CN111676183A - Preparation method of rape mesophyll protoplast - Google Patents

Preparation method of rape mesophyll protoplast Download PDF

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CN111676183A
CN111676183A CN202010704620.9A CN202010704620A CN111676183A CN 111676183 A CN111676183 A CN 111676183A CN 202010704620 A CN202010704620 A CN 202010704620A CN 111676183 A CN111676183 A CN 111676183A
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solution
protoplast
rape
mesophyll
ficoll
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丁广大
张�浩
李玉
石磊
汪社亮
徐芳森
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Huazhong Agricultural University
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Abstract

The invention belongs to the technical field of biology, and particularly relates to a preparation method of rape mesophyll protoplasts. The technology takes rape young leaf blades as test materials, analyzes the influence on the separation of mesophyll protoplasts under different Mannitol concentrations and determines the optimal concentration; in the purification process, by using 20% Ficoll Solution, the complete protoplast can stay between Solution A and 20% Ficoll Solution, while the damaged protoplast can sink to the bottom of the tube, and then the damaged protoplast can be re-suspended twice by using Solution A, thereby effectively ensuring the integrity of the obtained rape mesophyll protoplast, simultaneously, the number of the protoplasts can also meet the requirement of subsequent instantaneous transformation, and establishing a convenient, rapid and efficient preparation system of the rape protoplast.

Description

Preparation method of rape mesophyll protoplast
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a preparation method of rape mesophyll protoplasts.
Background
Plant cells remaining after the cell wall has been removed in plant cell engineering are called protoplasts. In the field of biological research, protoplasts are often used as plant transient expression systems and have the characteristics of simple transformation, high detection speed and the like.
Rape is one of the oil crops with the highest oil production efficiency in China, the research of rape in the field of molecular biology is deepened day by day, and a large number of important functional genes are excavated. However, although the rape genetic transformation system is improved day by day, the separation and purification of the protoplast homeopathic expression system, especially the protoplast homeopathic expression system, are obviously delayed, so that the effects of convenience, high efficiency and rapidness cannot be achieved, and related research reports are relatively few. In recent years, the protoplast transient expression system has become a powerful tool for many plant gene function studies. The analysis of the gene functions such as the subcellular localization of the rape gene is usually realized by establishing a transient expression system by using an arabidopsis protoplast or a tobacco leaf, and the genetic difference between different species necessarily exists between the arabidopsis protoplast and the tobacco leaf. Therefore, an efficient rape protoplast preparation system is established, and an important foundation is laid for carrying out protein subcellular localization, protein interaction, gene expression regulation and the like by utilizing a protoplast transient expression system.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a preparation method of rape mesophyll protoplasts and application thereof, aiming at solving part of the problems in the prior art or at least relieving part of the problems in the prior art.
The invention is realized in such a way that a preparation method of rape mesophyll protoplast is characterized by comprising the following steps
Shredding rape leaves, and placing the shredded rape leaves in an enzymolysis solution for enzymolysis until the leaves become transparent;
continuously carrying out enzymolysis in dark;
filtering with tea bag or nylon net to remove residue;
centrifuging and removing supernatant; resuspending the pellet with Ficoll solution;
adding Ficoll solutions with different concentrations into the heavy suspension solution, and centrifuging to ensure that the protoplast is positioned between two layers of Ficoll solutions with different concentrations;
transferring the protoplast layer into Solution A, resuspending, mixing, centrifuging to remove supernatant; the SolutionA solution comprises 0.4M Mannitol;
adding Solution A into the precipitate, resuspending, mixing, and centrifuging to remove supernatant;
the pellet was resuspended in protoplasts by adding MMG solution.
Further, the enzymatic Solution includes 1% CellulaseRS 0.1g, 0.1% Macerozyme R100.01g per 10mL of Solution A without pH adjustment.
Further, the enzymolysis solution also comprises 0.01 percent of Pectolysase 0.001 g.
Furthermore, the enzymolysis time is 2-3h in a dark place.
Further, the pellet was resuspended in 20% Ficoll solution; to the resuspension Solution was added 0% Ficoll Solution, Solution A (pH 6.0).
Further, the protoplast is positioned between two layers of Ficoll solutions with different concentrations, and the centrifugation condition in the step is that a swing arm type centrifuge centrifuger is used for centrifugation for 10min at 500 g.
The preparation method of the mesophyll protoplast is applied to the preparation of the plant mesophyll protoplast.
Further, the plant includes any one of arabidopsis thaliana and brassica napus.
In summary, the advantages and positive effects of the invention are:
(1) the present technique analyzed extraction of canola protoplasts at 0.3, 0.4, and 0.5M Mannitol, respectively. Under microscopic examination, the rape protoplasts are damaged in different quantities under the conditions of 0.3M and 0.5M Mannitol no matter after enzymolysis, separation and purification and final cleaning, but the protoplasts are kept in a very complete state under the condition of 0.4M Mannitol and are relatively more in quantity, so that the proper Mannitol concentration is determined for the preparation of the rape protoplasts, and the method is a decisive factor for ensuring the integrity of the rape protoplasts.
(2) In the purification process, by using 20% Ficoll Solution, the complete protoplast can stay between Solution A and 20% Ficoll Solution, while the damaged protoplast can sink to the bottom of the tube, and then the Solution A is used for twice heavy suspension, thereby effectively ensuring the integrity of the protoplast required by the establishment of the transient expression system. Under microscopic examination, the technology has more complete rape protoplasts after purification. The purification efficiency of the protoplast obtained by separation is higher, the protoplast is purified by using Ficoll solution, damaged protoplasts are screened out, and the protoplast is resuspended for multiple times to ensure the quality of the protoplast.
(3) The preparation of the rape protoplast by the technology only needs about 3.5h, ensures convenience and rapidness, and simultaneously greatly improves the preparation efficiency, which is a major breakthrough in the research of the rape biology field.
Drawings
FIG. 1 is a 0.3M Mannitol-10 fold mirror (left) and 0.3M Mannitol-20 fold mirror (right) image after separation chromatography;
FIG. 2 is a 0.4M Mannitol-10 fold mirror (left) and 0.4M Mannitol-20 fold mirror (right) image after separation chromatography;
FIG. 3 is a 0.5M Mannitol-10 fold mirror (left) and 0.5M Mannitol-20 fold mirror (right) image after separation chromatography;
FIG. 4 is a photograph of 0.3M Mannitol-10 fold mirror (left) and 0.3M Mannitol-20 fold mirror (right) after enzymatic hydrolysis;
FIG. 5 is a photograph of 0.4M Mannitol-10 fold mirror (left) and 0.4M Mannitol-20 fold mirror (right) after enzymatic hydrolysis;
FIG. 6 is a photograph of 0.5M Mannitol-10 fold mirror (left) and 0.5M Mannitol-20 fold mirror (right) after enzymatic hydrolysis;
FIG. 7 is a 0.3M Mannitol-20 fold mirror image after final wash;
FIG. 8 is a 0.4M Mannitol-20 times microscope image after final wash;
FIG. 9 is a 0.5M Mannitol-20 fold mirror image after final wash.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the equipment and reagents used in the examples and test examples are commercially available without specific reference. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.
Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit and scope of the appended claims. It is to be understood that the scope of the invention is not limited to the procedures, properties, or components defined, as these embodiments, as well as others described, are intended to be merely illustrative of particular aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be covered by the scope of the appended claims.
For a better understanding of the invention, and not as a limitation on the scope thereof, all numbers expressing quantities, percentages, and other numerical values used in this application are to be understood as being modified in all instances by the term "about". Accordingly, unless expressly indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. In the present invention, "about" means within 10%, preferably within 5% of a given value or range.
The normal temperature in the following embodiments of the present invention refers to a natural room temperature condition in four seasons, and is not subjected to additional cooling or heating treatment, and is generally controlled at 10 to 30 ℃, preferably 15 to 25 ℃.
The invention discloses a preparation method and application of rape mesophyll protoplasts, which are shown in the following embodiments.
Examples
First, preparation of experiment
1. Material preparation
3-4 weeks of size hydroponic rape (with normal fully developed leaves).
2. Preparing a mother solution:
1M mannitol (MW:182.17), 18.22g in ddH2O, fixing the volume to 100mL, and heating for dissolving;
1M MgCl2·6H2o (MW:203.3), 20.33g in ddH2O, fixing the volume to 100 mL;
0.1M MES (MW:195.2), 1.95g in ddH2O (heating and dissolving), metering to 100mL, and adjusting the pH value to 5.7 by using KOH;
1M MES (MW:195.2), 19.52g in ddH2O (heating and dissolving), metering to 100mL, and adjusting the pH value to 5.7 by using KOH;
1M CaCl2(MW:110.98), 11.1g in ddH2O, fixing the volume to 100 mL;
0.2M KCl (MW:74.55), 1.49g in ddH2O, fixing the volume to 100 mL;
1.54M NaCl (MW:58.44), 8.99g dissolved in ddH2O, and metering to 100 mL.
3. Solution A (two kinds: unadjusted pH and pH6.0)
Figure BDA0002594253290000041
Add ddH2O to 200mL, and adjusting the pH value to 6.0 with KOH.
4. Enzymolysis Solution (enzyme + Solution A without pH value, ready for use)
Figure BDA0002594253290000042
Added to 10mL of Solution A without pH adjustment, and adjusted to pH6.0 with KOH.
5. Ficoll (MW:400) solution
20% Ficoll Solution, 20g Ficoll dissolved in 100mL Solution A (pH6.0), without heating.
6. MMG solution (Room temperature preservation)
Figure BDA0002594253290000043
Add ddH2And O is metered to 20 mL.
Second, the operation steps
(one) extraction of protoplast
1. Taking 20 leaves, cutting into 0.5-1mm wide filaments with a blade, quickly transferring into a 50mL centrifuge tube containing 10mL enzymolysis solution, centrifuging at 6000 Xg for 2min, taking out, shaking once until the leaves are completely immersed in the enzymolysis solution, and centrifuging for 1-2 times until the leaves become transparent.
2. Placing on a shaking bed, performing enzymolysis for 2-3h at 40rpm in the dark, and then observing the enzymolysis effect under a microscope.
3. Filter to a 50mL centrifuge tube with a tea bag or paper towel or nylon mesh to remove the residue.
4. Centrifuging for 5min at 100 × g with a swing arm type centrifuge, carefully sucking off the supernatant, and precipitating the protoplast in the lower part (the supernatant is enzyme solution and can be recovered for 1-2 times).
5. 3-5mL of 20% Ficoll solution was added to resuspend the pellet (protoplasts).
6. Transfer to a 15mL centrifuge tube, gently add a layer of 0% Ficoll Solution (i.e., Solution A, pH6.0) about 2cM thick (about 2-3mL), centrifuge at 500 Xg with a swing arm centrifuge for 10min with protoplasts between 20% Ficoll and 0% Ficoll.
7. Transferring protoplasts to 50mL centrifuge tubes containing 15mL Solution A (pH6.0), resuspending, and mixing. (SolutionA dosage can be increased or decreased according to the amount of protoplast)
8. Centrifuging for 5min at 100 Xg with a swing arm type centrifuge, discarding the supernatant, and collecting the precipitate.
9. Then 15mL of Solution A (pH6.0) is added for heavy suspension, mixed evenly, centrifuged at 100 Xg for 5min, the supernatant is removed as far as possible, and some protoplast is rather lost.
10. Add appropriate amount (about 1mL, depending on the amount of protoplast) of MMG solution to resuspend the protoplasts to 2 × 105mL-1And placing the product at room temperature when the microscopic examination is good.
Note that:
1. manipulations involving protoplasts are gentle.
2. Avoid using the pipette to suck the protoplast directly as far as possible, cut off the pipette tip when actually using.
3. When the reagent is added to the centrifuge tube, the reagent is slowly and uniformly added.
4. The overnight culture of protoplasts requires placement in cell culture plates or microplates, preferably without the use of centrifuge tubes, or the cells tend to stick together and die.
5. The status of protoplasts can be confirmed by performing microscopic examination at intervals during the extraction and transformation of the protoplasts.
Thirdly, the extraction condition of the rape protoplast by the Mannitol with different concentrations is researched, and the specific method comprises the following steps: setting 0.3M Mannitol, 0.4M Mannitol and 0.5M Mannitol in the Solution A component respectively for preparing an enzymolysis Solution, separating and re-suspending the protoplast in the extraction process, and performing microscopic examination comparison on the extraction conditions of the protoplast under different Mannitol concentrations.
The results are shown in FIGS. 1 to 9.
The present technique analyzed extraction of canola protoplasts at 0.3, 0.4, and 0.5M Mannitol, respectively. Under microscopic examination, the rape protoplasts are damaged in different quantities under the conditions of 0.3M and 0.5M Mannitol no matter after enzymolysis, separation and purification and final cleaning, but the protoplasts are kept in a very complete state under the condition of 0.4M Mannitol and are relatively more in quantity, so that the proper Mannitol concentration is determined for the preparation of the rape protoplasts, and the method is a decisive factor for ensuring the integrity of the rape protoplasts.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims (8)

1. A preparation method of rape mesophyll protoplast is characterized by comprising the following steps
Shredding the leaves, and placing the shredded leaves in an enzymolysis solution for enzymolysis until the leaves become transparent;
continuously carrying out enzymolysis in dark;
filtering with tea bag or nylon net to remove residue;
centrifuging and removing supernatant; resuspending the pellet with Ficoll solution;
adding Ficoll solutions with different concentrations into the heavy suspension solution, and centrifuging to ensure that the protoplast is positioned between two layers of Ficoll solutions with different concentrations;
transferring the protoplast layer into Solution A, resuspending, mixing, centrifuging to remove supernatant; the Solution A Solution comprises 0.4M Mannitol;
adding Solution A into the precipitate, resuspending, mixing, and centrifuging to remove supernatant;
the pellet was resuspended in protoplasts by adding MMG solution.
2. The method of claim 1, wherein the protoplast of rape mesophyll is prepared by: the enzymatic Solution comprises 1% Cellulase RS 0.1g, 0.1% Macerozyme R100.01g per 10mL of Solution A without pH adjustment.
3. The method of claim 2, wherein the protoplast of rape mesophyll is prepared by: the enzymolysis solution also comprises 0.001g of 0.01 percent Pectolysase.
4. The method of claim 1, wherein the protoplast of rape mesophyll is prepared by: the enzymolysis time is 2-3h in dark.
5. The method of claim 1, wherein the protoplast of rape mesophyll is prepared by: resuspending the pellet with 20% Ficoll solution; to the resuspension Solution was added 0% Ficoll Solution, Solution A (pH 6.0).
6. The method of claim 2, wherein the protoplast of rape mesophyll is prepared by: the protoplast is positioned between two layers of Ficoll solutions with different concentrations, and the centrifugation condition in the step is a swing arm type centrifuge of 500 Xg
Centrifuge for 10 min.
7. Use of the method for the preparation of mesophyll protoplasts according to any one of claims 1 to 6 for the preparation of canola mesophyll protoplasts.
8. Use according to claim 7, characterized in that: the plant includes oilseed rape.
CN202010704620.9A 2020-07-21 2020-07-21 Preparation method of rape mesophyll protoplast Pending CN111676183A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104745523A (en) * 2015-04-03 2015-07-01 天津吉诺沃生物科技有限公司 Separation, transformation and regeneration system for oilseed rape protoplast
CN107488675A (en) * 2017-09-29 2017-12-19 中国农业科学院油料作物研究所 The separation of Rapeseed Protoplast and method for transformation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104745523A (en) * 2015-04-03 2015-07-01 天津吉诺沃生物科技有限公司 Separation, transformation and regeneration system for oilseed rape protoplast
CN107488675A (en) * 2017-09-29 2017-12-19 中国农业科学院油料作物研究所 The separation of Rapeseed Protoplast and method for transformation

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
RENFANG SHEN 等: "Compartmentation of aluminium in leaves of an Al-accumulator, Fagopyrum esculentum Moench", 《PLANTA》 *
周音 等: "油菜原生质体培养研究进展", 《上海农业学报》 *

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