Application of chitosan oligosaccharide amino methylthio butyramide derivative in plant salt stress resistance

Abstract

The invention provides an application of a chitosan oligosaccharide amino methylthio butyramide derivative in plant salt stress resistance, and belongs to the technical field of agricultural biologication.The derivative is used for relieving oxidative damage of salt stress to plants and specifically has the following effects that (1) the content of malondialdehyde in plants is reduced; (2) the activity of superoxide dismutase and peroxidase is improved; (3) promoting seed germination and seedling growth; the derivative is formed by connecting carboxyl of methionine and amino of chitosan oligosaccharide through an amido bond, the derivative is 2-amino-4-methylthio butyryl chitosan oligosaccharide, the chitosan oligosaccharide is methionine polymer chitosan oligosaccharide, and the deacetylation degree is larger than or equal to 90%; the polymerization degree of the methionine polymer chitosan oligosaccharide is 10 to 18; the invention provides a methionine carrier which is used for loading methionine into plants and improving the salt stress resistance of the plants.

Description

Technical Field

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

[0002] When plants are under salt stress, they will produce a large amount of reactive oxygen species (ROS) by themselves. Reactive oxygen species are free radical and non-free radical oxygen-containing molecules, including superoxide anion, hydrogen peroxide, singlet oxygen and hydroxyl radical. Because they contain unpaired electrons, free radicals are unstable. When reactive oxygen species are overproduced and exceed the self-cleaning ability of cells, it will lead 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 yield.

[0003] Methionine is the only sulfur-containing essential amino acid, and its residue is prone to redox reactions and is very sensitive to reactive oxygen species (ROS). Methionine reacts with reactive oxygen species to form methionine disulfone to scavenge reactive oxygen species in the body. Methionine disulfone can also be reduced to methionine under specific methionine sulfoxide reductase, so it can act cyclically to effectively reduce oxidative stress. At the same time, methionine can also form cysteine through the transsulfuration biochemical pathway, and then synthesize glutathione to enhance the activity of antioxidant enzymes. It plays a role in scavenging reactive oxygen species and reducing oxidative stress. However, when directly using methionine, the -COOH of methionine will cause some physiological toxicity to plants. Therefore, it is necessary to find a suitable carrier to load methionine into plants to achieve efficient delivery of methionine and avoid the physiological toxicity of its carboxyl group (-COOH) to plants. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides the application of a chitosan oligosaccharide aminomethanethiobutyramide derivative in plant salt stress resistance.

[0005] Abbreviations used in the text: HC (High molecular weight chitooligosaccharide): High molecular weight chitosan oligosaccharide LC (Low molecular weight chitooligosaccharide): Low molecular weight chitosan oligosaccharide COS (Chitooligosaccharide): Chitosan oligosaccharide Met (Methionine): Methionine EDC·HCl: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride NHS: N-Hydroxysuccinimide MES(2-Morpholinoethanesulphonic acid): 2-Morpholinoethanesulfonic acid SOD(superoxide dismutase): Superoxide Dismutase MDA(malonaldehyde): Malondialdehyde POD(peroxidase): Peroxidase MHC: Methionine high molecular weight chitosan oligosaccharide MLC: Methionine low molecular weight chitosan oligosaccharide The solution provided by the present invention is as follows: Application of a chitosan oligosaccharide carbamoylthio butyramide derivative in plant resistance to salt stress, wherein the derivative is used to alleviate the oxidative damage of salt stress to plants; 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: .

[0006] Moreover, the derivative is used to alleviate the oxidative damage of salt stress to plants, and specifically includes the following effects: (1) Reducing the content of malondialdehyde in plants; (2) Increasing the activities of superoxide dismutase and peroxidase; (3) Promoting seed germination and seedling growth; Moreover, the way the derivative is used to alleviate the oxidative damage of salt stress to plants is: applying it to the seeds of plants by the method of seed soaking, and the seed soaking time is 24 hours.

[0007] Moreover, the plant is oats, and the application concentration of the derivative is 0.1 mg / mL.

[0008] Moreover, the chitosan oligosaccharide is methionine high molecular weight chitosan oligosaccharide, and the degree of deacetylation is ≥90%; the degree of polymerization of the methionine high molecular weight chitosan oligosaccharide is 10 - 18.

[0009] Moreover, the substitution degree of the derivative is 74.15%, and the yield is 38.45%.

[0010] Moreover, the preparation method of the derivative is as follows: (1) Dissolve methionine in a 2-morpholinoethanesulfonic acid buffer solution with pH = 5.5, add EDC·HCl and NHS, and stir to activate the carboxyl group; (2) Add chitosan oligosaccharide to the reaction system in step (1), and react at 25 - 45 °C for 24 - 48 hours; After the reaction is completed, the product is dialyzed with a dialysis bag having a molecular weight cut-off of 500 Da for 3 - 5 days, concentrated and then freeze-dried to obtain a target derivative in the form of a pale yellow powder, namely a chitosan carbamoylthio butyramide derivative.

[0011] The specific reaction route is as follows: ; Among them, .

[0012] Moreover, in step (1), the molar ratio of methionine, EDC·HCl, and NHS is 1:3:3, and the molar ratio of chitosan to methionine is 1:1 - 1:3.

[0013] Moreover, when the molar ratio of chitosan to methionine is 1:3, the reaction temperature is 25 °C, and the reaction time is 48 hours.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. In the present invention, first, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC⋅HCl) reacts with -COOH on methionine to form an O-acylurea intermediate, namely intermediate 1; then, N-hydroxysuccinimide (NHS) is added, which can convert the O-acylurea intermediate in the reaction system into an active NHS ester capable of reacting with an amino group, namely intermediate 2; then, chitosan is added, and the C-O bond on the NHS ester breaks and combines with -NH2 on chitosan to form an amide bond, finally synthesizing a chitosan carbamoylthio butyramide derivative for plant salt stress resistance.

[0015] 2. The present invention provides a carrier for methionine, which is used to load methionine into plants to play a role in salt stress resistance. The present invention uses chitosan, an alkaline polysaccharide, to carry out an amide reaction between -COOH on methionine and -NH2 on chitosan, graft methionine onto chitosan, and obtain a chitosan carbamoylthio butyramide derivative. The generated amide bond can avoid the physiological toxicity of the carboxyl group (-COOH) of directly using methionine to plants, and at the same time can improve the activities of superoxide dismutase (SOD) and peroxidase (POD). These two enzymes play a key role in scavenging reactive oxygen species (ROS) and reducing oxidative stress; using this derivative to alleviate the oxidative damage of salt stress to plants specifically includes the following effects: (1) reducing the content of malondialdehyde in plants; (2) increasing the activities of superoxide dismutase and peroxidase; (3) promoting seed germination and seedling growth.

[0016] 3. The present invention provides an application of a chitosan carbamoylthio butyramide derivative in plant salt stress resistance. When applied to oats, when the application concentration of the derivative 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

[0017] Figure 1 is the preparation route of the chitosan carbamoylthio butyramide derivative; Figure 2 is the HC infrared spectrum; Figure 3 is the LC infrared spectrum; Figure 4 is the MHC infrared spectrum; Figure 5 is the MLC infrared spectrum; Figure 6 is the 13C nuclear magnetic resonance spectrum of MHC; Figure 7 is the germination potential of oats under salt stress with different derivatives; Figure 8 is the germination rate of oats under salt stress with different derivatives; Figure 9 is the MDA content of oats treated with MHC under salt stress; Figure 10 is the SOD activity of oats treated with MHC under salt stress; Figure 11 is the POD activity of oats treated with MHC under salt stress. DETAILED DESCRIPTION OF THE INVENTION

[0018] Example 1 Synthesis of Chitosan Carbamoylthio Butyramide Derivative 1.1 Chemicals Used The raw materials and reagents used are all conventional raw materials and reagents on the market. Among them, high-molecular chitosan (n = 10 - 18) was purchased from Qingdao Yunzhou Biotechnology Co., Ltd., with a DD>90%; low-molecular chitosan (n = 4 - 6) was purchased from Macklin Reagent Co., Ltd., with a DD>90%.

[0019] 1.2 Experimental Procedures (1) Prepare 1000 mL of 2-morpholinoethanesulfonic acid (MES) buffer solution with a concentration of 0.1 mol / L and a pH of 5.5.

[0020] (2) Weigh L-methionine (L-Met) and add it to 100 mL of 2-morpholinoethanesulfonic acid (MES) buffer solution, and stir continuously until it is completely dissolved.

[0021] (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), start timing and stir for 3 h to activate the carboxyl group.

[0022] (4) After 3 h, add chitosan oligosaccharide (molar ratio of chitosan oligosaccharide to methionine is 1:3), stir and react at 25 °C for 48 h. Dialyze using a dialysis bag with a molecular weight cut-off of 500 Da in distilled water for 3 - 5 days, rotary concentrate to about 30 mL, place in the refrigerator and freeze for 24 h, then perform freeze-drying for 60 h to obtain a pale yellow powdery sample, which is the target derivative.

[0023] In this step, the dialysis bag with a "molecular weight cut-off of 500 Da" is used to remove the unreacted part of methionine, EDC·HCl and NHS. Chitosan oligosaccharide itself is a macromolecule 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 dialysis with "distilled water" is to remove small molecule impurities more thoroughly. "Concentration" can remove excess water and reduce the volume. "Freezing" is because the compound obtained in the present invention is easily decomposed at high temperature, so water is thoroughly removed by low-temperature freeze-drying, and the resulting solid powder is the finished product.

[0024] The specific reaction route is as Figure 1 shown: First, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) reacts with -COOH on methionine to form an O-acylurea intermediate, that is, intermediate 1; then, add N-hydroxysuccinimide (NHS), which can convert the O-acylurea intermediate in the reaction system into an active NHS ester that can react with amino groups, that is, intermediate 2; then, add chitosan oligosaccharide, the C - O bond on the NHS ester breaks and combines with -NH2 on chitosan oligosaccharide to form an amide bond, and finally synthesize chitosan oligosaccharide carbamoylthio butyramide derivatives.

[0025] Example 2 Optimal preparation conditions of chitosan oligosaccharide carbamoylthio butyramide derivatives The experiment uses a three-factor three-level L9(3 3 ) to screen the optimal reaction conditions. According to the previous experiment, the raw material molar ratios of 1:1, 1:2, and 1:3 are used as the three levels; the temperatures of 25 °C, 35 °C, and 45 °C are used as the three levels; the times of 24 h, 36 h, and 48 h are used as the three levels to screen the optimal reaction conditions, as shown in Table 1.

[0026] Table 1 Screening of the optimal synthesis conditions of chitosan oligosaccharide carbamoylthio butyramide derivatives

[0027] According to the yield results, the influence of each factor on the yield of chitosan carbamoylthio butyramide derivatives is as follows: temperature A (ratio) > time C (h) > temperature B (°C). The optimal conditions for preparing chitosan carbamoylthio butyramide derivatives are A3B1C3, that is, the raw material molar ratio is 1:3, the temperature is 25 °C, and the time is 48 h. The highest yield of MHC can reach 38.45%.

[0028] Example 3 Characterization of chitosan carbamoylthio butyramide derivatives 3.1 Infrared spectra of chitosan carbamoylthio butyramide derivatives High molecular weight chitosan (HC), low molecular weight chitosan (LC), chitosan carbamoylthio butyramide derivatives prepared using high molecular weight chitosan (MHC), and chitosan carbamoylthio butyramide derivatives prepared using low molecular weight chitosan (MLC) were respectively subjected to infrared scanning, and the infrared spectra are shown in Figures 2 - 5 .

[0029] As Figure 2 shown, it is the infrared spectrum of high molecular weight chitosan (n = 10 - 18), and its characteristic infrared (cm -1 ) are: 3245, 2885, 1604, 1507, 1378, 1063.

[0030] As Figure 3 shown, it is the infrared spectrum of low molecular weight chitosan (n = 4 - 6), and its characteristic infrared (cm -1 ) are: 3245, 2887, 1613, 1512, 1380, 1065.

[0031] As Figure 4 shown, it is the infrared spectrum of 2-amino-4-methylthiobutyryl chitosan obtained by reacting high molecular weight chitosan (n = 10 - 18) with methionine, and its characteristic infrared (cm -1 ) are: 3281, 2917, 1636, 1531, 1375, 1238, 1059, 575.

[0032] As Figure 5 shown, it is the infrared spectrum of 2-amino-4-methylthiobutyryl chitosan obtained by reacting low molecular weight chitosan (n = 4 - 6) with methionine, and its characteristic infrared (cm -1 ) are: 3279, 2916, 1635, 1529, 1374, 1236, 1057, 574.

[0033] Figure 4 Compared with Figure 2 , in the range of 3500 - 3200 cm -1The characteristic broad peaks of O-H and N-H groups at [specific location] show an obvious shift, indicating that N-H may react; the characteristic absorption peak of -NH2 at 1507 cm -1 is strengthened, indicating that the amide reaction of -NH2 has occurred; Figure 4 In [specific compound], at 1636 cm -1 is the "amide I peak", which is the stretching vibration absorption peak of C=O of the amide compound; at 1531 cm -1 is the "amide II peak", which is the bending vibration absorption peak of C-N-H; at 1375 cm -1 is the "amide III peak", which is a "mixed peak" containing C-N stretching vibration and N-H bending vibration; at 575 cm -1 is the "amide VI peak", which is the out-of-plane bending vibration of the secondary amide carbonyl group; at 3281 cm -1 is the N-H stretching vibration; at 2917 cm -1 an obvious saturated C-H stretching vibration absorption peak appears.

[0034] Figure 5 Compared with Figure 3 , the characteristic broad peaks of O-H and N-H groups at 3500 - 3200 cm -1 show an obvious shift, indicating that N-H may react; the characteristic absorption peak of -NH2 at 1512 cm -1 is strengthened, indicating that the amide reaction of -NH2 has occurred; Figure 5 In [specific compound], at 1635 cm -1 is the "amide I peak", which is the stretching vibration absorption peak of C=O of the amide compound; at 1529 cm-1 is the "amide II peak", which is the bending vibration absorption peak of C-N-H; at 1374 cm -1 is the "amide III peak", which is a "mixed peak" containing C-N stretching vibration and N-H bending vibration; at 574 cm -1 is the "amide VI peak", which is the out-of-plane bending vibration of the secondary amide carbonyl group; at 3279 cm -1 is the N-H stretching vibration; at 2916 cm -1 an obvious saturated C-H stretching vibration absorption peak appears.

[0035] 3.2 Chitosan oligosaccharide carbamoylthio butyramide derivatives 13 13C nuclear magnetic resonance spectrum Using 13 13C nuclear magnetic resonance to further determine the structure of the derivative, Figure 6 for MHC's 1313C nuclear magnetic resonance spectrum. Analysis shows that: the chemical shift of the C=O carbon of the amide bond is 173.0 ppm; the chemical shifts of C1-C6 on the chitosan oligosaccharide molecule are at δ = 101.5, 54.3, 68.2, 75.0, 73.2, 62.9 ppm; the chemical shifts of the Met carbon chain are 44.6, 36.6, 31.0, 16.0.

[0036] 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 has been successfully synthesized.

[0037] 3.3 Elemental analysis In this experiment, the percentage contents of five elements, C, H, N, O, and S, in the derivative were determined, and the degree of substitution of each derivative was calculated, as shown in Table 2.

[0038] Table 2 Elemental analysis results and degree of substitution of the derivative

[0039] The degrees of substitution of MLC and MHC listed in Table 2 are 72.57% and 74.15% respectively; the yields are 36.45% and 38.45% respectively, which can prove the successful synthesis of the target compound.

[0040] Example 4 Application of chitosan oligosaccharide carbamoylthio butyramide derivatives in the salt stress resistance of oats 4.1 Study on the salt stress resistance of chitosan oligosaccharide carbamoylthio butyramide derivatives to oat seeds 4.1.1 Research method Select oat seeds with plump grains and similar growth degrees for disinfection treatment. Soak them in 0.1% HgCl2 solution for 10 min, then rinse them 3 times with deionized water, and blot the surface moisture of the seeds with absorbent paper. Using the paper germination bed method, add 100 mmol / L NaCl solution into the petri dish until the filter paper is saturated. Use distilled water soaking treatment as the control, and soak the seeds with chitosan oligosaccharide and its carbamoylthio butyramide derivatives. The concentrations are set at 0.05 mg / mL, 0.1 mg / mL, 0.3 mg / mL, 0.5 mg / mL, 0.7 mg / mL, 0.9 mg / mL for 24 h. Rinse the seeds 3 times with deionized water, blot the surface moisture with absorbent paper, put the soaked seeds into the pre-treated petri dish, place 30 seeds in each dish, and culture them in an artificial climate chamber. Observe and record the germination situation regularly every day. Measure the germination potential on the third day and the germination rate on the seventh day. Repeat each treatment 3 times and take the average value.

[0041] Germination potential (GE) = (number of germinated seeds on the 3rd day / number of tested seeds) × 100% Germination rate (GR) = (number of germinated seeds on the 7th day / total number of tested seeds) × 100% 4.1.2 Experimental results See Figure 7 : Oat germination potential under salt stress of different derivatives Figure 8 : Oat germination rate under salt stress of different derivatives

[0042] It can be seen from Figure 7 that the germination potential of almost all oat seeds soaked with chitosan oligosaccharide and its derivatives is higher than that of oat seeds under moderate salt stress. That is to say, under salt stress, chitosan oligosaccharide and its derivatives have the effect of promoting the germination and growth of oat seeds. Compared with the distilled water control, MHC shows a very good salt stress resistance effect. When the concentration is 0.1 mg / mL, its germination potential is the largest, which can reach 82.22%.

[0043] It can be seen from Figure 8 that the growth rate of almost all oat seeds soaked with chitosan oligosaccharide and its derivatives is higher than that of oat seeds under moderate salt stress. That is to say, under salt stress, these chitosan oligosaccharide and its derivatives have the effect of promoting the germination and growth of oat seeds. Compared with the distilled water control, MHC shows a very good salt stress resistance effect. When the concentration is 0.1 mg / mL, its germination rate can reach 86.67%.

[0044] 4.2 Physiological and biochemical study on the salt stress resistance of chitosan oligosaccharide carbamoylthio butyramide derivatives in oat seedlings 4.2.1 Seedling cultivation method Mix vermiculite of different sizes and place it in a seedling cultivation pot. Add 300 mL of nutrient solution, and sow the seeds soaked with the chitosan oligosaccharide derivative with the best salt resistance effect selected. Place it in an artificial climate chamber for cultivation, add nutrient solution once every 3 days, cultivate for 15 days, and collect the seedlings on the 15th day and store them in a -80°C ultra-low temperature refrigerator for later use.

[0045] 4.2.2 Determination method (1) Determination of malondialdehyde (MDA) Take about 0.1 g of oat seedling samples stored in a -80°C ultra-low temperature refrigerator and put them into a mortar. First, grind them with 2.5 mL of distilled water. After thorough grinding, transfer them to a centrifuge tube, and then rinse the mortar with 2.5 mL of distilled water. Add 5 mL of 0.5% thiobarbituric acid trichloroacetic acid solution and heat it at 100°C for 15 min, then immediately cool it in an ice bath. The cooling solution is centrifuged at 8000 r / min for 10 min, and the absorbance values of the supernatant are measured at 450 nm, 532 nm, and 600 nm respectively, and the MDA content is calculated according to the formula.

[0046] MDA concentration C (μmol / L) = 6.452×(A 532 -A 600 ) - 0.56×A 450 (2)Determination of SOD and POD The determination methods of SOD and POD activities refer to the instructions of the SOD and POD kits. First, weigh about 0.1 g of oat seedling samples stored in an ultra-low temperature freezer at -80 °C, add 1 mL of extraction solution and grind in an ice bath. After thorough grinding, transfer to a centrifuge tube, then rinse the mortar with 1 mL of extraction solution, centrifuge at 8000 r / min for 10 min at 4 °C, and take the supernatant and place it in an ice bath for further measurement. The subsequent measurement steps refer to the instructions of the SOD and POD kits.

[0047] 4.2.3 Experimental results (1)Malondialdehyde (MDA) Malondialdehyde is the peroxidation product of membrane lipid fatty acids and is often used as an important indicator of lipid peroxidation to reflect the oxidative damage of cells under stress conditions. As Figure 9 shown, compared with H2O (blank control), the MDA content in oat seedlings treated with NaCl increased significantly (P < 0.05), which was 18.9% higher than that of H2O (blank control), indicating that NaCl treatment could increase cell membrane permeability and lead to lipid peroxidation. Compared with the NaCl treatment group, HC, Met, and MHC could all significantly reduce the MDA content (P < 0.05), by 38.6%, 31.8%, and 37.4% respectively, indicating that MHC could reduce the MDA content in oat seedlings under salt stress and protect the oat cell membrane from oxidative damage. In addition, from the experimental results, the MDA content treated with MHC was lower than that of H2O (blank control), that is to say, the effect of MHC treatment was even higher than that of H2O (blank control), and its protective effect on cell membrane lipid peroxidation could even offset the adverse effect of salt stress, and the effect was very obvious.

[0048] (2)Superoxide dismutase (SOD) SOD is crucial for scavenging superoxide free radicals and preventing cell oxidative damage under abiotic stress. The up-regulation of SOD activity is an important measure for plants to combat oxidative stress. Figure 10It is the SOD activity of oats seedlings with MHC and its raw materials under salt stress. Compared with H2O (blank control), NaCl treatment significantly increased the SOD activity of oats seedlings (P < 0.05), which was 91.9% higher than that of H2O (blank control), indicating that oats increased SOD by itself to resist the oxidative stress caused by salt stress under salt stress. Compared with the NaCl treatment group, HC, Met, and MHC could all significantly increase the SOD activity (P < 0.05), increasing by 35.0%, 11.6%, and 38.4% respectively, indicating that MHC could increase the SOD activity of oats seedlings under salt stress, alleviate the production of ROS, and reduce the damage caused by oxidative stress. And the order of SOD activity was MHC > HC > Met > NaCl, indicating that the screened MHC treatment group had the strongest SOD activity, indicating that it had the strongest ability to scavenge the reactive oxygen species generated by membrane lipid peroxidation and the best salt tolerance effect.

[0049] (3)Peroxidase (POD) Under salt stress, the activity of peroxidase (POD) increased significantly, which was crucial for balancing oxygen free radicals and maintaining metabolic efficiency. As Figure 11 shown, compared with H2O (blank control), NaCl treatment significantly increased the POD activity of oats seedlings (P < 0.05), which was 22.2% higher than that of H2O (blank control), indicating that oats increased POD by itself to resist the oxidative stress caused by salt stress under salt stress. Compared with the NaCl treatment group, HC and MHC could both significantly increase the POD activity (P < 0.05), increasing by 20.7% and 160.6% respectively, and the MHC treatment was 2.6 times that of the NaCl treatment, indicating that MHC could significantly increase the POD activity of oats seedlings under salt stress, alleviate the production of ROS, and reduce the damage caused by oxidative stress. And the order of POD activity was MHC > HC > Met > NaCl, indicating that the MHC treatment group had the strongest POD activity, indicating that it could scavenge the reactive oxygen species generated by membrane lipid peroxidation, protect the membrane system, and improve the salt tolerance ability.

[0050] The above experiments comprehensively proved that chitosan oligosaccharide loaded with methionine (chitosan oligosaccharide carbamoylthio butyramide derivatives) could significantly reduce the MDA content of oats seedlings under salt stress, protect the membrane lipid peroxidation; could significantly increase the SOD and POD activities of oats, effectively scavenge reactive oxygen species, protect the membrane system, and had good salt stress resistance ability.

Claims

1. Application of a chitosan oligosaccharide carbamoylthio butyramide derivative in plant salt stress resistance, characterized in that, The derivative is used to alleviate the oxidative damage of salt stress to plants; The derivative is formed by connecting the carboxyl group of methionine with 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: 。 2. Use of a chitosan oligosaccharide carbamoylthiobutyramide derivative according to claim 1 in plant resistance to salt stress, characterized in that, The way in which the derivative is used to alleviate the oxidative damage of salt stress to plants is: applying it to the seeds of plants by the method of seed soaking, and the seed soaking time is 24 hours.

3. Use of a chitosan oligosaccharide aminomethanethiobutyramide derivative according to claim 1 in plant resistance to salt stress, characterized in that, The plant is oats, and the application concentration of the derivative is 0.1 mg / mL.

4. Use of a chitosan oligosaccharide aminomethanethiol butyramide derivative according to claim 1 in plant salt stress resistance, characterized in that, The derivative is used to alleviate the oxidative damage of salt stress to plants, and specifically includes the following effects: (1) Reducing the content of malondialdehyde in plants; (2) Improving the activities of superoxide dismutase and peroxidase; (3) Promoting seed germination and seedling growth.

5. Use of a chitosan oligosaccharide aminomethanethiol butyramide derivative according to claim 1 in plant salt stress resistance, characterized in that, The chitosan oligosaccharide is methionine high-molecular chitosan oligosaccharide, and the degree of deacetylation is ≥90%; the degree of polymerization of the methionine high-molecular chitosan oligosaccharide is 10-18.

6. Use of a chitosan oligosaccharide aminomethanethiol butyramide derivative according to claim 1 in plant resistance to salt stress, characterized in that, The substitution degree of the derivative is 74.15%, and the yield is 38.45%.

7. Use of a chitosan oligosaccharide aminomethanethiobutyramide derivative according to claim 1 in plant salt stress resistance, characterized in that, The preparation method of the derivative is as follows: (1) Dissolve methionine in 2-(N-morpholino)ethanesulfonic acid buffer solution with pH = 5.5, add EDC·HCl and NHS, and stir to activate the carboxyl group; (2) Add chitosan oligosaccharide to the reaction system in step (1), and react at 25-45 °C for 24-48 hours; (3) After the reaction is completed, dialyze the product with a dialysis bag with a molecular weight cut-off of 500 Da for 3-5 days, concentrate it, and then freeze-dry it to obtain the target derivative in the form of a pale yellow powder, that is, chitosan oligosaccharide aminomethionyl butyramide derivative.

8. Use of a chitosan oligosaccharide carbamoylthio butyramide derivative according to claim 7 in plant salt stress resistance, characterized in that, 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.

9. Use of a chitosan oligosaccharide aminomethanethiol butyramide derivative according to claim 8 in plant resistance to salt stress, characterized in that, The molar ratio of chitosan oligosaccharide to methionine is 1:3, the reaction temperature is 25 °C, and the reaction time is 48 hours.

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