Application of chitosan oligosaccharide aminomethylthiobutyramide derivatives in plant resistance to salt stress

Abstract

The present invention provides an application of chitosan oligosaccharide aminomethylthiobutyramide derivatives in plant resistance to salt stress, belonging to the field of agricultural biotechnology. The derivatives are used to alleviate the oxidative damage of plants caused by salt stress, specifically including the following effects: (1) reducing the malondialdehyde content in plants; (2) increasing the activities of superoxide dismutase and peroxidase; and (3) promoting seed germination and seedling growth. The derivatives are formed by connecting the carboxyl group of methionine with the amino group of chitosan oligosaccharide through an amide bond, and the derivatives are 2-amino-4-methylthiobutyryl chitosan oligosaccharide, which is a methionine high-molecular chitosan oligosaccharide with a deacetylation degree of ≥90%. The polymerization degree of the methionine high-molecular chitosan oligosaccharide is 10-18. The present invention provides a methionine carrier for loading methionine into plants to improve the effect of plant resistance to salt stress.

Description

Technical Field

[0001] The invention belongs to the field of agricultural biotechnology, and specifically relates to application of chitosan oligosaccharide aminomethylthiobutyramide derivatives in plant resistance to salt stress. Background Art

[0002] When plants are exposed to salt stress, they produce large amounts of reactive oxygen species (ROS). These are free radicals and non-radical oxygen-containing molecules, including superoxide anions, hydrogen peroxide, singlet oxygen, and hydroxyl radicals. Because they contain unpaired electrons, free radicals are unstable. When excessive ROS production exceeds the cell's ability to clear them, it leads to oxidative stress. Oxidative stress is caused by the inability of biological systems to neutralize excess free radicals. Excessive free radicals attack cells and cause cell damage. Continuous attacks can lead to cell and tissue death, ultimately affecting plant growth and crop yields.

[0003] Methionine is the only sulfur-containing essential amino acid. Its residues are susceptible to redox reactions and are highly sensitive to reactive oxygen species (ROS). Methionine reacts with ROS to form methionine disulfone, which scavenges ROS in the body. Methionine disulfone can then be reduced to methionine by the specific enzyme methionine sulfoxide reductase, allowing for recycling and effectively reducing oxidative stress. Methionine can also undergo a transsulfuration biochemical pathway to form cysteine, which is then synthesized into glutathione, enhancing antioxidant enzyme activity. This helps scavenge ROS and reduce oxidative stress. However, when methionine is used directly, the -COOH group of methionine can be physiologically toxic to plants. Therefore, finding a suitable carrier to deliver methionine into plants is crucial for efficient delivery and to avoid the physiological toxicity of the carboxyl group (-COOH). Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides an application of chitosan oligosaccharide aminomethylthiobutyramide derivatives in plant resistance to salt stress.

[0005] Abbreviations used in this text:

[0006] HC (High molecular weight chitooligosaccharide): High molecular weight chitooligosaccharide

[0007] LC (Low molecular weight chitooligosaccharide): low molecular weight chitooligosaccharide

[0008] COS (Chitooligosaccharide): chitosan oligosaccharide

[0009] Met (Methionine): Methionine

[0010] EDC·HCl: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride

[0011] NHS: N-hydroxysuccinimide

[0012] MES (2-Morpholinoethanesulphonic acid): 2-Morpholinoethanesulfonic acid

[0013] SOD (superoxide dismutase): Superoxide dismutase

[0014] MDA (malonaldehyde): malondialdehyde

[0015] POD (peroxidase): peroxidase

[0016] MHC: methionine high molecular weight chitosan oligosaccharide

[0017] MLC: methionine low molecular weight chitosan oligosaccharide

[0018] The solution provided by the present invention is as follows:

[0019] A use of chitosan oligosaccharide aminomethylthiobutyramide derivatives in plant resistance to salt stress, wherein the derivatives are used to alleviate the oxidative damage caused by salt stress to plants;

[0020] The derivative is formed by connecting the carboxyl group of methionine and the amino group of chitosan oligosaccharide through an amide bond. The derivative is 2-amino-4-methylthiobutyryl chitosan oligosaccharide, and its structural formula is as follows:

[0021] .

[0022] Moreover, the derivatives are used to alleviate the oxidative damage to plants caused by salt stress, specifically including the following effects:

[0023] (1) Reduce the content of malondialdehyde in plants;

[0024] (2) Increase the activity of superoxide dismutase and peroxidase;

[0025] (3) Promote seed germination and seedling growth;

[0026] Moreover, the derivative is used to alleviate the oxidative damage of plants caused by salt stress by applying the derivative to the seeds of the plants by soaking the seeds for 24 hours.

[0027] Furthermore, the plant is oat, and the derivative is applied at a concentration of 0.1 mg / mL.

[0028] Moreover, the chitosan oligosaccharide is a methionine high molecular weight chitosan oligosaccharide with a deacetylation degree of ≥90%; and the polymerization degree of the methionine high molecular weight chitosan oligosaccharide is 10-18.

[0029] Moreover, the degree of substitution of the derivative was 74.15%, and the yield was 38.45%.

[0030] Furthermore, the preparation method of the derivative is as follows:

[0031] (1) Dissolve methionine in 2-morpholineethanesulfonic acid buffer solution at pH 5.5, add EDC·HCl and NHS, and stir to activate the carboxyl group;

[0032] (2) adding chitosan oligosaccharide to the reaction system of step (1) and reacting at 25-45°C for 24-48 hours;

[0033] (3) After the reaction is completed, the product is dialyzed using a dialysis bag with a molecular weight cutoff of 500 Da for 3-5 days, concentrated, and then freeze-dried to obtain the target derivative as a light yellow powder, namely, chitosan oligosaccharide aminomethylthiobutyramide derivatives.

[0034] The specific reaction route is as follows:

[0035] ;

[0036] in, .

[0037] Moreover, in step (1), the molar ratio of methionine, EDC·HCl, and NHS is 1:3:3, and the molar ratio of chitosan oligosaccharide to methionine is 1:1-1:3.

[0038] Moreover, the molar ratio of chitosan oligosaccharide to methionine was 1:3, the reaction temperature was 25° C., and the reaction time was 48 hours.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] 1. In the present invention, first, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC⋅HCl) reacts with the -COOH on methionine to form an O-acylurea intermediate, i.e., intermediate product 1; then, N-hydroxysuccinimide (NHS) is added to convert the O-acylurea intermediate in the reaction system into an active NHS ester that can react with an amino group, i.e., intermediate product 2; then, chitosan oligosaccharide is added, and the CO bond on the NHS ester is broken and combined with the -NH2 on the chitosan oligosaccharide to form an amide bond, thereby finally synthesizing chitosan oligosaccharide aminomethylthiobutyramide derivatives for plant salt stress resistance.

[0041] 2. The present invention provides a methionine carrier for carrying methionine into plants to play a role in resisting salt stress. The present invention uses chitosan oligosaccharide, an alkaline polysaccharide, to react the -COOH on methionine with the -NH2 on chitosan oligosaccharide to undergo an amide reaction, and methionine is grafted onto chitosan oligosaccharide to obtain a chitosan oligosaccharide aminomethylthiobutyramide derivative. The generated amide bond avoids the physiological toxicity of the carboxyl group (-COOH) of methionine to plants when methionine is used directly, and at the same time can increase the activity of superoxide dismutase (SOD) and peroxidase (POD). These two enzymes play a key role in scavenging reactive oxygen species (ROS) and reducing oxidative stress. The derivative is used to alleviate the oxidative damage to plants caused by salt stress, specifically including the following effects: (1) reducing the content of malondialdehyde in plants; (2) increasing the activity of superoxide dismutase and peroxidase; and (3) promoting seed germination and seedling growth.

[0042] 3. The present invention provides an application of chitosan oligosaccharide aminomethylthiobutyramide derivatives in plant resistance to salt stress. When used on oats and the derivative application concentration is 0.1 mg / mL, the germination potential of oats can reach 82.22% and the germination rate can reach 86.67%. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is a preparation route for chitosan oligosaccharide aminomethylthiobutyramide derivatives;

[0044] Figure 2 is the infrared spectrum of HC;

[0045] Figure 3 is the LC infrared spectrum;

[0046] Figure 4 is the infrared spectrum of MHC;

[0047] Figure 5 is the infrared spectrum of MLC;

[0048] Figure 6 is the MHC 13C NMR spectrum;

[0049] Figure 7 The germination potential of oats under salt stress of different derivatives;

[0050] Figure 8 is the germination rate of oats under salt stress of different derivatives;

[0051] Figure 9 MDA content of oats treated with MHC under salt stress;

[0052] Figure 10 The SOD activity of oats treated with MHC under salt stress;

[0053] Figure 11 The POD activity of oats treated with MHC under salt stress. DETAILED DESCRIPTION

[0054] Example 1 Synthesis of Chitosan Oligosaccharide Aminomethylthiobutyramide Derivatives

[0055] 1.1 Drugs used

[0056] All raw materials and reagents used were commercially available. High-molecular-weight chitosan oligosaccharides (n=10-18) were purchased from Qingdao Yunzhou Biotechnology Co., Ltd., with a DD>90%, and low-molecular-weight chitosan oligosaccharides (n=4-6) were purchased from MacLean Reagent Co., Ltd., with a DD>90%.

[0057] 1.2 Experimental Procedure

[0058] (1) Prepare 1000 mL of 0.1 mol / L 2-morpholineethanesulfonic acid (MES) buffer solution with a pH of 5.5.

[0059] (2) Weigh L-methionine (L-Met), add 100 mL of 2-morpholineethanesulfonic acid (MES) buffer solution, and stir continuously until completely dissolved.

[0060] (3) Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC⋅HCl) and N-hydroxysuccinimide (NHS) (molar ratio of L-methionine: EDC⋅HCl: NHS = 1:3:3) and start timed stirring for 3 h to activate the carboxyl groups.

[0061] (4) After 3 hours, chitosan oligosaccharide (the molar ratio of chitosan oligosaccharide to methionine is 1:3) is added and stirred at 25°C for 48 hours. The product is dialyzed in distilled water using a dialysis bag with a molecular weight cutoff of 500 Da for 3-5 days. The product is concentrated by rotary stirring to about 30 mL and placed in a refrigerator for 24 hours. The product is then freeze-dried for 60 hours to obtain a light yellow powder sample, which is the target derivative.

[0062] In this step, a dialysis bag with a molecular weight cutoff of 500 Da is used to remove unreacted methionine, EDC⋅HCl, and NHS. Chitosan oligosaccharides are macromolecules with a molecular weight greater than 1000 Da, and the molecular weight of the target chitosan oligosaccharide derivative will increase after the reaction. The purpose of using distilled water for dialysis is to more thoroughly remove small molecule impurities. Concentration removes excess water and reduces volume. Freezing is performed because the compound obtained in this invention is easily decomposed at high temperatures. Therefore, low-temperature freeze-drying is used to completely remove water, and the resulting solid powder is the finished product.

[0063] The specific reaction route is as follows Figure 1As shown in the figure: First, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC⋅HCl) reacts with the -COOH on methionine to form an O-acylurea intermediate, namely intermediate 1; then, N-hydroxysuccinimide (NHS) is added to convert the O-acylurea intermediate in the reaction system into an active NHS ester that can react with amino groups, namely intermediate 2; then, chitosan oligosaccharide is added, the CO bond on the NHS ester is broken and combines with the -NH2 on the chitosan oligosaccharide to form an amide bond, and finally chitosan oligosaccharide aminomethylthiobutyramide derivatives are synthesized.

[0064] Example 2 Optimal Preparation Conditions of Chitosan Oligosaccharide Aminomethylthiobutyramide Derivatives

[0065] The experiment used three factors and three levels L9(3 3 ) were used to screen the optimal reaction conditions. According to the preliminary experiments, the raw material molar ratio was 1:1, 1:2, and 1:3 as three levels; the temperature was 25 ℃, 35 ℃, and 45 ℃ as three levels; and the time was 24 h, 36 h, and 48 h as three levels. The optimal reaction conditions were screened, as shown in Table 1.

[0066] Table 1 Screening of optimal synthesis conditions for chitosan oligosaccharide aminomethylthiobutyramide derivatives

[0067]

[0068] The yield results indicate that the influence of various factors on the yield of chitosan oligosaccharide aminomethylthiobutyramide derivatives follows the order: temperature A (ratio) > time C (h) > temperature B (°C). The optimal conditions for preparing chitosan oligosaccharide aminomethylthiobutyramide derivatives are A3B1C3, i.e., a raw material molar ratio of 1:3, a temperature of 25°C, and a reaction time of 48 h. The maximum MHC yield reached 38.45%.

[0069] Example 3 Characterization of chitosan oligosaccharide aminomethylthiobutyramide derivatives

[0070] 3.1 Infrared spectra of chitosan oligosaccharide aminomethylthiobutyramide derivatives

[0071] Infrared scanning was performed on high molecular weight chitosan oligosaccharide (HC), low molecular weight chitosan oligosaccharide (LC), chitosan oligosaccharide aminomethylthiobutyramide derivatives (MHC) prepared using high molecular weight chitosan oligosaccharide, and chitosan oligosaccharide aminomethylthiobutyramide derivatives (MLC) prepared using low molecular weight chitosan oligosaccharide. The infrared spectra are shown in Figure 2-5 .

[0072] like Figure 2 The following is the infrared spectrum of high molecular weight chitosan oligosaccharide (n=10-18), its characteristic infrared (cm -1): 3245, 2885, 1604, 1507, 1378, 1063.

[0073] like Figure 3 The following is the infrared spectrum of low molecular weight chitosan oligosaccharide (n=4-6), its characteristic infrared (cm -1 ): 3245, 2887, 1613, 1512, 1380, 1065.

[0074] like Figure 4 The following is the infrared spectrum of 2-amino-4-methylthiobutyryl chitosan oligosaccharide obtained by reacting high molecular weight chitosan oligosaccharide (n=10-18) with methionine. Its characteristic infrared (cm -1 ): 3281, 2917, 1636, 1531, 1375, 1238, 1059, 575.

[0075] like Figure 5 The following is the infrared spectrum of 2-amino-4-methylthiobutyryl chitosan oligosaccharide obtained by reacting low molecular weight chitosan oligosaccharide (n=4-6) with methionine. Its characteristic infrared (cm -1 ): 3279, 2916, 1635, 1529, 1374, 1236, 1057, 574.

[0076] Figure 4 and Figure 2 In comparison, the -1 The characteristic broad peaks of OH and NH groups at 1507 cm-1 were significantly shifted, indicating that NH may react; -1 The characteristic absorption peak of -NH2 at the position is strengthened, indicating that -NH2 has undergone amide reaction; Figure 4 At 1636 cm -1 The "amide I peak" is the stretching vibration absorption peak of C=O of the amide compound; 1531cm -1 The "amide II peak" is the bending vibration absorption peak of CNH; 1375 cm -1 The "amide III peak" is located at 575 cm, which is a mixed peak consisting of CN stretching vibration and NH bending vibration; -1 The peak at 3281 cm is the "amide VI peak", which is caused by the out-of-plane bending vibration of the secondary amide carbonyl group; -1 At 2917 cm is the NH stretching vibration; -1 An obvious saturated CH stretching vibration absorption peak appears at

[0077] Figure 5 and Figure 3 In comparison, the -1The characteristic broad peaks of OH and NH groups at 1512 cm -1 The characteristic absorption peak of -NH2 at the position is strengthened, indicating that -NH2 has undergone amide reaction; Figure 5 At 1635 cm -1 The "amide I peak" is the stretching vibration absorption peak of C=O of the amide compound; the "amide II peak" is at 1529 cm-1, which is the bending vibration absorption peak of CNH; the "amide II peak" is at 1374 cm-1, which is the bending vibration absorption peak of CNH; -1 The "amide III peak" is located at 574 cm, which is a "mixed peak" containing CN stretching vibration and NH bending vibration; -1 The peak at 3279 cm is the "amide VI peak", which is caused by the out-of-plane bending vibration of the secondary amide carbonyl group; -1 The NH stretching vibration is at 2916 cm -1 An obvious saturated CH stretching vibration absorption peak appears at

[0078] 3.2 Chitosan oligosaccharide aminomethylthiobutyramide derivatives 13 C NMR spectroscopy

[0079] use 13 C NMR further confirmed the structure of the derivative. Figure 6 For MHC 13 C NMR spectrum. Analysis showed that 173.0 ppm was the chemical shift of the C=O carbon of the amide bond; δ=101.5, 54.3, 68.2, 75.0, 73.2, and 62.9 ppm were the chemical shifts of C1-C6 on the chitosan oligosaccharide molecule; and 44.6, 36.6, 31.0, and 16.0 were the chemical shifts of the Met carbon chain.

[0080] Based on the above analysis, the characteristic peaks of each functional group of the target derivative are basically the same, and the absorption intensities of the characteristic peaks are different. It can be preliminarily determined that MHC was successfully synthesized.

[0081] 3.3 Elemental analysis

[0082] In this experiment, the percentages of the five elements C, H, N, O, and S in the derivatives were determined, and the degree of substitution of each derivative was calculated, as shown in Table 2.

[0083] Table 2 Elemental analysis results and degree of substitution of derivatives

[0084]

[0085] Table 2 shows that the substitution degrees of MLC and MHC are 72.57% and 74.15%, respectively; the yields are 36.45% and 38.45%, respectively, which proves that the target compounds were successfully synthesized.

[0086] Example 4 Application of chitosan oligosaccharide aminomethylthiobutyramide derivatives in the salt stress resistance of oats

[0087] 4.1 Study on the effect of chitosan oligosaccharide aminomethylthiobutyramide derivatives on the resistance of oat seeds to salt stress

[0088] 4.1.1 Research Methods

[0089] Oat seeds with plump grains and similar growth levels were selected for disinfection. They were then soaked in 0.1% HgCl₂ solution for 10 min, rinsed three times with deionized water, and dried with absorbent paper. Using the paper germination bed method, 100 mmol / L NaCl solution was added to each Petri dish until the filter paper was saturated. Seeds soaked in distilled water served as a control. Seeds were then soaked in chitosan oligosaccharides and their aminomethylthiobutyramide derivatives at concentrations of 0.05 mg / mL, 0.1 mg / mL, 0.3 mg / mL, 0.5 mg / mL, 0.7 mg / mL, and 0.9 mg / mL for 24 h. The seeds were rinsed three times with deionized water and dried with absorbent paper. The soaked seeds were placed in pre-treated Petri dishes, with 30 seeds per dish, and incubated in an artificial climate chamber. Germination was observed and recorded daily. Germination potential was measured on the third day, and germination rate was measured on the seventh day. Three replicates were performed for each treatment, and the average value was calculated.

[0090] Germination potential (GE) = (number of seeds germinated on the 3rd day / number of test seeds) × 100%

[0091] Germination rate (GR) = (number of seeds germinated on the 7th day / total number of test seeds) × 100%

[0092] 4.1.2 Experimental Results

[0093] See Figure 7 :Germination potential of oats under salt stress of different derivatives, Figure 8 : Germination rate of oats under salt stress with different derivatives.

[0094] from Figure 7 As can be seen from the results, almost all oat seeds soaked in chitosan oligosaccharides and their derivatives had higher germination potential than oat seeds under moderate salt stress conditions. In other words, chitosan oligosaccharides and their derivatives have a germination and growth promoting effect on oat seeds under salt stress. Compared with the distilled water control, MHC showed a very good salt stress resistance. At a concentration of 0.1 mg / mL, its germination potential was the highest, reaching 82.22%.

[0095] from Figure 8As can be seen in the results, the growth rate of oat seeds soaked in almost all chitosan oligosaccharides and their derivatives was higher than that of oat seeds under moderate salt stress conditions. In other words, under salt stress, these chitosan oligosaccharides and their derivatives have a germination and growth promoting effect on oat seeds. Compared with the distilled water control, MHC showed a very good salt stress resistance. At a concentration of 0.1mg / mL, its germination rate can reach 86.67%.

[0096] 4.2 Physiological and biochemical effects of chitosan oligosaccharide aminomethylthiobutyramide derivatives on salt stress resistance of oat seedlings

[0097] 4.2.1 Seedling cultivation methods

[0098] Vermiculites of different sizes were mixed and placed in a seedling pot, 300 mL of nutrient solution was added, seeds soaked in the best salt-resistant chitosan oligosaccharide derivatives were sown, and the seeds were cultured in an artificial climate chamber. Nutrient solution was added every 3 days, and the culture was carried out for 15 days. On the 15th day, the seedlings were collected and stored in a -80°C ultra-low temperature refrigerator for later use.

[0099] 4.2.2 Determination method

[0100] (1) Malondialdehyde (MDA) determination

[0101] Approximately 0.1 g of oat seedlings stored at -80°C were placed in a mortar and ground with 2.5 mL of distilled water. Once thoroughly ground, the sample was transferred to a centrifuge tube and rinsed with 2.5 mL of distilled water. The sample was then heated at 100°C for 15 minutes in 5 mL of 0.5% thiobarbituric acid-trichloroacetic acid solution and immediately cooled in an ice bath. The cooled solution was centrifuged at 8000 rpm for 10 minutes. The supernatant was collected and its absorbance was measured at 450 nm, 532 nm, and 600 nm. MDA content was calculated according to the formula.

[0102] MDA concentration C (μmol / L) = 6.452 × (A 532 -A 600 )-0.56×A 450

[0103] (2) SOD and POD determination

[0104] SOD and POD activity assays are based on the SOD and POD kit instructions. First, weigh approximately 0.1 g of oat seedling sample stored at -80°C, add 1 mL of the extract, and grind on ice. Once thoroughly ground, transfer the sample to a centrifuge tube. Rinse the mortar with 1 mL of the extract, centrifuge at 8000 rpm for 10 minutes at 4°C, and place the supernatant on ice for assay. Subsequent assay steps are based on the SOD and POD kit instructions.

[0105] 4.2.3 Experimental Results

[0106] (1) Malondialdehyde (MDA)

[0107] Malondialdehyde is a peroxidation product of membrane fatty acids and is often used as an important indicator of lipid peroxidation to reflect oxidative damage to cells under stress environments. Figure 9 As shown in the results, compared with H2O2 (blank control), NaCl treatment significantly increased the MDA content in oat seedlings (P < 0.05), by 18.9% compared to the H2O2 (blank control), indicating that NaCl treatment increases cell membrane permeability and leads to lipid peroxidation. Compared with the NaCl-treated group, HC, Met, and MHC all significantly reduced MDA content (P < 0.05), by 38.6%, 31.8%, and 37.4%, respectively. This suggests that MHC can reduce MDA content in oat seedlings under salt stress and protect oat cell membranes from oxidative damage. Furthermore, the experimental results show that MDA content in the MHC treatment was lower than that in the H2O2 (blank control), indicating that the MHC treatment was even more effective than the H2O2 (blank control). Its protective effect on cell membrane lipid peroxidation can even offset the adverse effects of salt stress, a highly significant effect.

[0108] (2) Superoxide dismutase (SOD)

[0109] SOD is crucial for scavenging superoxide radicals and preventing cellular oxidative damage under abiotic stress. Upregulation of SOD activity is an important measure for plants to combat oxidative stress. Figure 10 The results show the SOD activity of MHC and its raw materials in oat seedlings under salt stress. Compared with H₂O (blank control), NaCl treatment significantly increased SOD activity in oat seedlings (P < 0.05), reaching a 91.9% increase compared to the H₂O (blank control), indicating that oats, under salt stress, increase their own SOD activity to resist salt-induced oxidative stress. Compared with the NaCl treatment, HC, Met, and MHC all significantly increased SOD activity (P < 0.05), by 35.0%, 11.6%, and 38.4%, respectively. This indicates that MHC can enhance SOD activity in oat seedlings under salt stress, alleviate ROS production, and reduce the damage caused by oxidative stress. The order of SOD activity was MHC > HC > Met > NaCl, indicating that the MHC treatment group had the highest SOD activity, indicating its strongest ability to scavenge reactive oxygen species (ROS) produced by cell membrane peroxidation and the best salt tolerance.

[0110] (3) Peroxidase (POD)

[0111] Under salt stress, peroxidase (POD) activity increases significantly, which is crucial for balancing oxygen free radicals and maintaining metabolic efficiency. Figure 11As shown, compared with H₂O₂ (blank control), NaCl treatment significantly increased POD activity in oat seedlings (P < 0.05), by 22.2% compared to the H₂O₂ (blank control), indicating that oats, under salt stress, increase their POD activity to protect against salt-induced oxidative stress. Compared with the NaCl treatment, both HC and MHC significantly increased POD activity (P < 0.05), by 20.7% and 160.6%, respectively. The MHC treatment was 2.6 times more effective than the NaCl treatment, indicating that MHC significantly enhances POD activity in oat seedlings under salt stress, alleviating ROS production and reducing oxidative stress damage. The order of POD activity was MHC > HC > Met > NaCl, indicating that MHC treatment had the highest POD activity, suggesting that it can scavenge reactive oxygen species (ROS) produced by cell membrane peroxidation, protect the membrane system, and enhance salt tolerance.

[0112] The above experiments show that chitosan oligosaccharides loaded with methionine (chitosan oligosaccharide aminomethylthiobutyramide derivatives) can significantly reduce the MDA content of oat seedlings under salt stress and protect cell membrane lipid peroxidation; they can significantly increase the activity of oat SOD and POD, effectively eliminate reactive oxygen species, protect the cell membrane system, and have good resistance to salt stress.

Claims

1. An application of chitosan oligosaccharide aminomethylthiobutyramide derivatives in plant resistance to salt stress, characterized in that: The derivatives are used to alleviate the oxidative damage to plants caused by salt stress, specifically to increase the activity of peroxidase; The derivative is formed by connecting the carboxyl group of methionine and the amino group of chitosan oligosaccharide through an amide bond. The derivative is 2-amino-4-methylthiobutyryl chitosan oligosaccharide, and its structural formula is as follows: ； in, ; The plant is oat, and the application concentration of the derivative is 0.1 mg / mL; The chitosan oligosaccharide is a high-molecular-weight chitosan oligosaccharide with a deacetylation degree greater than 90%; and the polymerization degree of the high-molecular-weight chitosan oligosaccharide is 10-18.

2. The use of a chitosan oligosaccharide aminomethylthiobutyramide derivative in plant resistance to salt stress according to claim 1, characterized in that: The preparation method of the derivative is as follows: (1) Dissolve methionine in 2-morpholineethanesulfonic acid buffer solution at pH 5.5, add EDC·HCl and NHS, and stir to activate the carboxyl group; (2) adding chitosan oligosaccharide to the reaction system of step (1) and reacting at 25-45°C for 24-48 hours; (3) After the reaction is completed, the product is dialyzed using a dialysis bag with a molecular weight cutoff of 500 Da for 3-5 days, concentrated, and then freeze-dried to obtain the target derivative as a light yellow powder, namely, chitosan oligosaccharide aminomethylthiobutyramide derivatives.

3. The use of a chitosan oligosaccharide aminomethylthiobutyramide derivative in plant resistance to salt stress according to claim 1, characterized in that: The derivative is used to alleviate the oxidative damage to plants caused by salt stress by applying the derivative to the seeds of the plants by soaking the seeds for 24 hours.

4. The use of a chitosan oligosaccharide aminomethylthiobutyramide derivative in plant resistance to salt stress according to claim 1, characterized in that: The degree of substitution of the derivative is 74.15%, and the yield is 38.45%.

5. The use of a chitosan oligosaccharide aminomethylthiobutyramide derivative in plant resistance to salt stress according to claim 2, characterized in that: The molar ratio of methionine, EDC·HCl and NHS is 1:3:3, and the molar ratio of chitosan oligosaccharide and methionine is 1:1-1:

3.

6. The use of a chitosan oligosaccharide aminomethylthiobutyramide derivative in plant resistance to salt stress according to claim 5, characterized in that: The molar ratio of chitosan oligosaccharide to methionine was 1:3, the reaction temperature was 25°C, and the reaction time was 48 hours.

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