Jujube protoplast instantaneous transformation method and application thereof in protein subcellular localization analysis

Abstract

The invention discloses a jujube protoplast instantaneous transformation method and application thereof in protein subcellular localization analysis, and belongs to the technical field of biology. According to the method, tender leaves and pulp calluses of jujube sterile tissue culture seedlings are used as materials to prepare the protoplast, and compared with other existing jujube tree protoplast preparation technologies, the method has the advantages that the jujube pulp calluses are used as materials for preparing the protoplast for the first time; the problems of gene transient transformation taking fruits as research objects and research on protein subcellular localization are solved; the protoplast is prepared by using tender leaves of jujube sterile tissue culture seedlings as materials for the first time, a large amount of high-activity protoplast can be efficiently prepared all year round, instant transformation of genes is facilitated, the transformation rate is high, subcellular localization research of jujube body genes is successfully realized for the first time, and the accuracy of analyzing jujube body protein localization is improved; strong support is provided for research and gene editing of jujube gene functions.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for transient transformation of jujube protoplasts and application thereof in protein subcellular localization analysis. Background Art

[0002] Ziziphus Mill. is the most economically valuable genus in the Rhamnaceae family, and Ziziphus jujuba Mill. is the main cultivated species in the genus. It has important economic, medicinal and ornamental values ​​and plays an important role in the development of my country's agricultural economy. At present, the whole genome of jujube has been sequenced, and it is gradually entering the era of molecular breeding. Therefore, it is urgent to explore a large number of genes related to fruit quality and high resistance and analyze their functions, so as to provide a theoretical basis for jujube breeding.

[0003] Generally, in order to accurately understand the functions of some proteins, in addition to analyzing the protein structure, it is also necessary to understand the subcellular localization of the protein to determine the possible place where it exerts its function. However, at present, the transient transformation verification of jujube functional genes and the study of protein subcellular localization are mainly carried out with model plants such as tobacco as carriers. Although heterologous expression systems can be used for gene function research, due to differences in material background, abnormal results may occur. Therefore, it is urgent to establish an efficient jujube protoplast gene transient expression system, which is of great guiding significance for studying jujube gene functions and gene editing, clarifying the place where genes exert their functions and understanding the mechanism of gene action.

[0004] So far, there are very few literatures on the extraction and cultivation of jujube protoplasts. The reported jujube protoplast preparation uses mature jujube leaves and embryonic suspension cell lines as research materials. However, the characteristics of mature jujube leaves rich in polysaccharides and polyphenols increase the difficulty of protoplast extraction, which seriously limits the quantity and quality of extraction; and the preparation of embryonic suspension cell lines is time-consuming and complicated, which greatly reduces the preparation and transformation efficiency of jujube protoplasts. The obtained protoplasts cannot meet the 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, the present invention provides a method for transient transformation of jujube protoplasts, comprising the following steps:

[0006] (1) preparing jujube sterile seedling leaf and pulp callus protoplasts;

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

[0008] In certain embodiments, step (1) comprises the following steps:

[0009] S1, taking jujube leaves or jujube pulp callus and placing them in an enzyme hydrolyzate to obtain a mixture;

[0010] S2, adding the mixture obtained in step S1 into a conical flask, shaking and enzymolyzing in a dark environment to obtain an enzymolysis product;

[0011] S3, sieving the enzymatic hydrolysis product obtained in step S2, collecting the sieved material to obtain an enzymatically hydrolyzed jujube leaf solution, adding W5 solution, and obtaining jujube leaf protoplasts through centrifugation, washing, and precipitation.

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

[0013] In certain embodiments, the protoplast yield is calculated using a hemocytometer.

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

[0015] In certain embodiments, the enzymatic solution comprises cellulase and maceration enzyme.

[0016] In certain embodiments, when the test material is jujube leaves, the preparation method of the enzymatic hydrolyzate is as follows: add 1.5% cellulase and 0.3% macerate to a 10ml centrifuge tube, make the volume to 8ml with an osmotic pressure stabilizer containing MES and mannitol, mix well and place in a 55°C water bath for 10min, invert and mix 2-3 times during the period, cool to room temperature, add 100μl of 10% BSA and 3.57μl of β-mercaptoethanol, add deionized water to make the volume to 10ml, and filter into a conical flask using a 0.45μm microporous filter membrane.

[0017] In certain embodiments, when the test material is jujube pulp callus, the preparation method of the enzymatic hydrolyzate is as follows: add 1.5% cellulase and 0.3% maceration enzyme to a 50ml centrifuge tube, make the volume to 16ml with an osmotic pressure stabilizer containing MES and mannitol, mix well, and place in a 55°C water bath for 10min, invert and mix 2-3 times during the period, cool to room temperature, add 10% BSA 200μl, 0.1% AMP 10μl and β-mercaptoethanol reducing agent 7.14μl, add deionized water to make the volume to 20ml, and filter into a conical flask using a 0.45μm microporous filter membrane.

[0018] Secondly, the present 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] The present invention uses jujube pulp callus as material to prepare protoplasts for the first time, solving the problem of instantaneous gene transformation and protein subcellular localization research with fruit as the research object; it uses young leaves of jujube sterile tissue culture seedlings for the first time to efficiently prepare a large number of highly active protoplasts throughout the year, and is easy to instantaneously transform genes with a high transformation rate. It successfully realizes the subcellular localization research of jujube genes for the first time, and improves the accuracy of analyzing the localization of jujube proteins. The test shows that the concentration of protoplasts in sterile jujube seedling leaves is 3.85×10 6 protopl·mL -1 The concentration of protoplasts from jujube pulp callus was 0.85×10 6 , the concentration of endotoxin-free plasmid is 1-2 μL·mL -1 , incubated with 40% PEG-4000 for 16 hours, and used the protoplast transformation system to carry out the subcellular localization of GFP, Mcherry fluorescent protein label vectors and DAPI dye in jujube leaves and pulp. The protoplasts obtained can be used to study the transient expression, subcellular localization and gene editing of jujube gene functions, accelerating the research process of jujube functional genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The jujube leaf material of the present invention and the electron microscope image of the leaf protoplast yield and vitality detection, wherein Figure 1 C is the jujube sterile seedling leaf protoplasts extracted by the present invention, the leaf has all its cell walls removed and is in a spherical shape, and the jujube leaf protoplasts extracted by the present invention maintain relative integrity;

[0022] Figure 2 It is the electron microscope picture of the jujube pulp callus material and pulp protoplast yield and vitality detection of the present invention;

[0023] Figure 3 It is a fluorescence micrograph of the jujube leaf protoplasts of the present invention after co-transformation of pCAMBIA2300-GFP and pCAMBIA2300-Mcherry plasmids;

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

[0025] Figure 5 It is a fluorescence micrograph after the jujube pulp callus protoplasts of the present invention are co-transformed with pCAMBIA2300-GFP, pCAMBIA2300-Mcherry plasmids and DAPI dye. DETAILED DESCRIPTION

[0026] The embodiments of the present application will be described in detail below through examples.

[0027] The solution preparation method, storage conditions and usage instructions in the following examples are shown in Table 1 below:

[0028] Table 1 Protoplast extraction solution formula and use

[0029]

[0030]

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

[0032] The method for preparing protoplasts of leaves and pulp callus of jujube sterile seedlings comprises the following steps:

[0033] 1. Material cultivation

[0034] (1) Young leaves of sterile seedlings: Take the sterile jujube seedlings grown in the tissue culture bottle, cut the young leaves, carefully place the cut material on the glass culture dish covered with filter paper with sterile tweezers, cut the leaf edge with a sterile sharp blade, and cut the leaf thread with a width of 0.1-0.5 mm from the middle of the leaf for protoplast isolation ( Figure 1 A).

[0035] (2) Fruit pulp callus: In a clean bench, filter the jujube fruit pulp callus cultured in a conical flask with sterile gauze, filter and wash it with sterile water for 3-5 times, use sterile filter paper to absorb excess water, and gently use tweezers to pick up an appropriate amount of callus for protoplast isolation ( Figure 2 A)

[0036] 2. Protoplast preparation

[0037] S1. Preparation of enzymatic solution:

[0038] When the test material is jujube leaves, the preparation method of the enzymatic hydrolysate is as follows: add 1.5% cellulase and 0.3% macerate to a 10ml centrifuge tube, dilute to 8ml with an osmotic pressure stabilizer containing MES and mannitol, mix well, place in a 55℃ water bath for 10min, invert and mix 2-3 times during the mixing, cool to room temperature, add 100μl of 10% BSA and 3.57μl of β-mercaptoethanol, add deionized water to dilute to 10ml, and filter into a conical flask using a 0.45μm microporous filter membrane;

[0039] When the test material is jujube pulp callus, the preparation method of the enzymatic hydrolyzate is as follows: add 1.5% cellulase and 0.3% maceration enzyme into a 50ml centrifuge tube, make up to 16ml with an osmotic pressure stabilizer containing MES and mannitol, mix well and place in a 55℃ water bath for 10min, invert and mix 2-3 times during the mixing, cool to room temperature, add 10% BSA 200μl, 0.1% AMP 10μl and β-mercaptoethanol reducing agent 7.14μl, add deionized water to make up to 20ml, and filter into a conical flask using a 0.45μm microporous filter membrane.

[0040] S2. Preparation of protoplasts:

[0041] When the test material is jujube leaves, the leaves processed for protoplast isolation are quickly immersed in the prepared 10 ml enzymatic solution, wrapped in tin foil and placed in a 28°C light-proof shaker at a low speed (50 r / min). -1 ) and oscillate the enzymatic hydrolysis for 4 h until the enzymatic hydrolysis mixture turns light green and yellow-white, respectively, and precipitated cells are observed with the naked eye.

[0042] When the test material is jujube pulp callus, the processed pulp material is quickly immersed in the prepared 20 ml enzymatic solution, wrapped in tin foil and vacuum-infiltrated in the dark for 15 min, and then placed in a 28°C dark shaking incubator at a low speed (50 r / min) -1 ) Oscillate the enzymatic hydrolysis for 6 h until the enzymatic hydrolysis mixture turns light yellow and precipitated cells are observed with the naked eye.

[0043] S3. Protoplast enrichment: Filter the enzymatic hydrolysis product with a cell sieve (rinse the cell sieve with 1 ml of W5 solution and discard the waste liquid), gently squeeze the enzymatic hydrolyzate with tweezers or a sterile gun tip to help fully release the protoplasts. At this time, the leaf protoplasts can be seen dripping with a thick green liquid, while the pulp protoplasts can be observed as a yellow-white liquid. Rinse the enzymatic hydrolysis vessel and undigested leaves twice with 5 ml of W5 solution, and collect all the liquid into a 50 ml centrifuge tube. Use a horizontal rotor, centrifuge at room temperature at 150g (100g for pulp callus) for 3 minutes, increase the speed by 3, decrease the speed by 3, and remove the supernatant.

[0044] S4, separation and preparation of protoplasts: After the enzymatic hydrolysis time of two protoplast materials is reached, they are taken out from the shaker steadily, and an equal volume of W5 solution is slowly added along the wall of the centrifuge tube with a pipette to terminate the enzymatic hydrolysis reaction, and the enzymatic hydrolyzate is filtered into a 50mL centrifuge tube with a cell sieve. The filtrate is centrifuged at 23°C and 150g (100g of pulp callus) for 3min, the speed is increased by 3, the speed is decreased by 3, and the supernatant is removed; 1ml of W5 solution is added, the protoplasts are resuspended, and transferred to a 2ml centrifuge tube on ice and kept away from light for 30min. After obvious precipitation appears, the supernatant is removed as much as possible and placed in ice for use. Take the protoplast suspension and drop it on a slide, cover it with an 18mm×18mm cover glass, and use an optical microscope to collect pictures.

[0045] S5. Calculation of protoplast yield and vitality: Use a 0.1 mm blood cell counting plate to measure the protoplast yield, draw the protoplast resuspension solution and drop it into the counting chamber, observe the protoplast morphology and count it under a microscope, repeat 3 times, and follow the principle of "counting the top but not the bottom, counting the left but not the right", respectively count the number of cells in the four corners of the square and the central square, and take the average value. The results are shown in Table 2. Then, use the corresponding volume of MMG solution to resuspend the protoplasts and adjust the protoplast density to 2x10 5 / mL.

[0046] Protoplast yield (protopl·mL -1 ) = the average number of protoplasts in the four corners plus the five squares in the 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 protoplast preparation using mature leaves of jujube outdoors as materials, the protoplasts prepared from young leaves in tissue culture were more in quantity and had complete edges, followed by pulp callus.

[0048] Table 20.1mm blood cell counting plate for protoplast yield

[0049] Material <![CDATA[Yield (protopl·mL -1 )]]> Outdoor mature leaves <![CDATA[1.15×10 4 ]]> Tissue culture seedlings young leaves <![CDATA[3.85×10 6 ]]> Fruit callus <![CDATA[0.85×10 6 ]]>

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

[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 activity of traditional outdoor mature leaves, the protoplast activity prepared from tissue culture young leaves was the highest, followed by pulp callus.

[0053] Table 3 Protoplast activity statistics

[0054]

[0055] Example 2: Transformation of Jujube Protoplasts with Exogenous Plasmids

[0056] The transformation of exogenous plasmids into jujube leaf and pulp callus protoplasts includes the following steps:

[0057] S1, transient expression plasmid pCAMBIA2300-GFP and pCAMBIA2300-Mcherry all contain Kan resistance label, wherein pCAMBIA2300-GFP contains green fluorescent protein (GFP) label, and pCAMBIA2300-Mcherry contains red fluorescent protein (Mcherry) label, after successful conversion, cell can present green fluorescence under 488nm emission light irradiation, present red fluorescence under 610nm emission light irradiation.DAPI dye is a kind of blue fluorescent dye that can penetrate cell membrane, and after double-stranded DNA is combined with dyeing, can be under 454nm excitation light, with fluorescence microscope, cell nucleus is stained blue.In the present invention, DAPI dye and above-mentioned label plasmid are all used to the transient conversion of protoplast, observe conversion effect and conversion efficiency.

[0058] S2. The above plasmids were transformed into DH5α competent cells respectively. After transformation, they were cultured overnight in LB liquid culture medium containing 50 mg / mL kanamycin, and plasmids were extracted using a small plasmid recovery kit without endotoxin.

[0059] S3, transformation of protoplasts: All steps of PEG-mediated plasmid transformation of jujube protoplasts were carried out at 25-28°C. Add 10μl of freshly extracted endotoxin-free pCAMBIA2300-GFP plasmid, pCAMBIA2300-Mcherry plasmid and 1×DAPI dye to a 2ml sterile round-bottom centrifuge tube, then add 100μl of protoplasts adjusted to a good concentration, and flick to mix thoroughly. Add 110μl of PEG solution, flick to mix, avoid bubbles during the process, and incubate at room temperature for 15min. Add 500μl of W5 solution, gently invert the mud, and terminate the transformation. Centrifuge at room temperature for 3min at 150g, increase speed and decrease speed by 3, and remove the supernatant as much as possible without losing protoplasts. Add 500μL of W5 solution to resuspend the protoplasts, centrifuge at room temperature for 3min at 150g, increase speed by 3, decrease speed by 3, and remove the supernatant.

[0060] S4. Protoplast culture: Add 500 μl of W5 solution to culture and suspend the cells, place the centrifuge tube horizontally at room temperature away from light, and incubate for 16 hours.

[0061] S5. Protoplast collection: slowly pick up the centrifuge tube, gently suspend the cells attached to the tube wall with the tip of the pipette, let the tube stand vertically at room temperature for 3 minutes, then centrifuge at 100g for 3 minutes at room temperature, increase the speed by 3, decrease the speed by 3, remove most of the W5 solution, and collect the protoplasts.

[0062] S6. Fluorescence observation: The results of laser confocal microscopy showed that the protoplast cells of jujube leaves and pulp callus were successfully transformed. The green fluorescent protein contained in them showed green excitation light under 488nm emission light, red fluorescence under 610nm emission light, and blue color of the cell nucleus could be observed 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 professionals in the field to implement or use the present invention. It should be understood by those skilled in the art that the present invention is not limited by the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Modifications and changes made by those skilled in the art do not depart from the spirit and scope of the present application, and should be within the scope of protection of the claims attached to the present application.

Claims

1. A method for transient transformation of jujube protoplasts, characterized in that: The following steps are involved: (1) preparing jujube sterile seedling leaf and pulp callus protoplasts; (2) Transformation of jujube protoplasts with exogenous plasmids.

2. The method according to claim 1, characterized in that The step (1) comprises the following steps: S1, taking jujube leaves or jujube pulp callus and placing them in an enzyme hydrolyzate to obtain a mixture; S2, adding the mixture obtained in step S1 into a conical flask, shaking and enzymolyzing in a dark environment to obtain an enzymolysis product; S3, sieving the enzymatic hydrolysis product obtained in step S2, collecting the sieved material to obtain an enzymatically hydrolyzed jujube leaf solution, adding W5 solution, and obtaining jujube leaf protoplasts through centrifugation, washing, and precipitation.

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

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

5. The method according to claim 1, characterized in that The protoplast viability was evaluated by fluorescein diacetate (FDA) staining.

6. The method according to claim 2, characterized in that The enzymatic hydrolysis solution comprises cellulase and maceration enzyme.

7. The method according to claim 2, characterized in that: When the test material is jujube leaves, the preparation method of the enzymatic hydrolyzate is as follows: 1.5% cellulase and 0.3% macerate are added to a 10ml centrifuge tube, the volume is adjusted to 8ml by an osmotic pressure stabilizer containing MES and mannitol, the mixture is mixed and placed in a water bath at 55°C for 10min, during which the mixture is inverted and mixed 2-3 times, 10% BSA 100μl and β-mercaptoethanol 3.57μl are added after cooling to room temperature, deionized water is added to adjust the volume to 10ml, and the mixture is filtered into a conical flask using a 0.45μm microporous filter membrane.

8. The method according to claim 2, characterized in that: When the test material is jujube pulp callus, the preparation method of the enzymatic hydrolyzate is as follows: add 1.5% cellulase and 0.3% maceration enzyme into a 50ml centrifuge tube, make up to 16ml with an osmotic pressure stabilizer containing MES and mannitol, mix well and place in a 55℃ water bath for 10min, invert and mix 2-3 times during the mixing, cool to room temperature, add 10% BSA 200μl, 0.1% AMP 10μl and β-mercaptoethanol reducing agent 7.14μl, add deionized water to make up to 20ml, and filter into a conical flask using a 0.45μm microporous filter membrane.

9. Application of the method for transient transformation of jujube protoplasts as described in any one of claims 1 to 8 in protein subcellular localization analysis.

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