Tartary buckwheat protoplast separation and transient expression system construction method
By optimizing the protoplast isolation and transient transformation system of tartary buckwheat, the problem of immature protoplast isolation technology in tartary buckwheat was solved, achieving efficient transient gene expression and improving the ease of operation for gene function identification and application in tartary buckwheat.
Patent Information
- Application Number
- CN202510902575.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2025-03-21
- Filing Date
- 2025-07-01
- Publication Date
- 2025-11-07
AI Technical Summary
The immature technology for isolating protoplasts from tartary buckwheat makes the operation of transient gene expression systems in tartary buckwheat complex and inefficient, which limits gene function identification and application.
By treating tartary buckwheat hypocotyl tissue with specific concentrations of cellulase and pectinase, and combining vacuum aspiration and dark culture, the enzymatic hydrolysis conditions were optimized to establish a tartary buckwheat protoplast isolation and transient transformation system, including enzymatic hydrolysis, collection and transformation steps, and plasmid transformation was mediated by PEG4000 solution.
It improved the yield and transformation efficiency of tartary buckwheat protoplast cells, maintained good cell activity, and achieved a transformation efficiency of up to 65%, thus realizing efficient transient gene expression.
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Figure CN120905116A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of molecular biology, and particularly relates to a method for separating buckwheat protoplasts and constructing a transient expression system. BACKGROUND
[0002] Fagopyrum Mill. is a dicotyledonous plant of the Polygonaceae family, and is a characteristic plant integrating nutrition, medicine and health care functions. Buckwheat contains a large amount of rutin and rich copper, selenium, cystine and cysteine, and rutin has a good therapeutic effect on reducing intravascular cholesterol, high blood pressure and diabetes. Buckwheat has unique nutritional and medicinal values. In production, there are mainly two kinds of buckwheat, Fagopyrum tataricum Gaertn and Fagopyrum. esculentum Moench. Fagopyrum tataricum Gaertn is unique to China. However, due to the flowering habit and self-pollination characteristics of Fagopyrum tataricum Gaertn, artificial hybridization is difficult to succeed in production and breeding, which is an important reason why Fagopyrum tataricum Gaertn is difficult to make breakthroughs in breeding. At present, most of the researches on buckwheat by domestic scholars are about genetic diversity and phylogenetic relationship of buckwheat, and there are still few researches on molecular biology. Transgenic technology and genome editing technology are two widely used technologies in modern molecular biology. Transgenic technology is a technology that integrates a pre-designed foreign DNA with a complete expression unit into a recipient cell through biological technology and integrates it into the recipient genome, so that the foreign gene can be stably expressed in the recipient and has the ability to be inherited by the next generation. However, it is difficult to construct a stable genetic transformation system for Fagopyrum tataricum Gaertn. The application of Agrobacterium-mediated plant stable genetic transformation method in Fagopyrum tataricum Gaertn is complex and has low transformation efficiency. This greatly limits the basic research on gene function identification of Fagopyrum tataricum Gaertn and the implementation of related application promotion. Therefore, it is urgent to develop a rapid foreign DNA transformation and detection transient expression system suitable for Fagopyrum tataricum Gaertn for gene function identification and genetic engineering application.
[0003] Gene transient expression refers to the technology that, in a short time, foreign genes are introduced into recipient cells without integration of the foreign genes and the chromosomal DNA of the recipient cells, so that the foreign genes can be expressed at a high level for a short time. The gene transient transformation system has a short experimental period and convenient operation, which makes up for the shortcomings of long experimental period, complicated operation and low transformation efficiency in traditional genetic transformation methods.
[0004] Protoplasts, as a kind of artificially formed cells which have removed cell walls, can be used to uptake exogenous DNA, and are good receptor materials for gene transient expression detection after transformation. Although protoplast transient transformation technology has been very mature after decades of development, it has been successfully implemented in a variety of plants such as Arabidopsis, cucumber, carrot and the like, but the protoplast transformation gene transient expression method in tartary buckwheat is still relatively rare, and it is currently a problem to be solved in the art to establish a method with high cell yield and transformation efficiency. SUMMARY
[0005] The technical problem to be solved by the present application is that the protoplast separation technology of tartary buckwheat is not mature.
[0006] The technical solution of the present application is a method for separating tartary buckwheat protoplasts, comprising the following steps:
[0007] S1, enzymolysis: taking tartary buckwheat hypocotyl tissue into an enzyme solution and vacuum suctioning for 15-60 min, and then enzymolysis for 1-6 h; the concentration of mannitol in the enzyme solution is 0.2-0.8 M; the enzyme is cellulase and pectinase, the mass concentration of cellulase is 1.0-2.0%, the mass concentration of pectinase is 0.25-1.0%, and the ratio of tartary buckwheat hypocotyl tissue to enzyme solution is 0.1 g / mL;
[0008] S2, collection: adding W5 Buffer to terminate the enzyme reaction, filtering with 0.7 μm nylon membrane, adding W5 buffer again, filtering, collecting the filtrate twice, and combining; centrifuging the filtrate, discarding the supernatant, resuspending with W5 buffer, centrifuging, discarding the supernatant, and repeating once again, and the precipitate is the protoplast.
[0009] In particular, in step S1, the vacuum suctioning is performed for 30-60 min.
[0010] Preferably, in step S1, the vacuum suctioning is performed for 30 min.
[0011] In step S1, the enzymolysis is performed for 3-6 h.
[0012] Preferably, in step S1, the enzymolysis is performed for 3 h.
[0013] Further, in step S1, the enzymolysis is performed in a dark environment, at 24-26°C and 25-35 rpm.
[0014] In step S1, the mass concentration of cellulase is 1.5-2.0%, and the mass concentration of pectinase is 0.8-1.0%.
[0015] Preferably, in step S1, the mass concentration of cellulase is 1.5%, and the mass concentration of pectinase is 0.8%.
[0016] In step S2, the centrifugal condition is 70-90xg centrifugation for 5 min.
[0017] Further, in step S1, the preparation of the bitter buckwheat hypocotyl tissue comprises the following steps: washing the buckwheat seeds with water, oscillating and sterilizing the buckwheat seeds with 75% alcohol for 1 min, and washing the buckwheat seeds with sterilized water for 3 times; placing the buckwheat seeds in vermiculite, pouring the vermiculite with tap water, adding a 50 mg / L solution of paclobutrazol, and culturing the buckwheat seeds in darkness at 25±1℃ for 4-5 days.
[0018] In particular, the paclobutrazol solution uses pure water as a solvent.
[0019] The application further provides a transient transformation system of the bitter buckwheat protoplast, comprising the following steps:
[0020] S1, enzymolysis: taking the bitter buckwheat hypocotyl tissue into an enzymolysis solution and vacuum suctioning for 15-60 min, and then enzymolysis for 1-6 h; the concentration of mannitol in the enzymolysis solution is 0.2-0.8 M; the enzyme is cellulase and pectinase, the mass concentration of the cellulase is 1.0-2.0%, the mass concentration of the pectinase is 0.25-1.0%, and the ratio of the bitter buckwheat hypocotyl tissue to the enzymolysis solution is 0.1 g / mL;
[0021] S2, collection: adding W5 Buffer to terminate the enzymolysis reaction, filtering with a 0.7 μm nylon membrane, adding W5 buffer again, filtering, collecting the filtrate of the two times, and combining; centrifuging the filtrate, discarding the supernatant, resuspending with W5 buffer, centrifuging, discarding the supernatant, and repeating once again, and the precipitate is the protoplast cell;
[0022] S3, transformation: resuspending the protoplast cell obtained in step S2 with MMG Buffer, so that the final concentration is 1.5x10 6 ~3x10 6 ML; mixing the plasmid containing the target gene with the protoplast cell; adding a PEG4000 solution, and standing in darkness for 20 min; adding W5 Buffer to terminate the reaction; centrifuging, collecting the precipitate, resuspending with W5 Buffer, and incubating at 25℃ in darkness for 18-48 h; wherein, after adding the PEG4000 solution, the mass final concentration of PEG4000 is 20%.
[0023] In particular, in step S1, the vacuum suctioning is performed for 30-60 min.
[0024] Preferably, in step S1, the vacuum suctioning is performed for 30 min.
[0025] In step S1, the enzymolysis is performed for 3-6 h.
[0026] Preferably, in step S1, the enzymolysis 3h.
[0027] Further, in step S1, the enzymolysis is carried out in a dark environment, at 24-26℃ and 25-35 rpm.
[0028] In step S1, the mass concentration of cellulase is 1.5-2.0%, and the mass concentration of pectinase is 0.8-1.0%.
[0029] Preferably, in step S1, the mass concentration of cellulase is 1.5%, and the mass concentration of pectinase is 0.8%.
[0030] In step S2, the centrifugal condition is 70-90xg centrifugation for 5min.
[0031] Further, in step S1, the preparation of the bitter buckwheat hypocotyl tissue comprises the following steps: washing the buckwheat seeds with water, oscillating and sterilizing the buckwheat seeds with 75% alcohol for 1min, and washing the buckwheat seeds with sterilized water for 3 times; placing the buckwheat seeds in vermiculite, pouring the vermiculite with tap water, adding a solution of 50mg / L paclobutrazol, and culturing the buckwheat seeds in the dark at 25±1℃ for 4-5 days.
[0032] In particular, the solution of paclobutrazol uses pure water as a solvent.
[0033] In step S3, the centrifugal condition is 70-90xg centrifugation for 5min.
[0034] In step S3, the usage ratio of the plasmid to the protoplast cells is 10-30ug:200ul.
[0035] Preferably, in step S3, the usage ratio of the plasmid to the protoplast cells is 30ug:200ul.
[0036] The method of the application has the following advantages: by exploring the vacuum pump suction time, enzymolysis time, and enzyme concentration ratio in the enzymolysis solution in the bitter buckwheat protoplast transformation experiment, the method of the application uses the dark-cultured bitter buckwheat hypocotyl, which is easy to obtain and does not contain chloroplasts, does not affect observation, greatly improves the cell yield after vacuum pump suction, and does not affect the cell activity. On the basis of the foregoing protoplast separation, the application further establishes a set of protoplast transient transformation technology applicable to bitter buckwheat; by optimizing the transformation plasmid mass, the appropriate plasmid concentration improves the transformation efficiency to a certain extent, which is a short-cycle, time-saving, labor-saving, and cost-effective method for bitter buckwheat protoplast separation and transient expression. After the bitter buckwheat protoplast cells prepared by the method of the application are transformed with plasmids, the transformed cell activity is good, and the active cells can reach 85%; the transformation efficiency is the highest, and the cells expressing green fluorescent protein can reach 65%. BRIEF DESCRIPTION OF DRAWINGS
[0037] Figure 1 Effects of different vacuum suction time on the yield and activity of buckwheat protoplasts. (a) The diagram of protoplast density with the change of vacuum suction time; (b) The diagram of protoplast activity with the change of vacuum suction time.
[0038] Figure 2 Effects of different enzymolysis time on the yield and activity of buckwheat protoplasts. (a) The diagram of protoplast density with the change of enzymolysis time; (b) The diagram of protoplast activity with the change of enzymolysis time.
[0039] Figure 3 Effects of different enzymolysis liquid ratio on the yield and activity of buckwheat protoplasts. (a) The diagram of protoplast density with the change of enzymolysis liquid ratio; (b) The diagram of protoplast activity with the change of enzymolysis liquid ratio.
[0040] Figure 4 Effects of different plasmid quality on the yield and activity of buckwheat protoplasts.
[0041] Figure 5 The process of buckwheat protoplast preparation (a). The short strip of buckwheat hypocotyls with a length of about 1mm (b) after adding enzymolysis liquid (c) the collected buckwheat protoplasts.
[0042] Figure 6 The state of buckwheat protoplast cells observed under an optical microscope. (a) The state of protoplast cells separated after enzymolysis of buckwheat hypocotyls for 6h; (b) The state of cells after transient transformation of buckwheat protoplasts.
[0043] Figure 7 The observation of cell activity and transformation efficiency under a fluorescence microscope 48h after completion of transient transformation of buckwheat protoplasts. (a) Bright field, objective lens 10x; (b) Green fluorescence emitted by cells after transient transformation of buckwheat protoplasts. DETAILED DESCRIPTION
[0044] In the following examples, the method for culturing buckwheat hypocotyls is as follows:
[0045] The germination of buckwheat embryo aseptic seedlings is divided into three steps of seed disinfection, seed inoculation and illumination culture. The germinated buckwheat seedlings can provide abundant materials for the study of buckwheat protoplast transient transformation system. The buckwheat seeds are divided into 50mL centrifuge tubes, washed with tap water for 3 times, and then subjected to 75% alcohol shock disinfection for 1min. The disinfection is washed with sterilized tap water for 3 times. The seeds are placed in vermiculite, and then 20mL of pure water added with 50mg / L chlormequat are poured to the vermiculite. The vermiculite is cultured in darkness at 25±1℃ for 4-5 days.
[0046] In the following examples, the tartary buckwheat protoplast cell counting and activity detection method is as follows:
[0047] The number of tartary buckwheat protoplast cells is counted using a hemocytometer under a microscope. The hemocytometer is a commonly used tool for cell or microbial counting. The counting formula for a 25x16 type hemocytometer is:
[0048] Cell or microbial density (cells / mL) = total number of cells in 5 central squares ÷ 5 x 25 x 10 x dilution factor. 4
[0049] Take 5 μL of undiluted tartary buckwheat protoplast cell suspension and drop it into the central counting grid of a 25x16 type hemocytometer. After completely immersing it, cover it with a cover glass and count it under an inverted microscope. Since tartary buckwheat protoplast cells are large and not many, when counting, all protoplast cells contained in 25 central squares are counted, and the counting operation is repeated six times to take the average value. The final tartary buckwheat protoplast cell counting formula is:
[0050] Protoplast density (cells / mL) = average number of tartary buckwheat protoplast cells in 25 central squares x 10 4 .
[0051] The activity of tartary buckwheat protoplast cells is detected using the fluorescein diacetate (FDA) method for observation and identification. FDA is a live cell stain that can freely pass through the cell membrane and accumulate in live cells, and under excitation light, it can emit yellow-green fluorescence, while dead cells cannot emit fluorescence. The specific operation of FDA for tartary buckwheat protoplast cell activity detection is as follows: add 2.4 μL of FDA-acetone solution with a concentration of 5 mg / mL to 100 μL of tartary buckwheat protoplast cell suspension, and incubate it at room temperature in the dark for 5-10 min, then observe it under a fluorescence inverted microscope. Protoplasts with activity will emit fluorescence. Observe multiple fields and take the average value of protoplast activity. The tartary buckwheat protoplast cell activity detection formula is:
[0052] Tartary buckwheat protoplast cell activity = (total number of green fluorescent protoplast cells in the field ÷ total number of protoplast cells in the field) x 100%.
[0053] The MMG buffer used in the examples is "Mannitol-Magnesium Chloride-MES buffer" or "Mannitol-Magnesium Chloride-Morpholine Ethanesulfonic Acid buffer". The MMG buffer is also a commonly used buffer in plant cell engineering, mainly used for related experiments of plant protoplasts, and the main components usually contain magnesium chloride (MgCl2), mannitol (Mannitol) and MES, etc. The typical formula is 0.4M mannitol, 15mM MgCl2, 4mM MES, and the pH is generally adjusted to 5.7.
[0054] The W5 buffer used in the examples can be named "Sodium Chloride-Calcium Chloride-Potassium Chloride-Magnesium Chloride-Glucose Buffer" according to its components and functions. It is a commonly used buffer in plant cell engineering experiments, mainly used for washing and suspending plant protoplasts. The main components usually contain calcium chloride (CaCl2), potassium chloride (KCl), sodium chloride (NaCl), magnesium chloride (MgCl2) and other salts, and glucose and other sugar substances, and sometimes some buffers such as MES are added to maintain a stable pH environment. The typical formula is 154mM NaCl, 125mM CaCl2, 5mM KCl, 2mM MgCl2, 5mM glucose, and the pH is generally adjusted to about 5.6.
[0055] Example 1 Influence of vacuum pumping time on protoplast yield and protoplast activity
[0056] After adding the enzyme solution, the enzyme solution can be better infiltrated into the buckwheat hypocotyl tissue before dark enzymolysis by vacuum pumping to improve the yield of protoplasts. Therefore, this experiment first tests the influence of different vacuum pumping times on the density and activity of protoplasts. Four time gradients of pumping time of 0min, 15min, 30min and 60min are set in turn while keeping other experimental conditions unchanged.
[0057] The specific operation is: take 1g of buckwheat hypocotyl tissue material and add it to 10mL of enzyme solution, pump for 0min, 15min, 30min and 60min respectively, and take out 150μL from the enzyme solution for buckwheat protoplast cell counting and activity detection. Three parallel experiments were performed for statistics. Take out 150μL from the enzyme solution and divide it into two tubes, numbered 0-A, 0-B, 15-A, 15-B, 30-A, 30-B, 60-A, 60-B. Numbered A group is used to calculate the protoplast density under the hemocytometer. Numbered B group is used to detect the activity of protoplasts by FDA.
[0058] The results are as follows Figure 1As shown, the density of buckwheat protoplasts increases with increasing vacuum time, while the activity remains basically the same. Therefore, it is determined that the vacuum time for preparing buckwheat protoplast cells through isolation is preferably 15–60 min, with 30–60 min being better, and the optimal vacuum time is 30 min.
[0059] Example 2: Effect of enzymatic hydrolysis time on protoplast yield and protoplast activity
[0060] To explore the different effects of varying enzymatic hydrolysis times on the yield and activity of tartary buckwheat protoplasts during preparation, four time gradients were set up with hydrolysis times of 1 h, 2 h, 3 h, and 4 h, while keeping other experimental conditions constant.
[0061] The specific procedures were as follows: 1g of tartary buckwheat hypocotyl tissue was added to 10mL of enzymatic hydrolysis solution and vacuum-sealed for 30min. 150μL of the hydrolysis solution was collected at 1h, 2h, 3h, and 6h for protoplast cell counting and activity assay. Three parallel experiments were performed and the results were statistically analyzed. 1g of tartary buckwheat callus tissue was added to 10mL of enzymatic hydrolysis solution and enzymatically hydrolyzed at 30 rpm in a dark environment at 25℃. 150μL of the hydrolysis solution was collected at 1h, 2h, 3h, and 6h and aliquoted into two tubes, labeled 1-A, 1-B, 2-A, 2-B, 3-A, 3-B, 6-A, and 6-B. Group A was used to calculate protoplast density using a hemocytometer. Group B was used to detect protoplast activity using FDA assay.
[0062] Based on the experimental results, as follows Figure 2 As shown, it can be observed that the cell density of buckwheat protoplasts increases significantly with increasing enzymatic hydrolysis time, while the cell density at 3h and 6h is not significantly different. The activity of protoplast cells remains relatively stable at around 80%, showing minimal impact from enzymatic hydrolysis time. Therefore, Example 3 confirms the effect of different enzyme concentration ratios on the yield and activity of buckwheat protoplasts.
[0063] Cellulase and Macerozyme are the most critical enzymes for isolating protoplast cells from the tissue of F. tataricum Descr. de Candolle. Different contents and ratios of the two enzymes can affect the yield and activity of protoplasts. Therefore, in order to explore the effects of different concentrations of cellulase or pectinase in the enzyme solution on the yield and activity of protoplasts, three enzyme concentration gradients were set while keeping other experimental conditions unchanged, i.e. 1.0% Cellulase R-10 and 0.25% Macerozyme R-10, 1.5% Cellulase R-10 and 0.8% Macerozyme R-10, and 2.0% Cellulase R-10 and 1.0% Macerozyme R-10.
[0064] Specific operation: 1 g of F. tataricum Descr. de Candolle hypocotyls were added to 5 mL of enzyme solution with concentrations of 1.0% Cellulase R-10 and 0.25% Macerozyme R-10, 1.5% Cellulase R-10 and 0.8% Macerozyme R-10, and 2.0% Cellulase R-10 and 1.0% Macerozyme R-10, respectively. After 3 h of enzyme digestion, 150 μL was taken out for counting and activity detection of F. tataricum protoplast cells. Three parallel experiments were performed for statistical analysis.
[0065] The experimental results Figure 3 indicate that the density of F. tataricum protoplast cells increases slightly with the increase of enzyme content in the enzyme solution, and the activity is basically the same. It is found that the number of protoplast cells prepared using 2.0% + 1.0% and 1.5% + 0.8% enzyme solution can meet the experimental requirements. Therefore, considering the economic cost, the concentrations of Cellulase R-10 and Macerozyme R-10 in the enzyme solution used in the subsequent experiments are 1.5% and 0.8%, respectively.
[0066] Example 4 Effects of Different Plasmid Concentrations on Transformation Efficiency of F. tataricum Protoplasts
[0067] In order to explore the effects of different plasmid masses on the transformation efficiency of F. tataricum protoplasts, three different plasmid masses were set while keeping other experimental conditions unchanged, i.e. 10 μg, 20 μg, and 30 μg.
[0068] Specific operation: 10 μg, 20 μg, and 30 μg of plasmid DNA with green fluorescent reporter gene (total volume 30 μL, if the volume is less than 30 μL, add MMG Buffer to make up) and 200 μL of protoplast cells were mixed gently, numbered 10 μg, 20 μg, and 30 μg, and then 230 μL of 40% PEG4000-Ca 2+The solution (final concentration 20%) was mixed gently with the protoplast cells, and after standing in the dark for 20 minutes, 1 mL of W5 Buffer was added to terminate the reaction; the protoplasts were centrifuged at 80 x g for 5 min, the supernatant was discarded, and the protoplasts were resuspended in W5 buffer; the protoplast cells were incubated at 25°C in the dark for 18-48 hours.
[0069] The cell activity was detected under a fluorescence microscope with fluorescein diacetate, and the transient transformation efficiency of the buckwheat protoplasts was calculated.
[0070] According to the experimental results as shown in Figure 4 , it was found that the plasmid concentration of 10-30 μg had good transformation efficiency, especially the transformation efficiency was the highest when the plasmid concentration was 30 μg, so the buckwheat protoplast cells could be transformed with 30 μg of plasmid.
[0071] Example 5: Buckwheat protoplast transient transformation system
[0072] After a series of buckwheat protoplast transient transformation conditions were explored above, a set of buckwheat protoplast transient transformation system was established, and the specific method was as follows:
[0073] a) 30 buckwheat hypocotyls cultured for 5 days were taken, cut into short strips about 1 mm long, and transferred to the enzyme solution (1 g / 10 mL), and pumped for 30 min, and then enzymolyzed at 25°C in the dark for 3 h at 30 rpm;
[0074] b) After the enzymolysis was completed, 10 mL of W5 Buffer was added to the enzyme solution to terminate the reaction, and filtered with a nylon membrane (0.7 μm);
[0075] c) 10 mL of W5 Buffer was added again, and the liquid was transferred to a 50 mL centrifuge tube after the nylon membrane was filtered again;
[0076] d) Centrifuged at 80 x g for 5 min, and the supernatant was discarded;
[0077] e) 10 mL of W5 Buffer was used to resuspend the protoplast cells in the precipitate gently;
[0078] d) After centrifuged at 80 x g for 5 min again, the supernatant was discarded, and the protoplast cells were resuspended in 5 mL of W5 Buffer, and 100 μL was taken for counting on a hemocytometer;
[0079] e) The separated protoplast cells were placed in an ice bath and gravity precipitated for 10 min, and the supernatant was discarded;
[0080] f) According to the counting results, a certain amount of MMG Buffer was used to resuspend the protoplast cells, so that the final concentration was 2 x 106 0.5 x 106cells / mL.
[0081] g) Mix 30 μg plasmid DNA with green fluorescent reporter gene (total volume 30 μL, if less than 30 μL, supplement with MMG Buffer) and 200 μL protoplast cells gently;
[0082] h) Add 230 μL 40% PEG4000-Ca2+ solution (final concentration 20%) to protoplast cells gently, after 20 minutes incubation in dark, add 1 mL W5 Buffer to terminate the reaction; 2+
[0083] i) Centrifuge at 80 x g for 5 min again, discard supernatant, and resuspend protoplasts in W5 buffer gently;
[0084] j) Incubate protoplast cells at 25°C for 18-48 hours in dark;
[0085] k) Observe GFP fluorescence of cells under fluorescence microscope, and calculate the efficiency of transient transformation of buckwheat protoplasts.
[0086] Through this method, buckwheat protoplast cells in good condition can be isolated and collected for protoplast transformation gene transient expression experiment. The buckwheat hypocotyl material observed under an optical microscope, the state of protoplast cells isolated after enzymatic hydrolysis of the material in enzyme solution for 3 hours, and the state of cells after completion of buckwheat protoplast transient transformation are shown in Figure 5 . Figure 6 The cells are loose, round, undamaged, and evenly distributed, indicating that the cells remain in good condition before and after transformation.
[0087] After 48 hours of dark culture, buckwheat protoplast cells are observed by fluorescence inverted microscope, as shown in Figure 7 , the cells after transformation remain in good condition, and nearly 85% of the cells still maintain cell activity. By counting the number of GFP fluorescent cells, the efficiency of buckwheat protoplast transient transformation can reach up to 65%. The above experimental results prove that the buckwheat protoplast transient transformation in buckwheat cells is realized for the first time.
[0088] In order to establish and explore the best transient transformation system of tartary buckwheat protoplast, through exploring the four conditions of vacuum pump pumping time, enzymolysis time, enzyme concentration ratio in enzymolysis solution and plasmid quality in the experiment of tartary buckwheat protoplast transformation, a set of protoplast transient transformation technology applicable to tartary buckwheat was successfully established. The best transformation conditions are as follows: tartary buckwheat material is vacuum pumped for 30 min in the enzymolysis solution with the concentration ratio of Cellulase R-10 and Macerozyme R-10 being 1.5% and 0.8%, and then enzymolyzed for 3 h, and then centrifuged at 80xg to collect the protoplast cells, and then the cells are transformed by adding 30 μg plasmid under the mediation of PEG4000, and the best cell activity can reach 85%, and the highest transformation efficiency can reach 65% for the cells expressing green fluorescent protein. Finally, an efficient, economical and time-saving method for tartary buckwheat protoplast separation and transient expression is established.
Claims
1. A method for isolation of tartary buckwheat protoplasts, characterized by: It comprises the following steps: S1, enzymolysis: take the bitter buckwheat hypocotyl tissue into the enzymolysis solution and vacuum suction for 15-60 min, and then enzymolysis in the dark environment, 24-26℃ and 25-35 rpm vibration conditions for 1-6 h; the concentration of mannitol in the enzymolysis solution is 0.2-0.8 M; the enzyme is cellulase and pectinase, the mass concentration of cellulase is 1.0-2.0%, the mass concentration of pectinase is 0.25-1.0%, and the ratio of bitter buckwheat hypocotyl tissue to enzymolysis solution is 0.1 g / mL; S2, collection: add W5 Buffer to terminate the enzymolysis reaction, filter with 0.7 μm nylon membrane, add W5 buffer again, filter, collect the filtrate of the two times, and combine; centrifuge the filtrate, discard the supernatant, resuspend with W5 buffer, centrifuge, discard the supernatant, and repeat once again, and the precipitate is the protoplast.
2. The method of claim 1, wherein: In step S1, the vacuum suction is 30-60 min; Preferably, in step S1, the vacuum suction is 30 min.
3. The method of claim 1, wherein: In step S1, the enzymolysis is 3-6 h; Preferably, in step S1, the enzymolysis is 3 h.
4. The method of claim 1, wherein: In step S1, the mass concentration of cellulase is 1.5-2.0%, and the mass concentration of pectinase is 0.8-1.0%; Preferably, in step S1, the mass concentration of cellulase is 1.5%, and the mass concentration of pectinase is 0.8%.
5. The method of claim 1, wherein: In step S2, the centrifugation condition is 70-90 x g for 5 min.
6. The method of claim 1, wherein: In step S1, the preparation of bitter buckwheat hypocotyl tissue comprises the following steps: washing the buckwheat seeds with water, shaking and disinfecting with 75% alcohol for 1 min, and washing with sterile water for 3 times; placing the buckwheat seeds in vermiculite, pouring with tap water, adding a solution of 50 mg / L paclobutrazol, 25±1℃, 8 h dark culture for 4-5 days, and standby; In particular, the paclobutrazol solution uses pure water as the solvent.
7. A transient transformation system of F. tartaricum protoplasts, characterized in that: comprising the steps S1 and S2 according to any one of claims 1 to 6, further comprising S3, transforming: resuspending the protoplast cells obtained from step S2 with MMG Buffer to a final concentration of 1.5 x 10 6 ~ 3 x 10 6 plasmids containing the target gene; adding PEG4000 solution, and standing for reaction in the dark for 20 minutes; adding W5 Buffer to terminate the reaction; Centrifugation, collection of the precipitate, resuspension with W5 Buffer, and incubation at 25℃ in the dark for 18-48 hours; wherein after adding the PEG4000 solution, the mass final concentration of PEG4000 is 20%.
8. The transient transformation system of F. tataricum protoplast according to claim 7, characterized in that: In step S3, the centrifugation condition is 70-90 x g for 5 min.
9. The transient transformation system of F. tataricum protoplast according to claim 7, characterized in that: In step S3, the ratio of the amount of plasmid to protoplast cells is 10-30 μg:200 μL.
10. The transient transformation system of F. tataricum protoplast according to claim 9, characterized in that: In step S3, the ratio of the amount of plasmid to protoplast cells is 30 μg:200 μL.