Method for separating protoplast from plant tissue and plasmid transformation method

Abstract

The invention relates to the technical field of plant genetic engineering, and discloses a method for separating protoplast from plant tissue and a plasmid transformation method.The method for separating the protoplast from the plant tissue comprises the steps that lower epidermis of the plant tissue is removed, the plant tissue is placed in TDE-1 enzymatic hydrolysate, enzymolysis is conducted in a dark environment, and enzymatic hydrolysate is obtained; wherein the TDE-1 enzymatic hydrolysate is prepared from cellulase R-10, macerozyme R-10, a BSA (Bovine Serum Albumin) solution, AMP (Adenosine monophosphate) and a PMC (Protein Methyl Cellulose) culture solution; the PMC culture solution comprises B5 salt, MES, calcium chloride dihydrate, ammonium nitrate, sucrose and a potassium hydroxide solution, and the concentration of the sucrose in the PMC culture solution is 130-140 g / L; and centrifuging the enzymatic hydrolysate, and discarding the lower clear liquid to obtain the protoplast. The method for separating the protoplast from the plant tissue has the advantages that the process is simple, the efficiency is high, and the service life of the separated protoplast is long.

Description

Technical Field

[0001] The invention relates to the technical field of plant gene engineering, and in particular to a method for separating protoplasts from plant tissues and a plasmid transformation method. Background Art

[0002] Tomato (Solanum lycopersicum L.) is a herbaceous plant of the genus Solanum in the Solanaceae family. Tomato is rich in carotene, vitamin C and B vitamins, and has high nutritional value. It can be used as both a vegetable and a fruit, and can be eaten raw or cooked. As an important crop, tomato not only occupies an important position in the diet, but also has important value in plant science research. In 2012, the genome sequencing of the tomato variety "Heinz1706" (Heinz 1706) was completed (Sato et al. Nature 2012), which was an important milestone in tomato research and laid the foundation for subsequent gene function analysis and genetic improvement. Since the genome sequencing, nearly a thousand tomato germplasm resources have been genotyped, which helps to understand the genetic diversity of tomatoes and select beneficial genetic variations. At present, research on tomatoes mainly focuses on the size of tomato fruits, the flavor of fruits, and the disease resistance of tomatoes. For example, the Solyc10g079240 gene of tomatoes encodes a protein containing the IQ67 domain, which regulates the elongation of fruits by increasing the number of longitudinal cells in fruits and reducing the number of transverse cells. Although tomatoes are annual plants, the analysis of their gene functions still faces challenges, such as traditional transgenic methods that require a long time period and are affected by environmental factors. As a model plant and an important crop, the study of tomatoes not only helps improve agricultural production efficiency, but also helps promote the development of plant science and genetics. Through continuous research and improvement, consumers can be provided with healthier and more delicious tomato products.

[0003] Protoplasts are plant or microbial cells with the cell wall removed, surrounded by a cell membrane, containing all the organelles and cytoplasm. They are totipotent cells, possessing all the genetic information of their respective species, they can perform the metabolic activities of their respective tissues, and most importantly, can be used to regenerate whole plants, allowing for the rapid propagation of transgenic plant lines. Protoplasts are an important tool in cell biology and genetics research, and they provide a unique system to study various processes inside cells. Protoplasts are a very good transient expression system, with the advantages of being easy to prepare, and the single-cell population itself can also take up various macromolecules such as foreign organelles, viruses, plasmids, and DNA. The transient expression technology of protoplasts is widely used in the study of gene function (by transiently expressing foreign genes, researchers can quickly study the function and expression pattern of genes), the study of plant physiological and biochemical processes (protoplasts can be used to study physiological and biochemical processes such as metabolic pathways and signal transduction of plant cells) and the study of molecular mechanisms (protoplasts help to reveal molecular mechanisms such as gene expression regulation and protein interaction). At present, the experimental techniques based on protoplasts include protein subcellular localization analysis (determine the specific location of proteins in cells and understand their functions), gene transient expression analysis (evaluate the expression of specific genes in cells), promoter activity analysis (study the activity of specific promoters in cells and understand their ability to regulate gene expression) and protein interaction verification (such as BiFC, FRET and protein immunoprecipitation), etc. Most importantly, it can be used to produce target macromolecules, such as recombinant proteins, for research applications, diagnosis and treatment in biotechnology processes.

[0004] Protoplast transient expression technology provides a powerful platform for plant science research, allowing researchers to quickly and efficiently study the functions of genes and proteins at the cellular level. For functional research on tomatoes, it is of great significance to efficiently obtain the number of tomato leaf protoplasts, improve the efficiency of transient transformation, and increase the survival rate of protoplasts, providing strong experimental support for genetics, physiology and molecular biology research on tomatoes.

[0005] Protoplasts are obtained by enzymatic degradation of plant cell walls. Plant cells remove cell walls to obtain protoplasts. Without the protection of cell walls, exogenous molecules can more easily enter cells through transformation methods. However, how to efficiently transfer exogenous plasmids containing DNA into protoplasts has become an important limiting factor. The currently commonly used methods for obtaining protoplasts have serious shortcomings, especially for tomato leaf tissues. The process of obtaining protoplasts is cumbersome and the number of protoplasts obtained is relatively small. Currently, protoplasts are commonly used to be mixed with PEG / Ca 2+There are major defects in the transformation by incubation in solution and MMg solution. The transformation incubation time is long, it is greatly affected by the cell state, and it shows a high variation in the expression level of different biological replicates, and the transformation efficiency is low. This is because the MMg solution used in the traditional method contains mannitol: first, mannitol can only maintain the osmotic pressure of the cells, but cannot provide sufficient equilibrium density for the protoplasts to float on the surface of the solution. Over time, this prevents the gas exchange required by the protoplasts and causes the cells at the bottom of the solution to be in a state of suffocation, and severely limits the lifespan of the protoplasts, which generally does not exceed 12-18 hours, thereby severely limiting the experimental selection and not allowing comprehensive experiments with longer expression times to be carried out; secondly, the protoplasts isolated by the method of using a culture medium containing mannitol / sorbitol (MMg culture medium) have living protoplasts. Living protoplast cells are mixed with dead protoplast cells, requiring cumbersome activity detection steps, and the purification and subsequent incubation steps still contain a large number of dead cells and cell fragments, which hinder transformation and interfere with subsequent analysis, and directly and seriously affect the subsequent plasmid transformation efficiency; finally, the culture medium containing mannitol / sorbitol cannot provide an energy source for cells, which is particularly important for protoplasts and protoplast-based experiments from non-photosynthetically active tissues (such as roots, callus tissues, and suspension culture cells). This also leads to a shorter lifespan of transformed protoplasts, affecting the development of comprehensive experiments. Summary of the invention

[0006] The purpose of the present invention is to overcome the problems of complicated separation process, low efficiency and short life of separated protoplasts in the prior art, and to provide a method for separating protoplasts from plant tissues, which has the advantages of simple process, high efficiency and long life of separated protoplasts.

[0007] In order to achieve the above object, the present invention provides a method for isolating protoplasts from plant tissues, comprising:

[0008] The lower epidermis of the plant tissue is removed, and the plant tissue is placed in a TDE-1 enzymatic hydrolyzate, and enzymatic hydrolyzed in a dark environment to obtain an enzymatic hydrolyzate;

[0009] The TDE-1 enzymatic hydrolysate comprises cellulase R-10, macerate R-10, BSA solution, AMP and PMC culture solution; the PMC culture solution comprises B5 salt, MES, calcium chloride dihydrate, ammonium nitrate, sucrose and potassium hydroxide solution, and the concentration of sucrose in the PMC culture solution is 130 g / L-140 g / L;

[0010] The enzymatically hydrolyzed cell suspension is centrifuged, and the supernatant is discarded to obtain protoplasts.

[0011] Preferably, the enzymatic hydrolysis time is not less than 3 hours.

[0012] Preferably, the TDE-1 enzymatic hydrolysate comprises 1.25% cellulase R-10, 0.3% macerate R-10, 10% BSA solution, 100 mg / ml AMP, and the remainder PMC culture medium.

[0013] Preferably, the PMC culture medium comprises 3.05 g / L B5 salt, 0.5 g / L MES, 0.75 g / L calcium chloride dihydrate, 0.25 g / L ammonium nitrate, 130 g / L-140 g / L sucrose, and 5 M potassium hydroxide solution.

[0014] Preferably, the pH value of the PMC culture solution is 5.5-5.9.

[0015] Preferably, the plant tissue comprises tomato leaves, and before removing the lower epidermis, further comprises:

[0016] The tomato leaves were grown on a 16 / 8 light / dark cycle for 20-30 days.

[0017] A second aspect of the present invention provides a plasmid transformation method, comprising:

[0018] The protoplasts are washed 2-3 times with PTB electrotransfer buffer, and the PTB electrotransfer buffer is added again to obtain a resuspension, wherein the protoplasts are separated using the method described in the first aspect of the present invention;

[0019] PTB electrotransfer buffer containing DNA plasmid is added to the resuspension solution, and plasmid transformation is performed by electroporation transformation method.

[0020] Preferably, the PTB electrotransfer buffer comprises: 2.4 g / L HEPES, 0.6 g / L calcium chloride, 6 g / L potassium chloride, 130 g / L-140 g / L sucrose, and 5 M potassium hydroxide solution.

[0021] Preferably, the pH value of the PTB electrotransfer buffer is 6.9-7.4.

[0022] Preferably, the electroporation is performed using square wave pulses.

[0023] Preferably, the voltage of the square wave pulse is 140V-160V.

[0024] Preferably, the duration of the square wave pulse is 6ms-10ms.

[0025] Preferably, in the PTB electroporation buffer containing the DNA plasmid, the concentration of the DNA plasmid is 10 μg / 100 μL to 50 μg / 100 μL.

[0026] Preferably, the number of protoplasts is 2×10 5 -5×105 indivual.

[0027] Preferably, in the PTB electroporation buffer containing the DNA plasmid, the mass of the DNA plasmid is 1 μg-100 μg.

[0028] Preferably, after the step of using the electroporation transformation method to transform the plasmid, the method further comprises:

[0029] After the electroporation transformation is completed, the cells are incubated for 15-30 minutes to obtain an incubation solution;

[0030] PMC culture medium was added to the incubation solution, and the obtained cell suspension was cultured in the dark for 2 h to 48 h.

[0031] By the above technical scheme of the present invention, the sucrose concentration in the PMC culture fluid adopted is 130g / L-140g / L, it is ensured that the osmotic stability of protoplasts is ensured, and the density of the corresponding solution is provided for the protoplasts to float, and the living protoplasts float on the surface of the enzymolysis solution, thus achieving the best gas exchange. The culture fluid containing sucrose allows to separate living cells and dead cells by floating, and the method allows to separate living cells from dead cells and cell debris in protoplasts and downstream processes, which greatly prolongs the survival rate and the lifespan of protoplasts, allows the long-term experimental strategy (compared with traditional mannitol / sorbitol solution component culture medium) of up to 48h, and there is no need to separate living cells from dead cells and cell debris, and flow process is simple, with huge application potential. In summary, the method for separating protoplasts of the present application has the advantages of simple process, high efficiency, and long life of the isolated protoplasts. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a diagram showing the state of tomato leaf tissue in Example 1 of the present invention after the lower epidermis is removed and the tissue is in contact with the enzymatic hydrolysis solution;

[0033] Figure 2 is a state diagram of the method for separating protoplasts from plant tissues after centrifugation in Example 1 of the present invention;

[0034] Figure 3 It is a state diagram of the protoplasts after mixing with the plasmid containing DNA in Example 1 of the present invention;

[0035] Figure 4 is a process diagram of plasmid transformation by electroporation in Example 1 of the present invention;

[0036] Figure 5 is a state diagram of the cell suspension obtained in Example 1 of the present invention cultured in a culture dish in the dark for 24 hours and 48 hours;

[0037] Figure 6This is a graph of protein expression levels in transformed cells after culturing the cell suspension obtained in Example 1 of the present invention in a culture dish in the dark for 48 hours;

[0038] Figure 7 This is a diagram showing the expression of GFP-labeled protein in living cells after the cell suspension obtained in Example 1 of the present invention was cultured in a culture dish in the dark for 48 hours. DETAILED DESCRIPTION

[0039] The specific implementation of the present invention is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described here is only used to illustrate and explain the present invention, and is not used to limit the present invention.

[0040] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0041] A first aspect of the present invention provides a method for isolating protoplasts from plant tissues, comprising:

[0042] S1: Remove the lower epidermis of the plant tissue, place it in TDE-1 enzymatic hydrolysis solution, and perform enzymatic hydrolysis in a dark environment to obtain an enzymatic hydrolyzed cell suspension;

[0043] The TDE-1 enzymatic hydrolysate comprises cellulase R-10, macerate R-10, BSA solution, AMP and PMC culture solution; the PMC culture solution comprises B5 salt, MES, calcium chloride dihydrate, ammonium nitrate, sucrose and potassium hydroxide solution, and the concentration of sucrose in the PMC culture solution is 130 g / L-140 g / L;

[0044] As an example, the concentration of sucrose in the PMC culture medium can be 130 g / L, 132 g / L, 134 g / L, 135 g / L, 137 g / L, 139 g / L, 140 g / L, etc.

[0045] In some embodiments of the present invention, the plant tissue includes leaves, roots, callus tissue and suspension culture cells. The plant tissue includes tomato leaves, and before removing the lower epidermis, further includes:

[0046] S0: Tomato leaves were grown under a 16 / 8 hour light / dark cycle for 20-30 days.

[0047] Specifically, the tomato variety used in the present application is Micro-Tom, and the treated tomato leaves are seedling leaves grown in soil for 20-30 days with a 16 / 8 hour light and dark cycle, that is, after 16 hours of light growth, 8 hours of darkness growth, and this cycle for 20-30 days. The tomato leaves are fixed with tape using the "sandwich method" and the lower epidermis is removed.

[0048] In some embodiments of the present invention, the lower epidermis is removed by the following method: prepare sterile tape, first fix the upper epidermis of the plant tissue (tomato leaf) with tape, fix the lower epidermis with another tape, and tear off the lower epidermis with force ("sandwich method").

[0049] In some embodiments of the present invention, the enzymolysis time is not less than 3 hours. For example, the enzymolysis time can be 3 hours to 5 hours, etc., so that as many protoplasts in the plant tissue as possible can enter the enzymolysis solution, thereby increasing the amount of separated protoplasts.

[0050] In some embodiments of the present invention, the PMC culture medium includes 3.05 g / L B5 salt,

[0051] 0.5g / L MES, 0.75g / L calcium chloride dihydrate, 0.25g / L ammonium nitrate, 130g / L-140g / L sucrose, and 5M potassium hydroxide solution to adjust pH.

[0052] Specifically, the composition and configuration of the PMC culture solution are shown in Table 1:

[0053] Table 1 PMC culture medium

[0054] Total volume 1L B5 Salt 3.05g MES 0.5g Calcium chloride dihydrate 0.75g Ammonium Nitrate 0.25g sucrose 130g-140g 5M KOH solution Adjust pH to 5.5-5.9

[0055] Among them, B5 salt is the basic salt of B5 culture medium, MES is 2-morpholineethanesulfonic acid, that is, according to the ratio in Table 1, B5 salt, MES, calcium chloride dihydrate, ammonium nitrate and sucrose are mixed, and then 5M KOH solution is added to adjust the pH value to 5.5-5.9. The total volume of the solution is 1L, and it is filtered and sterilized using a 0.22μM filter membrane and stored in an environment of 4°C.

[0056] In some embodiments of the present invention, the pH value of the PMC culture solution is 5.5-5.9, for example, 5.5, 5.6, 5.7, 5.8, 5.9, etc.

[0057] Specifically, adopt above-mentioned PMC culture fluid, sucrose concentration is 130g / L-140g / L, ensure that protoplast osmotic stability, and provide corresponding solution density for protoplast floating, live protoplast floats on the surface of enzymolysis solution, thus realizes optimal gas exchange. Culture fluid containing sucrose allows to separate living cells and dead cells by floating, and this method allows living cells to be separated from dead cells and cell debris in protoplast and downstream process, which greatly prolongs the survival rate and life span of protoplast, allows long-term experimental strategy (compared with traditional mannitol / sorbitol solution component culture medium) of up to 48h, and no longer needs to separate living cells from dead cells and cell debris, and flow process is simple, with huge application potential.

[0058] In some embodiments of the present invention, the TDE-1 enzymatic hydrolysate includes 1.25% cellulase R-10, 0.3% macerate R-10, 10% BSA solution, 100 mg / ml AMP, and the remainder PMC culture solution.

[0059] Specifically, the composition and configuration of the TDE-1 enzymatic hydrolysate are shown in Table 2:

[0060] Table 2 TDE-1 enzymatic hydrolysate

[0061]

[0062]

[0063] Specifically, according to the ratio in Table 2, the above-mentioned cellulase R-10 (CAS: 9012-54-8), macerate R-10 (CAS No.: 9032-75-1), BSA (bovine serum albumin) solution, and AMP (adenosine monophosphate) were mixed evenly, and the volume was fixed to 100 mL with PMC culture medium to obtain TDE-1 enzymatic solution, which was sterilized by filtration using a 0.22 μM filter membrane and stored in an environment of 4° C. The TDE-1 enzymatic solution using the above-mentioned formula has a further improved and optimized formula, which can fully dissolve the cell wall in the plant tissue, release the protoplasts, and has a high separation efficiency.

[0064] S2: Centrifuge the enzymatic hydrolysate, discard the supernatant, and obtain protoplasts.

[0065] By the technique scheme, the sucrose concentration in the PMC culture fluid adopted is 130g / L-140g / L, it is ensured that the osmotic stability of protoplasts is ensured, and the density of the corresponding solution is provided for the protoplasts to float, and the living protoplasts float on the surface of the enzymolysis solution, thus achieving the best gas exchange. The culture fluid containing sucrose allows to separate living cells and dead cells by floating, and the method allows living cells to be separated from dead cells and cell debris in protoplasts and downstream processes, which greatly prolongs the survival rate and the lifespan of protoplasts, allows the long-term experimental strategy (compared with traditional mannitol / sorbitol solution component culture medium) of up to 48h, and there is no need to separate living cells from dead cells and cell debris, and flow process is simple, with huge application potential. In summary, the method for separating protoplasts of the application, has simple process, high efficiency, and the advantages of the long life of the isolated protoplasts.

[0066] In some embodiments of the present invention, after centrifugation, it can be seen that the live protoplast cells are floating and the dead protoplast cells are at the bottom of the centrifuge tube. The centrifuge speed is set to 0 to avoid deformation of the floating cell layer.

[0067] A second aspect of the present invention provides a plasmid transformation method, comprising:

[0068] S10: washing the protoplasts with PTB electrotransfer buffer for 2-3 times, and adding PTB electrotransfer buffer again to obtain a resuspension, wherein the protoplasts are separated using the method described in the first aspect of the present invention;

[0069] In some embodiments of the present invention, the PTB electrotransfer buffer comprises: 2.4 g / L HEPES, 0.6 g / L calcium chloride, 6 g / L potassium chloride, 130 g / L-140 g / L sucrose, and 5 M potassium hydroxide solution for adjusting pH.

[0070] Specifically, the composition and configuration of the PTB electrotransfer buffer are shown in Table 3:

[0071] Table 3 PTB electrotransfer buffer

[0072] Total volume 1L HEPES 2.4g Calcium chloride 0.6g Potassium chloride 6g sucrose 130g-140g 5M KOH solution Adjust pH to 6.9-7.4

[0073] HEPES, i.e. 4-hydroxyethylpiperazineethanesulfonic acid, is a biological buffer. In the specific configuration, HEPES, calcium chloride, potassium chloride, and sucrose are mixed according to the ratio in Table 3, and then 5M KOH solution is added to adjust the pH value to 5.5-5.9. The total volume of the solution is 1L, and it is sterilized by filtration using a 0.22μM filter membrane and stored in an environment of 4°C. Using the above buffer, the sucrose concentration is 130g / L-140g / L, which ensures the osmotic stability of the protoplasts and provides the density of the corresponding solution for the floating of the protoplasts. The living protoplasts float on the surface of the enzymatic solution, thereby achieving the best gas exchange. The culture medium containing sucrose allows the separation of living cells and dead cells by floating. This method allows the separation of living cells from dead cells and cell fragments in protoplasts and downstream processes, which greatly prolongs the survival rate and lifespan of protoplasts and allows long-term experimental strategies of up to 48 hours (compared with traditional mannitol / sorbitol solution component culture medium). There is no need to separate living cells from dead cells and cell fragments. The process is simple and has great application potential.

[0074] In some embodiments of the present invention, the pH value of the PTB electroporation buffer is 6.9-7.4, for example, 6.9, 7, 7.1, 7.2, 7.3, 7.4, etc.

[0075] S20: adding PTB electroporation buffer containing DNA plasmid into the resuspension solution, and performing plasmid transformation by electroporation transformation method.

[0076] The electroporation method can increase the expression level of enzyme-detectable reporter protein by 4 times, while greatly reducing the mortality rate.

[0077] In some embodiments of the present invention, the electroporation transformation method is performed using a square wave pulse. The electroporation transformation of protoplasts is performed using a square wave pulse instead of an exponential decay pulse. The results show that the square wave has a pure waveform, and the high level and the low level occupy an equal amount of time. The pulse width and period of the pulse wave are not equal to 1:2, which can increase the expression level of the enzyme detectable reporter protein by 4 times and greatly reduce the mortality rate of protoplasts.

[0078] In some embodiments of the present invention, the voltage of the square wave pulse is 140V-160V. For example, it can be 140V, 145V, 150V, 155V, 160V, etc., which can increase the expression level of the enzyme detectable reporter protein by 4 times and greatly reduce the mortality rate of protoplasts.

[0079] In some embodiments of the present invention, the square wave pulse duration is 6ms-10ms. For example, it can be 6ms, 7ms, 8ms, 9ms, 10ms, etc., which can increase the expression level of the enzyme detectable reporter protein by 4 times and greatly reduce the mortality rate of protoplasts.

[0080] In some embodiments of the present invention, the present invention uses a square wave pulse of 140V-160V, lasting 6-10 milliseconds, as the best choice for electro-transfection of protoplasts isolated with plasmid DNA according to the above protocol.

[0081] In some embodiments of the present invention, the concentration of the DNA plasmid in the PTB electroporation buffer containing the DNA plasmid is 10 μg / 100 μL to 50 μg / 100 μL. For example, 10 μg / 100 μL, 20 μg / 100 μL, 30 μg / 100 μL, 40 μg / 100 μL, 50 μg / 100 μL, etc. Within the above range, the plasmid can be fully transformed with good effect.

[0082] In some embodiments of the present invention, the number of protoplasts is 2×10 5 -5×10 5 For example, it can be 2×10 5 pcs, 3×10 5 pcs, 4×10 5 pcs, 5×10 5 In this way, the instantaneous transformation efficiency of protoplasts can be improved and the mortality rate of protoplasts can be greatly reduced.

[0083] In some embodiments of the present invention, the mass of the DNA plasmid in the PTB electroporation buffer containing the DNA plasmid is 1 μg-100 μg. For example, it can be 1 μg, 10 μg, 30 μg, 50 μg, 100 μg, etc., so that the instantaneous transformation efficiency of the protoplasts can be further improved and the mortality rate of the protoplasts can be greatly reduced.

[0084] In some embodiments of the present invention, after the step of using the electroporation transformation method to transform the plasmid, the method further comprises:

[0085] S30: After the electroporation transformation is completed, the cells are incubated for 15 to 30 minutes to obtain an incubation solution;

[0086] For example, the static incubation time can be 15 min, 20 min, 25 min, 30 min, etc., thereby further improving the instantaneous transformation efficiency of the protoplasts and greatly reducing the mortality rate of the protoplasts.

[0087] S40: Add PMC buffer to the incubation solution, and culture the obtained cell suspension in the dark for 2 h to 48 h.

[0088] For example, the culturing time in darkness can be 2 h, 10 h, 20 h, 30 h, 40 h, 48 h, etc., thereby further improving the instantaneous transformation efficiency of protoplasts and greatly reducing the mortality rate of protoplasts.

[0089] In summary, in a second aspect, the present invention uses square wave pulses instead of exponential decay pulses to electroporate protoplasts. The present invention uses 140V-160V square wave pulses for 6-10 milliseconds as the best choice for electrotransfection of protoplasts isolated with plasmid DNA according to the above scheme. The results show that this optimized transformation scheme can increase the expression level of enzyme-detectable reporter protein by 4 times, while greatly reducing the mortality rate of protoplasts.

[0090] The method of the present invention can efficiently obtain tomato leaf protoplasts with good activity, and transform the protoplasts with high quality plasmids by electric shock method to obtain positive cells with good activity, which is of great significance in providing strong experimental support for the physiological and molecular biological research of tomatoes.

[0091] The present invention will be described in detail below through examples, but the protection scope of the present invention is not limited thereto.

[0092] Example 1

[0093] (1) Sterile ultrapure water was used to prepare the solutions used for protoplast preparation, including PMC culture medium, TDE-1 enzymatic hydrolyzate, and PTB electroporation buffer (the compositions and configurations of the three are shown in Tables 1, 2, and 3). The concentration of sucrose in the PMC culture medium was 139.3 g / L, and the solution was sterilized by filtration using a 0.22 μM filter membrane and stored at 4°C.

[0094] (2) Prepare sterile tape and use it to fix the upper epidermis. Use another tape to fix the lower epidermis. Tear off the lower epidermis with force. Spread the tomato leaves without the lower epidermis flat in a culture dish containing enzyme solution (such as Figure 1 shown).

[0095] (3) Place the enzymatic hydrolyzate in a dark incubator at 24°C and shake it for at least 3 hours to promote the release of protoplasts.

[0096] (4) When the enzymatic hydrolysate turns green, gently shake the culture dish, collect the enzymatic hydrolysate containing protoplasts in a 50 ml centrifuge tube, and centrifuge at 700 rpm for 5 minutes (the centrifuge speed is set to 0).

[0097] (5) After centrifugation, you can see that the live protoplasts are floating, while the dead protoplasts are at the bottom of the centrifuge tube (e.g. Figure 2 shown).

[0098] (6) Use a sterile Pasteur tube to absorb the supernatant, discard the supernatant, add 20 ml of EPB electrotransfer buffer, gently resuspend the protoplasts, and centrifuge at 700 rpm for 5 minutes (set the centrifuge speed to 0).

[0099] (7) Discard the supernatant and wash the cells 2-3 times with PTB electroporation buffer. Finally, resuspend the cells with PTB electroporation buffer according to the cell quantity. (PTB solution is used for washing here because electroporation is required next. The rest of the cells are cultured in PMC medium.)

[0100] (8) Gently transfer 500 μL of protoplasts into a disposable 1 mL plastic cuvette. Add 100 μL of electroporation buffer containing 10-50 μg of DNA plasmid and gently shake to mix (e.g. Figure 3 shown).

[0101] (9) The cell mixture is incubated at room temperature for 5 minutes (usually the time required to complete the entire series of pipetting) and then electroporation can be performed. This study will use a homemade electroporation device in which a pair of stainless steel plate electrodes are embedded in a polytetrafluoroethylene insulator, which will be directly inserted into the cuvette. When performing electroporation, no metal parts are exposed on the surface, and a 155V square wave pulse is applied for 8 milliseconds (such as Figure 4 shown).

[0102] (10) After electroporation, incubate the cells at room temperature (without shaking) for 15-30 minutes.

[0103] (11) Add 2 ml of PMC culture medium and culture the obtained cell suspension in a culture dish in the dark for an appropriate period of time (2-48 hours).

[0104] Figure 5 The diagram is a state diagram of culturing the obtained cell suspension in a culture dish in the dark for 24 hours and 48 hours. It can be seen that after culturing for 24 hours and 48 hours, living protoplast cells float on the upper surface of the cell suspension, indicating that the isolated protoplasts have a long lifespan.

[0105] The obtained cell suspension was cultured in a culture dish in the dark for 48 h, and the upper suspended living cells were collected. The protein expression level in the transformed cells was detected by WB. Figure 6 ,from Figure 6 Obvious signal bands can be seen, 1 and 2 are the levels of protein expression after 160V electric shock transformation, 1 is the expression level of 1μg plasmid protein, 2 is the level of 10μg plasmid protein; 3, 4 and 5 represent the expression levels of 2μg plasmid protein after 140V, 150V and 160V electric shock transformation, indicating that the electric shock transformation of plasmid protein adopted in the present invention can stably express more proteins when more plasmids are transformed, and the expression efficiency of this system after transformation is high.

[0106] After the obtained cell suspension was cultured in a culture dish in the dark for 48 h, the expression of GFP-tagged protein in living cells was detected by laser confocal microscopy. Figure 7 ,from Figure 7It can be seen that the green channel is the endoplasmic reticulum marker protein signal marked by GFP (green fluorescent protein); the middle bright field channel is the complete cell morphology under a microscope; the merged image is the signal position of the expressed protein in the complete cell state, scale bar = 5μm.

[0107] Example 2

[0108] The method of Example 1 was followed, except that the concentration of sucrose in the PMC culture medium and the PTB electroporation buffer was 130 g / L. The results are shown in Table 4.

[0109] Example 3

[0110] The method of Example 1 was followed, except that the concentration of sucrose in the PMC culture medium and the PTB electrotransfer buffer was 140 g / L. The results are shown in Table 4.

[0111] Example 4

[0112] The method of Example 1 was followed, except that the voltage of the square wave pulse was 140V. The results are shown in Table 4.

[0113] Example 5

[0114] The method of Example 1 was followed, except that the voltage of the square wave pulse was 150V. The results are shown in Table 4.

[0115] Example 6

[0116] The method of Example 1 was followed, except that the voltage of the square wave pulse was 160V. The results are shown in Table 4.

[0117] Example 7

[0118] The method of Example 1 was followed, except that the time of the square wave pulse was 6 ms. The results are shown in Table 4.

[0119] Example 8

[0120] The method of Example 1 was followed, except that the time of the square wave pulse was 10 ms. The results are shown in Table 4.

[0121] Comparative Example 1

[0122] The method of Example 1 was followed, except that the concentration of sucrose in the PMC culture medium and the PTB electrotransfer buffer was 50 g / L. The results are shown in Table 4.

[0123] Comparative Example 2

[0124] The method of Example 1 was followed, except that the concentration of sucrose in the PMC culture medium and the PTB electrotransfer buffer was 180 g / L. The results are shown in Table 4.

[0125] Comparative Example 3

[0126] The method of Example 1 was followed, except that the electroporation conversion method used an exponential decay pulse. The results are shown in Table 4.

[0127] Table 4

[0128] Protoplast lifespan, protoplast activity after transformation, intracellular protein expression level, and expression of GFP-tagged proteins in living cells

[0129]

[0130]

[0131] It can be seen from the results in Table 4 that the embodiments of the present invention have significantly better effects than the comparative examples.

[0132] In summary, based on the research summary of the existing plant cell protoplast extraction method, the inventors combined the structural and morphological characteristics of tomato leaves, combined with the current needs of genetics, physiology and molecular biology research on tomatoes based on protoplasts, further improved and optimized the formula in the enzymatic preparation process, and combined with the electroporation transformation method for plasmid transformation. The number of protoplast viable cells prepared by the present method is large, the activity is high, the expression efficiency is high after plasmid transformation, and the survival time is long. Researchers can more efficiently prepare and transform tomato leaf protoplasts, providing a powerful tool for molecular biology research on tomatoes. This not only helps to speed up the process of tomato genetics research, but also provides a reference for improving the research of protoplasts and tissues (such as roots and suspension culture cells) of other plant species. And it provides a reference for improving the research of protoplasts and tissues (such as roots and suspension culture cells) of other plant species.

[0133] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A method for isolating protoplasts from plant tissues, characterized in that include: The lower epidermis of the plant tissue was removed, and the tissue was placed in TDE-1 enzymatic hydrolysis solution and enzymatically hydrolyzed in a dark environment to obtain an enzymatic hydrolyzed cell suspension; The TDE-1 enzymatic hydrolysate comprises cellulase R-10, macerate R-10, BSA solution, AMP and PMC culture solution; the PMC culture solution comprises B5 salt, MES, calcium chloride dihydrate, ammonium nitrate, sucrose and potassium hydroxide solution, and the concentration of sucrose in the PMC culture solution is 130 g / L-140 g / L; The enzymatically hydrolyzed cell suspension is centrifuged, and the supernatant is discarded to obtain protoplasts.

2. The method according to claim 1, characterized in that The enzymatic hydrolysis time is not less than 3 hours.

3. The method according to claim 1, characterized in that The TDE-1 enzymatic hydrolysate comprises 1.25% cellulase R-10, 0.3% macerate R-10, 10% BSA solution, 100 mg / ml AMP, and the remainder of PMC culture solution, and / or, The PMC culture medium includes 3.05 g / L B5 salt, 0.5 g / L MES, 0.75 g / L calcium chloride dihydrate, 0.25 g / L ammonium nitrate, 130 g / L-140 g / L sucrose, 5M potassium hydroxide solution to adjust pH, and / or, The pH value of the PMC culture solution is 5.5-5.

9.

4. The method according to any one of claims 1 to 3, characterized in that The plant tissue comprises tomato leaves, and before removing the lower epidermis, further comprises: Tomato leaves were grown on a 16 / 8 hour light / dark cycle for 20-30 days.

5. A plasmid transformation method, characterized in that: include: The protoplasts are washed 2-3 times with PTB electrotransfer buffer, and the PTB electrotransfer buffer is added again to obtain a resuspension, wherein the protoplasts are separated by the method according to any one of claims 1 to 4; PTB electrotransfer buffer containing DNA plasmid is added to the resuspension solution, and plasmid transformation is performed by electroporation transformation method.

6. The method according to claim 5, characterized in that The PTB electrotransfer buffer comprises: 2.4 g / L HEPES, 0.6 g / L calcium chloride, 6 g / L potassium chloride, 130 g / L-140 g / L sucrose, and 5 M potassium hydroxide solution to adjust the pH; and / or, The pH value of the PTB electrotransfer buffer is 6.9-7.

4.

7. The method according to claim 5, characterized in that The electroporation method was performed using square wave pulses.

8. The method according to claim 7, characterized in that The voltage of the square wave pulse is 140V-160V; The time of the square wave pulse is 6ms-10ms.

9. The method according to any one of claims 5 to 8, characterized in that: In the PTB electrotransfer buffer containing the DNA plasmid, the concentration of the DNA plasmid is 10 μg / 100 μL-50 μg / 100 μL; and / or, The number of protoplasts is 2×10 5 -5×10 5 and / or, In the PTB electrotransfer buffer containing the DNA plasmid, the mass of the DNA plasmid is 1 μg-100 μg.

10. The method according to any one of claims 5 to 8, characterized in that: After the step of using the electroporation transformation method to transform the plasmid, the method further comprises: After the electroporation transformation is completed, the cells are incubated for 15-30 minutes to obtain an incubation solution; PMC culture medium was added to the incubation solution, and the obtained cell suspension was cultured in the dark for 2 h to 48 h.

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