Preparation and transformation method suitable for protoplasts of different tissues of soybeans

Abstract

The invention discloses a preparation and transformation method suitable for protoplasts of different tissues of soybeans, and belongs to the technical field of biology. The method comprises the following steps: taking leaves, stems or root tissues of earth-cultured yellowing soybean seedlings of 10-12 days, chopping, carrying out enzymolysis in a mixed enzymatic hydrolysate in a dark place, filtering, centrifuging, collecting precipitated cells, and re-suspending the precipitated cells to obtain a protoplast suspension; uniformly mixing the protoplast suspension with plasmids, adding 50% PEG (polyethylene glycol) conversion liquid, and converting at room temperature for 15 minutes. According to the method, by optimizing an enzymolysis system and key parameters of PEG conversion liquid, the method suitable for preparing and converting the soybean multi-tissue and multi-genotype protoplast is obtained, and the problems that in the prior art, in the soybean protoplast preparation and conversion process, material limitation exists, the enzymolysis efficiency is low, and the conversion efficiency is poor are solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a preparation and transformation method suitable for different tissues of soybean protoplasts. BACKGROUND

[0002] Protoplast is a plant cell after removing the cell wall, which is an important tool for studying gene expression, protein localization, gene editing, cell fusion, etc. At present, the preparation of plant protoplast mainly adopts enzymatic method, and how to use cellulase, pectinase and other enzyme solutions to obtain high yield and high activity protoplast is the key technology. PEG-mediated transient transformation has become the mainstream method of protoplast transformation due to its simple operation, low cost and no genotype restriction. Soybean is an important oil and forage crop in the world, and the development of efficient protoplast preparation and transformation method is of great significance for soybean gene function research.

[0003] Invention patent CN201510597136.X (published on March 29, 2019) discloses a method for subcellular localization of soybean leaf protoplast separation, which takes 10-30 day-old Xiangdou No. 3 soybean seedling fully expanded young leaves as materials, cuts the leaves into 0.5-1 mm strips, and then places them in CPW+13% mannitol solution in dark conditions for 2 hours of protoplast separation. Then, it is transferred to an enzyme solution containing CPW+0.1% MES+0.5% cellulase Onozuka R-10+0.8% hemicellulase+0.8% macerozyme R-10+0.4% pectolyase Y-23+10% mannitol (pH 6.0), and then it is enzymolyzed at 27℃ and 45 rpm in the dark for 6 hours. After filtration through a 75 μm nylon mesh and washing with W5 solution, protoplasts with a yield of 1.75×10 7 protoplasts with a yield of 1.75×10

[0004] Invention patent CN201910480976.6 (published on September 17, 2019) takes soybean embryogenic callus or leaves as materials, uses an enzyme solution of 2% cellulase R-10+1% macerozyme R-10+0.1% pectolyase Y-23+MES (pH 5.8), and enzymolyzes at 25-26℃ and 50 rpm in the dark for 5h with a ratio of 0.2 g callus or leaves / 5 mL enzyme solution. After filtration through a 200 mesh cell sieve and centrifugal purification with W5-23% solution, the protoplast density is adjusted to 2.5×10 6afterwards, and the protoplasts were cultured in the dark for 24 hours. This method significantly improved the yield and transformation efficiency of protoplasts compared to the traditional cotyledon method, with a yield of 3.6 x 10 6 However, the embryogenic callus induction was dependent on soybean immature embryos, and the induction time was long and required sterile operation.

[0005] Invention patent CN202510958372.3 (published on October 17, 2025) optimized the preparation and transformation process for soybean stem protoplasts. The first internode stem segment of Williams 82 soybean sterile seedlings (collected when the first pair of trifoliolate leaves emerged) or the stem segment of soil-grown 10-day-old seedlings was used. An enzyme solution containing 1.75% cellulase R-10 + 0.75% macerozyme R-10 + 10 mM MES + 0.6 M mannitol + 0.1% BSA + 1 mM CaCl2+ 5 mM β-mercaptoethanol (pH 5.7) was used for 4 hours of dark enzymolysis at 28°C and 80 rpm (without vacuum filtration). After filtration through a 40 μm cell sieve and washing with W5 buffer, the yield of protoplasts reached 2.1 x 10 6 The transformation was achieved by incubating with 20% PEG4000 (containing 0.6 M mannitol and 1 mM CaCl2) at room temperature for 15-20 minutes. Protein expression could be detected after 12-16 hours of incubation in the dark or weak light at 28°C, but the transformation rate value was not specified.

[0006] Although there are some related technologies for soybean protoplast preparation and transformation, there are obvious defects: (1) strong material and genotype limitations, such as patent CN201510597136.X, which is only suitable for Xiangdou No. 3 leaf, CN202510958372.3 uses Williams 82 stem segments, and CN201910480976.6 requires induction of embryogenic callus, which depends on immature embryos, has a short window period for sampling, and requires long subculture time. (2) Low protoplast yield, such as patent CN201510597136.X, which has a protoplast yield of 1.75 x 10 7 However, there is a contradiction between yield and viability, with a survival rate of only 82.86%; CN201910480976.6 has a protoplast yield of 3.6 x 10 6 CN202510958372.3 has a protoplast yield of 2.1 x 10 6(3) Transformation efficiency is insufficient, for example, the patent CN201510597136.X and CN201910480976.6 do not explicitly indicate the transformation rate value, and the transformation rate of CN201910480976.6 is about 20%. Therefore, it is necessary to provide a method suitable for the preparation of soybean protoplasts and high transformation rate of multiple tissues and multiple genotypes. SUMMARY

[0007] The purpose of the present application is to provide a method suitable for the preparation and transformation of soybean protoplasts of different tissues. Specifically, the present application optimizes the key parameters of the enzyme system and PEG transformation solution to obtain a method suitable for the preparation and transformation of soybean protoplasts of multiple tissues and multiple genotypes, solving the problems of material limitation, low enzyme efficiency and poor transformation efficiency in the prior art.

[0008] To achieve the above-mentioned purpose, the present application provides the following scheme:

[0009] The present application provides a method suitable for the preparation of soybean protoplasts of different tissues, comprising the following steps:

[0010] The leaf, stem or root tissue of yellow soybean seedlings is cut and then enzymolyzed in a mixed enzyme solution in the dark. The precipitated cells are collected after filtration and centrifugation, and the precipitated cells are resuspended to obtain a protoplast suspension. The components of the mixed enzyme solution are 2% cellulase, 0.1% pectinase, 1% macerozyme, 0.6M mannitol, 10mM MES, 0.1% BSA and 3.4mM CaCl2, and ddH2O is used as the solvent.

[0011] Preferably, the mass-volume ratio of the tissue to the mixed enzyme solution is (0.1-0.4) g:5mL, and the enzyme digestion conditions are: first enzyme digestion at room temperature at 60r / min for 3-4 hours, and then enzyme digestion at 90r / min for 5 minutes.

[0012] Preferably, the filtration and centrifugation conditions are: the enzyme solution is filtered with a 70µm filter screen, the filtrate is then centrifuged at 150g for 2 minutes, and the supernatant is discarded to obtain the precipitated cells.

[0013] Preferably, the resuspension is: the precipitated cells are resuspended with W5 solution, then centrifuged at 150g for 2 minutes, the supernatant is discarded, and MMG solution is added to the precipitate to resuspend the cells.

[0014] The components of the W5 solution are: MES 2mM, CaCl2 125mM, NaCl 154mM and KCl 5mM, and ddH2O is used as the solvent.

[0015] The components of the MMG solution are: mannitol 0.4M, MgCl2 15mM and MES 4mM, with ddH2O as the solvent.

[0016] Preferably, the yellow soybean seedlings are soil-cultured yellow soybean seedlings for 10-12 days; the varieties of the yellow soybean seedlings include Zhonghuang 301, Heihe 43 and Chengdou 17. However, the soybean varieties are not limited to the above.

[0017] The application also provides a transformation method of soybean protoplasts, comprising the following steps:

[0018] The protoplast suspension is obtained by the preparation method, and then the protoplast suspension is uniformly mixed with a plasmid and PEG transformation liquid is added for 15min at room temperature; the components of the PEG transformation liquid are: 0.2M mannitol, CaCl2 100mM and PEG4000 50% w / v, with ddH2O as the solvent.

[0019] Preferably, the density of the protoplast suspension is 2×10 6 / mL, and the volume ratio of the protoplast suspension, the plasmid and the PEG transformation liquid is 10:1:11.

[0020] Preferably, after adding the PEG transformation liquid, the following steps are further included: 500μL W5 solution is added to terminate the transformation, centrifugation is performed, the supernatant is discarded, 1mL W5 solution is added to resuspend the cells, centrifugation is performed again, the supernatant is discarded, 500μL W5 solution is added to resuspend the cells, and the culture plate is transferred for overnight culture for 12-18 hours.

[0021] The application discloses the following technical effects:

[0022] (1) The application breaks through the material and genotype limitations, and develops a universal method suitable for soybean multi-organ tissues (roots, stems and leaves) and multiple cultivation varieties (such as Zhonghuang 301, Heihe 43 and Chengdou 17).

[0023] (2) The application improves the protoplast yield and survival rate by optimizing the enzymolysis system.

[0024] (3) The application clarifies the key parameters (such as PEG concentration, incubation time and Ca 2+ concentration) of PEG-mediated transformation, and improves the transformation efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present application, and for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 Soybean protoplasts prepared for enzymatic digestion;

[0027] Figure 2 Soybean protoplasts transformed with GFP plasmid;

[0028] Figure 3 Soybean protoplasts co-expressing GFP and mCherry. DETAILED DESCRIPTION

[0029] Various exemplary embodiments of the present application will now be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of some aspects, characteristics and embodiments of the present application.

[0030] It should be understood that the terms used in the present application merely describe particular embodiments and are not intended to limit the present application. In addition, for the numerical ranges in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or stated range, and any other stated value or stated range within the stated range is also included in the present application. The upper limit and the lower limit of these smaller ranges can be included or excluded independently from the range.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All publications mentioned in the specification are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference in the specification is not intended as an admission that the reference is prior art to the present application.

[0032] Many modifications and variations of the present application described in the specification are possible without departing from the scope or spirit of the present application. Other implementations of the present application will be apparent to those skilled in the art from consideration of the specification and practice of the present application. The specification and examples are illustrative only.

[0033] As used herein, "comprise", "comprising", "have", "having", "include", "including", "contain", "containing", and the like, are open-ended terms that are intended to mean including but not limited to.

[0034] Reagents involved in the following examples:

[0035] (1) Mixed enzyme solution: 2% cellulase, 0.1% pectolyase, 1% macerozyme, 0.6M mannitol, 10mM MES, dissolved in sterile water, pH 5.7, 65°C water bath for 10min, 0.22µm membrane filtration, after cooling to room temperature, add BSA to a final concentration of 0.1%, then add CaCl2 to a final concentration of 3.4mM.

[0036] (2) W5 solution: MES 2mM, CaCl2 125mM, NaCl 154mM, KCl 5mM, dissolved with ddH2O, pH=5.7.

[0037] (3) MMG solution: mannitol 0.4M, MgCl2 15mM, MES 4mM, dissolved with ddH2O, pH=5.7.

[0038] (4) PEG transformation solution: 0.2M mannitol, CaCl2 100mM, PEG4000 50%(w / v), dissolved with ddH2O, 65°C water bath for 10min to completely dissolve.

[0039] Example 1: Preparation and transformation of soybean protoplasts

[0040] 1. Preparation of soybean protoplasts

[0041] (1) Take the leaf, stem or root tissue of soybean (Zhonghuang 301) yellowing seedlings cultured for about 10 days, cut the tissue into 0.5mm thin strips or slices with a blade, and take about 0.3g of each sample;

[0042] (2) Add the cut sample to 5mL of mixed enzyme solution, and enzymatically digest at room temperature at 60r / min for about 4 hours, and then at 90r / min for 5min, avoiding light during the enzymatic digestion;

[0043] (3) Filter the enzyme solution using a 70µm filter screen, and transfer all the filtrate into a centrifuge tube and centrifuge at 150g for 2min;

[0044] (4) Discard the supernatant, resuspend the cells with 5mL of W5 solution, and centrifuge at 150g for 2min;

[0045] (5) Discard the supernatant, resuspend the cells with about 500µL of MMG solution, and use a microscope to check the number and morphology of protoplasts (see Figure 1 ).

[0046] 2. Transformation of soybean protoplasts

[0047] (1) Adjust the density of leaf protoplast suspension to 2 x 10 6 100 μL of MMG suspension cells were taken into a 2 mL round-bottom EP tube;

[0048] (2) Add 10 μL of plasmid to the protoplast cell suspension and mix well. The concentration of the plasmid was 1 μg / μL. The plasmid was a plasmid with 35S-driven green fluorescent protein GFP, referred to as pCAMBIA-GFP; or a plasmid for studying protein subcellular localization, and the empty vector was a plasmid with 35S-driven green fluorescent protein GFP and CmYLCV-driven red fluorescent protein mCherry, referred to as pCAMBIA-GFP-mCherry;

[0049] (3) Add 110 μL of PEG transformation solution to the cell suspension and mix gently. Incubate at room temperature for 15 min;

[0050] (4) Add 500 μL of W5 solution to terminate the transformation, and centrifuge at 150 g for 2 min.

[0051] (5) Remove the supernatant, add 1 mL of W5 solution to resuspend the cells, and centrifuge at 150 g for 2 min;

[0052] (6) Remove the supernatant, add 500 μL of W5 solution to resuspend the cells, and transfer to a 24-well plate for overnight culture for 12-18 h. The next day, observe the transformation efficiency under a microscope (see Figure 2 ) or protein subcellular localization (see Figure 3 ).

[0053] Example 2

[0054] The difference from Example 1 is that the leaves are replaced by stems; the other method steps are the same.

[0055] Example 3

[0056] The difference from Example 1 is that the leaves are replaced by roots; the other method steps are the same.

[0057] Example 4

[0058] The difference from Example 1 is that the soybean variety is replaced by Hehe 43; the other method steps are the same.

[0059] Example 5

[0060] The difference from Example 4 is that the leaves are replaced by stems; the other method steps are the same.

[0061] Example 6

[0062] The difference from Example 4 is that the leaves are replaced by roots; the other method steps are the same.

[0063] Example 7

[0064] The difference from Example 1 is that soybean variety is replaced by Chengdou 17; other method steps are the same.

[0065] Example 8

[0066] The difference from Example 7 is that leaf is replaced by stem; other method steps are the same.

[0067] Example 9

[0068] The difference from Example 7 is that leaf is replaced by root; other method steps are the same.

[0069] Comparative Example 1

[0070] The difference from Example 1 is that 0.1% pectinase is not added in mixed enzymatic hydrolysate, and the concentration of PEG4000 in PEG transformation solution is 30%; other method steps are the same.

[0071] Comparative Example 2

[0072] The difference from Example 1 is that 0.1% pectinase is not added in mixed enzymatic hydrolysate, and the concentration of PEG4000 in PEG transformation solution is 20%; other method steps are the same.

[0073] The results of the number, morphology and transformation rate of protoplast prepared by the above examples and comparative examples are shown in Table 1. The results show that the method of the present application can significantly improve the transformation rate, which can be as high as 31%.

[0074] Table 1

[0075]

[0076] The above examples only describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A method for preparing protoplasts from different tissues of soybean, characterized by, The method comprises the following steps: The leaf, stem or root tissue of the yellow soybean seedling is cut into pieces and then subjected to enzymolysis in a mixed enzyme solution in the dark. The precipitated cells are collected after filtration and centrifugation, and the precipitated cells are resuspended to obtain a protoplast suspension; wherein the components of the mixed enzyme solution are 2% cellulase, 0.1% pectinase, 1% macerozyme, 0.6M mannitol, 10mM MES, 0.1% BSA and 3.4mM CaCl2.

2. The production method according to claim 1, wherein The mass-volume ratio of the tissue to the mixed enzyme solution is (0.1-0.4) g:5mL, and the enzymolysis conditions are: first enzymolysis at room temperature at a speed of 60r / min for 3-4 hours, and then enzymolysis at a speed of 90r / min for 5 minutes.

3. The production method according to claim 1, wherein The filtration and centrifugation conditions are: the enzyme solution is filtered through a 70µm filter screen, and then the filtrate is centrifuged at 150g for 2 minutes, and the supernatant is discarded to obtain the precipitated cells.

4. The production method according to claim 1, wherein The resuspension is: the precipitated cells are resuspended in a W5 solution, and then centrifuged at 150g for 2 minutes, and the supernatant is discarded, and the MMG solution is added to the precipitate to resuspend the cells. The components of the W5 solution are: 2mM MES, 125mM CaCl2, 154mM NaCl and 5mM KCl. The components of the MMG solution are: 0.4M mannitol, 15mM MgCl2 and 4mM MES.

5. The production method according to claim 1, wherein The yellow soybean seedling is a soil-cultivated yellow soybean seedling for 10-12 days; and the varieties of the yellow soybean seedling include Zhonghuang 301, Heihe 43 and Chengdou 17.

6. A method for transforming a soybean protoplast, characterized by, The method comprises the following steps: The protoplast suspension is obtained by the preparation method of claim 1, and then the protoplast suspension is uniformly mixed with a plasmid and 500μL of a W5 solution is added to terminate the transformation, and then centrifuged, and the supernatant is discarded, and 1mL of a W5 solution is added to resuspend the cells, and then centrifuged, and the supernatant is discarded, and 500μL of a W5 solution is added to resuspend the cells, and then transferred to a culture plate for overnight culture for 12-18 hours.

7. The conversion process of claim 6 wherein, The density of the protoplast suspension is 2 x 10 6 The volume ratio of the protoplast suspension, the plasmid and the PEG transformation solution is 10:1:

11.

8. The conversion process of claim 6 wherein, ​

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