Efficient and simple tobacco protoplast instantaneous conversion method

Abstract

According to the efficient and simple instantaneous transformation method for the tobacco protoplast provided by the invention, agrobacterium carrying a target vector is utilized to firstly transform the Bensi tobacco leaves, then the protoplast is obtained through an enzymolysis method, the operation is simple and easy, the consumed time is short, and the transformation efficiency is greatly improved; and a PEG reagent does not need to be used. The yield of the obtained nicotiana benthamiana protoplast is 16.92 + / -0.27 * 10 < 6 > / g FW, and the instantaneous conversion efficiency reaches 85.57% or above. Compared with the prior art, the method has the advantages that the step of PEG conversion is omitted, the toxic action of a PEG chemical reagent on the protoplast is avoided, and a plurality of unstable factors in the PEG-mediated protoplast conversion process are also avoided. In addition, the method is low in cost, convenient and fast, the obtained protoplast is good in quality, and the conversion efficiency is high.

Description

Technical Field

[0001] The invention belongs to the field of biological science and technology, and in particular relates to an efficient and simple tobacco protoplast transient transformation method. Background Art

[0002] Plant protoplasts refer to living naked cells that have been freed of cell walls and are wrapped by cell membranes. The cell wall, as a protective structure for plant cells, is mainly composed of cellulose, hemicellulose, pectin and a small amount of protein, which supports and protects the cells. Protoplasts can be obtained by mechanical separation or enzymatic hydrolysis, of which enzymatic hydrolysis is the best. With the development of plant biology, protoplasts are increasingly used in the fields of cell biology, molecular genetics and plant physiology, becoming an ideal material for research on subcellular localization, gene expression and regulation, protein interaction, physiological and biochemical research, cell fusion and new variety breeding.

[0003] Nicotiana benthamiana is native to Australia and belongs to the Solanaceae family, along with peppers, tomatoes, potatoes and cultivated tobacco. In the past decade, Nicotiana benthamiana has been considered an important model plant in plant biology and biotechnology research, and is widely used in research fields such as functional genomics, plant-pathogen interactions, metabolic engineering, synthetic biology and genome editing.

[0004] In traditional protoplast transformation method, usually first utilize enzymolysis to obtain protoplasts, and then carry out protoplast transformation by PEG-mediated transformation method.For example, the patent of publication number CN104789518 A disclosed on July 22, 2015 discloses efficient separation, transformation and regeneration system of tobacco protoplasts, and the reagent combination includes 1 / 2MS culture medium, MS culture medium, enzymolysis solution, C culture medium, W5 washing liquid, Evans Blue dye solution, conversion liquid, polyethylene glycol solution, C: B culture medium, A culture medium, MS regeneration culture medium, and the transformation method includes the cultivation of sterile seedlings, the separation and purification of protoplasts, the transformation of protoplasts, the cultivation and regeneration of protoplasts and other steps.This method relates to multiple experimental steps and reagents, and has higher requirements on reagent quality, is affected by the concentration and transformation time of PEG, so the operation process is cumbersome and time-consuming, and has higher technical requirements.

[0005] Therefore, it is of great significance to establish an efficient and simple tobacco protoplast transient transformation system. Summary of the invention

[0006] The purpose of the present invention is to provide an efficient and simple method for transient transformation of tobacco protoplasts, which uses Agrobacterium carrying a target vector to first transform Nicotiana benthamiana leaves, and then obtains protoplasts by enzymatic hydrolysis. The operation is simple, time-saving, and the transformation efficiency is greatly improved, providing technical support for basic theoretical research and plant genetic improvement.

[0007] The specific technical solutions of the present invention are as follows:

[0008] An efficient and simple method for transient transformation of tobacco protoplasts comprises the following steps:

[0009] 1) Injecting the Agrobacterium infection solution carrying the target vector into the growing Nicotiana benthamiana leaves, culturing them in the dark, and then moving them to a growth chamber for culturing;

[0010] 2) Cut the Nicotiana benthamiana leaves treated in step 1), remove the main veins of the leaves, and then cut the leaves into thin strips. After enzymatic hydrolysis, filter, centrifuge the liquid, and discard the supernatant; add ice-bathed W5 solution to resuspend the cells to obtain.

[0011] In step 1), the Agrobacterium infection solution carrying the target vector refers to OD 600 The value is 0.6 to 0.8, and the Agrobacterium infection solution carrying the target vector is resuspended;

[0012] Specifically, in the step 1), the preparation method of the Agrobacterium infection solution is as follows: take Agrobacterium carrying the target vector, streak activation in LB solid culture medium containing three antibiotics: 50 μg / mL rifampicin, 50 μg / mL kanamycin and 25 μg / mL gentamicin, and culture in the dark at 28°C for 2 days; prepare the Agrobacterium infection solution (prepared and used immediately): add 0.2133g 2-(N-morpholino)ethanesulfonic acid (MES), 0.2032g MgCl2·6H2O, and 100μL of 200μmol / L acetosyringone (AS) to every 100mL ddH2O, adjust the pH to 5.0-5.4 with NaOH, and use it as the infection solution; resuspend the bacteria with the infection solution, centrifuge at 6000rmp for 10 minutes, remove the supernatant, add the invasion infection solution to each tube of precipitate and resuspend it, and dilute to OD 600 Value: 0.6~0.8.

[0013] The Agrobacterium carrying the target vector refers to Agrobacterium GV3101 carrying the green fluorescent protein (GFP) marker gene vector pBinGFP3 driven by the CaMV35S promoter;

[0014] In step 1), the method for obtaining the growing Nicotiana benthamiana leaves is as follows: soaking Nicotiana benthamiana seeds in 70% ethanol by volume for 30 seconds in a clean bench, and quickly rinsing with sterile water once; then disinfecting with 0.1% mercuric chloride (prepared by taking 1g of mercuric chloride and adding 1000mL of distilled water) for 6 minutes, during which continuous shaking is required to ensure thorough sterilization, and rinsing with sterile water 3 to 4 times; then pouring the sterilized seeds into a sterile culture dish containing 2 layers of filter paper and blowing dry for 5 minutes, picking the seeds and inoculating them in a culture bottle containing MS culture medium, and culturing them at 25°C, 70% relative humidity, 14h light / 10h dark conditions for 4 to 5 weeks to obtain growing Nicotiana benthamiana leaves; the formula of the MS culture medium is: 4.74g / L MS powder, 30g / L sucrose, 8g / L agar, pH 5.8;

[0015] Alternatively, in step 1), the method for obtaining the growing Nicotiana benthamiana leaves is: sowing mature seeds of Nicotiana benthamiana in soil, and culturing them in a growth chamber under conditions of 25° C., 70% relative humidity, and 14 h light / 10 h dark for 4 to 5 weeks to obtain growing Nicotiana benthamiana leaves.

[0016] The soil is a mixture of peat soil and vermiculite in a volume ratio of 3:1.

[0017] In step 1), the Agrobacterium infection solution carrying the target vector is injected into the leaves of Nicotiana benthamiana, and the injection volume is 2-3 ml;

[0018] In step 1), the dark culture conditions are: 25°C dark culture for 2h;

[0019] In step 1), the growth chamber culture conditions are: 25°C, 14h light / 10h dark in the growth chamber for 2 days;

[0020] In step 2), the enzymatic hydrolysis solution used is: 1.25% (wt / vol) cellulase R10, 0.3% (wt / vol) macrozyme R10, 2.0mM 2-(N-morpholine)ethanesulfonic acid (MES) (pH5.7), 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; wherein in wt / vol, the unit of wt is g and the unit of vol is ml.

[0021] In step 2), the amount of the enzymatic hydrolysis solution used in the enzymatic hydrolysis of the thin leaves is 0.1 g / mL; the thin leaves are completely immersed; the width of the thin leaves is 0.5 mm;

[0022] In step 2), the enzymatic hydrolysis conditions are: 23-25°C for 3-4h;

[0023] In step 2), the filtering refers to filtering using a 75 μm nylon mesh; the nylon mesh is rinsed with W5 solution before use;

[0024] In step 2), the liquid is centrifuged at 100 g for 2 min;

[0025] 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, and the solvent is ultrapure water.

[0026] All the above reagents need to be sterilized by filtration through a 0.22 μm microporous filter membrane.

[0027] The present invention uses Agrobacterium carrying a target vector to transform Nicotiana benthamiana leaves first, and then obtains protoplasts by enzymatic hydrolysis. The operation is simple, time-saving, and the transformation efficiency is greatly improved. The yield of Nicotiana benthamiana protoplasts is 16.92±0.27×10 6 / g FW, and the instantaneous conversion efficiency reached over 85.57%.

[0028] Compared with the prior art, the present invention saves the step of PEG conversion, avoids the toxic effect of PEG chemical reagents on protoplasts, and also avoids many unstable factors in the protoplast transformation process mediated by PEG. In addition, the invention is low in cost, convenient and fast, obtains good protoplast quality, and has high conversion efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 FIG. 1 is a diagram of transient transformation and protoplast preparation of Nicotiana benthamiana of the present invention; Figure 1 Middle A: The Agrobacterium infection solution carrying the pBinGFP3 vector was injected into the growing Nicotiana benthamiana leaves, and the leaves continued to grow after the 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: The leaves cultured for 2 days were cut into thin strips and placed in the enzymatic solution for enzymatic hydrolysis;

[0030] Figure 2 This is a diagram of the instantaneous transformation effect of Nicotiana benthamiana protoplasts of the present invention; GFP: green fluorescent protein signal diagram; Bright field: bright field diagram; Merged: merged diagram. DETAILED DESCRIPTION

[0031] 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.

[0032] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0033] 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.

[0034] Example 1

[0035] An efficient and simple method for transient transformation of tobacco protoplasts comprises the following steps:

[0036] 1) Preparation of donor materials:

[0037] Mature seeds of Nicotiana benthamiana were sown in soil, which was a mixed soil with a volume ratio of peat soil: vermiculite = 3:1; the soil was cultured in a growth chamber at 25°C, 70% relative humidity, 14h light / 10h dark conditions for 4 weeks to obtain growing Nicotiana benthamiana leaves.

[0038] 2) Transient expression of target gene:

[0039] 2-1) Take Agrobacterium GV3101 carrying the green fluorescent protein (GFP) marker gene vector pBinGFP3 driven by the CaMV35S promoter, streak it on LB solid medium containing three antibiotics: 50 μg / mL rifampicin, 50 μg / mL kanamycin, and 25 μg / mL gentamicin, and culture it in the dark at 28°C for 2 days to obtain Agrobacterium carrying the target vector;

[0040] 2-2) Preparation of Agrobacterium infection solution: 100 mL of infection solution formula is 0.2133 g 2-(N-morpholino)ethanesulfonic acid (MES), 0.2032 g MgCl2·6H2O, 100 μL of 200 μmol / L acetosyringone (AS), pH 5.2, as the infection solution. Resuspend the Agrobacterium carrying the target vector with the infection solution: centrifuge at 6000 rpm for 10 minutes, remove the supernatant, resuspend the infection solution added to each tube of precipitate, and dilute to OD 600 is 0.8, and the Agrobacterium infection solution carrying the target vector is obtained.

[0041] 2-3) Use the needle tip of a 1 mL syringe to gently make a small cut on the back of the leaf, then remove the needle of the syringe and draw up the infection suspension, slowly push the syringe piston, and inject the Agrobacterium infection solution carrying the target vector into the Nicotiana benthamiana leaf with a syringe (with the needle tip removed). The injection volume is 2-3 ml per leaf, depending on the size of each leaf, and each leaf is injected as a whole leaf ( Figure 1 (A) After culturing in the dark for 2 h, the cells were moved to a growth chamber at 25 °C with 14 h light / 10 h dark conditions and cultured for 2 days.

[0042] 3) Isolation and purification of protoplasts:

[0043] 3-1) Prepare the following reagents in advance:

[0044] Enzyme hydrolysate: 1.25% (wt / vol) cellulase R10, 0.3% (wt / vol) macrozyme R10, 2.0 mM 2-(N-morpholine)ethanesulfonic acid (MES) (pH 5.7), 0.4 M mannitol, 20 mM KCl, heated at 55°C for 10 min, cooled to room temperature, then added with 10 mM CaCl2, 5 mM β-mercaptoethanol, 0.1% (wt / vol) bovine serum albumin (BSA), and made up to 10 mL with ultrapure water, wherein in wt / vol, the unit of wt is g and the unit of vol is mL.

[0045] W5 solution: 2 mM 2-(N-morpholino)ethanesulfonic acid (MES) (pH 5.7), 154 mM NaCl, 125 mM CaCl2, 5 mM KCl.

[0046] The enzymatic solution and W5 solution need to be sterilized by filtration through a 0.22 μm microporous filter membrane.

[0047] 3-2) After culturing Nicotiana benthamiana for 2 days, the leaves of the uninjected tobacco turned red under ultraviolet light ( Figure 1 Middle B), leaves injected with Agrobacterium carrying the target vector pBinGFP3 show green fluorescence ( Figure 1 Middle C).

[0048] 3-3) Select tobacco leaves with high and uniform green fluorescence intensity, remove the main veins of the leaves with a sharp blade, and then cut the leaves into thin strips of about 0.5 mm. Take 1 g of the thin strips and quickly place them in 10 mL of enzymatic hydrolysis solution to completely immerse them ( Figure 1D), and enzymatic hydrolysis was performed at 25°C for 4 h; then, the cells were filtered through a 75 μm nylon mesh that had been rinsed with W5 solution in advance, and the liquid was transferred to a 50 mL round-bottom centrifuge tube; centrifuged at 100 g for 2 min, and the supernatant was discarded; the cells were resuspended in ice-bathed W5 solution, and observed and counted under a laser confocal microscope. The results showed that the protoplast yield was 16.92±0.27×10 6 / g FW, the instantaneous conversion efficiency is over 85.57% ( Figure 2 ).

[0049] 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 simple method for transient transformation of tobacco protoplasts, characterized in that: The efficient and simple tobacco protoplast transient transformation method comprises the following steps: 1) Injecting the Agrobacterium infection solution carrying the target vector into the growing Nicotiana benthamiana leaves, culturing them in the dark, and then moving them to a growth chamber for culturing; 2) Cut the Nicotiana benthamiana leaves treated in step 1), remove the main veins of the leaves, and then cut the leaves into thin strips. After enzymatic hydrolysis, filter, centrifuge the liquid, and discard the supernatant; add ice-bathed W5 solution to resuspend the cells to obtain.

2. The efficient and simple tobacco protoplast transient transformation method according to claim 1, characterized in that: In step 1), the Agrobacterium infection solution carrying the target vector refers to OD 600 Resuspend the Agrobacterium infection solution carrying the target vector with a value of 0.6 to 0.

8.

3. The efficient and simple tobacco protoplast transient transformation method according to claim 1 or 2, characterized in that: In step 1), the Agrobacterium carrying the target vector refers to Agrobacterium GV3101 carrying a green fluorescent protein (GFP) marker gene vector driven by the CaMV35S promoter.

4. The efficient and simple tobacco protoplast transient transformation method according to claim 1, characterized in that: In step 1), the method for obtaining the growing Nicotiana benthamiana leaves is as follows: soak the Nicotiana benthamiana seeds in 70% ethanol by volume for 30 seconds in a clean bench, and quickly rinse once with sterile water; then disinfect with 0.1% mercuric chloride for 6 minutes, during which continuous shaking is required to ensure thorough sterilization, and rinse with sterile water 3 to 4 times; then pour the sterilized seeds into a sterile culture dish containing 2 layers of filter paper and blow dry for 5 minutes, pick the seeds and inoculate them in a culture bottle containing MS culture medium, and culture them at 25°C, 70% relative humidity, 14h light / 10h dark conditions for 4 to 5 weeks to obtain the growing Nicotiana benthamiana leaves.

5. The efficient and simple tobacco protoplast transient transformation method according to claim 1, characterized in that: In step 1), the method for obtaining the Nicotiana benthamiana leaves is as follows: sowing mature Nicotiana benthamiana seeds in soil, culturing in a growth chamber under conditions of 25° C., 70% relative humidity, and 14 h light / 10 h dark for 4 to 5 weeks to obtain growing Nicotiana benthamiana leaves.

6. The efficient and simple tobacco protoplast transient transformation method according to claim 1, characterized in that: In step 1), dark culturing refers to culturing at 25° C. for 2 h.

7. The efficient and simple tobacco protoplast transient transformation method according to claim 1, characterized in that: In step 1), the culture conditions in the growth chamber are: 25° C., 14 h light / 10 h dark, and culture in the growth chamber for 2 days.

8. The efficient and simple tobacco protoplast transient transformation method according to claim 1, characterized in that: In step 2), the amount ratio of the thin leaf strips to the enzymatic hydrolysis solution used during enzymatic hydrolysis is 0.1 g / mL.

9. The efficient and simple tobacco protoplast transient transformation method according to claim 1, characterized in that: In step 2), the enzymatic hydrolysis conditions are: 23-25° C. for 3-4 hours.

10. The efficient and simple tobacco protoplast transient transformation method according to claim 1, characterized in that: The yield of Nicotiana benthamiana protoplasts obtained in step 2) was 16.92±0.27×10 6 / g FW, and the instantaneous conversion efficiency reached over 85.57%.

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