellulase (e.g., Cellulase (R-10)) and an eductase (e.g., Macerozyme (R-10) or an eductase having the same composition).
Further, the content of the cellulase in the buffer solution 2 is 150U/mL; the content of the eductase in the buffer 2 was 15.3U/mL.
In step S3, the osmotic pressure stabilizer in the buffer solution 2 is sucrose and mannitol, wherein the concentration of sucrose is 20g/L, and the concentration of mannitol is 130 g/L.
Further, the solvent of the buffer solution 2 is water, and the solutes and the concentrations are as follows: 150U/mL cellulase, 15.3U/mL macerase, 10g/L Bovine Serum Albumin (BSA), 130g/L D-mannitol, 20g/L sucrose, 0.2mM dipotassium hydrogen phosphate (KH)2PO4) 1.0mM potassium nitrate (KNO)3) 10.0mM calcium chloride dihydrate (CaCl)2·2H2O), 1.0mM magnesium sulfate heptahydrate (MgSO)4·7H2O), 1.0 μ M potassium iodide (KI), 0.1 μ M copper sulfate pentahydrate (CuSO)4·5H2O) and 2.0mM MES (2- (N-morpholino) ethanesulfonic acid) pH 5.7.
The buffer 2 was prepared as follows: in the buffer 1 (excluding 10.0mM of calcium chloride dihydrate (CaCl)2·2H2O) and Cellulase (Celluase (R-10)) at a final concentration of 150U/mL and Segrelase (Macerozyme (R-10)) at a final concentration of 15.3U/mL were added thereto, and the mixture was mixed in a water bath at 55 ℃ for 10 minutes, and then the mixture was thoroughly dissolved by inversion, cooled to room temperature, and calcium chloride dihydrate (CaCl) at a final concentration of 10.0mM was added thereto2·2H2O) and Bovine Serum Albumin (BSA) at a final concentration of 10g/L, and filtering and sterilizing the mixture for later use.
The research of the invention finds that the buffer solution 2 has better effects on the enzymolysis of the endosperm tissues of wheat and the release of protoplasts.
In step S3, the enzymatic hydrolysis is performed by placing the endosperm tissue in the buffer 2 and shaking for 90-120min (e.g., 120min) in the dark at 24 ℃.
In a specific embodiment of the present invention, the oscillation rate is 40rpm and the amplitude is 26 mm.
In step S3, the ratio of the endosperm tissue to the buffer 2 is 0.4-0.8g (e.g. 0.6 g): 2 mL.
In step S3, after the endosperm tissue is placed in the buffer solution 2 for enzymolysis, the method may further include the following steps: adding the buffer solution 1 with the same volume into the system after enzymolysis, and then filtering the mixture by using a 200-mesh cell sieve into a container (such as a centrifuge tube, specifically a 10.0mL round-bottom centrifuge tube) to obtain the crude wheat endosperm cell protoplast.
Wherein the cell sieve and centrifuge tube were previously rinsed with buffer 1. And adding the buffer solution 1 with the same volume into the system after enzymolysis to form a mixed solution, and transferring the mixed solution into the cell sieve through a 5.0mL gun head for removing the tip part.
During this period, the enzymolyzed tissue was microscopically examined, and materials or reagents used for subsequent testing, including 1-precooled buffer, 200-mesh cell sieve, 10.0mL round-bottom centrifuge tube wet, and 5.0mL tip treatment were included. Thereby improving the yield of protoplasts and reducing the damage rate caused by mechanical damage as much as possible.
Step S4 may proceed as follows:
s41: and (4) placing the container containing the crude wheat endosperm cell protoplast obtained in the step S3 in an ice water bath or pre-cooling at 4 ℃ for 10min, and discarding the supernatant, wherein the amount of the removed supernatant is half of the total volume (the upper part is removed, and the lower part is reserved as the buffer containing the crude endosperm cell protoplast).
S42: slowly adding the buffer solution 1 into a container containing the sample treated in the step S41 along the side wall, wherein the adding amount of the buffer solution 1 is 3.5 times of the volume of the crude wheat endosperm cell protoplast in the container, mixing (mixing by gentle inversion), then vertically standing in an ice water bath for 8min, discarding the supernatant, and the amount of the supernatant removed is the volume of the added buffer solution 1 (removing the upper part and keeping the lower part as the buffer solution containing the crude endosperm cell protoplast);
s43: slowly adding the buffer solution 1 into the container filled with the sample treated in the step S42 along the side wall, wherein the adding amount of the buffer solution 1 is 3.5 times of the volume of the crude wheat endosperm cell protoplast in the container, mixing (mixing by gentle inversion), vertically standing in an ice water bath for 6min, and discarding the supernatant, wherein the amount of the removed supernatant is the volume of the added buffer solution 1 (the upper part is removed, and the lower part is reserved as the buffer solution containing the crude endosperm cell protoplast).
Further, step S44 may be further included after step S43:
s44: and (4) vertically standing the container filled with the sample processed in the step (S43) in an ice water bath for 30min, and removing all supernatant to obtain the purified wheat endosperm cell protoplast.
That is, according to the principle that the sedimentation rate of the protoplast is faster than that of the starch granules, the starch granules are removed successively by adopting the sedimentation time of the protoplast: standing in ice water bath for 10min, and removing supernatant; adding buffer solution 1 for suspension, and then sequentially carrying out the steps of 8min ice bath standing-supernatant removal and 6min ice bath standing-supernatant removal. Therefore, the mechanical damage of the centrifugal force to the protoplast can be reduced, a large amount of starch granules can be removed more efficiently, and the effects of high efficiency, economy and quickness are achieved.
In a second aspect, the invention claims a method for transformation of wheat endosperm cell protoplasts.
The method for transforming the wheat endosperm cell protoplast claimed by the invention can comprise the following steps:
s5: resuspending wheat endosperm cell protoplasts prepared by the method of the first aspect in MMg buffer;
s6: PEG-induced transformation was performed.
In step S5, the MMg buffer solution is water, and the solutes and concentrations are as follows: 130g/L of D-mannitol, 15.0mM of magnesium chloride hexahydrate (MgCl)2·6H2O) and 4.0mM MES (2- (N-morpholino) ethanesulfonic acid) pH 5.7. The MMg buffer is used after filtration sterilization.
In step S5, after the wheat endosperm cell protoplast is resuspended in the MMg buffer, the content of the wheat endosperm cell protoplast in the resuspended solution is about 1 × 106one/mL.
In step S6, the solvent of the PEG-induced transformation solution used for the PEG-induced transformation is water, and the solutes and concentrations are as follows: 400g/L PEG4000, 0.2mol/L D-mannitol and 10.0mmol/L calcium chloride dihydrate (CaCl)2·2H2O). And filtering and sterilizing the PEG induced transformation liquid for later use.
Further, step S6 may be performed as follows:
s61: mixing the heavy suspension containing the wheat endosperm cell protoplast obtained in the step S5 with a plasmid to be transformed, and then adding the PEG to induce transformation liquid; the proportion of the resuspension, the plasmid to be transformed and the PEG induced transformation liquid is 200 mu L: 10 μ g: 220 mu L of the solution;
s62: turning, mixing, and standing at 22-24 deg.C in dark for 10-15min (such as 15 min);
s63: adding the buffer solution 1, slightly reversing, uniformly mixing, centrifuging at 4 ℃ in a soft mode for 1min at 100g, and removing supernatant; repeating this step 2-3 times (e.g., 3 times) until the PEG-induced transformation solution is completely removed;
s64: adding the buffer solution, and incubating for 12h at 1, 22-24 ℃ in a dark place.
Detection may then be performed. In a specific embodiment of the present invention, the plasmid to be transformed is a plasmid expressing GFP fluorescent protein, and the transformation effect is determined by observing the fluorescence of the protoplast with LSM780 fluorescence confocal microscope (Zeiss, Germany).
In a third aspect, the invention claims any of the following:
(A1) a method for the preparation and transformation of wheat endosperm cell protoplasts can comprise steps S1-S4 of the method of the first aspect above and steps S5 and S6 of the method of the second aspect above.
(A2) Wheat endosperm cell protoplasts prepared by the method described in the first aspect hereinbefore.
(A3) A suspension of wheat endosperm cell protoplasts obtained after resuspending the wheat endosperm cell protoplasts prepared as described in the first aspect above in an MMg buffer as described above.
(A4) Use of a method as described in the first aspect hereinbefore or (A2) said protoplast of a wheat endosperm cell or (A3) said suspension of said protoplast of a wheat endosperm cell in the genetic transformation of wheat.
(A5) Use of a method as described in the first aspect hereinbefore or a method as described in the second aspect hereinbefore or (a1) a method as described in (a2) a protoplast of a wheat endosperm cell or (A3) a suspension of a protoplast of a wheat endosperm cell in subcellular localization of a factor expressing wheat endosperm, in analysis of transcription factor activity, in identification of protein interactions and/or in analysis of enzyme activity.
The invention provides a method for preparing and transforming protoplast with high efficiency, economy and rapidness aiming at wheat endosperm cells containing a large amount of storage starch granules, the method can rapidly and effectively separate the endosperm cell protoplast of fresh wheat grains 8-11 days after flowering, can be used for subcellular localization, transcription factor activity analysis, protein interaction identification and enzyme activity analysis of wheat endosperm expression factors, and creates conditions for the in vivo molecular biology research of the factors. The method can effectively avoid experimental deviation caused by the error expression of the endosperm expression genes, particularly endosperm specific expression genes, in other cells, and improve the accuracy of experimental results. The invention can realize accurate positioning of the subcellular of the endosperm expression gene, thereby providing a complete technical system for further researching the molecular biological function of the wheat endosperm expression gene in the endosperm development process.
The invention has the following advantages:
(1) test materials: the method selects wheat grains 8-11 days after flowering, avoids the problem of small quantity of endosperm cells at the early development stage of grains, and also avoids the problem that the cells are seriously damaged and the complete endosperm protoplast is difficult to obtain in the preparation and transformation processes due to excessive storage starch in the endosperm cells at the middle and later stages;
(2) the operation is simple: the purification step of the protoplast adopts time gradient sedimentation, and the method is simple and easy to operate and is incomparable with the traditional protoplast density gradient purification method;
(3) the cost is low: the method replaces a density gradient centrifugation method with a time gradient sedimentation method in the process of purifying the endosperm protoplast, does not use expensive cesium chloride and other media, and reduces the cost;
(4) the potential value is high: the method can be used for the research of molecular biological functions of genes related to wheat grain development and nutrient synthesis, and is particularly suitable for endosperm specific expression genes: the method comprises some in vivo experiments such as subcellular localization, dual-luciferase reporter gene detection, Pull down and enzyme activity detection, and solves the problems of high difficulty in wheat genetic transformation, long in vivo experiment period and the like.
Drawings
FIG. 1 is a flow chart of the preparation and transient transformation of wheat endosperm cell protoplasts of the invention.
FIG. 2 is a graph showing the effect of removing starch granules by ice bath standing time gradient sedimentation.
FIG. 3 is a graph showing the effect of different osmotic pressures on the preparation of protoplasts from wheat endosperm cells.
FIG. 4 shows the effect of different enzymatic hydrolysis times on the preparation of protoplasts of wheat endosperm cells.
FIG. 5 is a graph of the effect of wheat endosperm at different developmental stages on endosperm cell protoplast preparation.
FIG. 6 shows the effect of different endosperm doses on the preparation of protoplasts from wheat endosperm cells.
Fig. 7 is wheat grain and endosperm cell protoplasts 8 days after flowering.
FIG. 8 shows the subcellular localization of the transcription factor TaABI5 in wheat endosperm cell protoplasts.
In the figure, FDA is fluorescein diacetate (excitation wavelength 488nm), GFP is green quartz (excitation wavelength 488nm), chlorophyllin is chloroplast fluorescence (excitation wavelength 633nm), Bright field is light, and Merged is superposition.
Detailed Description
The invention provides a method for preparing and transforming wheat endosperm cell protoplast, which mainly comprises the following steps: obtaining materials, separating and purifying protoplast, inducing and transforming PEG, observing and analyzing microscopically and the like. Stripping fresh wheat endosperm tissues in a specific period, soaking and washing the wheat endosperm tissues with a buffer solution 1 to remove starch granules released by endosperm cells due to mechanical damage; performing enzymolysis on the endosperm cells by using a buffer solution 2 under a set condition to release protoplasts; removing starch granules released by the broken cells by using an ice bath standing time gradient sedimentation method; followed by MMg buffer suspension, PEG-induced transformation, overnight incubation for microscopic observation. Protoplast fluorescence was observed using an LSM780 laser confocal microscope (Zeiss, Germany) to study the molecular biological function of the gene.
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The operating procedures in the examples which follow are not specifically indicated for temperature and are all at room temperature (about 22-24 ℃).
Various solutions used in the following examples:
1. buffer 1: the solvent is water, and the solutes and the concentrations are as follows: 130g/L of D-mannitol, 20g/L of sucrose, 0.2mM of dipotassium hydrogen phosphate (KH)2PO4) 1.0mM potassium nitrate (KNO)3) 10.0mM calcium chloride dihydrate (CaCl)2·2H2O), 1.0mM magnesium sulfate heptahydrate (MgSO)4·7H2O), 1.0 μ M potassium iodide (KI), 0.1 μ M copper sulfate pentahydrate (CuSO)4·5H2O) and 2.0mM MES (2- (N-morpholino) ethanesulfonic acid) pH 5.7. Filtering and sterilizing for later use.
2. Buffer 2: the solvent is water, and the solutes and the concentrations are as follows: 150U/mL cellulase, 15.3U/mL macerase, 10g/L Bovine Serum Albumin (BSA), 130g/L D-mannitol, 20g/L sucrose, 0.2mM dipotassium hydrogen phosphate (KH)2PO4) 1.0mM potassium nitrate (KNO)3) 10.0mM calcium chloride dihydrate (CaCl)2·2H2O), 1.0mM magnesium sulfate heptahydrate (MgSO)4·7H2O), 1.0 μ M potassium iodide (KI), 0.1 μ M copper sulfate pentahydrate (CuSO)4·5H2O) and 2.0mM MES (2- (N-morpholino) ethanesulfonic acid) pH 5.7.
Buffer 2 was prepared as follows: in buffer 1 (excluding 10.0mM calcium chloride dihydrate (CaCl)2·2H2O) and Cellulase (Celluase (R-10)) at a final concentration of 150U/mL and Segrelase (Macerozyme (R-10)) at a final concentration of 15.3U/mL were added thereto, and the mixture was mixed in a water bath at 55 ℃ for 10 minutes, and then the mixture was thoroughly dissolved by inversion, cooled to room temperature, and calcium chloride dihydrate (CaCl) at a final concentration of 10.0mM was added thereto2·2H2O) and Bovine Serum Albumin (BSA) at a final concentration of 10g/L, and filtering and sterilizing the mixture for later use.
Wherein, the Cellulase (Celluase (R-10)) is a product of Yakult company, and the product number is L0012-10 g; the enzyme activity is defined as: one unit of cellulase enzyme is capable of degrading cellulose at 25 ℃ per minute to release 1. mu. mol glucose. The isolation enzyme (Macerozyme (R-10) is a product of Yakult, Inc., and has a product number of L0021-10 g.
3. MMg buffer solution: the solvent is water, and the solutes and the concentrations are as follows: 130g/L of D-mannitol, 15.0mM of magnesium chloride hexahydrate (MgCl)2·6H2O) and 4.0mM MES (2- (N-morpholino) ethanesulfonic acid) pH 5.7. Filtering and sterilizing for later use.
4. PEG induced transformation solution: the solvent is water, and the solutes and the concentrations are as follows: 400g/L PEG4000, 0.2M D-mannitol and 10.0mM calcium chloride dihydrate (CaCl)2·2H2O). Filtering and sterilizing for later use.
GFP (16318hGFP) is commercially available from a company (e.g., Biovector NTCC type culture Collection (Biovector Co., LTD)). 35S: TaABI5 GFP was self-constructed by the laboratory: taking wheat endosperm cDNA as a template, amplifying by primers 5'-TATCTCTAGAGGATCCATGGCGTCGGAGATGAGCA-3' and 5'-TGCTCACCATGGATCCACCAGATGCAGCTGCCGCA-3' to obtain a TaABI5 gene, and recombining to a BamHI site in a 16318hGFP vector by using a homologous recombination method.
Example 1 preparation and transformation of wheat endosperm cell protoplasts
First, experiment method
The flow chart of the preparation and transient transformation of wheat endosperm cell protoplasts of this example is shown in FIG. 1. The method mainly comprises the following steps:
s1: harvesting fresh wheat grains 8-11 days after blooming, and splitting along abdominal seams to obtain endosperm tissues;
s2: cutting endosperm tissues into thin strips, soaking in a buffer solution 1, and washing;
s3: placing the washed endosperm tissues in a buffer solution 2 to obtain crude wheat endosperm cell protoplasts;
s4: treating the crude endosperm cell protoplast by adopting an ice bath standing time gradient sedimentation method to obtain a purified wheat endosperm cell protoplast;
s5: suspending the purified wheat endosperm cell protoplast by using MMg buffer solution;
s6: performing PEG induced transformation on the obtained wheat protoplast; after overnight dark culture, fluorescence was observed using LSM780 laser confocal microscope (Zeiss, Germany).
In the embodiment, the influence of wheat grains with different development times, different osmotic pressures, different enzymolysis times and different endosperm dosage on the finally obtained wheat endosperm cell protoplast is explored. Wherein the different osmotic pressures were achieved by adjusting the mannitol concentrations in buffer 1 and buffer 2 as described above, and the mannitol concentrations were set at four gradients of 11%, 12%, 13% and 14% (% represents g/100mL), and the remaining components were unchanged.
1. Materials obtained from
Seeds developed from Chinese spring wheat varieties are used as test materials, and fresh seeds at different time (5, 8, 11 and 14 days) after flowering are used for separating and instantaneously transforming wheat endosperm protoplasts.
2. Isolation and transient transformation of protoplasts
(1) Taking 15 grains of wheat endosperm, cutting into 0.5mm strips, soaking in 1.5mL buffer solution 1 for 45min, slightly reversing, mixing, and removing supernatant; gently wash 3 times with buffer 1; and removing the supernatant.
(2) Taking endosperm (0.2, 0.4, 0.6 and 0.8g) with different dosages after the treatment of the supplement (1), adding 2.0mL of enzymolysis buffer solution 2, placing in a constant temperature shaking table at 24 ℃, at 40rpm, with the amplitude of 26mm, and carrying out enzymolysis for different time (0.5, 1, 2 and 3h) in a dark place; during the period, 20. mu.L of the lysed tissue was subjected to microscopic examination, and the state and amount of protoplasts were observed.
(3) An equal volume of buffer 1 was added and slowly filtered through a 200 mesh cell sieve into a 10.0mL round bottom centrifuge tube. (all vessels were rinsed with buffer 1 in advance). This step was performed by transferring crude endosperm cell protoplasts into a cell sieve using a treated 5.0mL tip (tip removal). The crude wheat endosperm cell protoplast is obtained by the steps.
(4) And (4) carrying out ice-water bath on the round-bottom centrifuge tube filled with the crude wheat endosperm cell protoplasts in the step (3) for 10min, and removing the supernatant until 2.0mL of buffer solution containing the crude protoplasts is reserved at the bottom of the tube.
(5) Add slowly 7.0mL buffer 1 along the side wall, mix gently by inversion, stand vertically in ice water bath for 8min, discard the supernatant until 2.0mL buffer containing protoplasts is left at the bottom of the tube.
(6) Slowly adding 7.0mL of buffer solution 1 along the side wall, slightly reversing and uniformly mixing, vertically standing in an ice water bath for 6min, and removing the supernatant until 2.0mL of buffer solution containing the protoplast is left at the bottom of the tube; 20 μ L of the purified protoplast was microscopically observed.
(7) Standing in ice bath for 30min to completely remove supernatant.
(8) Slowly adding appropriate amount of MMg buffer solution along the side wall, and resuspending the wheat endosperm cell protoplast to a concentration of 1 × 106one/mL.
(9) 2 centrifuge tubes of 2.0mL are taken, 200 μ L of protoplast MMg suspension and 10 μ g of plasmid (35S:: GFP or 35S:: TaABI5: GFP) are respectively added in sequence, mixed gently, 220 μ L of PEG induced transformation liquid is added, mixed gently and inverted, and kept stand for 15min at room temperature in a dark place.
(10) Add slowly 880. mu.L buffer 1 along the tube wall, mix well by gentle inversion and centrifuge for 1min at 4 ℃ soft mode 100 g.
(11) Repeating the step (10)3 times until the PEG-induced transformation solution is completely removed.
(12) Remove supernatant, add 220. mu.L buffer 1, resuspend gently by inversion, place at room temperature, wrap with tinfoil, and incubate overnight in the dark.
(13) The GFP fluorescence of the transiently transformed wheat endosperm cell protoplasts was observed using LSM780 fluorescent confocal microscope (Zeiss, Germany).
Second, results and analysis
1. Effect of removing starch granules by ice bath standing time gradient sedimentation method
The effect of removing starch granules by the ice-bath standing time gradient sedimentation method is shown in figure 2. a is crude protoplast (not purified); b is purified protoplast (after purification). The dotted circles and arrows indicate starch granules.
Starch granules gradually accumulate in the wheat endosperm cells as the kernel develops. During sample processing, a large number of starch granules are released from damaged cells, on the one hand causing mechanical damage to the fragile protoplasts, and on the other hand adsorbing a large number of vectors during transformation, which is not conducive to the uptake of foreign DNA fragments by the protoplasts. FIG. 2 shows that prior to the present purification, a large number of starch granules filled the protoplast space (shown by the dashed circles); after purification, only a small amount of starch granules remained (arrow).
2. Influence of different osmotic pressures on preparation of protoplasts of wheat endosperm cells
The effect of different osmotic pressures on the preparation of wheat endosperm cell protoplasts according to the invention is shown in FIG. 3.
The osmotic pressure of the buffer determines the quality and yield of the protoplasts. The too high osmotic pressure of the buffer solution can cause the protoplast to shrink, which is not beneficial to the division of the protoplast; too low can lead to disruption of the protoplasts. Taking 0.5g of wheat endosperm tissue 8 days after flowering, and placing the wheat endosperm tissue in 2.0mL of buffer solution 2 for enzymolysis for 2.0 h. Both buffers 1 and 2 used sucrose and mannitol as osmotic pressure stabilizers, with sucrose at 2% (i.e., 20 g/L); mannitol is provided with four concentration gradients of 11%, 12%, 13% and 14%. Removing most starch granules by an ice-bath standing time gradient sedimentation method; adding FDA (fluorescein diacetate) with the final concentration of 0.05g/mL, standing for 5-10min at normal temperature in the dark, and performing microscopic examination. The most active protoplasts were obtained when mannitol was used at 13%.
3. Influence of different enzymolysis time on preparation of wheat endosperm cell protoplast
The influence of different enzymolysis times on the preparation of the wheat endosperm cell protoplast is shown in figure 4.
0.5g of wheat endosperm tissue 8 days after flowering is placed in 2.0mL of buffer solution 2 containing 13% mannitol for enzymolysis, and four time gradients of 0.5, 1.0, 2.0 and 3.0h are set. Removing most starch granules by an ice-bath standing time gradient sedimentation method; adding FDA with final concentration of 0.05g/mL, standing at normal temperature in dark place for 5-10min, and performing microscopic examination. The effect of the endosperm tissue enzymolysis is optimal for 2.0h, and the yield of the active protoplast is highest. The enzymolysis time is too short, and the number of free protoplasts is small; the enzymolysis time is too long, and the protoplast is broken more.
4. Effect of wheat endosperm at different developmental stages on endosperm cell protoplast preparation
The effect of wheat endosperm at different developmental stages on endosperm cell protoplast preparation is shown in FIG. 5.
Wheat endosperm at different developmental stages affects the yield of protoplasts. The endosperm cells are small in number in the early development stage of endosperm; in the middle and later period of endosperm development, the starch granules are too much and too large, which easily causes protoplast damage. 0.5g of wheat endosperm tissue at 5, 8, 11 and 14 days after flowering is placed in 2.0mL of buffer 2 containing 13% mannitol for enzymolysis for 2 h. Removing most starch granules by an ice-bath standing time gradient sedimentation method; adding FDA with final concentration of 0.05g/mL, standing at normal temperature in dark place for 5-10min, and performing microscopic examination. The number of active protoplasts isolated from endosperm tissue 8 and 11 days after flowering was high.
5. Influence of different endosperm dosage on preparation of wheat endosperm cell protoplast
The effect of different endosperm dosages on the preparation of protoplasts from wheat endosperm cells is shown in FIG. 6.
The amount of endosperm tissue has an effect on the yield of protoplasts. 0.2 g, 0.4 g, 0.6g and 0.8g of wheat endosperm tissues 8 days after flowering are respectively put into 2.0mL of buffer solution 2 containing 13% mannitol for enzymolysis for 2 h. Removing most starch granules by an ice-bath standing time gradient sedimentation method; adding FDA with final concentration of 0.05g/mL, standing at normal temperature in dark place for 5-10min, and performing microscopic examination. 0.6g of endosperm tissue released the most active protoplasts. The using amount of endosperm tissues is too small or too much, and the prepared protoplast has lower concentration.
6. Microscopic examination of protoplasts of refined wheat endosperm cells
Taking 0.6g of wheat endosperm 8 days after flowering, and 2.0mL of buffer solution 2 containing 13% mannitol for enzymolysis for 2 h. Most starch granules are removed by ice bath standing and time gradient sedimentation method, and microscopic examination is carried out. As shown in fig. 7, wheat grain and endosperm cell protoplasts 8 days after flowering. Wherein a is wheat grains 8 days after flowering; b and c are field-refined endosperm cell protoplasts at 40X and 20X, respectively. Arrows indicate type A starch grains.
7. Subcellular localization of transcription factor TaABI5 in wheat endosperm cell protoplast
Taking 0.6g of wheat endosperm 8 days after flowering and 2.0mL of 13% mannitol-containing buffer solution 2 for enzymolysis for 2h to prepare the wheat endosperm cell protoplast, and inducing and transforming 35S (GFP) or 35S (TaABI 5) GFP by PEG. The results are shown in FIG. 8, which shows the subcellular localization of the transcription factor TaABI5 in wheat endosperm cell protoplasts. As can be seen, the transcription factor TaABI5 fused with GFP is expressed in wheat endosperm cell protoplast under the drive of 35S promoter. TaABI5 was clearly localized in the nucleus, consistent with prior reports.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Claims (10)
1. A method for preparing wheat endosperm cell protoplasts, comprising the steps of:
s1: taking fresh wheat seeds 8-11 days after flowering, splitting along abdominal seams to obtain endosperm tissues;
s2: soaking and washing endosperm tissues in a buffer solution 1;
s3: placing the washed endosperm tissues in a buffer solution 2 for enzymolysis to obtain crude wheat endosperm cell protoplasts;
s4: and (3) treating the crude wheat endosperm cell protoplast by adopting an ice bath standing time gradient sedimentation method to obtain the purified wheat endosperm cell protoplast.
2. The method of claim 1, wherein: in step S2, the osmotic pressure stabilizer in the buffer solution 1 is sucrose and mannitol, wherein the concentration of the sucrose is 20g/L, and the concentration of the mannitol is 130 g/L;
further, the solvent of the buffer solution 1 is water, and the solutes and the concentrations are as follows: 130g/L of D-mannitol, 20g/L of sucrose, 0.2mM of dipotassium hydrogen phosphate, 1.0mM of potassium nitrate, 10.0mM of calcium chloride dihydrate, 1.0mM of magnesium sulfate heptahydrate, 1.0. mu.M of potassium iodide, 0.1. mu.M of copper sulfate pentahydrate and 2.0mM of MES with pH 5.7.
3. The method according to claim 1 or 2, characterized in that: in step S2, the endosperm tissue is cut into thin strips, soaked in the buffer 1 for 30-60min, and then washed 3-5 times with the buffer 1.
4. A method according to any one of claims 1-3, characterized in that: in step S3, the buffer solution 2 contains cellulase and macerozyme;
further, the content of the cellulase in the buffer solution 2 is 150U/mL; the content of the eductase in the buffer solution 2 is 15.3U/mL;
and/or
In step S3, the osmotic pressure stabilizer in the buffer solution 2 is sucrose and mannitol, wherein the concentration of the sucrose is 20g/L, and the concentration of the mannitol is 130 g/L;
and/or
The solvent of the buffer solution 2 is water, and the solutes and the concentrations are as follows: 150U/mL cellulase, 15.3U/mL macerase, 10g/L Bovine Serum Albumin (BSA), 130g/L D-mannitol, 20g/L sucrose, 0.2mM dipotassium phosphate, 1.0mM potassium nitrate, 10.0mM calcium chloride dihydrate, 1.0mM magnesium sulfate heptahydrate, 1.0. mu.M potassium iodide, 0.1. mu.M copper sulfate pentahydrate, and 2.0mM MES pH 5.7.
5. The method according to any one of claims 1-4, wherein: in step S3, the enzymolysis is to place the endosperm tissue in the buffer solution 2 and shake the endosperm tissue for 90-120min in a dark place at 24 ℃; and/or
In step S3, the ratio of the endosperm tissue to the buffer 2 is 0.4-0.8 g: 2 mL.
6. The method according to any one of claims 1-5, wherein: in step S3, after the endosperm tissue is placed in the buffer solution 2 for enzymolysis, the method further includes the following steps: adding the buffer solution 1 with the same volume into the system after enzymolysis, and then filtering the mixture into a container by using a 200-mesh cell sieve to obtain the crude wheat endosperm cell protoplast.
7. The method according to any one of claims 1-6, wherein: step S4 proceeds as follows:
s41: placing the container containing the crude wheat endosperm cell protoplast obtained in the step S3 in an ice water bath or precooling for 10min at 4 ℃, and removing the supernatant, wherein the amount of the removed supernatant is half of the total volume;
s42: adding the buffer solution 1 into a container filled with the sample treated in the step S41 along the side wall, wherein the adding amount of the buffer solution 1 is 3.5 times of the volume of the crude wheat endosperm cell protoplast in the container, vertically standing in an ice water bath for 8min after uniformly mixing, and discarding the supernatant, wherein the removed amount of the supernatant is the volume of the added buffer solution 1;
s43: adding the buffer solution 1 into the container filled with the sample processed in the step S42 along the side wall, wherein the adding amount of the buffer solution 1 is 3.5 times of the volume of the crude wheat endosperm cell protoplast in the container, vertically standing in an ice-water bath for 6min after uniformly mixing, and discarding the supernatant, wherein the removed amount of the supernatant is the volume of the added buffer solution 1;
further, the following step S44 is also included after step S43:
s44: and (4) vertically standing the container filled with the sample processed in the step (S43) in an ice water bath for 30min, and removing all supernatant to obtain the purified wheat endosperm cell protoplast.
8. A transformation method of wheat endosperm cell protoplast comprises the following steps:
s5: resuspending wheat endosperm cell protoplasts prepared by the method of any one of claims 1-7 in MMg buffer;
s6: PEG-induced transformation was performed.
9. The method of claim 8, wherein: in step S5, the MMg buffer solution is water, and the solutes and concentrations are as follows: 130g/L of D-mannitol, 15.0mM of magnesium chloride hexahydrate and 4.0mM of MES pH 5.7; and/or
In step S5, after the wheat endosperm cell protoplast is resuspended in the MMg buffer, the content of the wheat endosperm cell protoplast in the resuspended solution is 1 × 106Per mL;
and/or
In step S6, the solvent of the PEG-induced transformation solution used for the PEG-induced transformation is water, and the solutes and concentrations are as follows: 400g/L PEG4000, 0.2M D-mannitol and 10.0mM calcium chloride dihydrate;
further, step S6 proceeds as follows:
s61: mixing the heavy suspension containing the wheat endosperm cell protoplast obtained in the step S5 with a plasmid to be transformed, and then adding the PEG to induce transformation liquid; the proportion of the resuspension, the plasmid to be transformed and the PEG induced transformation liquid is 200 mu L: 10 μ g: 220 mu L of the solution;
s62: standing at 22-24 deg.C in dark for 10-15 min;
s63: adding the buffer solution of any one of claims 1-7 at 1, 4 deg.C and 100g, centrifuging for 1min, and discarding the supernatant;
s64: adding the buffer solution, and incubating for 12h at 1, 22-24 ℃ in a dark place.
10. Any of the following:
(A1) a method for preparing and transforming wheat endosperm cell protoplast, comprising steps S1-S4 in the method of any one of claims 1 to 7 and steps S5 and S6 in the method of claim 8 or 9;
(A2) wheat endosperm cell protoplasts prepared by the method of any one of claims 1 to 7;
(A3) a suspension of wheat endosperm cell protoplasts obtained by resuspending the wheat endosperm cell protoplast prepared by the method of any one of claims 1 to 7 in the MMg buffer of claim 8 or 9;
(A4) use of the method of any one of claims 1 to 7 or (A2) the protoplast of a wheat endosperm cell or (A3) the protoplast heavy suspension of a wheat endosperm cell in the genetic transformation of wheat;
(A5) use of the method of any one of claims 1 to 9 or (a1) the method or (a2) the protoplast of a wheat endosperm cell or (A3) the suspension of the protoplast of a wheat endosperm cell in subcellular localization, transcription factor activity assay, protein interaction identification and/or enzyme activity assay of a factor expressed in wheat endosperm.
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