Preparation method and application of artemisia selengensis protoplast

Abstract

The invention provides a preparation method and application of artemisia selengensis protoplast, and belongs to the technical field of plant cell research. The preparation method of the Artemisia selengensis protoplast comprises the following steps: S1, cutting tender leaves of Artemisia selengensis into filaments, and completely immersing the filaments in a 13% CPW salt solution for pretreatment for 1 h; and S2, adding the artemisia selengensis tissues pretreated in the step S1 into an enzymatic hydrolysate for enzymolysis, centrifuging after enzymolysis is finished, collecting protoplast, resuspending the protoplast by using a W5 salt solution, and superposing the obtained resuspension on a sucrose solution for centrifugation to obtain the purified artemisia selengensis protoplast. Wherein the enzymatic hydrolysate contains 3wt% of cellulase and 0.8 wt% of macerozyme. The method successfully prepares a large amount of high-activity Artemisia selengensis protoplast. Based on the protoplast, an efficient and stable Artemisia selengensis protoplast instantaneous transformation system is established, subcellular localization of Artemisia selengensis AsPAL gene is successfully realized, and an important technical basis is provided for Artemisia selengensis gene function research and gene editing application.

Description

Technical Field

[0001] This invention belongs to the field of plant cell research technology, specifically relating to a method for preparing Artemisia annua protoplasts and their application. Background Technology

[0002] Protoplasts are totipotent and viable naked plant cells capable of various metabolic activities. As a unique single-celled system, protoplasts are ideal materials for basic research and crop improvement, and can be used in plant cell physiology and genetics research, such as cell wall formation, plant regeneration, somatic cell hybridization, and transient gene expression. Plant protoplasts can be extracted from materials such as leaves, cotyledons, petals, roots, hypocotyls, suspension-cultured cells, and callus tissue. The main separation methods include mechanical, chemical, and enzymatic methods. Studies have shown that enzymatic hydrolysis is the most widely used method due to its high yield and good integrity. However, the enzymatic separation of protoplasts is affected by various factors, including explant type, enzyme type, enzyme concentration, and hydrolysis time. The optimal protoplast preparation system varies for different plant tissue materials. For example, tobacco (… Nicotiana tabacum The enzymatic hydrolysis time for L. leaf protoplasts is only 4 hours; kenaf ( Hibiscus cannabinus The enzymatic hydrolysis time for hypocotyl protoplasts needs to be extended to 12 hours to achieve the highest yield, while a hydrolysis time of 9 hours results in a lower yield. The optimal preparation conditions for maize leaf protoplasts are 1.5% cellulase and 0.5% cleavage enzyme, with hydrolysis lasting 5 hours. The optimal enzyme solution combination for Arabidopsis leaf protoplasts is 1.5% cellulase and 0.4% cleavage enzyme. Because the cell walls of protoplasts have been enzymatically or mechanically removed, they are more readily accepting exogenous organelles and nucleic acids. Transient gene expression technology provides a rapid and effective means for cellular, molecular, biochemical, and genetic research, and is particularly suitable for the rapid assessment of gene function, such as subcellular protein localization, protein interactions, protein activity, and signal transduction studies.

[0003] Artemisia selengensis ( Artemisia selengensis Artemisia is a perennial herb belonging to the genus Artemisia in the family Asteraceae. It possesses properties such as clearing heat and detoxifying, calming the liver and extinguishing wind, dispelling wind and dampness, and reducing inflammation and relieving cough. It also exhibits lipid-lowering and blood pressure-lowering effects, making it valuable for the dietary therapy of cardiovascular diseases. (Example: Artemisia annua) AsPALThe gene encodes phenylalanine ammonia-lyase, the rate-limiting enzyme in the phenylpropane metabolic pathway. It catalyzes the production of trans-cinnamic acid from L-phenylalanine, providing a precursor for the synthesis of caffeoylquinic acid derivatives, thereby positively regulating the biosynthesis of the antioxidant 1,4-dicaffeoylquinic acid (1,4-DCQA). 1,4-DCQA effectively inhibits the activity of hepatic xanthine oxidase (XO), reducing uric acid production and thus exerting a uric acid-lowering effect. Therefore, developing an efficient transient gene expression system for *Artemisia argyi* protoplasts is of great significance. However, currently, there is a lack of well-established methods for the preparation and transformation of *Artemisia argyi* protoplasts, making it difficult to meet the needs of subsequent genetic transformation and gene function research. Therefore, establishing an efficient *Artemisia argyi* protoplast preparation and transformation system is crucial for studying *Artemisia argyi* gene function and gene editing. Summary of the Invention

[0004] The first objective of this invention is to provide a method for preparing Artemisia annua protoplasts, through which high-yield and high-vitality Artemisia annua protoplasts are obtained.

[0005] Specifically, the present invention achieves the above objectives through the following technical solutions: A method for preparing Artemisia annua protoplasts includes the following steps: S1. Cut the tender leaves of Artemisia argyi into thin strips, and immerse the strips completely in a 13% CPW salt solution for 1 hour for pretreatment; S2. Add the pretreated Artemisia argyi tissue from step S1 to the enzymatic hydrolysis solution for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, centrifuge and collect the protoplasts. Resuspend the protoplasts in W5 salt solution. Stack the resuspended solution on sucrose solution and centrifuge to obtain purified Artemisia argyi protoplasts. The enzymatic hydrolysate contains 3 wt% cellulase and 0.8 wt% ionizing enzyme.

[0006] In the preferred embodiment, in step S1, the tender leaves of Artemisia argyi are cut into thin strips 0.1~1mm wide.

[0007] In a preferred embodiment, the enzymatic hydrolysate comprises the following components: 3 wt% cellulase, 0.8 wt% dissociative enzyme, 0.4 M mannitol, 20 mM KCl, 10 mM CaCl2, 20 mM MES, and 0.1 wt% BSA.

[0008] In the preferred embodiment, the enzymatic hydrolysis conditions in step S2 are 24-26°C and incubation in the dark for 4-12 hours.

[0009] In the preferred embodiment, the centrifugation conditions after enzymatic hydrolysis in step S2 are: centrifugation at 4°C with a centrifugal force of 100g for 6-10 minutes.

[0010] In a preferred embodiment, the W5 salt solution in step S2 comprises the following components: 2 mM MES, 154 mM NaCl, 125 mM CaCl2, and 5 mM KCl.

[0011] In the preferred embodiment, in step S2, the obtained resuspended solution is superimposed on a 0.55M sucrose solution and centrifuged at 100g for 6-10 minutes at 4°C.

[0012] In a preferred embodiment, the tender leaves of Artemisia annua mentioned in step S1 are obtained by inoculating Artemisia annua stem segments into MS medium and aseptically culturing for 2-3 weeks to obtain robust Artemisia annua leaves.

[0013] In the preferred embodiment, the ratio of the mass of the young leaves of Artemisia annua in step S1 to the volume of the enzymatic hydrolysate in step S2 is 1g:20mL.

[0014] A second objective of this invention is to provide the application of Artemisia annua protoplasts prepared according to any of the above schemes in the subcellular localization of Artemisia annua AsPAL protein.

[0015] In a preferred embodiment, the subcellular localization of the Artemisia argyi AsPAL protein includes the following steps: AsPAL The nucleotide sequence of the gene was cloned into a vector containing fluorescent protein to construct a gene containing... AsPAL A recombinant expression vector for the gene; the expression vector was transiently transformed into the Artemisia annua protoplasts, and after being cultured at 23-25°C in the dark for 2-6 hours, the subcellular localization of the protein in the Artemisia annua protoplasts was detected under a fluorescence microscope.

[0016] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: (1) The method for preparing Artemisia annua protoplasts provided in this invention successfully prepared a large number of highly viable Artemisia annua protoplasts, with a maximum yield of 2.88 × 10⁻⁶. 7 The viability rate of Artemisia annua leaf tissue can reach up to 91% per gram.

[0017] (2) This invention establishes an efficient and stable transient transformation system for Artemisia annua protoplasts, and uses this system to perform subcellular localization of the Artemisia annua AsPAL gene. The results show that AsPAL is located in the cytoplasm. This approach provides technical support for the study of Artemisia annua gene function and gene editing applications. Attached Figure Description

[0018] Figure 1 The images show the observation results of Artemisia annua protoplasts prepared at different enzymatic hydrolysis times under a fluorescence microscope in Example 1 of the present invention, as well as a comparison of yield and viability. Figure 2The images show the observation results of Artemisia annua protoplasts prepared using different enzymatic hydrolysates in Example 1 of the present invention under a fluorescence microscope, as well as a comparison of yield and viability. Figure 3 The images shown in Example 2 of this invention are images of protoplasts isolated and extracted from different varieties of Artemisia argyi using the preparation method of Example 1, observed under a fluorescence microscope, along with a comparison of yield and viability. Figure 4 This is a comparison image of protoplasts before and after purification using sucrose solution in Example 1 of the present invention; Figure 5 This is a diagram showing the results of subcellular localization analysis of the AsPAL gene in Artemisia annua protoplasts in Example 3 of the present invention. Detailed Implementation

[0019] The following description, in conjunction with embodiments, clearly and completely describes the technical solutions of this application, so that those skilled in the art can fully understand this application. Obviously, the described embodiments are merely some preferred embodiments of this application, and not all embodiments. Any equivalent modifications or substitutions made by those skilled in the art to the following embodiments without creative effort are within the protection scope of this application.

[0020] The full names of the English abbreviations used in the following embodiments are as follows: CPW: Cell Protoplast Wash Medium; mainly includes the following components: 27.2 mg / L KH2PO4, 101.0 mg / L KNO3, 1480.0 mg / L CaCl2·2H2O, 246.0 mg / L MgSO4, 0.16 mg / L KI, 0.025 mg / L CuSO4, 13% (w / v) mannitol, pH 6.0; MES: 2-(N-methylamino)ethanesulfonic acid; BSA: Bovine serum albumin; FDA: Fluorescein diacetate; GFP: Green Fluorescent Protein; CDS: Coding sequence.

[0021] In the following embodiments, L106, L30, L33, and L39 are numbers given for the convenience of recording in this application, and are not plant variety names.

[0022] Example 1 A method for preparing Artemisia annua protoplasts includes the following steps: S1, Plant material pretreatment The *Artemisia argyi* var. *mongolica* L106 from Wangdian Village, Funan County, Fuyang City, Anhui Province was selected. This variety has a wide planting range and strong adaptability to stress. The stem segments were processed, including leaf removal, rinsing, and disinfection. First, the harvested stem segments were cleaned of leaves, then rinsed with tap water for 20 minutes and drained. The stem segments were rinsed twice with sterile water on a clean bench, then disinfected with 75% alcohol for 30 seconds, rinsed four to five times with sterile water, disinfected with 3.7mM HgCl2 solution for 15 minutes, and rinsed four to five times with sterile water. The surface moisture of the stem segments was absorbed with filter paper, and the segments with buds were cut into 2-3cm segments with sterile blades. The segments were inoculated onto MS medium (main components: 30g / L sucrose, 8g / L agar, 4.4g / L commercially available MS medium), the bottle was sealed, and cultured under sterile conditions at 26℃, 71% humidity, and a photoperiod of 16h light (5000-10000 lux) / 8h darkness for 2-3 weeks until each seedling had 4-6 leaves. The young leaves of the robust seedlings were used for subsequent protoplast extraction.

[0023] S2, Protoplast Isolation and Purification S2-1. Preparation of Enzymatic Hydrolysate: First, prepare a solution containing 20 mM KCl, 10 mM CaCl2, 20 mM MES, 0.1 wt% BSA, and 400 mM mannitol using ddH2O. Then, add a mixture of cellulase R-10 and analyte R-10 to construct three different combinations of enzymatic hydrolysate. These three hydrolysate are designated as 1.5 wt% cellulase R-10 + 0.4 wt% analyte R-10 (1×), 2.25 wt% cellulase R-10 + 0.6 wt% analyte R-10 (1.5×), and 3 wt% cellulase R-10 + 0.8 wt% analyte R-10 (2×). Adjust the pH of all three hydrolysate to 5.8, then filter them through a 0.22 μm syringe and store them at 4°C for later use.

[0024] S2-2, Enzymatic hydrolysis and protoplast collection: The sterile young leaves of Artemisia annua from step S1 were cut into 0.5–1 mm wide filaments and completely immersed in a 13% CPW salt solution for 1 hour for pretreatment. Then, they were placed in a 3 wt% cellulase R-10 + 0.8 wt% cleavage enzyme R-10 hydrolysate solution (mass of young leaves to volume of hydrolysate: 1 g: 20 mL) and incubated in the dark at 24–26°C for 4–12 hours (e.g., 10 hours) for enzymatic hydrolysis. After hydrolysis, the protoplasts were collected by centrifugation at 4°C and 100 g for 6–10 minutes (e.g., 10 minutes). The protoplasts were resuspended in a W5 salt solution (main components: 2 mM MES, 154 mM NaCl, 125 mM CaCl2, and 5 mM KCl) and gently mixed to obtain a protoplast resuspension. Then, at 4℃ and 100g centrifugation, the protoplast resuspension was stacked on a 0.55M sucrose solution and centrifuged for 6-10 minutes (e.g., 8 minutes) for purification. The volume ratio of the resuspension to the sucrose solution was 1:9, yielding Artemisia annua protoplasts. Comparison of protoplasts before and after purification is shown below. Figure 4 As shown in the figure, it can be seen that after purification, incompletely digested cells, broken cells, and other impurities were removed, resulting in a sample with high purity and high survival rate.

[0025] Enzymatic hydrolysis was performed at different times (6h, 8h, 10h, and 12h) according to the method in step S2-2. The yield and viability of protoplasts were measured, and the results are as follows: Figure 1 As shown. Protoplast yield was counted using a dual-chamber hematology analyzer under a Zeiss fluorescence microscope. Protoplast yield = number of protoplasts produced in the enzyme solution / fresh weight of plant leaves used in the enzyme solution. Protoplast viability was determined by FDA staining. FDA was dissolved in acetone to prepare a 5 mg / mL FDA solution. Protoplasts were diluted 5-fold with W5 salt solution, and 5 mg / mL FDA solution was added to bring the final FDA concentration to 100 μg / mL. The solution was then placed in the dark at room temperature for 20 min and observed under a fluorescence microscope. Viable protoplasts emitted yellow-green fluorescence, while inactive protoplasts did not emit fluorescence. Three fields of view were randomly selected for observation and photography, with each sample repeated three times. The number of cells emitting yellow-green fluorescence was counted against the total number of cells, and the protoplast viability rate was calculated. Protoplast viability rate = (number of yellow-green fluorescent protoplasts / total number of protoplasts) × 100%.

[0026] from Figure 1 As can be seen above, both the yield and viability of protoplasts initially increased and then decreased with increasing enzymatic hydrolysis time. The protoplast yield reached 0.74 × 10⁻⁶ after 4–12 hours of enzymatic hydrolysis. 7The yield of protoplasts reached 1.31 × 10⁶ per gram of Artemisia annua leaf tissue; after 6-10 hours of enzymatic hydrolysis, the yield of protoplasts reached 1.31 × 10⁶ per gram of leaf tissue. 7 The yield of protoplasts reached 1.89 × 10⁶ per gram of Artemisia annua leaf tissue; after 8-10 hours of enzymatic hydrolysis, the yield of protoplasts reached 1.89 × 10⁶ per gram of leaf tissue. 7 The yield of protoplasts was highest at 2.88 × 10⁶ per gram of Artemisia annua leaf tissue; after 8 hours of enzymatic hydrolysis, the protoplast yield reached 2.88 × 10⁶. 7 Protoplasts were analyzed at 1 / g per gram of *Artemisia argyi* leaf tissue. After 4–12 hours of enzymatic hydrolysis, the protoplast viability exceeded 50%; after 6–12 hours, it exceeded 55%; after 6–8 hours, it exceeded 65%; and after 8 hours, the highest protoplast viability, reaching 91%, was achieved. Figure 1 The results also show that after 8 hours of enzymatic hydrolysis, the cell morphology is most intact, with the most free cells and the fewest broken cells. After 4-6 hours of enzymatic hydrolysis, some cells clump together and fail to separate.

[0027] Following the method in step S2-2, the enzymatic hydrolysis time was fixed at 8 hours. Three different enzymatic hydrolysates prepared in step S2-1 were used for hydrolysis. Protoplasts were collected, and the yield and viability of the protoplasts were measured. The results are as follows: Figure 2 As shown. From Figure 2 As can be seen above, the number of protoplasts obtained from enzymatic hydrolysis varies significantly under different enzyme concentrations. The optimal hydrolysis solution is: 20 mM KCl, 10 mM CaCl2, 20 mM MES, 0.1 wt% BSA, 400 mM mannitol, 3 wt% cellulase R-10, and 0.80 wt% dissociative enzyme R-10, i.e., 3 wt% cellulase R-10 + 0.8 wt% dissociative enzyme R-10 in step S2-1. Using this hydrolysis solution, the highest number of protoplasts obtained from *Artemisia argyi* leaf tissue was 2.88 × 10⁻⁶. 7 The highest viability rate was 91% for *Artemisia argyi* leaf tissue per gram. Therefore, subsequent enzymatic hydrolysis was performed using a solution of 3 wt% cellulase R-10 + 0.8 wt% analyte R-10.

[0028] In summary, the optimal conditions for obtaining high-yield, high-vitality, and high-purity *Artemisia argyi* protoplasts using the above method are as follows: *Artemisia argyi* leaf tissue is enzymatically hydrolyzed for 8 hours using a 3wt% cellulase R-10 + 0.8wt% analyte R-10 hydrolysate solution; the protoplasts are then resuspended in W5 salt solution at a centrifugation speed of 100g; and the resuspended solution is purified by centrifugation with 0.55M sucrose at 100g centrifugation. Using these optimal conditions, the total protoplast yield can reach 2.88 × 10⁻⁶. 7 The viability rate reached 91% with 1 protoplast per gram of Artemisia annua leaf tissue.

[0029] Example 2 This embodiment investigated the applicability of the protoplast preparation method described in Example 1. *Artemisia argyi* germplasm resources from four different locations were collected: L30 (collected from Xishui, Huanggang City, Hubei Province), L33 (collected from Baishazhou, Wuhan City, Hubei Province), L39 (collected from Panjiawan, Xianning City, Hubei Province), and L106 (collected from Fuyang City, Anhui Province). Protoplasts were extracted from *Artemisia argyi* under the optimal conditions determined in Example 1, and the yield and viability of the protoplasts were measured. The results are as follows: Figure 3 As shown. From Figure 3 As can be seen above, the total protoplast yield of L30 reached 1.38 × 10⁻⁶. 7 The protoplast viability of L33 was 78.5% based on FDA staining of *Artemisia argyi* leaf tissue per gram; the total protoplast yield of L33 reached 1.53 × 10⁻⁶. 7 The number of protoplasts per gram of Artemisia annua leaf tissue was 69%; the total protoplast yield of L39 reached 1.68 × 10⁶. 7 The protoplast viability rate of Artemisia annua leaf tissue was as high as 81.5%; the total protoplast yield of L106 reached 1.25 × 10⁶. 7 The number of protoplasts per gram of Artemisia annua leaf tissue was as high as 86.3%. This indicates that the method for preparing Artemisia annua protoplasts in Example 1 can yield high-yield and highly active protoplasts when applied to different Artemisia annua germplasm resources.

[0030] Example 3 A method for transient transformation of Artemisia annua protoplasts includes the following steps: P1, Amplification of Artemisia annua AsPA L gene Total RNA was extracted from Artemisia annua leaves using a total RNA extraction kit (Nanjing Novizan Biotechnology Co., Ltd.), following the kit's instructions. The total RNA was reverse transcribed into cDNA using a reverse transcription kit (Beijing TransGen Biotech Co., Ltd.). The CDS sequence of the AsPAL gene was amplified from the cDNA using specific primer pair F1-R1, as shown in SEQ ID No. 1.

[0031] The reaction system for PCR amplification of the AsPAL gene is as follows: 2×Phanta Max Buffer 2μL, dNTPMix (10mM) 1μL, forward primer F1 (10μM) 2μL, reverse primer R1 (10μM) 2μL, Phanta Max Super-Fidelity DNA Polymerase 1μL, cDNA 2μL, ddH2O 17μL.

[0032] AsPALThe gene PCR amplification program is as follows: 95℃ for 3 min; 95℃ for 15 s, 54℃ for 15 s, 72℃ for 60 s, for 35 cycles; 72℃ for 10 min; 20℃ for 5 min.

[0033] Forward primer F1 for amplification of the AsPAL gene in Artemisia annua: 5'-agcagatctatcgattctagaatggctactaaaaatattgagaac-3'; Reverse primer R1 for amplifying the AsPAL gene of Artemisia annua: 5'-tcctttgcccatggctctagaacaaatcggaagtggaacac-3'.

[0034]

[0035] P2. Constructing the transformation carrier The amplification product from step P1 was cloned into the pm999 vector and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. After the sequencing results were returned, they were compared with the original sequence information. If the comparison was correct, the gene fragment containing the AsPAL gene CDS sequence with the stop codon removed was amplified by PCR using a pair of specific primers F2-R2.

[0036] The PCR amplification reaction system is as follows: 2×Phanta Max Buffer 2μL, dNTPMix (10mM) 1μL, forward primer F2 (10μM) 2μL, reverse primer R2 (10μM) 2μL, Phanta Max Super-Fidelity DNA Polymerase 1μL, template DNA 2μL, ddH2O 17μL.

[0037] The PCR amplification program was as follows: 95°C for 3 min; 95°C for 15 s, 54°C for 15 sec, 72°C for 60 sec) for 35 cycles, 72°C for 10 min, 20°C for 5 min.

[0038] Forward primer F2: 5'-agcagatctatcgattctagaatggctactaaaaatattgagaac-3'; Reverse primer R2: 5'-tcctttgcccatggctctagaacaaatcggaagtggaacac-3'.

[0039] The amplified product was cloned into the pm999 vector with a GFP tag and a 35S promoter via the XbaI site to obtain the recombinant plasmid 35S::AsPAL-GFP.

[0040] The constructed recombinant plasmid 35S::AsPAL-GFP was transformed into *E. coli* DH5α using a heat shock transformation method and cultured at 37°C. Positive clones were selected for plasmid extraction. Plasmid extraction was performed using the SanPrep column-based plasmid DNA mini-extraction kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.). The obtained plasmid DNA was analyzed using an ultra-micro nucleic acid and protein analyzer, and the concentration was adjusted to 1000 ng / μL with ddH2O. The recovered plasmid DNA was stored at -20°C for later use as the target plasmid.

[0041] P3, Protoplast Transformation Following the method in Example 1, Artemisia annua protoplasts were prepared under optimal conditions and placed on ice for 30 minutes by gravity sedimentation. The supernatant was drained, and the protoplasts were resuspended in MMG solution (main components: 0.2 M mannitol, 15 mM MgCl2, and 4 mM MES) at room temperature. AsPAL The gene fluorescently labeled fusion expression vector (the target plasmid obtained in step P2) was used as the experimental group. Artemisia annua protoplasts were used as the transformation recipients, and transfection was performed using a plant protoplast transfection kit (purchased from Shanghai Beyotime Biotechnology Co., Ltd., product number C0563S or C0563M). The procedure was performed according to the kit instructions. Specific steps are as follows: Transfer 10 μL of plasmid DNA (10-20 μg) into a 2 mL round-bottom centrifuge tube. The recommended plasmid size is 5-10 kb, and the plasmid concentration is 1-2 μg / μL. Add 100 μL of a (1.5-2.5) × 10⁻⁶ ppm plasmid. 5 Add a protoplast suspension of approximately 20,000 protoplasts per mL and mix gently. Add 110 μL of transfection reagent solution prepared at least 1 hour in advance, and gently tap the bottom of the centrifuge tube to mix thoroughly. Incubate at 25°C for 5–15 min. Add 440 μL of stop solution, mix gently, and centrifuge at 100 g for 1–2 min at 25°C, removing as much supernatant as possible and collecting the protoplasts. Add 500 μL of stop solution, gently resuspend the protoplasts, and centrifuge at 100 g for 1 min at 25°C, removing as much supernatant as possible. Add 1 mL of culture medium, carefully resuspend the protoplasts, and then incubate horizontally at 23–25°C in the dark for 2–6 h. All reagents and culture media used in the experiment were products provided with the kit.

[0042] P4 AsPAL Subcellular localization analysis of genes The subcellular localization of the protein in Artemisia annua protoplasts was detected under a fluorescence microscope to verify the transient expression system. The results are as follows: Figure 5 As shown. From Figure 5 As can be seen above, when the AsPAL-GFP fusion protein is co-expressed in the protoplasts of Artemisia annua leaves, a clear GFP signal is specifically detected in the cytoplasm of Artemisia annua, indicating that AsPAL is expressed in the cytoplasm.

[0043] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Various modifications and variations can be made to the present invention by any person skilled in the art. Any simple equivalent changes and modifications made based on the scope of protection of this invention and the content of the specification should be included within the scope of protection of the present invention.

Claims

1. A method for preparing Artemisia annua protoplasts, characterized in that, Includes the following steps: S1. Cut the tender leaves of Artemisia argyi into thin strips, and immerse the strips completely in a 13% CPW salt solution for 1 hour for pretreatment; S2. Add the pretreated Artemisia argyi tissue from step S1 to the enzymatic hydrolysis solution for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, centrifuge and collect the protoplasts. Resuspend the protoplasts in W5 salt solution. Stack the resuspended solution on sucrose solution and centrifuge to obtain purified Artemisia argyi protoplasts. The enzymatic hydrolysate contains 3 wt% cellulase and 0.8 wt% ionizing enzyme.

2. The method for preparing Artemisia protoplasts according to claim 1, characterized in that, The enzymatic hydrolysate comprises the following components: 3 wt% cellulase, 0.8 wt% dissociative enzyme, 0.4 M mannitol, 20 mM KCl, 10 mM CaCl2, 20 mM MES and 0.1 wt% BSA.

3. The method for preparing Artemisia protoplasts according to claim 1, characterized in that, The enzymatic hydrolysis conditions in step S2 are 24-26°C and incubation in the dark for 4-12 hours.

4. The method for preparing Artemisia protoplasts according to claim 1, characterized in that, The centrifugation conditions after enzymatic hydrolysis in step S2 are: centrifuge at 4℃ with a centrifugal force of 100g for 6~10min.

5. The method for preparing Artemisia protoplasts according to claim 1, characterized in that, The W5 salt solution in step S2 comprises the following components: 2 mM MES, 154 mM NaCl, 125 mM CaCl2, and 5 mM KCl.

6. The method for preparing Artemisia protoplasts according to claim 1, characterized in that, In step S2, the obtained resuspension is stacked on a 0.55M sucrose solution and centrifuged at 100g for 6-10 minutes at 4°C.

7. The method for preparing Artemisia protoplasts according to claim 1, characterized in that, The tender leaves of Artemisia annua mentioned in step S1 are obtained by inoculating Artemisia annua stem segments into MS medium and aseptically culturing for 2-3 weeks to obtain robust Artemisia annua leaves.

8. The method for preparing Artemisia protoplasts according to claim 1, characterized in that, The ratio of the mass of the young leaves of Artemisia annua in step S1 to the volume of the enzymatic hydrolysate in step S2 is 1g:20mL.

9. The application of Artemisia protoplasts prepared by the preparation method according to any one of claims 1 to 8 in the subcellular localization of Artemisia asPAL protein in Artemisia.

10. The application according to claim 9, characterized in that, The subcellular localization of the Artemisia argyi AsPAL protein includes the following steps: The nucleotide sequence of the AsPAL gene was cloned into a vector containing a fluorescent protein to construct a recombinant expression vector containing the AsPAL gene. The expression vector was transiently transformed into Artemisia annua protoplasts and cultured at 23-25°C in the dark for 2-6 hours. The subcellular localization of the protein in Artemisia annua protoplasts was then detected under a fluorescence microscope.

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