A method for instantaneously transforming jujube protoplasts and application thereof in protein subcellular localization analysis

Abstract

The application discloses a kind of jujube protoplast transient transformation method and its application in protein subcellular localization analysis, and the application belongs to biotechnology field.The present application uses jujube aseptic tissue culture seedling young leaf and pulp callus as material preparation protoplast, compared with other jujube protoplast preparation technology, first use jujube pulp callus as material preparation protoplast, solve the gene transient transformation and in protein subcellular localization research with fruit as research object;First use jujube aseptic tissue culture seedling young leaf as material preparation protoplast, can make a large number of high activity protoplasts year-round, efficiently, and easy to gene transient transformation, conversion rate is high, first successfully realized jujube ontology gene subcellular localization research, improves the accuracy of analysis jujube ontology protein positioning, provides strong support for jujube gene function research and gene editing.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a method for transient transformation of jujube protoplasts and its application in protein subcellular localization analysis. Background Technology

[0002] The genus *Ziziphus* Mill. is one of the most economically valuable genera in the family Rhamnaceae. *Ziziphus jujuba* Mill. is the main cultivated species within this genus, possessing significant economic, medicinal, and ornamental value, and holding an important position in my country's agricultural economic development. Currently, the entire genome of the jujube has been sequenced, and the field is gradually entering the era of molecular breeding. Therefore, there is an urgent need for extensive and in-depth research into genes related to fruit quality and high resistance, and for elucidating their functions, thereby providing a theoretical basis for jujube breeding.

[0003] To accurately understand the function of proteins, in addition to analyzing their structure, it is also necessary to clarify their subcellular localization to determine the possible sites of their function. However, current research on transient transformation verification of jujube functional genes and subcellular localization of proteins mainly uses model plants such as tobacco as vectors. Although heterologous expression systems can be used for gene function studies, abnormal results may occur due to differences in the background of the materials. Therefore, there is an urgent need to establish an efficient transient gene expression system for jujube protoplasts. This is of great guiding significance for studying jujube gene function and gene editing, clarifying the sites of gene function, and understanding the mechanisms of gene action.

[0004] To date, there is very little literature on the extraction and culture of jujube protoplasts. Existing reports on jujube protoplast preparation have used mature jujube leaves and embryogenic suspension cell lines as research materials. However, the high polysaccharide and polyphenol content of mature jujube leaves increases the difficulty of protoplast extraction, severely limiting the quantity and quality of extracted protoplasts. Furthermore, the preparation of embryogenic suspension cell lines is time-consuming and complex, all of which significantly reduce the efficiency of jujube protoplast preparation and transformation. The resulting protoplasts are also unsuitable for subsequent genetic transformation and other gene function studies. Therefore, establishing an efficient jujube protoplast preparation and transformation system is crucial for studying jujube gene function and gene editing. Summary of the Invention

[0005] First, this invention provides a method for transient transformation of jujube protoplasts, comprising the following steps:

[0006] (1) Preparation of callus protoplasts from leaves and pulp of sterile jujube seedlings;

[0007] (2) Transformation of jujube protoplasts with exogenous plasmids.

[0008] In some embodiments, step (1) includes the following steps:

[0009] S1. Take jujube leaf or jujube fruit callus tissue and place it in enzymatic hydrolysis solution to obtain a mixture;

[0010] S2. Add the mixture obtained in step S1 to an Erlenmeyer flask, and perform enzymatic hydrolysis by shaking in the dark to obtain the enzymatic hydrolysis product;

[0011] S3. The enzymatic hydrolysis product obtained in step S2 is sieved, and the sieved material is collected to obtain the enzymatically hydrolyzed jujube leaf solution. Then, W5 solution is added, and after centrifugation, washing, and precipitation, jujube leaf protoplasts are obtained.

[0012] In some embodiments, step (1) further includes: step S4, protoplast yield calculation and protoplast viability assessment.

[0013] In some embodiments, the protoplast yield calculation is performed using a hemocytometer.

[0014] In some embodiments, the protoplast viability is assessed using fluorescein diacetate (FDA) staining.

[0015] In some embodiments, the enzymatic hydrolysate contains cellulase and dissociation enzyme.

[0016] In some embodiments, when the test material is jujube leaves, the preparation method of the enzymatic hydrolysate is as follows: add 1.5% cellulase and 0.3% ionizing enzyme to a 10ml centrifuge tube, bring the volume to 8ml with an osmotic stabilizer containing MES and mannitol, mix well, and incubate in a 55℃ water bath for 10min, inverting and mixing 2-3 times during the incubation period. After cooling to room temperature, add 100μl of 10% BSA and 3.57μl of β-mercaptoethanol, bring the volume to 10ml with deionized water, and filter into an Erlenmeyer flask using a 0.45μm microporous membrane.

[0017] In some embodiments, when the test material is jujube pulp callus, the enzymatic hydrolysate is prepared as follows: 1.5% cellulase and 0.3% ionizing enzyme are added to a 50ml centrifuge tube, and the volume is adjusted to 16ml with an osmotic stabilizer containing MES and mannitol. After mixing, the mixture is incubated in a 55℃ water bath for 10min, inverting and mixing 2-3 times during the incubation period. After cooling to room temperature, 200μl of 10% BSA, 10μl of 0.1% AMP, and 7.14μl of β-mercaptoethanol reducing agent are added. The volume is adjusted to 20ml with deionized water, and the mixture is filtered into an Erlenmeyer flask using a 0.45μm microporous membrane.

[0018] Secondly, this invention provides an application of the above-mentioned method for transient transformation of jujube protoplasts in protein subcellular localization analysis.

[0019] Compared with the prior art, the present invention has at least the following beneficial effects:

[0020] This invention is the first to use jujube fruit callus tissue as material to prepare protoplasts, solving the problems of transient gene transformation and subcellular protein localization research using fruit as the research object. It is also the first to use young leaves from aseptic jujube seedlings to efficiently prepare large quantities of highly viable protoplasts year-round, with easy and high-efficiency transient gene transformation. Furthermore, it successfully achieved subcellular gene localization research on jujube, improving the accuracy of analyzing jujube protein localization. Experiments showed that the protoplast concentration in aseptic jujube seedling leaves was 3.85 × 10⁻⁶. 6 protopl·mL -1 The concentration of protoplasts in the callus tissue of jujube fruit pulp was 0.85 × 10⁻⁶. 6 The concentration of non-endotoxin plasmids was 1-2 μL·mL. -1 The protoplasts were incubated with 40% PEG-4000 for 16 hours, and subcellular localization of GFP, McCormick fluorescent protein tag vectors, and DAPI dye in jujube leaves and pulp was performed using a protoplast transformation system. The obtained protoplasts can be used to study transient expression, subcellular localization, and gene editing of jujube genes, accelerating the research process of jujube functional genes. Attached Figure Description

[0021] Figure 1 These are electron micrographs of jujube leaf materials and leaf protoplast yield and viability detection from the present invention. Figure 1 C represents the jujube aseptic seedling leaf protoplasts extracted in this invention. The leaves have lost all cell walls and are spherical in shape. The extracted jujube leaf protoplasts maintain relative integrity.

[0022] Figure 2 These are electron microscope images of the jujube pulp callus material and the yield and viability of the pulp protoplasts of the present invention.

[0023] Figure 3 This is a fluorescence micrograph of jujube leaf protoplasts co-transformed with pCAMBIA2300-GFP and pCAMBIA2300-Mcherry plasmids according to the present invention.

[0024] Figure 4 This is a fluorescence micrograph of the jujube leaf protoplasts stained with DAPI according to the present invention.

[0025] Figure 5 This is a fluorescence micrograph of the jujube pulp callus protoplasts co-transformed with pCAMBIA2300-GFP, pCAMBIA2300-Mcherry plasmids, and DAPI dye according to the present invention. Detailed Implementation

[0026] The implementation methods of this application will be described in detail below through examples.

[0027] The solution preparation methods, storage conditions, and uses described in the following examples are shown in Table 1 below:

[0028] Table 1. Formulation and uses of protoplast extraction solution

[0029]

[0030]

[0031] Example 1: Preparation of protoplasts from leaf and fruit pulp callus of sterile jujube seedlings

[0032] A method for preparing protoplasts from leaf and fruit pulp callus of sterile jujube seedlings includes the following steps:

[0033] 1. Material cultivation

[0034] (1) Sterile seedling tender leaves: Take sterile jujube seedlings growing in tissue culture bottles, cut tender leaves, carefully place the cut material on a glass culture dish covered with filter paper with sterile forceps, cut off the leaf edge with a sterile sharp blade, and cut leaf filaments 0.1-0.5 mm wide from the middle part of the leaf for protoplast isolation. Figure 1 A).

[0035] (2) Fruit pulp callus: In a clean bench, the jujube fruit pulp callus cultured in suspension in an Erlenmeyer flask was filtered through sterile gauze, rinsed 3-5 times with sterile water, and then the excess water was absorbed with sterile filter paper. A suitable amount of callus was gently picked up with tweezers for protoplast isolation. Figure 2 A)

[0036] 2. Protoplast preparation

[0037] S1. Preparation of enzymatic hydrolysate:

[0038] When the experimental material is jujube leaves, the preparation method of the enzymatic hydrolysate is as follows: Add 1.5% cellulase and 0.3% dissociative enzyme to a 10ml centrifuge tube, and bring the volume to 8ml with an osmotic stabilizer containing MES and mannitol. After mixing, incubate in a 55℃ water bath for 10min, inverting and mixing 2-3 times during the incubation period. After cooling to room temperature, add 100μl of 10% BSA and 3.57μl of β-mercaptoethanol, and bring the volume to 10ml with deionized water. Filter the solution into an Erlenmeyer flask using a 0.45μm microporous membrane.

[0039] When the experimental material is jujube pulp callus, the preparation method of the enzymatic hydrolysate is as follows: Add 1.5% cellulase and 0.3% dissociative enzyme to a 50ml centrifuge tube, and bring the volume to 16ml with an osmotic stabilizer containing MES and mannitol. After mixing, incubate in a 55℃ water bath for 10min, inverting and mixing 2-3 times during the incubation period. After cooling to room temperature, add 200μl of 10% BSA, 10μl of 0.1% AMP and 7.14μl of β-mercaptoethanol reducing agent. Add deionized water to bring the volume to 20ml and filter through a 0.45μm microporous membrane into an Erlenmeyer flask.

[0040] S2. Preparation of protoplasts:

[0041] When the experimental material is jujube leaves, the processed leaf material for protoplast isolation is quickly immersed in 10 ml of prepared enzymatic hydrolysate, wrapped in aluminum foil, and placed in a 28°C, light-protected shaker at a low speed (50 r·min). -1 Shake and hydrolyze for 4 hours until the hydrolysate turns light green and yellowish-white, respectively, and precipitated cells can be observed with the naked eye.

[0042] When the experimental material was jujube pulp callus, the treated pulp material was quickly immersed in 20 ml of prepared enzymatic hydrolysate, wrapped in tin foil, and vacuum-permeated in the dark for 15 min. Then it was placed in a 28℃ light-protected shaker at a low speed (50 r / min). -1 Shake and hydrolyze for 6 hours until the hydrolysate turns pale yellow and precipitated cells can be observed with the naked eye.

[0043] S3. Protoplast Enrichment: Filter the enzymatically digested product through a cell sieve (rinse the sieve with 1 ml of W5 solution and discard the waste liquid). Gently squeeze the digest with tweezers or a sterile pipette tip to help fully release the protoplasts. At this point, leaf protoplasts will show visible dark green liquid dripping down, while fruit pulp protoplasts will show a yellowish-white liquid state. Rinse the digestion vessel and undigested leaves twice with 5 ml of W5 solution, and collect all the liquid in a 50 ml centrifuge tube. Use a horizontal rotor and centrifuge at 150 g (100 g of fruit pulp callus) for 3 min at room temperature, with an ascending speed of 3 and a descending speed of 3. Remove the supernatant.

[0044] S4. Isolation and Preparation of Protoplasts: After the enzymatic hydrolysis time of the two protoplast materials is reached, they are steadily removed from the shaker. An equal volume of W5 solution is slowly added along the wall of the centrifuge tube using a pipette to terminate the enzymatic hydrolysis reaction. The hydrolysate is filtered through a cell sieve into a 50mL centrifuge tube. The filtrate is centrifuged at 23℃ and 150g (100g of fruit pulp callus) for 3 minutes, with an acceleration of 3 and a deceleration of 3. The supernatant is discarded. 1ml of W5 solution is added to resuspend the protoplasts, which are then transferred to a 2ml centrifuge tube and placed on ice in the dark for 30 minutes. After a clear precipitate appears, as much supernatant as possible is aspirated, and the tube is placed on ice for later use. A drop of the protoplast suspension is placed on a glass slide, covered with an 18mm×18mm coverslip, and an image is captured using an optical microscope.

[0045] S5. Protoplast Yield and Viability Calculation: Protoplast yield was measured using a 0.1 mm hemocytometer. Protoplast resuspension was added to the counting chamber, and protoplast morphology was observed and counted under a microscope. This was repeated three times, following the principle of "counting the top but not the bottom, counting the left but not the right." The number of cells at the four vertices and the central square of each square was counted, and the average value was taken. The results are shown in Table 2. The protoplasts were then resuspended in the corresponding volume of MMG solution, and the protoplast density was adjusted to 2 x 10⁻⁶. 5 / mL.

[0046] Protoplast yield (protopl·mL) -1 = Average number of protoplasts in the five square cells (four corners and center) × 25 × 10⁴ × dilution factor.

[0047] The results are shown in Table 2. Figure 1 C and Figure 2 C. The results showed that, compared with the traditional method of preparing protoplasts from mature outdoor jujube leaves, tissue cultured young leaves produced more protoplasts with more intact edges, followed by fruit pulp callus.

[0048] Table 20.1 mm Hemocytometer Statistics on Protoplast Production

[0049] Mature leaves outdoors <![CDATA[1.15×10 4 ]]> Tender leaves of tissue culture seedlings <![CDATA[3.85×10 6 ]]> Fruit pulp callus tissue <![CDATA[0.85×10 6 ]]>

[0050] Protoplast viability was assessed using fluorescein diacetate (FDA) staining. FDA was dissolved in acetone to prepare an FDA solution (5 mg / mL). -1 ). Use 25 μL·mL -1 The FDA-approved protoplasts were stained and cultured in the dark at room temperature for 20 minutes. The stained protoplasts were then observed under a fluorescence microscope. Three regions were randomly selected for observation and photography, with each sampling repeated three times. Fluorescent cells and the total number of cells were counted to determine protoplast viability.

[0051] Protoplast viability (%) = (Number of protoplasts emitting yellow-green fluorescence under dark field / Total number of protoplasts under bright field) × 100%.

[0052] The results are shown in Table 3. Figure 1 B and Figure 2 B. The results showed that, compared with the protoplast viability of traditional outdoor mature jujube leaves, the protoplast viability prepared from tissue-cultured young leaves was the highest, followed by the callus tissue from the fruit pulp.

[0053] Table 3 Protoplast Viability Statistics

[0054]

[0055] Example 2: Transformation of jujube protoplasts with exogenous plasmids

[0056] Transforming jujube leaf and fruit pulp callus protoplasts with exogenous plasmids involves the following steps:

[0057] S1. Both the transient expression plasmids pCAMBIA2300-GFP and pCAMBIA2300-Mcherry contain Kan resistance tags. pCAMBIA2300-GFP contains a green fluorescent protein (GFP) tag, while pCAMBIA2300-Mcherry contains a red fluorescent protein (Mcherry) tag. After successful transformation, cells exhibit green fluorescence under 488nm emission light and red fluorescence under 610nm emission light. DAPI dye, a blue fluorescent dye that can penetrate cell membranes, binds to double-stranded DNA and stains it. Under 454nm excitation light, the cell nucleus is observed to be stained blue using a fluorescence microscope. In this invention, both DAPI dye and the aforementioned tag plasmids were used for transient transformation of protoplasts to observe the transformation effect and efficiency.

[0058] S2. The above plasmids were transformed into DH5α competent cells respectively. After transformation, the cells were cultured overnight in LB liquid medium containing 50 mg / mL kanamycin. Plasmids were extracted using an endotoxin-free plasmid extraction kit.

[0059] S3. Protoplast Transformation: All steps of PEG-mediated plasmid transformation of jujube protoplasts were performed at 25–28°C. In 2 ml sterile round-bottom centrifuge tubes, add 10 μl of freshly extracted endotoxin-free pCAMBIA2300-GFP plasmid, pCAMBIA2300-Mcherry plasmid, and 1×DAPI dye, respectively. Then add 100 μl of protoplasts of adjusted concentration and gently tap to mix thoroughly. Add 110 μl of PEG solution, gently tap to mix, avoiding air bubbles, and incubate at room temperature for 15 min. Add 500 μl of W5 solution, gently invert to mix, and terminate the transformation. Centrifuge at 150g for 3 min at room temperature, increasing and decreasing speed by 3, discarding the supernatant as much as possible without losing protoplasts. Resuspend the protoplasts in 500 μL of W5 solution, centrifuge at 150g for 3 min at room temperature, increasing and decreasing speed by 3, and discard the supernatant.

[0060] S4. Culture of protoplasts: Add 500 μl of W5 solution to culture and suspend cells, place the centrifuge tube horizontally at room temperature in the dark, and incubate for 16 h.

[0061] S5. Collection of protoplasts: Slowly pick up the centrifuge tube and gently suspend the cells attached to the tube wall with the pipette tip. Let the centrifuge tube stand vertically at room temperature for 3 minutes before centrifuging. Centrifuge at 100g for 3 minutes at room temperature, with a speed increase of 3 and a decrease of 3 to remove most of the W5 solution and collect the protoplasts.

[0062] S6. Fluorescence Observation: Laser confocal microscopy revealed successful transformation of protoplast cells in the callus tissue of jujube leaves and pulp. The green fluorescent protein contained within these cells exhibited green excitation light under 488nm emission light, red fluorescence under 610nm emission light, and blue fluorescence was observed in the cell nuclei under 454nm excitation light. Figure 3 , Figure 4 , Figure 5 The conversion efficiency can reach 40%.

[0063] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of this application should be within the protection scope of the appended claims.

Claims

1. A method for transient transformation of jujube protoplasts, characterized in that, Includes the following steps: (1) Preparation of leaf protoplasts and fruit pulp callus protoplasts of sterile jujube seedlings; (2) Transformation of jujube protoplasts with exogenous plasmids; Step (1) includes the following steps: S1. Take jujube leaf and jujube fruit callus tissue respectively and place them in an enzymatic hydrolysate to obtain a mixture; the enzymatic hydrolysate contains cellulase and ionizing enzyme; S2. Add the mixture obtained in step S1 to an Erlenmeyer flask, and perform enzymatic hydrolysis by shaking in the dark to obtain the enzymatic hydrolysis product; S3. The enzymatic hydrolysis product obtained in step S2 is sieved, and the sieved material is collected to obtain the enzymatically hydrolyzed jujube leaf solution. Then, W5 solution is added, and after centrifugation, washing, and precipitation, jujube leaf protoplasts are obtained. When the experimental material is jujube leaves, the preparation method of the enzymatic hydrolysate described in S1 is as follows: Add 1.5% cellulase and 0.3% ionizing enzyme to a 10ml centrifuge tube, bring the volume to 8ml with an osmotic stabilizer containing MES and mannitol, mix well, and incubate at 55℃ for 10min, inverting and mixing 2-3 times during this period. After cooling to room temperature, add 100μl of 10% BSA and 3.57μl of β-mercaptoethanol, bring the volume to 10ml with deionized water, and filter through a 0.45μm microporous membrane into an Erlenmeyer flask; the shaking enzymatic hydrolysis time described in S2 is 4h; the centrifugal force described in S3 is 150g; When the experimental material is jujube pulp callus, the preparation method of the enzymatic hydrolysate in S1 is as follows: add 1.5% cellulase and 0.3% dissociative enzyme to a 50ml centrifuge tube, bring the volume to 16ml with an osmotic stabilizer containing MES and mannitol, mix well, and incubate at 55℃ for 10min, inverting and mixing 2-3 times during the incubation period. After cooling to room temperature, add 200μl of 10% BSA, 10μl of 0.1% AMP, and 7.14μl of β-mercaptoethanol reducing agent. Add deionized water to bring the volume to 20ml and filter through a 0.45μm microporous membrane into an Erlenmeyer flask. After the jujube pulp callus is placed in the enzymatic hydrolysate, it is first subjected to dark vacuum permeation for 15min. The shaking enzymatic hydrolysis time in S2 is 6h. The centrifugal force in S3 is 100g.

2. The method according to claim 1, characterized in that, The step (1) also includes: step S4, protoplast yield calculation and protoplast viability assessment.

3. The method according to claim 2, characterized in that, The protoplast yield was calculated using a hemocytometer.

4. The method according to claim 2, characterized in that, The protoplast viability was assessed using the fluorescein diacetate (FDA) staining method.

5. The application of the method for transient transformation of jujube protoplasts as described in any one of claims 1-4 in protein subcellular localization analysis.

Images