Method for increasing pectin content of plants
By using GA3 treatment during the plant seedling and flowering period, the pectin content of Arabidopsis seedlings and seeds was significantly improved, and the problems of high cost and seasonal limitations of traditional pectin extraction were solved, and efficient and low-cost pectin production was achieved.
Patent Information
- Application Number
- CN202510469626.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-15
- Publication Date
- 2025-07-04
AI Technical Summary
In the prior art, traditional pectin extraction depends on fruit raw materials, is costly and seasonally limited, and lacks a strategy for improving plant pectin content.
The pectin content of Arabidopsis seedlings and seeds was significantly increased by spraying 100 μM GA3 medium during the plant seedling stage and spraying 100 μM GA3 solution during the flowering stage, including GA3 treatment during the seedling stage and GA3 spray during the flowering stage.
The pectin content of Arabidopsis seedlings was significantly improved by 15%, the mucus pectin content of seeds was 14%, and the thickness of the seed coat pectin layer increased by 23%, reducing production costs, breaking through the bottleneck of traditional pectin sources, and providing a non-seasonal high-effect glue production pathway.
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Abstract
Description
Technical Field
[0001] The present invention relates to the fields of plant physiology and biotechnology, and particularly to a method for increasing the pectin content of plants by exogenous application of gibberellin (GA3). Background Art
[0002] Pectin, as an important component of the plant cell wall, plays a crucial role in plant growth and development, cell recognition and signal transduction, and maintaining the structural stability of the cell wall. In addition, due to its unique gelation properties, pectin is widely used in fields such as food thickeners and pharmaceutical excipients. However, the extraction of traditional pectin mainly relies on fruit raw materials such as citrus and apples, which is not only costly but also limited by seasonality. Therefore, increasing the pectin content in plants is of great significance for reducing production costs, enhancing plant stress resistance, and improving the quality of plant products.
[0003] Gibberellin (GA3), as a common plant growth regulator, plays a key role in promoting plant growth and development. Although gibberellic acid (GA3) has been widely used in agricultural production practices, there have been no reports on its increasing the content of pectin components in the plant cell wall through exogenous regulation strategies. Notably, there are still significant gaps in the pectin synthesis technology system based on exogenous chemical regulation, and key technical links such as the analysis of its action mechanism, the optimization of application parameters, and the construction of the metabolic regulation network have not yet formed a complete technical system. Currently, research on regulating the pectin content in plants mostly focuses on genetic engineering methods, and there is a relative lack of effective strategies and technical solutions for increasing the pectin content through simple and efficient exogenous chemical methods.
[0004] In view of this, exploring and developing a method based on exogenous application of gibberellin to systematically increase the pectin content of plants at different developmental stages not only has scientific significance but also has broad application prospects. The invention will help to break through the limitations of traditional pectin production, provide a more economical and stable pectin source for the food industry and the pharmaceutical field, and provide a new perspective for further understanding the role mechanism of gibberellin in plant cell wall synthesis. Summary of the Invention
[0005] Object of the Invention: The object of the present invention is to provide a method for increasing the pectin content of plants by exogenous application of gibberellin, which is simple to operate and low in cost. By treating plants with gibberellin, the pectin content of plant seedlings and seeds can be significantly increased.
[0006] Technical Solution: To achieve the above object of the invention, the technical solution adopted by the present invention is as follows: Preparation of gibberellin solution: Prepare a GA3 solution with a concentration of 100 μM.
[0007] For Arabidopsis thaliana seedlings: After surface sterilization of wild-type Arabidopsis thaliana seeds, they were germinated on 1 / 2 MS medium containing 100 μM GA3, and the control group was germinated on 1 / 2 MS medium without added GA3. After 10 days, the seedlings were taken to measure the pectin content. The results showed that the pectin content of the seedlings in the GA3 treatment group (about 106 ± 5 μg / mg) was significantly higher than that of the control group (Mock) without added GA3 (about 92 ± 2 μg / mg) (Appendix Figure 1 ), indicating that exogenous application of GA3 has a significant promoting effect on the pectin content of Arabidopsis thaliana seedlings.
[0008] For Arabidopsis thaliana seeds: After they grew to the flowering stage, the experimental group was sprayed with 100 μM GA3 solution daily, and the control group was sprayed with distilled water daily. At the end of flowering, after maturity, the seeds were harvested to measure the pectin content. The results showed that the mucilage pectin content of Arabidopsis thaliana seeds after GA3 treatment (about 112 ± 2 μg / mg) was significantly higher than that of the control group (Mock) without added GA3 (about 98 ± 2 μg / mg) (Appendix Figure 2 ), indicating that exogenous application of GA3 has a significant promoting effect on the mucilage pectin content of Arabidopsis thaliana seeds. According to ruthenium red staining combined with microscopic observation, it can be seen that the thickness of the seed coat pectin layer increased significantly (Appendix Figure 3 ). At the same time, statistical analysis of the thickness of the seed coat pectin layer was carried out. The results showed that the thickness of the seed coat pectin layer of the seeds sprayed with GA3 was 0.10 ± 0.01 mm, which was about 40% higher than that of the control group (Mock) without spraying GA3 (0.08 ± 0.007 mm) (Appendix Figure 4 ), further confirming that spraying GA3 can significantly increase the content of pectin in the seed coat of seeds.
[0009] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention provides a method for efficiently and low-costly increasing the pectin content of plants by exogenous application of gibberellin (GA3), significantly breaking through the yield bottleneck of traditional pectin sources. After treating Arabidopsis thaliana seedlings with 100 μM GA3, the pectin content increased by about 15%. Spraying GA3 during the flowering period can increase the mucilage pectin content of seeds by 14% and the thickness of the seed coat pectin layer by 23%. This method does not require complex genetic modification and can achieve pectin accumulation only by adding to the medium or foliar spraying, greatly reducing the technical threshold and production cost, and is especially suitable for large-scale application. At the same time, the present invention first clarifies the positive regulatory effect of exogenous GA3 on pectin synthesis, provides a new research direction for analyzing the molecular mechanism of gibberellin in cell wall metabolism, and opens up a new way for the efficient utilization of non-traditional raw materials such as herbaceous plants and seeds, alleviating the seasonal and resource limitation problems of traditional pectin raw materials. In summary, the present invention combines technological innovation and practical value, and provides an efficient solution for the field of pectin industrial production and plant physiological regulation. Description of the Drawings
[0010] Appendix Figure 1 Comparison of pectin content in Arabidopsis thaliana seedlings treated with exogenous GA3; Statistics show that exogenous GA3 can increase the pectin content in Arabidopsis thaliana seedlings; Appendix Figure 2 Comparison of pectin content in the seed coat cells of Arabidopsis thaliana sprayed with GA3; Statistics show that the pectin content in the seed coat cells of Arabidopsis thaliana sprayed with GA3 is significantly higher than that of the control group (Mock).
[0011] Appendix Figure 3 Comparison of pectin thickness in the seed coat cells of Arabidopsis thaliana sprayed with GA3; As can be seen from the figure, the pectin thickness in the seed coat cells of Arabidopsis thaliana sprayed with GA3 is significantly thickened.
[0012] Appendix Figure 4 Comparison of pectin thickness in the seed coat cells of Arabidopsis thaliana sprayed with GA3; Statistics show that the pectin thickness in the seed coat cells of Arabidopsis thaliana sprayed with GA3 is significantly higher than that of the control. Detailed implementation manners
[0013] The present invention will be further described below in conjunction with specific embodiments. For the molecular biology experimental methods not specifically described in the following embodiments, they can be referred to the methods listed in "Molecular Cloning: A Laboratory Manual" (Third Edition) by J. Sambrook or the conventional methods in the art, or carried out according to the kits and product specifications. The reagents and consumables used, unless otherwise specified, can be obtained from commercial channels.
[0014] Example 1: Exogenous GA3 increases the pectin content in Arabidopsis thaliana seedlings
[0015] 1. Seed treatment
[0016] Select wild-type Arabidopsis thaliana seeds and first disinfect them with 10% sodium hypochlorite for 8 minutes. Subsequently, rinse them 3 times with sterile water to ensure complete removal of sodium hypochlorite residues and avoid interference with subsequent experiments.
[0017] 2. Germination and culture
[0018] Sow the strictly disinfected Arabidopsis thaliana seeds on 1 / 2 MS medium respectively. One group of the medium is added with 100 μM GA3, and the other group is not added with GA3 as a control (Mock). After sowing, place the medium in a light incubator, and the culture conditions are set as: temperature 22°C, light and dark cycle of 16 hours of light / 8 hours of darkness, and light intensity of 80 μmol / m²·s to ensure the growth of Arabidopsis thaliana seedlings under suitable environmental conditions.
[0019] 3. Seedling treatment and pectin content determination
[0020] Ten days after sowing, the seedlings grew to an appropriate stage. The seedlings were carefully removed from the culture medium, and a series of operations such as cell wall extraction, pectin extraction, and content determination were carried out on the seedlings according to the pectin content determination method in Example 2. After a rigorous experimental process and data analysis, the results showed that the pectin content of the seedlings in the GA3 treatment group (about 106 ± 5 μg / mg) was significantly higher than that of the control group (Mock) without GA3 addition (about 92 ± 2 μg / mg) ( Figure 1 ), indicating that exogenous GA3 has a significant promoting effect on the pectin content of Arabidopsis seedlings.
[0021] Example 2 Determination of Pectin Content in Seedlings 1. Extraction of Cell Wall Components After grinding Arabidopsis seedlings with liquid nitrogen, the cell wall was extracted according to the following steps: extracted 3 times with 70% ethanol, centrifuged at 5,000 g for 15 minutes, discarded the supernatant, added chloroform: methanol (1:1, v / v) and extracted 2 times, centrifuged at 5,000 g for 15 minutes, discarded the supernatant, added 100% acetone and extracted 2 times, centrifuged at 10,000 g for 15 minutes, discarded the supernatant, and finally suspended the precipitate with 100% acetone and dried at 55 °C. The solid powder obtained after treating the dried white or grayish-white solid powder with α-amylase to remove starch is the cell wall.
[0022] 2. Pectin Extraction Weigh 2 mg of cell wall powder, add 1 mL of 0.5% ammonium oxalate, heat in a water bath at 100 °C for 1 hour, shake intermittently to mix evenly, centrifuge, transfer the supernatant to a new tube, add 1 mL of 0.5% ammonium oxalate to the precipitate again, heat in a water bath at 100 °C for 30 minutes, shake intermittently to mix evenly, centrifuge, and combine the two supernatants to obtain a pectin extract (2 mL in total).
[0023] 3. Pectin Content Determination The m-hydroxybiphenyl-sulfuric acid method was used to determine the pectin content (1) Take an appropriate amount of pectin extract in a 2 mL centrifuge tube, dilute it with water to 100 μL (to make its concentration about 0.2 - 10 μg / 100 μL, and after extracting the primary cell wall without starch AIR (Alcohol Insoluble Residue) according to the above steps, dilute it 5 - 10 times); (2) Add 0.6 mL of 0.0125 M sodium tetraborate / sulfuric acid solution (operate on ice), mix well, heat in a boiling water bath for 5 minutes, and cool in an ice bath; (3) Add 10 μL of 1.5 mg / mL m-hydroxybiphenyl solution (prepared with 5 mg / mL NaOH solution), mix well and shake for 5 minutes (showing pink), and let it stand. Pipette 200 μL of the reaction solution into an enzyme-linked immunosorbent assay (ELISA) plate and measure the absorbance at 520 nm.
[0024] (4) Standard curve preparation: Dilute the 10 mg / mL GalA (D-galacturonic acid) standard solution to the following concentrations: 0, 0.01, 0.02, 0.03, 0.04, 0.05 μg / μL. Take 100 μL of each and place them in 2 mL centrifuge tubes, and perform the above operations on them together with the samples to be tested.
[0025] Example 3: Spraying GA3 to increase the pectin content of Arabidopsis thaliana seeds Plant wild-type Arabidopsis thaliana plants in an incubator, set it to 16 hours of light / 8 hours of darkness, temperature 22°C, and light intensity 100 μmol / m²·s.
[0026] After the plants grow to the flowering stage, the experimental group is sprayed with 100 μM GA3 solution every day, and the control group (Mock) is sprayed with distilled water every day until the flowering ends.
[0027] Seed treatment and pectin content determination: After the seeds mature, harvest and dry them. After staining with 0.01% ruthenium red solution using the method described in Example 5, measure and count the thickness of the pectin layer of the seed coat. It can be seen that the mucilage pectin layer released from the seeds of the plants sprayed with GA3 is significantly thickened (attached Figure 3 ). Then, according to the method for determining the pectin content of Arabidopsis thaliana seeds in Example 4, extract, weigh, and calculate the pectin of Arabidopsis thaliana seeds. The results show that the mucilage pectin content of the seeds of the plants sprayed with GA3 is significantly increased (attached Figure 4 ).
[0028] Example 4: Determination of the pectin content of Arabidopsis thaliana seeds Accurately weigh 100 mg of mature and dry Arabidopsis thaliana seeds, put them into a 15 mL centrifuge tube, add 5 mL of distilled water, shake at 220 rpm and 16°C for 1 hour, centrifuge and transfer the supernatant. Wash the precipitate once with 2 mL of distilled water, and combine the supernatants in a 50 mL centrifuge tube, which is the outer mucilage extract; resuspend the precipitate with 5 mL of distilled water, perform ultrasonic treatment (200 W, ~2 minutes), centrifuge and transfer the supernatant. Wash the precipitate once with 2 mL of distilled water, and combine the supernatants in a 50 mL centrifuge tube, which is the inner mucilage extract. Combine the inner and outer extracts and transfer them to a dialysis bag (cut-off molecular weight 1000 Da), and dialyze with running water for 72 hours. Transfer the dialyzed extract to a new 50 mL centrifuge tube, freeze-dry, and weigh.
[0029] Example 5: Observation and statistics of the thickness of the seed pectin layer Put the dried and mature Arabidopsis thaliana seeds into a centrifuge tube, add 0.01% ruthenium red staining solution and stain for 1 hour, while shaking at 200 rpm at room temperature to remove the outer mucilage. Observe the thickness of the inner mucilage under a microscope and take pictures. Measure and calculate the thickness of the mucilage using Image J software. It can be seen under the microscope that the pectin layer thickness of the seed coat of the seeds sprayed with GA3 is significantly thicker than that of the seed coat of the seeds not sprayed with GA3 ( Figure 3 ).
[0030] Measurement using ImageJ software shows that the pectin layer thickness of the seed coat of the seeds sprayed with GA3 is 0.10 ± 0.01 mm, which is about 23% higher than that of the control group (Mock) not sprayed with GA3 (0.08 ± 0.007 mm), ( Figure 4 ); it can be seen that spraying GA3 can significantly increase the pectin content of the seed coat.
[0031] By externally applying gibberellin GA3, the present invention can significantly increase the pectin content of plants, providing a new method for plant growth and development regulation and quality improvement, and having broad application prospects.
[0032] In summary, the present invention innovatively develops a plant pectin technology based on chemical regulation of exogenous gibberellic acid (GA3), breaking through the bottleneck of the seasonal limitation of raw material supply in traditional pectin production. By establishing an application system of GA3, directional enrichment of pectin is achieved during the seedling stage and reproductive growth stage of Arabidopsis thaliana, with significant advantages such as a short operation cycle, low technical threshold, and environmental friendliness.
Claims
1. A method for increasing the pectin content of plants, characterized in that, Treat plants by exogenous spraying of gibberellin (GA3).
2. The method according to claim 1, wherein Exogenous spraying of GA3 on plants can significantly increase the pectin content of plant seedlings.
3. The method according to claim 1, characterized in that, Exogenous spraying of GA3 on plants can significantly increase the pectin content of plant seeds.
4. The method according to claim 1, wherein Exogenous spraying of GA3 on plants can significantly increase the thickness of the pectin layer of plant seeds.
5. The method according to any one of claims 1-4, characterized in that, The treatment concentration of the GA3 is 100 μM.
6. The method according to claim 1, wherein The plant is Arabidopsis thaliana.
7. The method according to claim 1, wherein The spraying is carried out daily from the flowering stage to the end of flowering of the plant.
8. The method according to claim 3, wherein After surface sterilization of plant seeds, sow them in a medium containing gibberellin GA3.
9. The method according to claim 1, characterized in that, Exogenous spraying of GA3 treatment can increase the pectin content of Arabidopsis thaliana seedlings by 15%. Spraying GA3 during the flowering period can increase the mucilage pectin content of seeds by 14% and increase the thickness of the pectin layer of the seed coat by 40%.
10. Application of a method for increasing the pectin content of plants, including but not limited to the fields of food industry, pharmaceutical excipients or improvement of plant stress resistance.