A method for isolating and transiently transforming pepper protoplasts

Abstract

The application discloses a method for separating and transiently transforming pepper protoplasts, comprising the following steps: taking 5th-7th sterile true leaves of a pepper plant for enzymolysis to obtain an enzymolysis mixed solution containing pepper leaf parenchyma cell protoplasts, filtering the enzymolysis mixed solution, centrifuging the filtrate to obtain a protoplast precipitate, resuspending the protoplast precipitate to obtain a pepper protoplast suspension, and using a PEG mediation method to perform transient transformation of an exogenous gene. Experiments prove that the yield of the pepper protoplasts obtained by using the separation method is as high as (11.09-11.79) x 10 6 The exogenous plasmid is transformed into the pepper protoplasts extracted by the method to perform expression, and the highest transient transformation efficiency can reach 64%.

Description

Technical Field

[0001] The invention belongs to the technical field of biological science, and particularly relates to a method for separating and transiently transforming pepper protoplasts. Background Art

[0002] Plant protoplasts are viable, naked cells with cell walls removed and surrounded by a plasma membrane. Large numbers of relatively uniform protoplasts can be isolated from the same plant tissue. Due to their lack of cell walls, protoplasts readily absorb exogenous genetic material. Transformed protoplasts are cultured using solid embedding techniques, resulting in transgenic plants that develop from a single cell, effectively avoiding the formation of chimeras and providing excellent receptor material for plant genetic improvement.

[0003] Plant protoplasts are also ideal for transient expression analysis and have been widely used in research such as gene expression, subcellular localization, promoter activity detection, protein interaction, single-cell sequencing, and gene editing vector screening, providing a convenient and effective analysis system for plant functional genomics. Precisely because they lack the support and protection of a cell wall, protoplast isolation and transformation processes can easily lead to cell rupture and death. Therefore, the use of protoplasts for basic and applied research must be based on the establishment of an effective protoplast isolation and transformation system.

[0004] chili( Capsicum annuum Pepper (Ciprida oleracea) is an important vegetable and industrial raw material crop worldwide. With the completion of the pepper genome sequencing, functional genomic research on pepper has made rapid progress in recent years. Compared with other Solanaceae plants such as tobacco and tomato, pepper protoplast research started later, and preparation conditions are still not perfect. Existing studies have shown that optimal protoplast isolation and transformation conditions vary significantly between different plants and are influenced by multiple factors such as explant type, enzyme type and concentration, enzymatic hydrolysis time, PEG concentration, and transformation time. Therefore, it is particularly important to establish an efficient protoplast isolation and transient transformation system suitable for pepper. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for separating and transiently transforming pepper protoplasts, thereby providing technical support for pepper genetic improvement and basic theoretical research.

[0006] In order to achieve the above-mentioned purpose, the present invention adopts the following technical measures:

[0007] A method for separating and transiently transforming pepper protoplasts comprises the following steps:

[0008] S1: Select mature pepper seeds with full grains and sterilize them in a clean bench. First, soak them in an appropriate amount of 70% ethanol for 30 seconds, quickly rinse them with sterile water once, and then sterilize them with 0.1% mercuric chloride for 6 minutes (shaking continuously during the period to ensure thorough sterilization). Rinse them with sterile water 3-4 times. Pour the sterilized seeds into a sterile Petri dish containing two layers of sterile filter paper and blow dry them for about 5 minutes. Then, inoculate them on GM medium for culture. When the seventh true leaf of the pepper plant is fully expanded, cut the fifth to seventh sterile true leaves as test materials;

[0009] S2: The main veins of the sterile true leaves were removed, and the leaves were cut into thin strips with a width of 0.5 mm. The strips were immersed in an enzymatic hydrolysis solution and enzymatically hydrolyzed at 23°C in the dark for 4 hours to obtain an enzymatic hydrolysis mixture containing pepper mesophyll cell protoplasts; wherein the enzymatic hydrolysis solution formula is: 1.25% cellulase R-10, 0.3% maclotase R-10, 20 mM pH = 5.7 MES buffer, 0.4 M mannitol, 20 mM KCl, 10 mM CaCl2, 5 mM β-mercaptoethanol, and 0.1% BSA;

[0010] S3: filtering and centrifuging the enzymatic hydrolyzed mixture containing pepper mesophyll cell protoplasts to obtain a protoplast precipitate, and resuspending the mixture to obtain a pepper protoplast suspension;

[0011] S4: Transient transformation of exogenous genes using the PEG-mediated method; wherein the transformation system is: 20 μg of plasmid, 100 μL of pepper protoplast suspension, and 110 μL of PEG solution; the transformation conditions are: gently mix the transformation system and incubate at room temperature for 20 minutes; the PEG solution is prepared by weighing 3.5 g of PEG4000, adding 2.5 mL of 0.8 M mannitol and 1 mL of 1 M CaCl2, and adding ultrapure water to a volume of 10 mL.

[0012] The GM culture medium formula is: 2.2g / L MS powder + 15g / L sucrose + 2.7g / L plant gel, pH = 5.72.

[0013] The method for obtaining the pepper protoplast suspension is as follows: the enzymatic hydrolysis mixture containing pepper mesophyll cell protoplasts is filtered through a nylon mesh with a pore size of 75 mm, the filtrate is centrifuged at 100 g for 2 min, the supernatant is removed, the protoplast precipitate is resuspended with W5 solution, and the protoplasts are counted using a hemocytometer under an ordinary optical microscope. The protoplast density is adjusted to 2×10 6 / mL, then placed on ice for 30min, centrifuged at 100g for 2min, removed the supernatant, and resuspended with MMG solution to maintain the protoplast density at 2×10 6 / mL, and a pepper protoplast suspension is obtained; wherein, the nylon mesh needs to be rinsed with W5 solution in advance.

[0014] Among them, the transient transformation of exogenous genes using the PEG-mediated method is specifically as follows: 20 μg of plasmid, 100 μL of pepper protoplast suspension and 110 μL of PEG solution are added to a centrifuge tube, the transformation system is gently mixed and incubated at room temperature for 20 minutes, then 440 μL of W5 solution is added, and the reaction is terminated by gentle inversion. Then, the mixture is centrifuged at 100 g for 2 minutes, the supernatant is removed, 1 mL of WI solution is added to resuspend the precipitate, and the mixture is incubated in the dark at 23°C for 16 hours to complete the transformation.

[0015] Wherein, the plasmid is pBinGFP2 plant expression plasmid.

[0016] The W5 solution formula is: 2mM pH=5.7 MES buffer, 154mM NaCl, 125mM CaCl2, 5mM KCl, and 5mM glucose.

[0017] The MMG solution formula is: 4mM pH=5.7 MES buffer, 0.4M mannitol, and 15mM MgCl2.

[0018] The WI solution formula is: 4mM pH=5.7 MES buffer, 0.5M mannitol, and 20mM KCl.

[0019] The above-mentioned pepper protoplast isolation and transient transformation method is used in the expression of exogenous genes.

[0020] The significant advantages of the present invention are:

[0021] 1) The present invention provides an efficient method for isolating pepper mesophyll protoplasts. Using this method, the yield of pepper protoplasts obtained is about 11×10 6 / g FW, with activity above 85%.

[0022] 2) The present invention provides a method suitable for transient transformation of pepper protoplasts. Using this method, about 64% of the protoplasts can be successfully transformed. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 : The effect of pepper protoplast separation under the 100μm scale in the bright field channel.

[0024] Figure 2 : Effects of PEG4000 concentration and transformation time on transient transformation efficiency of pepper protoplasts.

[0025] Figure 3 : Schematic diagram of transient expression of GFP reporter gene vector in pepper protoplasts under 10 μm scale in the fluorescence channel. DETAILED DESCRIPTION

[0026] In order to make the contents of the present invention easier to understand, the technical solutions of the present invention are further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0027] Example 1: Isolation of pepper protoplasts

[0028] The present invention provides an efficient method for isolating pepper protoplasts, which comprises the following steps:

[0029] 1) Preparation of donor material

[0030] Select mature pepper seeds with full grains, first soak them in an appropriate amount of 70% ethanol for 30 seconds in a clean bench, quickly rinse them with sterile water once, each time for 2 minutes; then soak them in 0.1% mercuric chloride for 6 minutes (shake continuously during the period to ensure thorough sterilization), and then rinse them with sterile water 3-4 times, each time for 2 minutes. After that, pour the sterilized seeds into a sterile culture dish containing 2 layers of sterile filter paper and blow dry them on the clean bench for about 5 minutes; pick the blow-dried seeds and inoculate them into a culture bottle containing GM medium (2.2g / L MS+15g / L sucrose+2.7g / L plant gel; pH=5.72) at 25℃, 70% relative humidity, and 14h light (70μM / m 2 / s) / 10h dark conditions. When the seventh true leaf of the pepper plant is just fully expanded, the 5th to 7th sterile true leaves are cut as test materials. If only for transient expression research, mature pepper seeds can also be directly sown in a soil matrix composed of peat soil and vermiculite in a mass ratio of 2:1. The medium is kept at 25℃, 70% relative humidity, 14h light (70μM / m 2 / s) / 10h dark conditions in the greenhouse. When the seventh true leaf of the pepper plant was just fully unfolded, the fifth to seventh true leaves were cut off and used as test materials.

[0031] 2) Isolation and purification of pepper protoplasts

[0032] Prepare the following reagents in advance:

[0033] Enzyme hydrolysate:

[0034] Enzyme solution A: 1% (wt / vol) Cellulose R10, 0.5% (wt / vol) Macerozyme R10, 20 mM MES buffer (pH = 5.7), 0.4 M mannitol, 20 mM KCl, 10 mM CaCl2, 5 mM β-mercaptoethanol, 0.1% (wt / vol) BSA;

[0035] Enzyme solution B: 1.25% (wt / vol) Cellulose R10, 0.3% (wt / vol) Macerozyme R10, 20 mM MES buffer (pH = 5.7), 0.4 M mannitol, 20 mM KCl, 10 mM CaCl2, 5 mM β-mercaptoethanol, 0.1% (wt / vol) BSA;

[0036] Enzyme solution C: 1.5% (wt / vol) Cellulose R10, 0.4% (wt / vol) Macerozyme R10, 20 mM MES buffer (pH=5.7), 0.4 M mannitol, 20 mM KCl, 1 mM CaCl2, 5 mM β-mercaptoethanol, 0.1% (wt / vol) BSA.

[0037] W5 solution: 2 mM MES buffer (pH = 5.7), 154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 5 mM glucose.

[0038] MMG solution: 4 mM MES buffer (pH=5.7), 0.4 M mannitol, 15 mM MgCl2.

[0039] All the above reagents need to be sterilized by filtration through a 0.22 μm microporous filter membrane.

[0040] Firstly, the 5th-7th sterile true leaves of Capsicum annuum L. cv. CM334 were used as donor materials, and the main veins of the leaves were removed with a sharp blade, then the middle part of the leaves was cut into thin strips with a width of 0.5 mm, 1 g of leaf strips was quickly placed in the above-mentioned different 10 mL enzyme solution to completely immerse, and the enzyme hydrolysis was carried out at 23℃ in the dark for 3 h to obtain the enzyme hydrolysis mixed solution containing Capsicum annuum L. leaf mesophyll cell protoplasts. Then the enzyme hydrolysis mixed solution containing Capsicum annuum L. leaf mesophyll cell protoplasts was filtered with a nylon mesh with a pore size of 75 mm (the nylon mesh needs to be pre-washed with W5 solution), and the filtrate was transferred into a 50 mL round bottom centrifuge tube; 100 g centrifugation for 2 min, the supernatant was discarded; the precipitate (protoplasts) was resuspended with ice bath W5 solution, observed under a microscope, counted with a hemocytometer, and the protoplast density was adjusted to 2×10 6 cells / mL, and placed on ice for 30 min; 100 g centrifugation for 2 min, the supernatant was removed, and the protoplast density was maintained at 2×10 6 cells / mL by resuspending with MMG solution to obtain a Capsicum annuum L. protoplast suspension, and the activity of the protoplasts was determined by using fluorescein diacetate (FDA) method.

[0041] The results show that: under the condition of enzyme solution A, the yield of Capsicum annuum L. leaf protoplasts is about 5.23×10 6 cells / g FW, and the viable protoplasts account for 85.46%; under the condition of enzyme solution B, the yield of protoplasts is significantly improved, reaching 9.72×10 6 cells / g FW, and the viable protoplasts account for 84.38%; under the condition of enzyme solution C, although the yield of Capsicum annuum L. leaf protoplasts is greatly improved, reaching about 17.95×10 6 cells / g FW, but the proportion of viable protoplasts is greatly reduced, accounting for only 63.07% (Table 1). Figure 1

[0042] Table 1 Effect of different enzyme solutions on the separation of Capsicum annuum L. leaf protoplasts.

[0043]

[0044] Note: The same column and different letters represent significant differences between treatments at the 0.01 level; the same below.

[0045] ​Based on the above results, next, a unified enzyme solution (enzyme solution B containing 1.25% Cellulose R10 and 0.3% Macerozyme R10) was set, and then different enzyme hydrolysis times (3h, 4h, 5h and 6h) were compared and analyzed. The results show that: with the extension of enzyme hydrolysis time, the yield of pepper leaf protoplast gradually increases, but the viable protoplast gradually decreases; among them, when the enzyme hydrolysis time is 4h, the yield of protoplast is about 11.11×10 6 6 / g FW, and the viable protoplast is about 85.70% (Table 2). Therefore, by comprehensively considering the yield and activity of protoplast, we think that under the enzyme hydrolysis of enzyme solution B containing 1.25% Cellulose R10 and 0.3% Macerozyme R10, when the enzyme hydrolysis time is 4h, the protoplast separation effect of pepper true leaf is best.

[0046] Table 2 Influence of different enzyme hydrolysis times on the separation effect of pepper leaf protoplast

[0047]

[0048] The present application also selects other 4 different genotypes of pepper varieties, and compares and analyzes their protoplast yield and activity when they are hydrolyzed in enzyme solution B containing 1.25% Cellulose R10 and 0.3% Macerozyme R10 for 4h. The results show that: under the same separation conditions, the protoplast yield and activity of these different genotypes of pepper true leaves do not have significant difference, and the protoplast yield is more than 11×10 6 6 / g FW, and the protoplast activity can reach more than 85% (Table 3).

[0049] Table 3 Influence of different genotypes on the separation effect of pepper true leaf protoplast

[0050]

[0051] Example 2: Transient transformation of pepper protoplast

[0052] The present application provides a method suitable for transient transformation of pepper protoplast, and the specific steps are as follows:

[0053] First, the 5th-7th sterile true leaves of Capsicum annuum L. cv. CM334 were used as donor materials, and the main veins of the leaves were removed with a sharp blade. Then, the middle part of the leaves was cut into thin strips with a width of 0.5 mm, and 1 g of the leaf strips was quickly placed in an enzyme solution B to be completely immersed. Enzymolysis was performed at 23°C in the dark for 4 h to obtain an enzyme solution mixed with Capsicum annuum L. leaf mesophyll cell protoplasts. Then, the enzyme solution mixed with Capsicum annuum L. leaf mesophyll cell protoplasts was filtered through a nylon mesh with a pore size of 75 mm (the nylon mesh was previously rinsed with W5 solution), and the filtrate was transferred into a 50 mL round-bottom centrifuge tube. Centrifugation was performed at 100 g for 2 min, and the supernatant was discarded. The precipitate (protoplasts) was resuspended with an ice bath W5 solution, observed under a microscope, and counted with a hemocytometer. The density of the protoplasts was adjusted to 2×10 6 copies / mL, and the protoplasts were placed on ice for 30 min. Centrifugation was performed at 100 g for 2 min, and the supernatant was removed. The protoplasts were resuspended with an MMG solution to maintain a density of 2×10 6 copies / mL, and a Capsicum annuum L. protoplast suspension was obtained.

[0054] The following reagents were prepared in advance:

[0055] PEG solution:

[0056] PEG solution A (containing 30% PEG4000): 3 g of PEG4000 was accurately weighed, 2.5 mL of 0.8M mannitol and 1 mL of 1M CaCl2 were added, and ultrapure water was added to make up to 10 mL;

[0057] PEG solution B (containing 35% PEG4000): 3.5 g of PEG4000 was accurately weighed, 2.5 mL of 0.8M mannitol and 1 mL of 1M CaCl2 were added, and ultrapure water was added to make up to 10 mL;

[0058] PEG solution C (containing 40% PEG4000): 4 g of PEG4000 was accurately weighed, 2.5 mL of 0.8M mannitol and 1 mL of 1M CaCl2 were added, and ultrapure water was added to make up to 10 mL;

[0059] PEG solution D (containing 45% PEG4000): 4.5 g of PEG4000 was accurately weighed, 2.5 mL of 0.8M mannitol and 1 mL of 1M CaCl2 were added, and ultrapure water was added to make up to 10 mL.

[0060] WI solution: 4mM MES buffer (pH=5.7), 0.5M mannitol, 20mM KCl.

[0061] The above reagents need to be filtered and sterilized by 0.22 μm microporous filter membrane.

[0062] Take 2 mL centrifuge tube, add 20 μg plasmid vector pBinGFP2, 100 μL of the prepared pepper protoplast suspension, 110 μL PEG solution, gently and thoroughly mix, incubate at room temperature according to the set time for transformation. Add 440 μL W5 solution, gently invert and mix to terminate the reaction. 100g centrifugation for 2min, discard the supernatant, add 1mL WI solution to resuspend the precipitate, 23℃ dark culture for 16h, complete the transformation, then use laser confocal microscope to observe green fluorescence.

[0063] The present application first sets up PEG solution containing different concentrations of PEG4000 for transformation, and the transformation time is 15min. The results show that: when the concentration of PEG4000 in the PEG solution is 30%, the transient transformation efficiency of pepper protoplast is about 47%; when the concentration of PEG4000 in the PEG solution is 35%, the transformation efficiency is significantly improved, about 62%; when the concentration of PEG4000 in the PEG solution is further increased, the transformation efficiency decreases accordingly. Figure 2 A).

[0064] Next, the present application uniformly sets the PEG solution B containing 35% PEG4000 for transformation, and then compares and analyzes different transformation times (10min, 15min, 20min and 25min). The results show that: when the transformation time is 20min, the transient transformation efficiency of pepper protoplast is the highest, about 64% of the protoplast can successfully express green fluorescent protein. Figure 2 B and Figure 3 )。

[0065] The above only describes the preferred embodiments of the present application, and any changes and modifications made within the scope of the present application should be included in the scope of the present application.

Claims

1. A method for separating and transiently transforming pepper protoplasts, characterized in that: The following steps are involved: S1: Select mature pepper seeds with full grains, sterilize them in a clean bench, blow dry them on the clean bench, and inoculate them on GM medium for culture. When the seventh true leaf of the pepper plant is fully expanded, cut the fifth to seventh sterile true leaves as test materials; S2: The main veins of the sterile true leaves were removed, and the leaves were cut into thin strips with a width of 0.5 mm. The strips were immersed in an enzymatic hydrolysis solution and enzymatically hydrolyzed at 23°C in the dark for 4 hours to obtain an enzymatic hydrolysis mixture containing pepper mesophyll cell protoplasts; wherein the enzymatic hydrolysis solution formula is: 1.25% cellulase R-10, 0.3% mechanase R-10, 20 mM pH = 5.7 MES buffer, 0.4 M mannitol, 20 mM KCl, 10 mM CaCl2, 5 mM β-mercaptoethanol, and 0.1% BSA; S3: filtering the enzymatic hydrolysis mixture containing pepper mesophyll cell protoplasts, centrifuging the filtrate to obtain a protoplast precipitate, and resuspending the filtrate to obtain a pepper protoplast suspension; S4: Transient transformation of exogenous genes using PEG-mediated method; wherein the transformation system is: 20 μg plasmid, 100 μL pepper protoplast suspension, 110 μL PEG solution; the transformation conditions are: gently mix the transformation system and incubate at room temperature for 20 minutes; the PEG solution is prepared by weighing 3.5 g PEG4000, adding 2.5 ml 0.8 M mannitol and 1 mL 1 M CaCl2, and adding ultrapure water to 10 mL; In step S1, the GM culture medium formula is: 2.2 g / L MS powder + 15 g / L sucrose + 2.7 g / L plant gel, pH = 5.72; In step S3, the protoplast density of the pepper protoplast suspension is 2×10 6 / mL; In step S4, the plasmid is pBinGFP2.

2. The method for separating and transiently transforming pepper protoplasts according to claim 1, wherein: The method for obtaining the pepper protoplast suspension comprises the following steps: filtering the enzymatic hydrolysis mixture containing pepper mesophyll cell protoplasts through a nylon mesh with a pore size of 75 mm, centrifuging the filtrate at 100 g for 2 minutes, removing the supernatant, resuspending the protoplast precipitate with W5 solution, observing and counting the protoplasts with a hemocytometer under an ordinary optical microscope, and adjusting the protoplast density to 2×10 6 After that, the cells were placed on ice for 30 min and centrifuged at 100 g for 2 min. The supernatant was removed and the cells were resuspended in MMG solution to maintain a protoplast density of 2 × 10 6 / mL, and a pepper protoplast suspension is obtained; wherein, the nylon mesh needs to be rinsed with W5 solution in advance.

3. The method for separation and transient transformation of pepper protoplasts according to claim 1, wherein: The transient transformation of exogenous genes using the PEG-mediated method is specifically as follows: 20 μg of plasmid, 100 μL of pepper protoplast suspension and 110 μL of PEG solution are added to a centrifuge tube, the transformation system is gently mixed and incubated at room temperature for 20 minutes, then 440 μL of W5 solution is added, and the reaction is terminated by gentle inversion. Then, the mixture is centrifuged at 100 g for 2 minutes, the supernatant is removed, 1 mL of WI solution is added to resuspend the precipitate, and the mixture is incubated in the dark at 23°C for 16 hours to complete the transformation.

4. The method for separation and transient transformation of pepper protoplasts according to claim 2, wherein: The W5 solution formula is: 2 mM pH=5.7 MES buffer, 154 mM NaCl, 125 mM CaCl2, 5 mM KCl, and 5 mM glucose.

5. The method for separation and transient transformation of pepper protoplasts according to claim 2, wherein: The MMG solution formula is: 4mM pH=5.7 MES buffer, 0.4M mannitol, and 15mM MgCl2.

6. The method for separation and transient transformation of pepper protoplasts according to claim 3, wherein: The WI solution formula is: 4 mM pH=5.7 MES buffer, 0.5 M mannitol, and 20 mM KCl.

7. Use of the method for separation and transient transformation of pepper protoplasts as claimed in claim 1 in exogenous gene expression.

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