Method for high-efficiency separation of musa acuminata callus protoplast and application thereof

Abstract

This invention provides a method for efficient isolation of callus protoplasts from Brazilian bananas and its application, belonging to the field of plant cell biology. The method for isolating Brazilian banana protoplasts involves: cutting unexpanded heart leaves of Brazilian bananas into strips and mixing them with an enzymatic hydrolysate at a ratio of 4-5 g: 15 mL for enzymatic hydrolysis. The enzymatic hydrolysate comprises the following components at the following concentrations: cellulase 1-1.5 w / v%, pectinase 0.2-0.4 w / v%, mannitol 0.35-0.45 M, KCl 18-22 mM, MES 18-22 mM, and CaCl₂. 2 8~12 mM, BSA bovine serum albumin 0.08~0.12%. This invention, by improving the enzymatic hydrolysate formulation, significantly enhanced the viability of protoplasts from the Brazilian banana plantation, achieving a protoplast yield of 3.1 × 10⁻⁶. 6 g ‑1 The viability of the protoplasts prepared by this invention is as high as 92%. These protoplasts can be used for subcellular localization and bimolecular fluorescence complementation experiments, laying a solid foundation for gene function research and molecular breeding work in the Musa genus, and also demonstrating good potential feasibility for their application in plant regeneration.

Description

Technical Field

[0001] This invention relates to the field of plant cell biology, and in particular to a method for efficient isolation of callus protoplasts from Brazilian banana and its application. Background Technology

[0002] Protoplasts are the naked, active structures of plant cells after the cell wall is removed. They can directly take up exogenous DNA, organelles, plasmids, and other substances, making them ideal recipients for plant genetic transformation and somatic cell hybridization. Protoplast fusion technology can effectively overcome breeding challenges such as incompatibility in distant hybridization, asynchronous flowering, and male-female sterility, and has irreplaceable application value in the creation of new species and the utilization of superior wild germplasm resources. Meanwhile, transient expression technology using plant protoplasts does not require the integration of exogenous genes into the cellular genome, and features short operation cycles and high expression efficiency. It is widely used in gene function research fields such as protein subcellular localization, transient gene expression, protein-protein interactions, and promoter activity analysis, and is also a core technology platform for verifying gene editing effects and advancing molecular design breeding.

[0003] Efficient banana protoplast preparation and transformation technology is not only a key platform for elucidating banana gene function, verifying gene editing effects, and conducting molecular design breeding, but it can also overcome problems such as abnormal expression regulation, subcellular localization errors, and protein-protein interaction interference that may exist when studying banana genes in heterologous plant systems. Although there are existing methods for preparing banana protoplasts, the enzyme composition or component ratio of protoplasts from different varieties of Brazilian bananas varies greatly, as do the buffer concentrations and digestion times.

[0004] "Brazilian banana" belongs to the genus *Musa* (Musa L.) of the family Musaceae, and is a perennial evergreen large herbaceous fruit tree. However, systematic research and mature programs in this important crop are still relatively limited. Therefore, establishing a stable and efficient protoplast isolation and transient expression technology system for "Brazilian banana" is of significant theoretical and practical importance for further elucidating the molecular mechanisms of disease resistance and stress tolerance in "Brazilian banana," as well as for accelerating germplasm innovation and variety improvement of "Brazilian banana" using modern biotechnology. Summary of the Invention

[0005] In view of this, the present invention provides a method for efficient separation of callus protoplasts from Brazilian bananas and its application, in order to solve the above problems.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for preparing protoplasts of Brazilian banana, comprising the following steps:

[0008] (1) Take the unopened heart leaves of the Brazilian banana and cut them into strips 0.5-1 mm wide with a knife;

[0009] (2) The leaf strips are mixed with the enzymatic hydrolysate and enzymatically hydrolyzed under light-protected conditions to obtain the hydrolyzed solution;

[0010] (3) Mix the enzymatically hydrolyzed solution with an equal volume of W5 solution, filter and collect the filtrate;

[0011] (4) Centrifuge the filtrate at 90-110g for 1-2 minutes, collect the precipitate, and obtain the final product;

[0012] The enzymatic hydrolysate comprises the following components at the following concentrations: cellulase 1-1.5 w / v, pectinase 0.2-0.4 w / v, mannitol 0.35-0.45 M, KCl 18-22 mM, MES 18-22 mM, CaCl2 8-12 mM, and BSA (bovine serum albumin) 0.08-0.12%.

[0013] Preferably, the mass-to-volume ratio of the leaf strip to the enzymatic hydrolysate is 4-5 g / 15 mL.

[0014] Preferably, the enzymatic hydrolysis temperature is 27~29℃, the rotation speed is 40~60rpm, and the enzymatic hydrolysis time is 4.5~5.5h.

[0015] Preferably, the filtration is carried out in a cell filter screen with a pore size of 35~75μm.

[0016] Preferably, after obtaining protoplasts in step (4), they are washed and resuspended with W5 solution and then centrifuged. The composition of the W5 solution is as follows: NaCl 150~160mM, CaCl2 120~130mM, KCl 3~7mM, glucose 4~6 mM, MES 0.03w / v%.

[0017] Preferably, the centrifugal force is 90-110g and the centrifugation time is 8-10min.

[0018] The present invention also provides a method for transient transformation of the protoplasts of the Brazilian banana, wherein the protoplasts are transformed with a PEG solution containing mannitol, wherein the concentration of mannitol is 0.8~1.2g / 1.2mL.

[0019] The present invention also provides the application of protoplasts obtained according to the preparation method in subcellular localization.

[0020] By adopting the above technical solution, the present invention has the following beneficial effects: The method for extracting protoplasts from Brazilian bananas provided by the present invention has advantages such as short cycle time, high efficiency, good stability and repeatability. The protoplasts separated by this method have a large yield, strong activity, and good integrity, effectively solving the problems of incomplete separation, easy breakage, and low yield of protoplasts derived from callus tissue in existing technologies. Through PEG-Ca... 2+ The method enables transient transformation, achieving a GFP protein transfection efficiency of 35%–48%, which can be used for protein-protein interaction studies. The efficient protoplast isolation and transient transformation system established in this invention not only provides stable and efficient technical support for in-depth analysis of the molecular mechanisms of growth, development, quality formation, and regulation of *Musa brasiliensis*, but also overcomes the shortcomings of traditional homologous genetic transformation, which is characterized by long cycles, low efficiency, and heavy workload. The *Musa brasiliensis* protoplasts prepared by this invention can be used for subcellular localization and bimolecular fluorescence complementation experiments, laying a solid foundation for gene function research and molecular breeding work in *Musa* species, and demonstrating good potential feasibility for application in plant regeneration. Attached Figure Description

[0021] Figure 1 The image shows a protoplast of a Brazilian banana as observed under a microscope.

[0022] Figure 2 Subcellular localization of MaDGAT3 and MaF3'5'H and BiFC interaction results in protoplasts of banana plantations in Brazil. Scale bar is 50 μm. Detailed Implementation

[0023] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0024] In this embodiment of the invention, the cellulase is Cellulase R10 (YaKult Honsha) dry powder, purchased from YaKult Honsha; the pectinase is Mecerozyme R10 (YaKult Honsha) dry powder, purchased from YaKult Honsha; and the mannitol is mannitol dry powder, purchased from Solarbio.

[0025] The preparation method of the enzymatic hydrolysate is as follows: 0.225g cellulase dry powder, 0.045g pectinase dry powder, 1.09g mannitol dry powder, 1mL 0.3M KCl, and 1mL 0.3M MES are added to 10mL distilled water, heated in a water bath at 55℃ for 10min, cooled to room temperature, and then 1mL 0.15M CaCl2 and 1mL 1.5% BSA are added. The final concentrations of each component in the enzymatic hydrolysate are shown in Table 1.

[0026] Table 1. Composition of the enzymatic hydrolysate

[0027] reagents Final concentration 1 Cellulase R10 (YaKult Honsha) 1-1.5﹪ 2 Mecerozyme R10 (YaKult Honsha) pectinase 0.2-0.4﹪ 3 Mannitol 0.4M 4 KCl 20mM 5 MES 20mM 6 <![CDATA[CaCl2]]> 10mM 7 BSA bovine serum albumin 0.1﹪

[0028] PEG4000 solution should be prepared fresh before use. The preparation method of PEG4000 solution is as follows: take 1g of PEG4000, 0.625mL of 0.8M mannitol, 0.25mL of 1M CaCl2 and 0.75mL of ddH2O and mix them evenly.

[0029] W5 solution comprises the following components at the following concentrations: NaCl 154mM, CaCl2 125mM, KCl 5mM, glucose 5mM, MES 0.03 w / v%, adjusted to pH 5.8, sterilized at high temperature, and stored at 4°C.

[0030] The MMG solution comprises the following components at the following concentrations: 15 mM MgCl2, 0.1 w / v% MES, 0.4 M mannitol, adjusted to pH 5.6, and sterilized at high temperature before storage at 4°C.

[0031] Example 1

[0032] In mid-April, unopened heart leaves of 4-leaf-aged Brazilian bananas (Musa acuminata AAA Group cv. 'Baxi') were taken and cut into 0.5-1 mm wide strips. The strips were then immersed in an enzymatic hydrolysate, with 5 g of leaves soaked in 15 mL of the hydrolysate. The mixture was enzymatically hydrolyzed at 28°C and 50 rpm in the dark for 5 hours. After hydrolysis, the solution was diluted with an equal volume of W5 solution, filtered through a 100-mesh sieve, and the filtrate was collected. The filtrate was centrifuged at 100 g for 1.5 min at 4°C to collect protoplasts. The protoplasts were resuspended in W5 solution and incubated on ice for 30 min. They were then centrifuged again at 100 g for 10 min to precipitate the protoplasts, and the filtrate was removed. The protoplasts were resuspended in MMG solution, and the protoplast concentration was adjusted to 2 × 10⁻⁶. 5 The concentration of protoplasts was measured at 1 / mL, yielding a purified protoplast suspension. 40 μL was taken for microscopic examination; the microscopic field of view is as shown in the figure. Figure 1 As shown. The yield of harvested protoplasts reached 3.1 × 10⁻⁶. 6 g -1 FW, with protoplast viability as high as 92%.

[0033] Example 2. Application of protoplasts in subcellular localization

[0034] This example is a bimolecular fluorescence complementation (BiFC) experiment based on the YFP tag, used to verify the direct interaction between MaDGAT3 and MaF3'5'H proteins in cells. The principle of the experiment is as follows: yellow fluorescent protein (YFP) is split into non-fluorescent N-terminal (YFPN) and C-terminal (YFPC) fragments, which are then fused with target proteins for expression. Only when the two target proteins interact will YFPN and YFPC approach each other and reassemble into a complete YFP, thereby emitting yellow fluorescence.

[0035] Take 100 μL of the protoplast suspension prepared in Example 1 (approximately 2 × 10⁻⁶). 4 Gently mix 15 μg of plasmid DNA with 15 μg of the protoplast, add 110 μL of freshly prepared PEG solution, gently tap to mix, and incubate at 25°C in the dark for 25 min. Then terminate the transformation with 400 μL of W5 solution, centrifuge to remove the supernatant, wash once more with W5, resuspend the protoplast in W5 solution, and incubate at 25°C in the dark for 24 h. Observe the fluorescence expression of GFP or YFP tags under a laser confocal microscope.

[0036] In the subcellular localization assay, empty vector GFP was used as a negative control (diffuse cytoplasmic fluorescence), and ER-GFP was used as a positive control (endoplasmic reticulum fluorescence). In the bimolecular fluorescence complementation (BiFC) assay, YFP was split into N-terminal (YFPN) and C-terminal (YFPC) fragments, and expression vectors fusing the target protein (MaDGAT3-YFPN, MaCYP75A1-YFPC) were constructed respectively. Simultaneously, co-transformation with empty vectors pANR-580-nYFP and pANR-580-cYFP served as a blank control. After co-transformation into protoplasts, yellow fluorescence signals were detected to verify the protein-protein interaction between MaDGAT3 and MaCYP75A1 and to exclude false-positive interference caused by YFP fragment self-assembly. The results are as follows: Figure 2 As shown.

[0037] Depend on Figure 2 As shown in the upper part, in the GFP group, green fluorescence is diffused in the cytoplasm and does not co-localize with chloroplasts (red), indicating that GFP alone does not specifically localize to a certain organelle.

[0038] In the ER-GFP group, the green fluorescence exhibits a typical endoplasmic reticulum network structure, which is clearly distinguishable from the ring structure of chloroplasts (red), serving as a positive localization control for the endoplasmic reticulum.

[0039] In the MaDGAT3-GFP group, the green fluorescence is mainly concentrated around or inside the chloroplasts, and highly overlaps with the red chloroplast signal (the Merge plot shows yellow-green), indicating that the protein is located in the chloroplasts.

[0040] In the MaF3'5'H-GFP group, the green fluorescence also showed significant colocalization with the red signal of the chloroplast, indicating that this protein is also located in the chloroplast.

[0041] When MaDGAT3-YFPN and MaF3'5'H-YFPC are co-expressed, clear yellow fluorescence can be detected in the cells, and this signal is completely colocalized with the autoradiographic red fluorescence of chloroplasts (appearing orange-yellow in the Merge plot), indicating that there is a protein-protein interaction between MaDGAT3 and MaF3'5'H in chloroplasts.

[0042] Depend on Figure 2 The lower part shows that when MaDGAT3-YFPN and MaF3'5'H-YFPC are co-expressed, obvious yellow fluorescence appears in the cells, and the fluorescence signal co-localizes with the red signal of chloroplasts (the Merge plot shows orange-yellow). This result proves that there is a protein-protein interaction between MaDGAT3 and MaF3'5'H in chloroplasts.

[0043] As can be seen from the above embodiments, the present invention provides a method for efficient isolation of callus protoplasts from Brazilian bananas and its application. The Brazilian banana protoplasts prepared by the present invention have a large yield and high viability, and can be used for subcellular localization studies.

[0044] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing protoplasts of the Brazilian banana, characterized in that, Includes the following steps: (1) Take the unopened heart leaves of the Brazilian banana and cut them into strips 0.5-1mm wide with a knife; (2) The leaf strips are mixed with the enzymatic hydrolysate and enzymatically hydrolyzed under light-protected conditions to obtain the hydrolyzed solution; (3) Mix the enzymatically hydrolyzed solution with an equal volume of W5 solution, filter and collect the filtrate; (4) Centrifuge the filtrate at 90-110g for 1-2 minutes, collect the precipitate, and obtain the final product; The enzymatic hydrolysate comprises the following components at the following concentrations: cellulase 1-1.5 w / v, pectinase 0.2-0.4 w / v, mannitol 0.35-0.45 M, KCl 18-22 mM, MES 18-22 mM, CaCl2 8-12 mM, and BSA (bovine serum albumin) 0.08-0.12%.

2. The preparation method according to claim 1, characterized in that, The mass-to-volume ratio of the leaf strips to the enzymatic hydrolysate is 4-5 g: 15 mL.

3. The preparation method according to claim 1, characterized in that, The enzymatic hydrolysis temperature is 27~29℃, the rotation speed is 40~60rpm, and the enzymatic hydrolysis time is 4.5~5.5h.

4. The preparation method according to claim 1, characterized in that, The filtration is carried out in a cell filter screen with a pore size of 35~75μm.

5. The preparation method according to claim 1, characterized in that, After obtaining protoplasts in step (4), wash and resuspend them with W5 solution, and then centrifuge. The composition of the W5 solution is as follows: NaCl 150~160mM, CaCl2 120~130mM, KCl 3~7mM, glucose 4~6 mM, MES 0.03 w / v%.

6. The preparation method according to claim 5, characterized in that, The centrifugal force is 90-110g, and the centrifugation time is 8-10min.

7. The method for transient transformation of protoplasts of the Brazilian banana according to claims 1-6, characterized in that, It is converted by a PEG solution containing mannitol, wherein the concentration of mannitol is 0.8~1.2 g / 1.2 mL.

8. The application of protoplasts obtained by the preparation method according to claims 1 to 6 in subcellular localization.

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