A method for isolating and transiently transforming original protoplasts of dracocephalum tanguticum

Abstract

The application discloses a method for separating and transiently transforming luffin protoplasts, and determines the method for separating luffin protoplasts from cellulase R-10, dissociation enzyme R-10, different concentrations, different temperatures, different osmotic pressures and different enzymolysis times, so that the luffin young leaves are enzymolyzed by using the optimal formula, and then the luffin protoplasts are transiently expressed, and it is found that the plasmid with green fluorescent protein GFP can be normally expressed. The method can provide a reference for the function research of luffin related genes and luffin crop breeding.

Description

Technical Field

[0001] This invention relates to a method for the separation and instantaneous transformation of Aconitum carmichaelii protoplasts. Background Technology

[0002] Yellow aconite ( Aconitum vilmorinianum *Aconitum* L. (Kom.) is a perennial vine belonging to the genus *Aconitum* of the family Ranunculaceae. It is mainly distributed in Yunnan, Sichuan, and Guizhou provinces, growing in mountainous areas at altitudes of 2100-3000 meters in cool, damp environments. As a distinctive traditional Chinese medicine, the root is the primary medicinal part, possessing anti-inflammatory, analgesic, and antipyretic effects. It is currently a key ingredient in several traditional Chinese medicine preparations, including Yunnan Baiyao, *Aconitum carmichaelii* tablets, Baibaodan, Sanwujiao, and Huli San. Due to the long-term influence of its unique natural and cultural environment, *Aconitum carmichaelii* has become a widely used and authentic traditional Chinese medicine in Yunnan. In Yunnan, there is a tradition of cooking and consuming the tuberous roots of *Aconitum carmichaelii* in winter to enhance immunity, hence its reputation as a powerful tonic. With the depletion of wild *Aconitum carmichaelii* resources, research has begun on its artificial domestication and cultivation, as well as its biological characteristics, chemical composition, pharmacological toxicity, pharmacognosy, molecular biology, endophytic fungal isolation, and ornamental horticulture. With the increasing demand for Aconitum carmichaelii and the continuous improvement of its planting scale and level, Aconitum carmichaelii is becoming an important Chinese medicinal herb for increasing the income and wealth of farmers in mountainous and semi-mountainous areas.

[0003] Plant protoplasts are viable, naked plant cells surrounded by a plasma membrane after the cell wall has been removed. Isolated protoplasts can survive for several days under suitable conditions and possess a certain reproductive capacity. Protoplasts are a superior transient expression system, and protoplast transient expression technology is widely used in gene function research, plant physiological and biochemical process research, molecular mechanism research, as well as for producing useful metabolites, constructing plant fusion cells, and creating polyploids. Existing protoplast-based experimental techniques include protein subcellular localization analysis, gene transient expression analysis, promoter activity analysis, ion absorption experiments, and protein-protein interaction verification. In plant single-cell research, protoplasts are the preferred material; however, protoplast preparation often faces challenges such as the difficulty in decomposing plant cell walls and high fragmentation rates. The characteristics of protoplasts make them more efficient in transformation, which is of great significance for genetic transformation research. Summary of the Invention

[0004] This invention provides a method for the separation and transient transformation of Aconitum carmichaelii protoplasts. The steps of this method are as follows: (1) Total RNA was extracted from the leaves of Aconitum carmichaelii. cDNA was obtained by reverse transcription of RNA. The AvUXS gene was amplified using cDNA as a template. The plant expression vector pRI-101-GFP-AvUXS was constructed using homologous recombination. The nucleotide sequence of the AvUXS gene is shown in SEQ ID NO:1. (2) Select large and plump Aconitum carmichaelii seeds, treat them at low temperature, place them on moist filter paper, then put them in a petri dish, and place them in a constant temperature incubator at 24-26℃. Spray water every day. After the Aconitum carmichaelii seeds grow cotyledons, transplant them into nutrient soil and continue to place them in a constant temperature incubator at 24-26℃ for cultivation. During cultivation, there is 8 hours of darkness and 16 hours of light. (3) Weigh cellulase R-10, ionase R-10, mannitol, potassium chloride, and morpholine ethanesulfonic acid monohydrate into ultrapure water and mix well. Incubate at 50-60℃ for 10 min, then cool to room temperature. Add calcium chloride and bovine serum albumin to dissolve and prepare the enzyme hydrolysate. Store at -20℃ in the dark for later use. (4) Take the young leaves of Aconitum carmichaelii from step (2), rinse them with tap water, wipe them dry, then peel off the epidermis, avoiding the veins, cut the young leaves of Aconitum carmichaelii into small pieces with scissors, put them into centrifuge tubes containing enzyme hydrolysate, and culture them in the dark at 26-32℃ for 3-5 hours on a shaker. After filtration through a 100-mesh filter, centrifuge the liquid and discard the supernatant. Add W5 washing solution to resuspend the precipitate, centrifuge again and discard the supernatant, and then add W5 washing solution to suspend it again to obtain protoplast suspension for later use. Among them, W5 washing solution is an aqueous solution containing 5 mmol / L potassium chloride, 154 mmol / L sodium chloride, 125 mmol / L calcium chloride, 2 mmol / L 2-morpholine ethanesulfonic acid monohydrate and 5 mmol / L glucose, with a pH of 5.7. (5) Take the protoplast suspension from step (4) onto a hemocytometer and count the number of protoplasts per milliliter of suspension. Then, after staining with fluorescein diacetate, observe under an inverted microscope. Viable protoplasts are green and inactive protoplasts are red. Count the number of green protoplasts and calculate the protoplast viability value. A viability value of 80% or higher can be used for transformation experiments. Protoplast viability = [number of protoplasts emitting green fluorescence / total number of protoplasts] × 100% (6) Take the protoplast suspension from step (4) into a centrifuge tube, add the plant expression vector pRI-101-GFP and the plant expression vector pRI-101-GFP-AvUXS respectively, and add 40% polyethylene glycol solution. Mix gently with a pipette, let stand, add W5 washing buffer to stop the reaction, centrifuge and discard the supernatant, add W5 washing buffer again to resuspend, let stand in the dark, prepare slides, stain with 4,6-biamidine-2-phenylindole (DAPI) for 5-10 min, observe with a laser confocal microscope, and compare with untransformed protoplasts. If the protoplasts with the transformed plasmid emit green fluorescence, the transformation is successful, and the position of the green fluorescence can be used to determine the location of the transformed gene in the protoplast.

[0005] The AvUXS (UDP-glucuronide decarboxylase) gene used in this invention encodes an enzyme found in the perinuclear Golgi apparatus that catalyzes the synthesis of UDP-xylose. AvUXS, as a catalytic enzyme, participates in plant cell wall synthesis and plays a crucial role in plant growth and development. This invention uses *Aconitum carmichaelii* as material and employs cellulase and ionizing enzymes for enzymatic hydrolysis. This method not only shortens the experimental time but also facilitates the isolation of *Aconitum carmichaelii* protoplasts with a low fragmentation rate, yielding a large number of intact protoplasts. A plant expression vector was constructed using homologous recombination, transiently transfected into *Aconitum carmichaelii* protoplasts, and the localization of the AvUXS gene was observed.

[0006] The protoplasts of Aconitum carmichaelii prepared according to this invention were used for subcellular localization analysis of Aconitum carmichaelii protein. Obvious fluorescence was observed under laser confocal microscopy, indicating that the GFP fusion protein was successfully expressed in the protoplasts, and the localization results were consistent with the predicted protein localization results. Therefore, the method for subcellular localization analysis of Aconitum carmichaelii protein using protoplasts provided by this invention can greatly improve the accuracy of Aconitum carmichaelii protein localization analysis, and at the same time provide a theoretical basis for clarifying the function of the Aconitum carmichaelii gene. Attached Figure Description

[0007] Figure 1 Figure A shows the total RNA of Aconitum carmichaelii, amplification of the AvUXS gene of Aconitum carmichaelii (Figure B), restriction enzyme digestion of pRI-101-GFP plasmid (Figure C), single clones on the recombinant reaction transformation plate (Figure D), and PCR verification of pRI-101-GFP-AvUXS recombinant plasmid (Figure E). Figure 2 Pre-culture of Aconitum carmichaelii seeds (Fig. A), Aconitum carmichaelii plants cultured for 3 months (Fig. B), young leaves of Aconitum carmichaelii (Fig. C), and treatment of young leaves of Aconitum carmichaelii (Fig. D). Figure 3The number of protoplasts digested by cellulase R-10 and cleavage enzyme R-10 at different concentrations (Figure A), the number of protoplasts digested by mannitol at different concentrations (Figure B), the number of protoplasts digested at different temperatures (Figure C), and the number of protoplasts digested at different times (Figure D). Figure 4 Protoplasts under a 20x microscope (Fig. A) and protoplasts under a 4x microscope (Fig. B) with FDA staining. Figure 5 Figure A shows unstained protoplasts of untransformed plasmid (CK), protoplasts transformed with pRI-101-GFP empty vector and stained with DAPI (Figure B), and protoplasts transformed with pRI-101-GFP-AvUXS recombinant plasmid and stained with DAPI (Figure C). Detailed Implementation

[0008] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings. However, the scope of protection of the present invention is not limited to the contents described. Unless otherwise specified, the methods in the embodiments shall be carried out in accordance with conventional operations. Unless otherwise specified, the reagents used shall be conventionally purchased reagents or reagents prepared in accordance with conventional methods. In the embodiments, a hemocytometer is used to count the number of protoplasts in the suspension. Example 1: Construction of a plant expression vector for the AvUXS gene of Aconitum carmichaelii Leaf samples of Aconitum carmichaelii were collected in liquid nitrogen, and total RNA was extracted using Trizol reagent (purchased from Aike Rui Company). First, the leaves were ground into powder in a sterilized mortar. 1 mL of Trizol reagent was added, and the mixture was ground further until liquid. The liquid was poured into a 2 mL centrifuge tube, 200 μL of chloroform was added, and the mixture was incubated on ice for 5 min. The mixture was then centrifuged at 12000 rpm and 4°C for 15 min. 400 μL of the supernatant was transferred to a new centrifuge tube, 400 μL of chloroform was added, and the mixture was incubated on ice for 5 min. The mixture was then centrifuged at 12000 rpm and 4°C for 15 min. 400 μL of the supernatant was transferred to a new centrifuge tube, 400 μL of isopropanol was added, and the mixture was incubated at -20°C for 30 min. The mixture was then centrifuged at 12000 rpm and 4°C for 30 min. The supernatant was discarded, and 500 μL of 75% ethanol was added, and the mixture was centrifuged at 7500 rpm and 4°C for 15 min. The supernatant was discarded, the liquid in the tube was dried, and 20 μL of... 75% diethyl pyrocarbonate (DEPC) in water, detected by agarose gel electrophoresis ( ) Figure 1 A).

[0009] Using the extracted 25 μL RNA as a template, cDNA was obtained by reverse transcription using the M-MLV Reverse Transcriptase kit (purchased from Ferments). Using 2 μL of cDNA as a template, PCR amplification was performed using specific primers F-TCTTCACTGTTGATACATATGATGGGTTCTTCAGAGCTCATT; R-GTTGATTCATAATTCGGATCCTTAGGAGGTAGAACCAGCCTGC. The total amplification system was 50 μL: 17 μL ddH2O, 25 μL 2×Phanta Max Buffer, 1 μL dNTP Mix, 1 μL Phanta Max Super-Fidelity DNA Polymerase (purchased from Novizan), 2 μL upstream primer, 2 μL downstream primer, and 2 μL template DNA. The PCR reaction program was: 95℃ for 3 min pre-denaturation; 95℃ for 30 s denaturation, 66.7℃ for 30 s annealing, and 72℃ for 1 min 40 s extension, for 32 cycles. Complete extension at 72℃ for 5 min, followed by PCR and gel electrophoresis detection. The AvUXS gene fragment (1284 bp) of Aconitum carmichaelii was recovered from the gel. Figure 1 B).

[0010] The pRI-101-GFP plasmid (provided by Kunming Institute of Botany) was digested overnight at 37°C using a double enzyme digestion method. Figure 1 C), Enzyme digestion system: 20 μL : 1.5 μL Nde Ⅰ, 1.5μL Bam The recombinant plasmid was prepared using the following reagents: HⅠ, 5 μL 10×Buffer K, 10 μL pRI-101-GFP plasmid, and 32 μL ddH2O. The AvUXS gene fragment was recovered from gel and recombined with the restriction enzyme plasmid using the ClonExpress II One Step Cloning Kit (purchased from Novizan). The reaction mixture consisted of 4 μL linearized vector, 1 μL insert fragment, 4 μL 5×CE II Buffer, 2 μL Exnase II, and 9 μL ddH2O. The reaction was carried out at 37°C for 30 min on ice.

[0011] Add 100 μL of competent DH5α cells to the 10 μL recombinant product, mix gently, and incubate on ice for 30 min. Heat shock in a 42℃ metal bath for 45 s, then incubate on ice for 2-3 min. Add 900 μL of LB liquid medium, shake at 37℃ and 220 rpm for 1 h, centrifuge at 2400g for 5 min, discard the supernatant, and spread approximately 100 μL onto LB solid medium containing kanamycin (final concentration 50 ug / mL) using magnetic beads. Incubate upside down at 37℃ for 12 h. Wait until a single colony grows on the LB solid medium (…). Figure 1 D) Afterwards, a single colony was picked from the clean bench and placed in LB liquid medium containing Kan. After incubation at 37°C for 5 hours, 2 μL of the bacterial culture was taken for PCR and sequencing verification (Sangon Biotech Co., Ltd.).

[0012] PCR validation ( Figure 1 E) After successful sequencing verification, the verified bacterial culture was placed in a 50mL centrifuge tube and cultured in a shaker at 37°C until the bacterial culture OD reached its limit. 600 When the concentration is 0.8-1.0, recombinant plasmids are extracted using the SanPrep column-based plasmid DNA mini-extraction kit (purchased from Sangon Biotech Co., Ltd.). The operation steps are performed according to the instructions. The extracted recombinant plasmids can be used for protoplast transformation.

[0013] Example 2: Method for Isolation and Transient Transformation of Aconitum carmichaelii Protoplasts 1. Select large and plump Aconitum carmichaelii seeds, first place them at 4℃ for two weeks, then place the Aconitum carmichaelii seeds in a petri dish with three layers of moist filter paper, and then place it in a 25℃ constant temperature incubator, spraying water daily. Figure 2 A) After the seeds of Aconitum carmichaelii have sprouted cotyledons, transplant them into nutrient soil and continue to cultivate them in a 25℃ constant temperature incubator, with 8 hours of darkness and 16 hours of light during cultivation. Harvest samples after 3 months of cultivation. Figure 2 B) The leaves of Aconitum carmichaelii were rinsed with tap water for 3 minutes and then dried. Figure 2 C), weigh 0.1g of the leaf using a balance and cut it into strips about 1mm wide. Figure 2 D), place in a petri dish for later use.

[0014] 2. Prepare cellulase R-10 solutions (purchased from Maokang Biotechnology Co., Ltd.) at concentrations of 5 g / L, 7.5 g / L, 10 g / L, and 12.5 g / L, and analyte R-10 solutions (purchased from Maokang Biotechnology Co., Ltd.) at concentrations of 2 g / L, 3 g / L, 4 g / L, and 5 g / L. Combine the above cellulase R-10 and analyte R-10 concentrations in pairs, add 0.4 mol / L mannitol, 20 mM potassium chloride, and 20 mM morpholine ethanesulfonic acid monohydrate (MES monohydrate), and cool in a 55°C water bath for 10 min. Then add 10 mM calcium chloride and 1% bovine serum albumin (BSA), followed by the leaves from step 1. Incubate in the dark at 29°C on a shaker for 3 h. When the cellulase R-10 concentration is 10 g / L and the analyte R-10 concentration is 3 g / L, the number of protoplasts obtained from enzymatic hydrolysis of Aconitum carmichaelii is the highest and significantly higher than other groups. Figure 3 A); When the concentration of cellulase R-10 was 10 g / L and the concentration of dissociative enzyme R-10 was 3 g / L, and the concentrations of mannitol were set at 0.4 mol / L, 0.6 mol / L, 0.8 mol / L, and 1.0 mol / L, with other conditions remaining unchanged, the number of protoplasts obtained by enzymatic hydrolysis at a mannitol concentration of 0.6 mol / L was greater than that in other groups. Figure 3 B); When the concentration of cellulase R-10 is 10 g / L, the concentration of anabolase R-10 is 3 g / L, and the concentration of mannitol is 0.6 mol / L, the enzymatic hydrolysis time is set to 2 h, 3 h, 4 h, and 5 h, with other conditions remaining unchanged. The number of protoplasts obtained after 4 h of enzymatic hydrolysis is ( Figure 3 C); When the concentration of cellulase R-10 was 10 g / L, the concentration of dissociative enzyme R-10 was 3 g / L, and the concentration of mannitol was 0.6 mol / L, the enzymatic hydrolysis temperatures were 26℃, 28℃, 30℃, and 32℃, respectively, with other conditions remaining unchanged. The number of protoplasts obtained at the enzymatic hydrolysis temperature of 30℃ was significantly higher than that in the other groups. Figure 3 D).

[0015] 3. Take 1 mL of protoplast suspension resuspended in W5 ( Figure 4 A) Add 1 μL of fluorescein diacetate solution (FDA dissolved in acetone to prepare a 5 mg / mL solution), mix well, let stand at room temperature for 5 min, and observe under a fluorescence microscope. Viable protoplasts emit green fluorescence under green fluorescence excitation light, while inactive protoplasts emit red fluorescence. Figure 4 B). Protoplasts with a viability of over 80% as determined by FDA staining can be used for protoplast plasmid transformation experiments; 4. Add 10 μL of pRI-101-GFP plasmid and pRI-101-GFP-AvUXS plasmid to 1 mL of protoplast suspension, respectively. The plasmid was mixed with 120 μL of 40% polyethylene glycol solution and gently pipetted to mix. The mixture was allowed to stand at room temperature for 5 min, and the reaction was terminated by adding 500 μL of W5 washing buffer. After centrifugation at 200 g for 1 min, the supernatant was discarded, and the precipitate was resuspended in 100 μL of W5 washing buffer. The mixture was then incubated at 24 °C for 16 h in the dark. For observation under a laser confocal microscope, 4',6-diamidinyl-2-phenylindole (DAPI) was added to a concentration of 10 μg / mL. A protoplast suspension without plasmid was used as a control. The results are shown in the figure. Figure 5 As can be seen from the figure, the control protoplasts do not emit green fluorescence, and the cell nuclei do not emit blue fluorescence. The protoplasts transformed with pRI-101-GFP plasmid are green overall, and the cell nuclei are blue. The protoplasts transformed with pRI-101-GFP-AvUXS plasmid are green at the edges, do not emit light in the middle, and the cell nuclei are still blue.

[0016] This study constructed the plant expression vector pRI-101-GFP-AvUXS using homologous recombination for transformation. Factors affecting protoplast preparation were investigated, and the optimal formulation for enzymatic hydrolysis of *Aconitum carmichaelii* leaves was determined by considering factors influencing protoplast yield, including cellulase, cleavage enzyme, osmotic pressure, time, and temperature. The plasmid was then transferred into *Aconitum carmichaelii* protoplasts using a PEG-mediated method, resulting in normal expression. Experimental results showed that the optimal protoplast separation and yield were achieved when the enzymatic hydrolysate contained 10 g / L cellulase, 3 g / L cleavage enzyme, 0.6 g / L mannitol, 20 mM potassium chloride, 20 mM morpholine ethanesulfonic acid monohydrate, 10 mM calcium chloride, and 1% of the total volume of bovine serum albumin, and when cultured in a shaker at 30℃ in the dark for 4 h.

Claims

1. A method for the separation and transient transformation of Aconitum carmichaelii protoplasts, characterized in that, The steps are as follows: (1) Select large and plump Aconitum carmichaelii seeds, treat them at low temperature, place them on moist filter paper, then put them in a petri dish, and place them in a constant temperature incubator at 24-26℃. Spray water every day. After the Aconitum carmichaelii seeds grow cotyledons, transplant them into nutrient soil and continue to place them in a constant temperature incubator at 24-26℃ for cultivation. During cultivation, there is 8 hours of darkness and 16 hours of light. (2) Weigh cellulase R-10, ionase R-10, mannitol, potassium chloride, and morpholine ethanesulfonic acid monohydrate into ultrapure water and mix well. Incubate at 50-60℃ for 10 min, then cool to room temperature. Add calcium chloride and bovine serum albumin to dissolve and prepare the enzyme hydrolysate. Store at -20℃ in the dark for later use. (3) Take the young leaves of Aconitum carmichaelii from step (1), rinse them with tap water, wipe them dry, then peel off the epidermis, avoiding the veins, cut the young leaves of Aconitum carmichaelii into small pieces with scissors, put them into centrifuge tubes containing enzyme hydrolysate, and culture them in the dark at 26-32℃ for 3-5 hours on a shaker. After filtration through a 100-mesh filter, centrifuge the liquid and discard the supernatant. Add W5 washing solution to resuspend the precipitate, centrifuge again and discard the supernatant. Then add W5 washing solution sterilized at high temperature to suspend the precipitate to obtain protoplast suspension. The W5 washing solution is an aqueous solution containing 5 mmol / L potassium chloride, 154 mmol / L sodium chloride, 125 mmol / L calcium chloride, 2 mmol / L 2-morpholine ethanesulfonic acid monohydrate and 5 mmol / L glucose, with a pH of 5.

7. (4) Take the protoplast suspension from step (3) onto a hemocytometer and count the number of protoplasts per milliliter of suspension. Then, after staining with fluorescein diacetate, observe under a microscope. Viable protoplasts are green and inactive protoplasts are red. Count the number of green protoplasts and calculate the protoplast viability value. A viability value of 80% or higher can be used for transformation experiments. (5) Take the protoplast suspension from step (3) into a centrifuge tube, add the recombinant plasmid pRI-101-GFP-AvUXS, and add 40% polyethylene glycol solution. Gently pipette and mix well. After standing, add W5 washing buffer to stop the reaction. Centrifuge and discard the supernatant. Add W5 washing buffer again to resuspend and stand in the dark. Stain with 4,6-biamidine-2-phenylindole for 5-10 min. Observe with a laser confocal microscope. Compared with the untransformed protoplasts, the protoplasts that have been transformed with the plasmid emit green fluorescence, which means that the transformation is successful. The position of the green fluorescence can be used to determine the location of the transformed gene in the protoplast. The nucleotide sequence of the AvUXS gene in the recombinant plasmid pRI-101-GFP-AvUXS is shown in SEQ ID NO:

1.

2. The method for separating and transiently transforming Aconitum carmichaelii protoplasts according to claim 1, characterized in that: Step (2) The concentration of cellulase R-10 in ultrapure water is 5-12.5 g / L, the concentration of anabolase R-10 is 2-5 g / L, the concentration of mannitol is 0.4-0.8 mol / L, the concentration of potassium chloride is 20 mmol / L, the concentration of morpholine ethanesulfonic acid monohydrate is 20 mmol / L, the concentration of calcium chloride is 10 mmol / L, and the volume concentration of bovine serum albumin is 1%.

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