N-acetylated chitosan oligosaccharide for inducing salt-resistant activity of plants as well as preparation method and application of N-acetylated chitosan oligosaccharide
The preparation of N-acetylated chitin oligosaccharides of specific sequences by the chitin deacetylase ArCE4 enzyme method solves the problem of unstable anti-salt activity of chitin oligosaccharides, and achieves efficient and environmentally friendly plant-induced anti-agent preparation, which is suitable for agricultural biological products.
Patent Information
- Application Number
- CN202510309342.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-17
- Publication Date
- 2025-07-18
AI Technical Summary
The existing chitin oligosaccharides are unstable in inducing plant anti-salt activity, and the preparation method is complex, resulting in the inability to accurately control the dosage, limiting their application as a plant-induced anti-agent.
The N-acetylated chitin oligosaccharide with specific sequence arrangement was prepared by the chitin deacetylase ArCE4 enzyme method. By reacting the whole N-acetylated chitin deacetylase ArCE4 in a specific buffer under low temperature conditions, the reaction was desalted and lyophilized to obtain the N-acetylated chitin oligosaccharide with stable anti-salt activity.
The specific sequence structure preparation of chitin oligosaccharides is achieved, by-product generation and environmental pollution are avoided, and high-purity plant-inducing anti-agents are obtained, with good water solubility and efficient plant-resistant salt activity, and are suitable for agricultural biological products.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of marine bioengineering, and particularly relates to an N-acetylated chitosan oligosaccharide for inducing plant salt tolerance activity, a preparation method thereof, and an application thereof. Background Art
[0002] Salt stress is a common abiotic stress and is becoming one of the important factors affecting world food security. Salt stress can cause ion toxicity and interfere with the absorption of essential elements by plants. The osmotic stress caused by the high-salt environment in the soil leads to a decrease in plant stomatal conductance and a sharp reduction in photosynthesis. At the same time, the oxidative stress induced by salt stress generates a large amount of reactive oxygen species (ROS), causing lipid peroxidation and protein damage, and ultimately resulting in plant growth stagnation or even death.
[0003] Chitin is a natural polysaccharide widely present in the shells of marine crustaceans. Its stock in nature is second only to cellulose, making it the second largest natural polymer compound and a recyclable resource. As a degradation product of chitin, chitosan oligosaccharide has good solubility, biocompatibility, and various biological activities such as antibacterial and inducing plant stress resistance. In particular, the salt tolerance activity of chitosan oligosaccharide in inducing plants has attracted extensive attention from domestic and foreign scholars, and it is a very promising plant inducer.
[0004] The molecular structure of chitosan oligosaccharide determines its biological activity. Chitosan oligosaccharide is composed of N-acetylglucosamine and glucosamine alternatingly, and the degree of acetylation (DA) is an important parameter affecting the biological activity of chitosan oligosaccharide. Some studies have shown that chitosan oligosaccharide with DA = 50% has better salt tolerance activity in inducing plants. However, in previous research reports on the salt tolerance activity of chitosan oligosaccharide in inducing plants, chitosan oligosaccharides with different degrees of acetylation were used. This chitosan oligosaccharide was prepared by degrading chitosan or chemically N-acetylating and modifying it, which is a very complex mixture. N-acetylglucosamine and glucosamine are randomly distributed in the chitosan oligosaccharide mixture, resulting in unstable salt tolerance activity of chitosan oligosaccharide in inducing plants. Moreover, the components of chitosan oligosaccharide prepared by different preparation methods are not exactly the same, resulting in inaccurate control of its dosage in application, which limits its application as a plant inducer. Summary of the Invention
[0005] To solve the above technical problems, the present invention provides an N-acetylated chitosan oligosaccharide for inducing plant salt tolerance activity, a preparation method thereof, and an application thereof, so as to achieve the purpose of using an enzymatic method to specify a chitosan oligosaccharide with a specific sequence arrangement, which has stable salt tolerance activity and helps to promote the development of efficient plant inducers.
[0006] To achieve the above purpose, the technical solution of the present invention is as follows:
[0007] An N-acetylated chitosan oligosaccharide for inducing plant salt tolerance activity has the following structural formula:
[0008]
[0009] A method for preparing N-acetylated chito-oligosaccharides with induced plant salt tolerance activity is as follows: reacting fully N-acetylated chito-oligosaccharides with chitin deacetylase ArCE4, desalting after the reaction, and freeze-drying to obtain N-acetylated chito-oligosaccharides with induced plant salt tolerance activity.
[0010] In the above scheme, the preparation method of chitin deacetylase ArCE4 is as follows: expressing the plasmid of chitin deacetylase ArCE4 heterologously in Escherichia coli, and obtaining the purified ArCE4 protein by means of low-temperature induction at 15-20 °C.
[0011] In a further technical scheme, at 34-40 °C, reacting fully N-acetylated chito-oligosaccharides and chitin deacetylase ArCE4 in a buffer solution with a cobalt chloride concentration of 5-20 μM, a concentration of tris(hydroxymethyl)aminomethane hydrochloride of 30-70 mM and a pH of 7-9 for 24-48 h, desalting after the reaction, and freeze-drying to obtain N-acetylated chito-oligosaccharides.
[0012] In a further technical scheme, the mass ratio of chitin deacetylase ArCE4 to fully N-acetylated chito-oligosaccharides is not less than 1:200, and 100-500 μL of buffer solution is added per milligram of fully N-acetylated chito-oligosaccharides.
[0013] In the above scheme, the preparation method of the fully N-acetylated chito-oligosaccharides is as follows: putting 1 g of chito-oligosaccharides into 100 mL of absolute ethanol, standing overnight, centrifuging to take the supernatant, and freeze-drying to obtain alcohol-precipitated fully N-acetylated chito-oligosaccharides.
[0014] An application of N-acetylated chito-oligosaccharides with induced plant salt tolerance activity as described above in the preparation of plant elicitors.
[0015] In the above scheme, the plant elicitor is used for spraying on the leaf surface of plants.
[0016] In the above scheme, the concentration of N-acetylated chito-oligosaccharides in the plant elicitor is 1-100 mg / L.
[0017] Through the above technical scheme, an N-acetylated chito-oligosaccharide with induced plant salt tolerance activity provided by the present invention, its preparation method and application have the following beneficial effects:
[0018] 1. The present invention uses chitin deacetylase to specifically remove the N-acetyl groups inside and at the non-reducing end of chito-oligosaccharides, avoiding the complexity and uncertainty of directly separating chito-oligosaccharides with different degrees of polymerization and chito-oligosaccharide isomers with different sequence arrangements, and can directly prepare the target product of chito-oligosaccharides with a specific sequence structure.
[0019] 2. In the present invention, the enzyme reaction conditions are mild, avoiding the generation of by-products and environmental pollution. It is a preparation method of an environmentally friendly and highly efficient chitosan oligosaccharide plant elicitor.
[0020] 3. The chitosan oligosaccharide with a specific sequence obtained in the present invention contains no nucleic acid and protein, has high purity, good water solubility, and high plant elicitor activity, and can be used for the development of agricultural biological products. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art.
[0022] Figure 1 It is the mass spectrometry diagram of fully N-acetylated chitosan oligosaccharide (CHOS) provided in Example 1 of the present invention.
[0023] Figure 2 It is the mass spectrometry diagram of N-acetylated chitosan oligosaccharide (A-COS) prepared by enzymatic hydrolysis with ArCE4 in Example 1 of the present invention.
[0024] Figure 3 It is the mass spectrometry diagram of N-acetylated chitosan oligosaccharide (N-COS) prepared by enzymatic hydrolysis with NodB in Example 1 of the present invention.
[0025] Figure 4 It is the mass spectrometry diagram of N-acetylated chitosan oligosaccharide (C-COS) prepared by enzymatic hydrolysis with VcCOS in Example 1 of the present invention.
[0026] Figure 5 It is the screening diagram of the salt tolerance induction activity of three N-acetylated chitosan oligosaccharides prepared in the present invention; (a) is the fresh weight index, and (b) is the change in malondialdehyde MDA. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0027] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention.
[0028] The present invention provides an N-acetylated chitosan oligosaccharide with salt tolerance induction activity in plants, its preparation method and application. The specific embodiments are as follows:
[0029] Example 1
[0030] I. Preparation of chitin deacetylase ArCE4:
[0031] Chitin deacetylase ArCE4 plasmid was added to competent cells BL21(DE3) respectively, and cultured with shaking at 37 °C for 2.5 - 5 h. The OD600 value was measured to be about 0.6 - 0.8. Then the temperature was reduced to 16 °C, and IPTG (isopropyl-β-D-thiogalactoside) was added to make the final concentration of IPTG 0.1 mM. After induction for 15 h, the bacterial cells were collected by centrifugation, and the cells were broken by ultrasound. Centrifugation was carried out at 8000 rpm for 100 min at 4 °C, and the supernatant was collected and passed through a nickel column to purify the protein, obtaining the purified chitin deacetylase ArCE4 protein.
[0032] II. Preparation of fully N-acetylated chito-oligosaccharides (CHOS)
[0033] 1 g of chito-oligosaccharides was put into 100 mL of absolute ethanol, left standing overnight, the supernatant was taken by centrifugation, and freeze-dried to obtain alcohol-precipitated fully N-acetylated chito-oligosaccharides (CHOS), which mainly contained fully N-acetylated chitobiose - chitopentaose, with molecular weights of 424 Da, 627 Da, 830 Da, and 1033 Da respectively. The characterization map is shown in Figure 1 , and the structural formula is as follows:
[0034]
[0035] III. Preparation of N-acetylated chito-oligosaccharides (A-COS) with plant salt tolerance induction activity
[0036] At 37 °C, fully N-acetylated chito-oligosaccharides (CHOS) and chitin deacetylase ArCE4 were reacted in a buffer solution with a concentration of 50 mM of tris(hydroxymethyl)aminomethane hydrochloride and a concentration of 10 μM of CoCl2, the pH was adjusted to 8, and the reaction was carried out for 40 h. After the reaction, desalting and freeze-drying were carried out to obtain A-COS; among them, the mass ratio of chitin deacetylase ArCE4 to fully N-acetylated chito-oligosaccharides was 1:160. The first-order mass spectrometry characterization of A-COS is shown in Figure 2 , its reducing-end sugar residue is N-acetylglucosamine, and at the same time, the structure with a residue of glucosamine in the middle of the sugar chain is retained, and the structural formula is as follows:
[0037]
[0038] Comparative Example 1
[0039] During the preparation of chitin deacetylase ArCE4 in Example 1, chitin deacetylase NodB plasmid was added to competent cells BL21(DE3), and chitin deacetylase NodB protein was prepared by the same method.
[0040] The fully N-acetylated chito-oligosaccharides (CHOS) prepared in Example 1 were used.
[0041] In the preparation process of N-acetylated chitosan oligosaccharide (A-COS) with plant salt tolerance induction activity in Example 1, chitin deacetylase ArCE4 was replaced with chitin deacetylase NodB, and the rest was the same as in Example 1, to obtain N-acetylated chitosan oligosaccharide (N-COS) with one acetyl group removed from the non-reducing sugar; the primary mass spectrometry characterization of N-COS is shown in Figure 3 , and its structural formula is as follows:
[0042]
[0043] Comparative Example 2
[0044] In the preparation process of chitin deacetylase ArCE4 in Example 1, chitin deacetylase VcCOD plasmid was added to competent cells BL21(DE3), and chitin deacetylase VcCOD protein was prepared by the same method.
[0045] The fully N-acetylated chitosan oligosaccharide (CHOS) prepared in Example 1 was used.
[0046] In the preparation process of N-acetylated chitosan oligosaccharide (A-COS) with plant salt tolerance induction activity in Example 1, chitin deacetylase ArCE4 was replaced with chitin deacetylase VcCOD, and the rest was the same as in Example 1, to obtain N-acetylated chitosan oligosaccharide (C-COS) with one acetyl group removed at the second position at the non-reducing end. The primary mass spectrometry characterization of C-COS is shown in Figure 4 , and its structural formula is as follows:
[0047]
[0048] Comparative Example 3
[0049] The fully N-acetylated chitosan oligosaccharide (CHOS) prepared in Example 1 was used. At 30 °C, CHOS and chitin deacetylase ArCE4 were reacted in a buffer solution with a concentration of 50 mM of tris(hydroxymethyl)aminomethane hydrochloride and a concentration of 10 μM of CoCl2. The mass ratio of chitin deacetylase ArCE4 to the fully N-acetylated chitosan oligosaccharide was 1:160, the pH was adjusted to 8, and the reaction was carried out for 40 h. After the reaction, desalting and freeze-drying were carried out. The reaction product was characterized by primary mass spectrometry analysis, and the reaction product contained chito-oligosaccharide products and a mixture of chitosan oligosaccharides with one or two acetyl groups removed. The reaction was incomplete, and the oligosaccharide sequence arrangement was still very complex.
[0050] Comparative Example 4
[0051] The fully N-acetylated chitosan oligosaccharide (CHOS) prepared in Example 1 was used. At 37 °C, CHOS and chitin deacetylase ArCE4 were reacted in a buffer solution with a concentration of 50 mM tris(hydroxymethyl)aminomethane hydrochloride and a concentration of 10 μM CoCl2. The mass ratio of chitin deacetylase ArCE4 to the fully N-acetylated chitosan oligosaccharide was 1:160, the pH was adjusted to 8, and the reaction was carried out for 20 h. After the reaction, desalting and freeze-drying were performed. The obtained N-acetylated chitosan oligosaccharide was characterized by first-order mass spectrometry. The reaction product contained a mixture of chitosan oligosaccharides with one to three acetyl groups removed, and the oligosaccharide sequence arrangement was still very complex.
[0052] Comparative Example 5
[0053] The fully N-acetylated chitosan oligosaccharide (CHOS) prepared in Example 1 was used. At 37 °C, CHOS and chitin deacetylase ArCE4 were reacted in a buffer solution with a concentration of 50 mM tris(hydroxymethyl)aminomethane hydrochloride and a concentration of 1 μM CoCl2. The mass ratio of chitin deacetylase ArCE4 to the fully N-acetylated chitosan oligosaccharide was 1:160, the pH was adjusted to 8, and the reaction was carried out for 40 h. After the reaction, desalting and freeze-drying were performed. The obtained N-acetylated chitosan oligosaccharide was characterized by first-order mass spectrometry. The reaction product contained chitooligosaccharide products and a mixture of chitosan oligosaccharides with one to two acetyl groups removed. The reaction was incomplete, and the oligosaccharide sequence arrangement was still very complex.
[0054] Comparative Example 6
[0055] The fully N-acetylated chitosan oligosaccharide (CHOS) prepared in Example 1 was used. At 37 °C, CHOS and chitin deacetylase ArCE4 were reacted in a buffer solution with a concentration of 50 mM tris(hydroxymethyl)aminomethane hydrochloride and a concentration of 10 μM CoCl2. The mass ratio of chitin deacetylase ArCE4 to the fully N-acetylated chitosan oligosaccharide was 1:160, the pH was adjusted to 10, and the reaction was carried out for 40 h. After the reaction, desalting and freeze-drying were performed. The obtained N-acetylated chitosan oligosaccharide was characterized by first-order mass spectrometry. The reaction product still mainly contained chitooligosaccharide products, and chitin deacetylase ArCE4 had almost no activity.
[0056] Salt resistance activity test:
[0057] Wheat seeds were germinated in the dark at 25°C for 24 h and then transferred to culture cups. The temperature was maintained at 25°C / 20°C, the day-night cycle was 14 / 10 h, the humidity was kept at 65 - 70%, and the nutrient solution was changed every other day. When the second leaf was fully developed, the wheat seedlings were randomly grouped, with three replicates in each experimental group, including a control group (CK, without NaCl but sprayed with distilled water), a negative control group (NaCl, treated with 100 mM NaCl and sprayed with distilled water), a positive control group (SA, treated with 100 mM NaCl and sprayed with 100 mg / L salicylic acid), and four sample groups (CHOS, N-COS, C-COS, and A-COS) all treated with 100 mM NaCl and sprayed with the corresponding samples at a concentration of 10 mg / L. After 10 days of treatment, the growth index (fresh weight) and the content of malondialdehyde (MDA) in the leaves of wheat seedlings were measured. The results are as Figure 5 shown, and it can be seen that A-COS has good activity in inducing plant salt stress resistance.
[0058] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. An N-acetylated chito-oligosaccharide for inducing plant salt resistance activity, characterized in that, Its structural formula is as follows:
2. An N-acetylated chitosan oligosaccharide for inducing plant salt resistance activity according to claim 1, characterized in that, The preparation method is as follows: React fully N-acetylated chitosan oligosaccharide with chitin deacetylase ArCE4, desalt and freeze-dry after the reaction to obtain N-acetylated chitosan oligosaccharide with the activity of inducing plant salt resistance.
3. The N-acetylated chito-oligosaccharide for inducing plant salt tolerance activity according to claim 2, characterized in that, The preparation method of the chitin deacetylase ArCE4 is as follows: The plasmid of chitin deacetylase ArCE4 is heterologously expressed in Escherichia coli, and the purified ArCE4 protein is obtained by the way of low-temperature induction at 15-20 °C.
4. The N-acetylated chito-oligosaccharide for inducing plant salt resistance activity according to claim 2, wherein At 34-40 °C, react fully N-acetylated chitosan oligosaccharide and chitin deacetylase ArCE4 in a buffer solution with a cobalt chloride concentration of 5-20 μM, a concentration of tris(hydroxymethyl)aminomethane hydrochloride of 30-70 mM and a pH of 7-9 for 24-48 h, desalt and freeze-dry after the reaction to obtain N-acetylated chitosan oligosaccharide.
5. An N-acetylated chitosan oligosaccharide for inducing plant salt resistance activity according to claim 4, characterized in that, The mass ratio of the chitin deacetylase ArCE4 to the fully N-acetylated chitosan oligosaccharide is not less than 1:200, and 100-500 μL of buffer solution is added per milligram of fully N-acetylated chitosan oligosaccharide.
6. The N-acetylated chito-oligosaccharide for inducing plant salt tolerance activity according to claim 2, wherein The preparation method of the fully N-acetylated chitosan oligosaccharide is as follows: Put 1 g of chito-oligosaccharide into 100 mL of absolute ethanol, let it stand overnight, centrifuge to take the supernatant, and freeze-dry to obtain alcohol-precipitated fully N-acetylated chitosan oligosaccharide.
7. Use of N-acetylated chitosan oligosaccharide with the activity of inducing plant salt resistance as described in claim 1 in the preparation of a plant elicitor.
8. The application according to claim 7, wherein The plant elicitor is used for spraying on the leaf surface of plants.
9. The application according to claim 7, wherein The concentration of N-acetylated chitosan oligosaccharide in the plant elicitor is 1-100 mg / L.