Efficient and rapid pepper protoplast transformation method

Abstract

The invention provides an efficient and rapid pepper protoplast transformation method. According to the method disclosed by the invention, the agrobacterium containing the target vector is utilized to infect the pepper leaves, the lower epidermis of the leaves is torn off after the target gene is successfully expressed, and then the transformed protoplast is obtained by an enzymolysis method, so that a PEG (Polyethylene Glycol) transformation step is omitted, and a plurality of unstable factors in the protoplast transformation process are avoided; and the influence of mechanical damage on the protoplast is also avoided. Compared with the prior art, the method is low in cost, convenient and fast, and the obtained protoplast is good in quality and high in conversion efficiency.

Description

Technical Field

[0001] The invention belongs to the field of biological science and technology, and particularly relates to an efficient and fast pepper protoplast transformation method. Background Art

[0002] Pepper (Capsicumannuum L.) is a widely grown vegetable and condiment crop in the world, with important nutritional and economic value. In recent years, my country's pepper industry has developed rapidly, and its planting area has remained stable at 2×10 6 hm 2 The above accounts for 8% to 10% of the total vegetable planting area in the country, and its planting area and output value benefits rank first among vegetables.

[0003] Plant protoplasts are living naked cells that have been stripped of their cell walls and are wrapped by plasma membranes. Protoplast transient expression detection is a rapid, high-throughput analysis system that is widely used in basic theoretical research on subcellular localization, protein interaction, gene expression and regulation, transmembrane transport of substances, signal transduction, cell wall regeneration, etc. In addition, due to the lack of cell walls, protoplasts can easily absorb exogenous genetic material, and under certain conditions, they can regenerate cell walls and redevelop into complete plants. These characteristics make plant protoplasts a good experimental material for biological breeding research.

[0004] Enzymatic separation is currently a common method for preparing protoplasts of higher plants. Usually, the plant leaves are cut into thin filaments and then enzymatically hydrolyzed. After a sufficient number of protoplasts are obtained, the exogenous gene is transformed using polyethylene glycol (PEG)-mediated transformation and other methods. For example, the Chinese invention patent with publication number CN 115896000A published on April 4, 2023, discloses a method for separating and transiently transforming pepper protoplasts: the 5th to 7th true leaves of the pepper plant are enzymatically hydrolyzed to obtain an enzymatic hydrolysis mixture containing pepper mesophyll cell protoplasts, the enzymatic hydrolysis mixture is filtered, and the filtrate is centrifuged to obtain a protoplast precipitate, which is resuspended to obtain a pepper protoplast suspension, and then the exogenous gene is transiently transformed using the PEG-mediated method. Experiments have shown that the yield of pepper protoplasts obtained using the separation method of the invention is as high as 11.09 to 11.79×10 6 / gFW, the highest instantaneous conversion efficiency reached 64%. However, the leaves in this invention are cut into filaments, which are easily mechanically damaged, and then produce a large amount of cell fragments, which is not conducive to the subsequent experiments of protoplasts. In addition, the protoplast transformation efficiency in the PEG-mediated method is affected by many factors, such as PEG concentration, plasmid dosage, protoplast viability, transformation time, etc., which leads to unstable transformation effect. Therefore, it is very important to establish a complete set of efficient and simple pepper protoplast separation and transformation methods. Summary of the invention

[0005] The invention aims to provide an efficient and fast pepper protoplast transformation method, which comprises the following steps: firstly, pepper leaves are infected with Agrobacterium containing a target vector, and when the target gene is successfully expressed, the lower epidermis of the leaves is torn off, and then the transformed protoplasts are obtained by enzymatic hydrolysis, thereby eliminating the step of PEG transformation, avoiding many unstable factors in the protoplast transformation process, and avoiding the influence of mechanical damage on the protoplasts.

[0006] The specific technical solutions of the present invention are as follows:

[0007] An efficient and fast method for transforming pepper protoplasts comprises the following steps:

[0008] 1) Injecting the Agrobacterium infection solution carrying the target vector into pepper leaves, culturing them in the dark, and then moving them to a greenhouse for culturing;

[0009] 2) Step 1) The upper epidermis of the cultured pepper leaf is pressed against the tape, and after pressing, the lower epidermis is forcefully torn off along the main vein, and the upper epidermis is adhered to the tape; the side of the tape with the leaf exposed to the mesophyll cells is immersed in an enzymatic solution, and after enzymatic hydrolysis, it is filtered, the filtrate is centrifuged, the supernatant is discarded, and the cells are resuspended in W5 solution.

[0010] In step 1), the Agrobacterium infection solution carrying the target vector is injected into the pepper leaves using a syringe with the needle tip removed, and the injection site is marked;

[0011] In step 1), the OD of the Agrobacterium infection solution carrying the target vector is 600 The value is 0.6~0.8.

[0012] In step 1), the Agrobacterium carrying the target vector refers to Agrobacterium carrying the pBinGFP3 vector;

[0013] In step 1), the injection volume of the Agrobacterium infection solution carrying the target vector is full leaf injection, generally 2 to 3 mL per leaf according to the leaf size;

[0014] In step 1), the culturing under dark conditions refers to: culturing the injected pepper plants in the dark at 25° C. for 2 h;

[0015] The greenhouse cultivation in step 1) means that the pepper plants are moved to a greenhouse at 25° C. and 14 h of light / 10 h of darkness and cultured for 2 days.

[0016] In step 1), the preparation method of the Agrobacterium infection solution carrying the target vector is as follows: take the Agrobacterium carrying the target vector, perform streaking activation, and culture in the dark at 28°C for 2 days; the infection solution (prepared and used immediately): 10mM 2-(N-morpholino)ethanesulfonic acid (MES), 10mM MgCl2, 200μmol / L acetosyringone (AS), and adjust the pH to 5.0-5.4 with NaOH; resuspend the Agrobacterium cells with the infection solution, centrifuge at 6000rmp for 10 minutes to precipitate the cells, remove the supernatant, add an appropriate amount of infection solution to each tube to resuspend the Agrobacterium again, and dilute to OD 600 The value is 0.6~0.8.

[0017] In step 1), mature pepper seeds with full seeds are selected, soaked in 70% ethanol for 30 seconds in a clean bench, and quickly rinsed with sterile water once; then disinfected with 0.1% mercuric chloride for 6 minutes, and constantly shaken during the period to ensure thorough sterilization; rinsed with sterile water 3 to 4 times; then the sterilized seeds are poured into a sterile culture dish containing 2 layers of filter paper and blown dry for 5 minutes, and the seeds are picked and inoculated into a culture bottle containing GM culture medium, and cultured at 25°C, 70% relative humidity, 14h light / 10h dark conditions. When the fourth true leaf of the pepper plant is just fully unfolded, the first to second sterile true leaves are used as test materials; the formula containing GM culture medium is: 2.2g / L MS powder, 15g / L sucrose and 2.7g / L plant gel (Phytagel), pH 5.72;

[0018] Alternatively, in step 1), the method for obtaining pepper leaves is: sowing mature pepper seeds in soil, growing in a greenhouse under 25°C, 70% relative humidity, 14h light / 10h dark conditions, and when the fourth true leaf of the pepper plant is just fully unfolded, the first to second true leaves are used as test materials. The soil is a mixture of peat soil and vermiculite in a volume ratio of 3:1;

[0019] In step 2), the enzymatic hydrolysis conditions are: 25° C. for 3 to 4 hours.

[0020] In step 2), the filtration is performed by filtration through a 75 μm nylon mesh rinsed with W5 solution;

[0021] In step 2), the enzymatic solution formula used is: 1.25% (wt / vol) cellulase R10, 0.3% (wt / vol) macrozyme R10, 20mM 2-(N-morpholino)ethanesulfonic acid (MES) (pH 5.7), 0.4M mannitol, 20mM KCl, heated at 55°C for 10min, cooled to room temperature, then added with 10mM CaCl2, 5mM β-mercaptoethanol, 0.1% bovine serum albumin (BSA), and made up to 10mL with ultrapure water; wherein wt / vol, wt is in g, and vol is in mL;

[0022] In step 2), the W5 solution used is: 2 mM 2-(N-morpholine)ethanesulfonic acid (MES) (pH 5.7), 154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 5 mM glucose, and the solvent is ultrapure water.

[0023] All the above reagents need to be sterilized by filtration through a 0.22 μm microporous filter membrane.

[0024] Compared with the prior art, the present invention first infects pepper leaves with Agrobacterium containing a target vector, and then tears off the lower epidermis of the leaves after the target gene is successfully expressed, and then obtains the transformed protoplasts by enzymatic hydrolysis, thereby eliminating the step of PEG transformation, avoiding many unstable factors in the protoplast transformation process, and also avoiding the influence of mechanical damage on the protoplasts. The method of the present invention is low in cost, convenient and fast, obtains good quality protoplasts, and has high transformation efficiency, providing technical support for pepper genetic improvement and basic theoretical research. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Figure 2 shows transient transformation and protoplast preparation of pepper leaves; Figure 1 Middle A: When the fourth true leaf of the pepper plant was fully expanded, the Agrobacterium infection solution carrying the pBinGFP3 vector was injected into the first to second growing true leaves. The leaves continued to grow for 2 days after injection. Figure 1 Middle B: Under ultraviolet light, leaves that were not injected with Agrobacterium appear red and do not express green fluorescence; Figure 1 Middle C: Leaves injected with Agrobacterium carrying the pBinGFP3 vector showed green fluorescence under ultraviolet light after 2 days of cultivation; Figure 1 Middle D: Preparation of pepper protoplasts using the chopping method; Figure 1 Middle E: The preparation of pepper protoplasts by peeling the epidermis;

[0026] Figure 2 For microscopic observation of protoplasts obtained by the mincing method and the epidermal tearing method; Figure 2 Middle A: Protoplasts obtained by mincing method and counting using a hemocytometer; Figure 2 Middle B: Protoplasts obtained by the epidermal tearing method were counted using a blood cell counting chamber, the scale bar is 100 μm;

[0027] Figure 3 To observe the activity of protoplasts after fluorescein diacetate (FDA) staining under a fluorescence microscope; Figure 3 Middle A: protoplasts obtained by mincing method; Figure 3 Middle B: protoplasts obtained by the epidermis tearing method; Fluorescence: fluorescence signal image; Bright field: bright field image; scale bar is 50μm;

[0028] Figure 4 Comparison of protoplast yield and activity obtained by two different methods; Figure 4 Middle A: Comparison of protoplast yields obtained by the mincing method and the epidermis tearing method; Figure 4 Middle B: Comparison of protoplast activity obtained by the mincing method and the epidermis tearing method;

[0029] Figure 5 This is a diagram of the instantaneous transformation effect of pepper protoplasts; Figure 5 Middle A: Protoplasts obtained by the mincing method were observed using a fluorescence microscope; Figure 4 Middle B: Protoplasts obtained by the epidermis tearing method were observed using a fluorescence microscope; GFP: green fluorescent protein signal image; Chlorophyll: chloroplast autofluorescence signal image; Merged: merged image; Bright field: bright field image; scale bar is 50μm. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0032] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0033] Example 1

[0034] An efficient and fast method for transforming pepper protoplasts comprises the following steps:

[0035] 1) Preparation of donor materials:

[0036] The mature seeds of pepper were sown in the soil with a volume ratio of peat soil and vermiculite of 3:1, and grown in a greenhouse at 25°C, 70% relative humidity, 14h light / 10h dark conditions. When the fourth true leaf of the pepper plant was fully unfolded, the first to second true leaves were used as test materials ( Figure 1 (A).

[0037] 2) Agrobacterium-mediated transient expression:

[0038] 2-1) Streak the Agrobacterium carrying the pBinGFP3 vector and culture it in the dark at 28°C for 2 days. Prepare the infection solution (prepared and used immediately): 10mM 2-(N-morpholino)ethanesulfonic acid (MES), 10mM MgCl2, 200μmol / L acetosyringone (AS), and adjust the pH to 5.2 with NaOH. Resuspend the Agrobacterium carrying the pBinGFP3 vector with the infection solution, centrifuge at 6000rmp for 10 minutes to precipitate the bacteria, remove the supernatant, add an appropriate amount of infection solution to each tube and resuspend again, and calculate the OD 600 The value was adjusted to 0.8 to obtain the Agrobacterium infection solution carrying the pBinGFP3 vector.

[0039] 2-2) Use the needle tip of a 1mL syringe to gently make a small cut on the back of the first or second true leaf of the growing pepper, then use the syringe without the needle to absorb the infection suspension prepared in step 2-1), slowly push the syringe piston to inject the infection suspension into the leaves, inject 2-3mL into each leaf, and inject all the leaves. Continue to culture the pepper plants in the dark at 25℃ for 2h, then move to a greenhouse at 25℃ with 14h light / 10h dark and culture for 2 days. Under ultraviolet light, the uninjected pepper leaves turn red ( Figure 1 B), pepper leaves injected with Agrobacterium carrying the target vector pBinGFP3 showed green fluorescence ( Figure 1 Middle C).

[0040] 3) Protoplast preparation

[0041] 3-1) Prepare the following reagents in advance:

[0042] Enzyme hydrolysate: 1.25% (wt / vol) Cellulose R10, 0.3% (wt / vol) Macerozyme R10, 20mM 2-(N-morpholino)ethanesulfonic acid (MES) (pH 5.7), 0.4M mannitol, 20mM KCl, heated at 55°C for 10min, cooled to room temperature, then added with 10mM CaCl2, 5mM β-mercaptoethanol, 0.1% (wt / vol) bovine serum albumin (BSA), and made up to 10mL with ultrapure water.

[0043] W5 solution: 2 mM 2-(N-morpholino)ethanesulfonic acid (MES) (pH 5.7), 154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 5 mM glucose, the solvent is ultrapure water.

[0044] All the above reagents need to be sterilized by filtration through a 0.22 μm microporous filter membrane.

[0045] 3-2) Two different protoplast preparation methods

[0046] Chopping method: After cutting the pepper leaves, use a sharp blade to cut the leaves into about 1mm thin wires and soak them in enzyme solution ( Figure 1 (middle D).

[0047] Peeling method: Cut the pepper leaves, press the upper epidermis of the leaves against the tape, press gently, and use tweezers to tear off the lower epidermis along the main vein. The upper epidermis sticks to the tape. Cut the tape with the leaves into about 1cm 2 size, and the side with the mesophyll cells exposed was immersed in the enzymatic solution ( Figure 1 Middle E).

[0048] The two culture dishes were placed at 25°C for 4 hours of enzymatic hydrolysis. Afterwards, the cells were filtered through a 75 μm nylon mesh rinsed with W5 solution in advance, and the filtrate was gently transferred along the tube wall into a 50 mL round-bottom centrifuge tube; centrifuged at 100 g for 5 minutes, the supernatant was discarded, and the cells were resuspended in ice-bathed W5 solution, observed under a microscope, and counted with a hemocytometer. The results showed that the protoplast yield obtained by the mincing method was 9.22 ± 0.38 × 10 6 pcs / g FW( Figure 2 Middle A, scale bar, 100 μm; Figure 4 In A), the protoplast yield obtained by the epidermis peeling method was 14.70±0.24×10 6 pcs / g FW( Figure 2 Middle B, scale bar, 100 μm; Figure 4 (A).

[0049] 3-3) Protoplast activity analysis

[0050] The protoplasts obtained in step 3-2) were stained with 0.01% fluorescein diacetate (FDA) dye at 25°C for 10 minutes and observed under a fluorescence microscope. The activity of the protoplasts was statistically analyzed. The results showed that the activity of the protoplasts obtained by the mincing method was 73.95±2.08% ( Figure 3 Middle A, scale bar, 50 μm; Figure 4 B), while the protoplast activity obtained by peeling the epidermis was 88.39±2.69% ( Figure 3 Middle B, scale bar, 50 μm; Figure 4 (B) The preparation method of the fluorescein diacetate (FDA) dye solution used is as follows: 1 mg of FDA powder is dissolved in 1 mL of acetone as a mother solution, and 0.1 mL of the mother solution is added to 10 mL of freshly prepared 0.6 mol / L mannitol.

[0051] 3-4) Analysis of transformation efficiency of protoplasts

[0052] At the same time, the protoplasts obtained in step 3-2) were observed and statistically analyzed using a fluorescence microscope. The results showed that the transformation efficiency of the protoplasts obtained by the mincing method was 68.38±3.16% ( Figure 5 A, scale bar is 50 μm), while the protoplast transformation efficiency obtained by the epidermis peeling method was 75.33±1.85% ( Figure 5 In B, the scale bar is 50 μm).

[0053] The description of the above embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. An efficient and fast method for transforming pepper protoplasts, characterized in that: The efficient and fast pepper protoplast transformation method comprises the following steps: 1) Injecting the Agrobacterium infection solution carrying the target vector into pepper leaves, culturing them in the dark, and then moving them to a greenhouse for culturing; 2) Step 1) The upper epidermis of the cultured pepper leaf is pressed against the tape, and after pressing, the lower epidermis is forcefully torn off along the main vein, and the upper epidermis is adhered to the tape; the side of the tape with the leaf exposed to the mesophyll cells is immersed in an enzymatic solution, and after enzymatic hydrolysis, it is filtered, the filtrate is centrifuged, the supernatant is discarded, and the cells are resuspended in W5 solution.

2. The efficient and fast pepper protoplast transformation method according to claim 1, characterized in that: In step 1), the OD of the Agrobacterium infection solution carrying the target vector is 600 The value is 0.6~0.

8.

3. The efficient and fast pepper protoplast transformation method according to claim 1 or 2, characterized in that: In step 1), the Agrobacterium carrying the target vector refers to Agrobacterium carrying the pBinGFP3 vector.

4. The efficient and fast pepper protoplast transformation method according to claim 1, characterized in that: In step 1), the culturing under dark conditions refers to culturing the pepper plants in the dark at 25° C. for 2 hours.

5. The efficient and fast pepper protoplast transformation method according to claim 1 is characterized in that: The greenhouse cultivation in step 1) refers to: transplanting the pepper plants to a greenhouse at 25° C. with 14 h of light and 10 h of darkness and cultivating them for 2 days.

6. The efficient and fast pepper protoplast transformation method according to claim 1 is characterized in that: In step 2), the enzymatic hydrolysis conditions are: 25° C. for 3 to 4 hours.

7. The efficient and fast pepper protoplast transformation method according to claim 1 is characterized in that: In step 2), the filtration is performed using a 75 μm nylon mesh rinsed with W5 solution.

8. The efficient and fast pepper protoplast transformation method according to claim 1 or 6, characterized in that: In step 2), the enzymatic solution formula used is: 1.25% cellulase R10, 0.3% chromatase R10, 20mM 2-(N-morpholine)ethanesulfonic acid pH 5.7, 0.4M mannitol, 20mM KCl, heated at 55°C for 10min, cooled to room temperature, then added with 10mM CaCl2, 5mM β-mercaptoethanol, 0.1% bovine serum albumin, and made up to 10mL with ultrapure water.

9. The efficient and fast pepper protoplast transformation method according to claim 1 or 7, characterized in that: In step 2), the W5 solution used is: 2mM 2-(N-morpholine)ethanesulfonic acid pH 5.7, 154mM NaCl, 125mM CaCl2, 5mM KCl, 5mM glucose, and the solvent is ultrapure water.

10. The efficient and fast pepper protoplast transformation method according to claim 1, characterized in that: The obtained protoplast transformation efficiency was 75.33±1.85%.

Images