A method for efficiently separating angelica dahurica protoplast

Abstract

The application belongs to the field of cell biology and particularly relates to a high-efficiency separation method of Angelica dahurica protoplasts. The method comprises the following steps: S1, culture of A. dahurica: selecting A. dahurica containing more than 3 basal leaves for dark culture, and the dark culture time is 20-60 days; S2, selection of protoplast material: selecting the yellowing leaf of A. dahurica treated in step S1 as the protoplast material; S3, separation of protoplasts: performing enzymolysis on the yellowing leaf obtained in step S2, wherein the enzymolysis solution used in the enzymolysis contains 1.3-1.7% cellulase, 0.6-0.8% macerozyme and 0.35-0.45 mol / L mannitol. The A. dahurica protoplasts obtained by the separation method provided in the application have the characteristics of high yield, cell integrity, few impurities and high gene transformation rate.

Description

Technical Field

[0001] This invention belongs to the field of cell biology, specifically relating to a method for efficient separation of Angelica dahurica protoplasts. Background Technology

[0002] Protoplasts are the viable parts of plant cells after the cell wall is removed. They include the cell membrane, cytoplasm, and nucleus, and are the smallest surviving unit of a plant cell. Because protoplasts lack the barrier of the cell wall, they can more easily take up exogenous genetic materials such as DNA, viral particles, organelles, and chromosomes. This makes them ideal materials for research in plant physiology, cell biology, and molecular biology. They are mainly used in cell fusion, subcellular localization, protein interactions, protein activity analysis, gene expression regulation, and new variety breeding. To date, the preparation and transformation of protoplasts have been widely used in model plants such as tobacco (Nicotiana tabacum Linn.) and Arabidopsis thaliana, as well as in major crops such as rice (Oryzasativa) and maize (Zea mays).

[0003] Angelica dahurica, a perennial plant of the Apiaceae family, is a traditional Chinese medicine with a long history of use, serving both as food and medicine. It contains various active ingredients such as coumarins, possessing high medicinal value and broad application prospects. With the accumulation of transcriptome sequencing resources, research on the molecular biology, secondary metabolism, and regulation of Angelica dahurica has advanced to a new stage. Currently, research on Angelica dahurica at the cellular and molecular levels is relatively lagging, and Agrobacterium-mediated gene function studies are particularly difficult to implement in Angelica dahurica. This is mainly due to difficulties in genetic transformation, in vitro regeneration, and long lifespan. Furthermore, the low efficiency and long time consumption of genetic transformation severely restrict the development and exploration of Angelica dahurica biological research. Therefore, an efficient, convenient, and comprehensive biotechnology research system is crucial. Transient expression in protoplasts provides a rapid and efficient technique for gene-related research, significantly shortening the culture time of Angelica dahurica and improving genetic transformation efficiency. It also provides a convenient and effective experimental system for studying protein subcellular localization and gene expression regulation. Therefore, the isolation, transformation, and application of protoplasts are of great significance for Angelica dahurica research. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for efficient separation of Angelica dahurica protoplasts.

[0005] The objective of this invention is achieved through the following technical solution: a method for efficient separation of Angelica dahurica protoplasts, comprising the following steps:

[0006] S1. Cultivation of Angelica dahurica: Select Angelica dahurica with more than 3 basal leaves for dark cultivation. The dark cultivation time is 20-60 days, preferably 25-55 days, and more preferably 25-35 days.

[0007] S2. Selection of protoplast material: The etiolated leaves of Angelica dahurica after treatment in step S1 are selected as protoplast material; the etiolated leaves are newly grown etiolated leaves that have emerged from the basal leaves of Angelica dahurica.

[0008] S3. Protoplast isolation: The etiolated leaves obtained in step S2 are subjected to enzymatic hydrolysis, wherein the enzymatic hydrolysis solution contains 1.3-1.7% cellulase, 0.6-0.8% dissociation enzyme and 0.35-45 mol / L mannitol.

[0009] Furthermore, in step S1, the Angelica dahurica contains 3-5 basal leaves.

[0010] Furthermore, in step S1, Angelica dahurica containing 3 or more basal leaves is obtained by culturing Angelica dahurica seeds under light conditions for 20-40 days, preferably 25-35 days.

[0011] Furthermore, in step S3, the enzymatic hydrolysis time is 5 h ± 30 min.

[0012] Furthermore, in step S3, the amount of enzymatic hydrolysate used is 10-80 mL per gram of etiolated leaf.

[0013] The beneficial effects of the present invention are: the Angelica dahurica protoplasts obtained by the separation method provided by the present invention have the characteristics of high yield, intact cells, few impurities, and high gene conversion rate. Attached Figure Description

[0014] Figure 1 Plant materials prepared from Angelica dahurica protoplasts;

[0015] Figure 2 Preparation of protoplasts from etiolated leaves of Angelica dahurica;

[0016] Figure 3 Table 3 shows the protoplast yield and viability of experiment 6 (protoplasts were stained with FDA, and those with GFP fluorescence were viable protoplasts).

[0017] Figure 4 Protoplast yield and viability under different factor treatments;

[0018] Figure 5 The images show fluorescence after protoplasts were transformed into the NAC20-3301 plasmid (protoplasts with GFP fluorescence were successfully transformed).

[0019] Figure 6The expression level of the NAC20 gene after protoplast transformation into the NAC20-3301 plasmid;

[0020] Figure 7 The protoplast cells were from the Angelica dahurica variety Chuanzhi No. 3 (protoplasts were stained with FDA, and those with GFP fluorescence were viable protoplasts).

[0021] Figure 8 The protoplasts were from the BZB003 Angelica dahurica strain (protoplasts were stained with FDA; those with GFP fluorescence were viable protoplasts). Detailed Implementation

[0022] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings, but the scope of protection of the present invention is not limited to the following description.

[0023] 1. Experimental Materials and Methods

[0024] 1.1 Culture of the tested plant materials

[0025] This invention uses the "Chuanzhi No. 2" Angelica dahurica variety as the test material. Its original plant is Angelica dahurica (Fisch. ex Hoffm.) Benth. et Hook. f. var. formosana (Boiss.) Shan et Yuan.

[0026] The growing conditions are as follows: after the emergence of four basal leaves, cultivate in darkness for one month. See also... Figure 1 In the image, the two leaves in the upper left corner of the plant material are distinctly yellow. (Note: Methods such as directly growing seeds in the dark or transferring tissue culture seedlings to a dark environment cannot successfully produce etiolated seedlings of Angelica dahurica.)

[0027] 1.2 Plasmid Vectors

[0028] The plant expression vector used in the experiment was pCAMBIA3301-35S-eGFP-kana.

[0029] The construction method is as follows: the NAC20 gene is constructed into the plant expression vector pCAMBIA3301-35S-eGFP-kana plasmid to form the NAC20-3301 plasmid.

[0030] 1.3 Main Reagents

[0031] The cellulase used was Yakult Cellulase R-10 from Japan, the dissociation enzyme used was Yakult Macerozyme R-10 from Japan, PEG4000 (polyethylene glycol 4000) was purchased from Chengdu Kelong Chemical Co., Ltd., and CaCl2, MgCl2, NaCl, KCl, MES, mannitol, etc. were purchased from Xilong Chemical Co., Ltd.

[0032] 1.4 Preparation of Angelica dahurica leaf mesophyll protoplasts

[0033] Green leaves, yellowed leaves, green stems, yellowed stems, and roots of Angelica dahurica in good growth condition were selected as experimental materials for protoplast extraction.

[0034] 1.5 Preparation conditions of Angelica dahurica leaf mesophyll protoplasts

[0035] Weigh 0.1g of plant tissue and cut it into 0.5-1.0mm fine filaments using the slicing method. Mix the filaments thoroughly with 5mL of enzymatic hydrolysis solution. Vacuum dry using a circulating water pump for 30min, then immediately transfer to darkness for protoplast isolation. After enzymatic hydrolysis, filter through a 70μm cell sieve to remove larger tissue fragments. Transfer the filtrate to a round-bottom centrifuge tube and centrifuge at 100g, 4℃, and a vertical speed of 5 for 2min to collect the protoplasts. Discard the supernatant. Wash the protoplasts three times with 2mL of W5 solution (154mmol / L NaCl + 125mmol / L CaCl2 + 5mmol / L KCl + 5mmol / L glucose + 2mmol / L MES). Centrifuge and discard the supernatant. Resuspend the protoplasts in 1mL of MMG solution (0.4mol / L mannitol, 15mmol / L MgCl2, 4mmol / L MES, pH 5.7) to obtain purified protoplasts.

[0036] To investigate the effects of cellulase content, ionase content, mannitol concentration, and enzymatic hydrolysis time on the yield of protoplasts from Angelica dahurica leaf mesophyll, an orthogonal experimental design was used to investigate these four factors at three levels. The design scheme is shown in Table 1.

[0037] Table 1. Orthogonal experimental factor levels for the preparation of Angelica dahurica protoplasts

[0038]

[0039] 1.6 Protoplast Transformation

[0040] Add 10 μL of purified NAC20-3301 plasmid (1 μg / μL) to a 2 mL centrifuge tube, followed by 100 μL of a plasmid with a concentration of 2 × 10⁻⁶ g / μL. 5 Add protoplasts per mL, mix gently, then add PEG-Ca. 2+Add 110 μL of solution and gently aspirate with a blue pipette tip until well mixed, avoiding air bubbles. Incubate at 23°C in the dark for 20 min. Add 480 μL of W5 solution and gently mix to terminate the transformation. Centrifuge at 100 × g for 2 min at room temperature with a ramp rate of 3 and a deceleration rate of 3. Discard the supernatant. Finally, add 1 mL of W5 solution to resuspend the protoplasm at the bottom of the tube and incubate in the dark at 28°C for 16 h. Observe under a fluorescence microscope afterward.

[0041] 1.7 Yield Measurement

[0042] Protoplasts were counted using a hemocytometer. The collected protoplasts were resuspended in W5 solution, and a small amount was dropped onto a 0.1 mm hemocytometer. The protoplasts were observed under an optical microscope. Protoplasts with intact membrane structures were selected for counting. Each sample was counted in triplicate, and the average value was taken to calculate the protoplast yield.

[0043] Protoplast yield (units / (g·FW)) = Total number of protoplasts in 25 squares / 0.1 × 1000 × 10

[0044] 1.8 Vitality Assay

[0045] Stain with 0.01% fluorescein diacetate (FDA), take a small amount of protoplast resuspension, and record the protoplasts emitting green fluorescence and the total number of protoplasts under a fluorescence microscope. Calculate the average number of protoplasts in three representative fields of view.

[0046] Protoplast viability is expressed as the percentage of viable protoplasts in a field of view relative to the total number of protoplasts in that field of view.

[0047] Protoplast viability (%) = (Number of protoplasts emitting green fluorescence / Total number of protoplasts) × 100

[0048] 2. Results and Analysis

[0049] 2.1 Selection of parts for preparing Angelica dahurica leaf protoplasts

[0050] Experimental results show that protoplasts are extracted from etiolated leaves with the best results, meaning that the cell integrity and cell viability are the highest.

[0051] Table 2 Summary of protoplast yield from different parts

[0052] Green leaves yellowed leaves Green stems yellowed stems Root system Hypocotyl <![CDATA[Protoplast yield (×10 6 cells / mL)]]> 33.47 46.73 30.63 14.26 16.25 4.76

[0053] Protoplasts prepared from etiolated leaves Figure 2 The concentration was 46.73 × 10⁻⁶. 6 per mL.

[0054] 2.2 Screening of preparation conditions for Angelica dahurica leaf mesophyll protoplasts

[0055] 2.2.1 Screening of conditions for the yield of Angelica dahurica leaf mesophyll protoplasts

[0056] A four-factor, three-level orthogonal experiment was used to conduct range analysis on the factors affecting the yield of protoplasts from Angelica dahurica leaf mesophyll. The results showed that the R value of mannitol concentration was the largest, indicating that mannitol concentration had the greatest impact on the separation of protoplasts from Angelica dahurica leaf mesophyll among the four factors. The next largest factors were cellulase concentration and enzymatic hydrolysis time. The R value of dissociative enzyme concentration was the smallest, indicating that dissociative enzyme concentration had little impact on the separation of protoplasts from etiolated seedlings of Angelica dahurica (Table 3).

[0057] By comparing the protoplast yield and activity of different combinations under orthogonal experiments, it was found that the most ideal protoplasts were obtained with a cellulase concentration of 1.5%, a dissociation enzyme concentration of 0.7%, a mannitol concentration of 0.4 mol / L, and an enzymatic hydrolysis time of 5 h.

[0058] Table 3. Range analysis of protoplast yield of Angelica dahurica mesophyll cells obtained under different experimental conditions.

[0059]

[0060] Note: K1, K2 and K3 represent the total protoplast yield at each level of different factors; k1, k2 and k3 represent the average protoplast yield at each level of different factors; R represents the range.

[0061] Table 4. Analysis of variance of protoplast yield of Angelica dahurica mesophyll cells obtained under different experimental conditions.

[0062]

[0063] Analysis of variance (ANOVA) was performed on the protoplast yield of etiolated Angelica dahurica seedlings (Table 4). The results showed that cellulase, mannitol, and enzymatic hydrolysis time all had significant effects on protoplast yield (P<0.05), while the concentration of the dissociative enzyme had no significant effect on protoplast yield. Among the factors, mannitol concentration had the greatest impact on protoplast yield, while the concentration of the dissociative enzyme had the least impact, consistent with the results of the range analysis.

[0064] 2.2.2 Screening of protoplast viability conditions for Angelica dahurica leaf mesophyll

[0065] A four-factor, three-level orthogonal experiment was used to conduct range analysis on the enzyme concentration, mannitol concentration, and enzymatic hydrolysis time that affected the protoplast viability of Angelica dahurica leaf mesophyll. The results showed that the R value of mannitol concentration was the largest, indicating that mannitol concentration had the greatest impact on the separation of Angelica dahurica leaf mesophyll protoplasts among the four factors; followed by enzymatic hydrolysis time and cellulase concentration; the R value of the dissociation enzyme concentration was the smallest, indicating that the dissociation enzyme concentration had little impact on the protoplast viability of Angelica dahurica etiolated seedlings among the four factors (Table 5).

[0066] Table 5. Analysis of the extremely poor viability of protoplasts in Angelica dahurica mesophyll cells obtained under different experimental conditions.

[0067]

[0068] Table 6. Analysis of variance of protoplast viability of Angelica dahurica mesophyll cells obtained under different experimental conditions.

[0069]

[0070] Analysis of variance (ANOVA) was performed on the protoplast viability of etiolated Angelica dahurica seedlings (Table 6). The results showed that cellulase, mannitol, and enzymatic hydrolysis time all had significant effects on protoplast yield (P<0.05), while the concentration of the dissociative enzyme had no significant effect on protoplast yield. Among the factors, mannitol concentration had the greatest impact on protoplast yield, while the concentration of the dissociative enzyme had the least impact, consistent with the results of the range analysis.

[0071] 2.2.3 The statistical analysis of the protoplast cell status of Angelica dahurica leaf mesophyll is as follows:

[0072] The cell states are shown in Table 7 below:

[0073] Table 7. Description of the protoplast cell states of Angelica dahurica mesophyll cells obtained under different experimental conditions.

[0074] Processing Number Cell state description No. 1 The number of cells is relatively small, with fewer intact and broken cells and fewer impurities. No. 2 The cell count is extremely high, with few intact or broken cells and minimal impurities. No. 3 The cell count is low, cell fragmentation is high, and there are many impurities. No. 4 There are many cells, few intact or broken cells, and many impurities. No. 5 The number of cells is relatively small, with few intact or broken cells and moderate impurities. No. 6 The cell count is extremely high, with few intact or broken cells and minimal impurities. No. 7 The number of cells is extremely small, with few intact or broken cells and few impurities. No. 8 The cell count is high, with few intact or broken cells and few impurities. No. 9 No intact cells, only broken cells, and many impurities.

[0075] 2.3 Protoplast Transformation of Angelica dahurica

[0076] The plant expression vector pCAMBIA3301-35S-eGFP-kana plasmid was transformed into Angelica dahurica leaf mesophyll protoplasts using the PEG-Ca2+ mediated method. After culturing at 28℃ for 15 h, the expression of the GFP gene on the pCAMBIA3301 vector was observed using a fluorescence microscope (Olympus CKX53). The transformation efficiency reached 47.37%, and the gene expression level of the transformed protoplasts was almost unaffected.

[0077] 3. Repeatability verification

[0078] The optimal operating method obtained by screening in this invention was repeatedly verified using "Chuanzhi No. 3" and "BZB003". Specifically, the growth conditions were the same as those described in 1.1; the preparation part was: yellowed leaves of Angelica dahurica in good growth condition were selected as experimental material for protoplast extraction; the preparation method was the same as those described in 1.5, with cellulase concentration of 1.5, cleavage enzyme concentration of 0.7%, mannitol concentration of 0.4 mol / L, and enzymatic hydrolysis time of 5 h. The yield and activity of "Chuanzhi No. 3" and "BZB003" were determined as follows: Figure 7 and Figure 8 , Figure 3 , Figure 7 and Figure 8 The yield and viability of mesoprotoplasts are comparable, indicating that the present invention is also applicable to other species of Angelica dahurica.

[0079] The above description is merely a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the concept described herein through the above teachings or related technologies or knowledge. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present invention should be within the protection scope of the appended claims.

Claims

1. A method for efficient separation of Angelica dahurica protoplasts, characterized in that, Includes the following steps: S1. Cultivation of Angelica dahurica: Select Angelica dahurica with more than 3 basal leaves for dark cultivation for 25 to 35 days. S2. Selection of protoplast material: Select the yellowed leaves of Angelica dahurica after treatment in step S1 as protoplast material. S3. Protoplast isolation: The etiolated leaves obtained in step S2 are subjected to enzymatic hydrolysis, wherein the enzymatic hydrolysis solution contains 1.3-1.7% cellulase, 0.6-0.8% dissociation enzyme and 0.35-0.45 mol / L mannitol.

2. The method for efficient separation of Angelica dahurica protoplasts according to claim 1, characterized in that, In step S1, the Angelica dahurica contains 3-5 basal leaves.

3. The method for efficient separation of Angelica dahurica protoplasts according to claim 2, characterized in that, In step S1, Angelica dahurica containing 3 or more basal leaves is obtained by culturing Angelica dahurica seeds under light conditions for 20-40 days.

4. The method for efficient separation of Angelica dahurica protoplasts according to claim 1, characterized in that, In step S3, the enzymatic hydrolysis time is 5 h ± 30 min.

5. The method for efficient separation of Angelica dahurica protoplasts according to claim 1, characterized in that, In step S3, the amount of enzymatic hydrolysate used is 10-80 mL per gram of etiolated leaf.

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