Plant stem cell nucleus wall breaking method and application

Abstract

The invention discloses a nuclear wall breaking method for plant stem cells and application, belongs to the technical field of plants, and aims to provide a nuclear wall breaking method which is efficient and mild and can maintain the activity of the plant stem cells to the maximum extent so as to overcome the defects in the prior art. By adopting the method disclosed by the invention, the wall breaking rate can reach 95% or above and is far higher than that of a traditional method; through optimized process parameters and a mild wall breaking mode, the cell survival rate can reach 85%, and the biological activity of the cell nucleus extract is effectively guaranteed; through multi-stage purification treatment, the purity of the cell nucleus extract can reach 80% or above, and the impurity content is remarkably reduced.

Description

Technical Field

[0001] The invention belongs to the field of plant technology, and specifically refers to a method for breaking the nuclear wall of plant stem cells and its application. Background Art

[0002] Plant stem cells are of great importance and have broad application prospects. They have the ability to self-renew and differentiate into various plant cell types, playing a key role in plant growth and development, genetic improvement, and biopharmaceuticals. For example, in the pharmaceutical field, plant stem cell extracts may have anti-cancer, anti-inflammatory and antioxidant activities; in the beauty industry, their ingredients can be used in the development of anti-aging skin care products; in agriculture, they help to cultivate superior varieties and improve crop yields and quality.

[0003] Existing plant stem cell extraction and processing technologies have many limitations. Traditional cell wall breaking methods such as mechanical crushing and chemical treatment often have low cell wall breaking efficiency and are difficult to fully release the effective components in the cell nucleus. At the same time, these methods may cause significant damage to cell activity, leading to cell death or loss of function, affecting subsequent application effects. In addition, existing technologies are also difficult to meet requirements in terms of product purity, and the impurity content is high, which increases the difficulty of subsequent separation and purification.

[0004] In view of the important value of plant stem cells and the shortcomings of existing technologies, it is urgent to solve the problem of plant stem cell nuclear cell wall breaking. An efficient, gentle cell wall breaking method that can maintain cell activity to the greatest extent will open up new avenues for the research and application of plant stem cells and promote the rapid development of related industries. Summary of the invention

[0005] The purpose of the present invention is to provide a method and application of plant stem cell nuclear wall breaking. The present invention aims to provide an efficient, gentle nuclear wall breaking method that can maintain the activity of plant stem cells to the greatest extent, so as to overcome the shortcomings of the prior art. Through innovative wall breaking technology, the wide application of plant stem cells in the fields of medicine, beauty, agriculture, etc. is expanded, bringing significant economic and social benefits to related industries. The present invention uses enzymatic hydrolysis to partially degrade cell wall components, increase the exposure of the cell nucleus, and improve the wall breaking efficiency. At the same time, the local high temperature, high pressure and microjet generated by the cavitation effect of ultrasound are used to cause the cell nuclear membrane to be broken by mechanical force. The low temperature environment can inhibit the activity of enzymes and cell metabolism, and reduce cell damage. Centrifugal separation can separate cell fragments and supernatant based on the density difference of different substances. Ultrafiltration and chromatography techniques can purify and concentrate the target components according to molecular size and charge characteristics.

[0006] In order to achieve the above-mentioned object, the technical solution adopted by the present invention is as follows: a method for breaking the nuclear wall of plant stem cells and its application, the method for breaking the nuclear wall of plant stem cells comprises the following steps:

[0007] (1) Pretreatment: First, fresh plant tissue is selected and the stem cell-rich part is obtained through aseptic operation. It is placed in a specific buffer for washing to remove surface impurities and contaminants. Then, enzymatic hydrolysis is used to remove components such as pectin and cellulose on the outer layer of the cell wall to create favorable conditions for subsequent cell wall breaking operations;

[0008] (2) Wall breaking operation: an ultrasound-assisted non-contact physical wall breaking method is used. The pretreated plant stem cell sample is placed in a specially designed ultrasonic reaction container and ultrasonic waves of a specific frequency and intensity are applied. The cavitation effect and mechanical vibration of the ultrasonic wave can cause tiny cracks in the cell nuclear membrane, thereby breaking the cell nuclear wall while avoiding excessive damage to the internal structure of the cell;

[0009] (3) Subsequent processing: After cell wall disruption, the sample is immediately placed in a low temperature environment to slow down cell metabolism and chemical reaction rates and prevent further reduction of cell activity. Next, centrifugation is used to remove cell debris and impurities, and the supernatant containing cell nuclear components is collected. Finally, the supernatant is purified and concentrated by ultrafiltration and chromatography to obtain a high-purity plant stem cell nuclear extract.

[0010] Furthermore, the buffer in step (1) is composed of one or more of a salt buffer and ethylenediaminetetraacetic acid.

[0011] Furthermore, the enzymatic hydrolysis reagent in step (1) is composed of one or more of cellulase and pectinase.

[0012] Furthermore, the enzymatic hydrolysis temperature in step (1) is 30-50°C.

[0013] Furthermore, the ultrasonic frequency in step (2) is 20-100 kHz.

[0014] Furthermore, in the step (2), the ultrasonic power is 1-3 kW, and the ultrasonic time is 20-30 min.

[0015] Furthermore, the chromatography medium used in the purification process in step (3) is composed of one or more of agarose and dextran.

[0016] Furthermore, the centrifugal speed during the purification process in step (3) is 800-1000 rpm / min.

[0017] Furthermore, in step (3), the pore size of the ultrafiltration membrane is 50-80 nm.

[0018] Furthermore, the present invention can be applied to the fields of medicine, food and cosmetics.

[0019] The beneficial effects achieved by the present invention using the above structure are as follows: (1) The present invention has a significant improvement in the cell wall breaking efficiency. By using the method of the present invention, the cell wall breaking rate can reach more than 95%, which is much higher than the cell wall breaking rate of the traditional method; (2) By optimizing the process parameters and the mild cell wall breaking method, the cell survival rate can reach 85%, which effectively ensures the biological activity of the cell nuclear extract; (3) By multi-stage purification treatment, the purity of the cell nuclear extract can reach more than 80%, and the impurity content is significantly reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The effect of a method for nuclear disruption of plant stem cells on the nuclear disruption rate.

[0021] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention. DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0023] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only and are not intended to limit the content of this application.

[0024] The experimental methods in the following examples are conventional methods unless otherwise specified; the experimental materials and test strains used in the following examples are purchased from commercial channels unless otherwise specified.

[0025] Example 1

[0026] Preprocessing

[0027] Fresh plant tissues were selected and the stem cell-rich part was obtained through aseptic operation. It was placed in hydrochloric acid buffer and ethylenediaminetetraacetic acid for cleaning to remove surface impurities and pollutants. Then, at 40°C, cellulase and pectinase were used to remove components such as pectin and cellulose on the outer layer of the cell wall, creating favorable conditions for subsequent cell wall breaking operations.

[0028] Example 2

[0029] Wall breaking operation

[0030] The ultrasonic-assisted non-contact physical cell wall breaking method is used. The pre-treated plant stem cell samples are placed in a specially designed ultrasonic reaction container, and ultrasonic waves are applied for 20-30 minutes at an ultrasonic frequency of 20-100kHz and an ultrasonic power of 1-3kW. The cavitation effect and mechanical vibration of the sound waves can cause tiny cracks in the cell nuclear membrane, thereby breaking the cell nuclear wall while avoiding excessive damage to the internal structure of the cell.

[0031] Example 3

[0032] Subsequent processing

[0033] After cell wall breaking, the sample is immediately placed in a low temperature environment to slow down cell metabolism and chemical reaction speed and prevent further reduction of cell activity. Then, centrifugal separation is used to remove cell debris and impurities, and the supernatant containing cell nuclear components is collected. Finally, an ultrafiltration membrane with a pore size of 50-80nm is selected for ultrafiltration, and chromatography reactions are performed in agarose and dextran media to purify and concentrate the supernatant to obtain a high-purity plant stem cell nuclear extract.

[0034] Experimental Example 1

[0035] The liver cell nuclei of soybean, wheat and corn were used as experimental materials, recorded as three groups A, B and C, and the cell wall breaking experiment was carried out using the plant stem cell nucleus breaking method provided in Examples 1-3. After the experiment, the cell wall breaking rates of the three groups A, B and C were calculated using a microscope observation method.

[0036] Result analysis:

[0037] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

[0038] The present invention and its embodiments are described above, and such description is not restrictive. The drawings show only one embodiment of the present invention, and the actual structure is not limited thereto. In short, if ordinary technicians in the field are inspired by it, without departing from the purpose of the invention, they can design a structure and embodiment similar to the technical solution without creativity, which should belong to the protection scope of the present invention.

Claims

1. A method for breaking the nuclear wall of plant stem cells, Features: A method for breaking the nuclear wall of plant stem cells and its application, the method for breaking the nuclear wall of plant stem cells comprising the following steps: (1) Pretreatment: First, fresh plant tissue is selected and the stem cell-rich part is obtained through aseptic operation. It is placed in a specific buffer for washing to remove surface impurities and contaminants. Then, enzymatic hydrolysis is used to remove components such as pectin and cellulose on the outer layer of the cell wall to create favorable conditions for subsequent cell wall breaking operations; (2) Cell wall breaking operation: Using an ultrasound-assisted non-contact physical cell wall breaking method, the pre-treated plant stem cell sample is placed in a specially designed ultrasonic reaction container and ultrasonic waves of a specific frequency and intensity are applied. The cavitation effect and mechanical vibration of the ultrasonic waves can cause tiny cracks in the cell nuclear membrane, thereby breaking the cell nucleus while avoiding excessive damage to the internal structure of the cell. (3) Subsequent processing: After cell wall disruption is completed, the sample is immediately placed in a low-temperature environment to slow down the cell metabolism and chemical reaction rate and prevent further reduction of cell activity. Then, centrifugation is used to remove cell debris and impurities, and the supernatant containing cell nuclear components is collected. Finally, the supernatant is purified and concentrated by ultrafiltration and chromatography to obtain a high-purity plant stem cell nuclear extract.

2. A method for breaking the nucleus of plant stem cells according to claim 1, wherein the buffer in step (1) is composed of one or more of a salt buffer and ethylenediaminetetraacetic acid.

3. A method for breaking the nucleus of plant stem cells according to claim 2, wherein the enzymatic hydrolysis reagent in step (1) is composed of one or more of cellulase and pectinase.

4. A method for disrupting the nucleus of plant stem cells according to claim 3, wherein the enzymatic hydrolysis temperature in step (1) is 30-50°C.

5. The method for disrupting the nucleus of plant stem cells according to claim 4, wherein the ultrasonic frequency in step (2) is 20-100 kHz.

6. A method for breaking the nuclear wall of plant stem cells according to claim 5, wherein the ultrasonic power in step (2) is 1-3 kW and the ultrasonic time is 20-30 min.

7. A method for breaking the nuclear wall of plant stem cells according to claim 6, wherein the chromatography medium used in the purification process in step (3) is composed of one or more of agarose and dextran.

8. A method for breaking the nuclear wall of plant stem cells according to claim 7, wherein the centrifugal speed during the purification process in step (3) is 800-1000 rpm / min.

9. A method for breaking the nucleus of plant stem cells according to claim 8, wherein the pore size of the ultrafiltration membrane in step (3) is 50-80 nm.

10. Use of the method for breaking the nuclear wall of plant stem cells according to claim 9, which can be applied to the fields of medicine, food and cosmetics.

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