Preparation method of p-hydroxymethylpeptide and application of p-hydroxymethylpeptide in agriculture

Abstract

The invention discloses a preparation method of p-hydroxymethylpeptide and application of the p-hydroxymethylpeptide in agriculture, and relates to the technical field of biochemistry and agriculture, and the p-hydroxymethylpeptide is prepared by carrying out reflux reaction on L-serine and ethylene glycol in anhydrous DMF (Dimethyl Formamide) at 130 DEG C for 10 hours, cooling, filtering, alternately washing with ethanol and water, and carrying out vacuum drying; the p-hydroxymethylpeptide can also be prepared by the following steps: carrying out fermentation culture on the trichonema, centrifuging, washing, freeze-drying, carrying out methanol ultrasonic extraction to obtain a crude extract, and carrying out macroporous resin and reversed-phase chromatography separation and purification; the p-hydroxymethylpeptide provided by the invention is simple in synthesis process, mild in reaction condition, high in product purity and suitable for large-scale preparation; the p-hydroxymethylpeptide-based potassium composition for promoting crop absorption has specificity on crop potassium synergism, shows good stability on crops such as wheat and the like, has a good effect and has a good crop popularization and application prospect.

Description

Technical Field

[0001] This invention relates to the fields of biochemistry and agricultural technology, specifically to a method for preparing p-hydroxymethylpeptide and its application in agriculture. Background Technology

[0002] Potassium is an essential macronutrient for plant growth and development, participating in various physiological metabolic processes such as osmotic regulation, enzyme activation, and protein synthesis. Currently, agricultural production commonly suffers from problems such as irrational potassium fertilizer application and low utilization rates, leading to resource waste, increased production costs, and environmental risks such as soil salinization. Therefore, developing bioactive substances that can efficiently improve the absorption and utilization of potassium in plants, thus achieving potassium-reduced cultivation, has become an important direction for sustainable agricultural development, possessing significant economic and ecological implications.

[0003] Currently, some plant growth regulators, such as brassinolide and sodium nitrophenolate, have been used to enhance crop stress resistance and nutrient absorption. However, their specific promoting effect on potassium absorption is limited, and their stability under different crop and soil conditions is insufficient. Furthermore, there are no reports in the existing technology of significantly enhancing potassium absorption at low concentrations by regulating the plant potassium transport system through specific cyclic ester peptide structures.

[0004] Therefore, there is an urgent need to propose a method for preparing hydroxymethyl peptides and their application in agriculture to solve the problems of low potassium fertilizer utilization rate in existing technologies and the poor specificity and limited effect of existing plant growth regulators on potassium enhancement. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing p-hydroxymethyl peptide and its application in agriculture, in order to solve the problems of low potassium fertilizer utilization rate in the prior art and the poor specificity and limited effect of existing plant growth regulators on potassium enhancement.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing p-hydroxymethylpeptide, comprising the following steps:

[0007] S1. Place L-serine and ethylene glycol in a molar ratio of 1:1.5-2 in a single-necked round-bottom flask, then add anhydrous N,N-dimethylformamide as a solvent, and stir magnetically until the solid is completely dissolved to obtain a mixed solution.

[0008] S2. The mixed solution was reacted in an oil bath at 130°C under reflux for 10 hours. After the reaction was completed, it was cooled to room temperature to obtain the reactants.

[0009] S3. Transfer the reactants to a sand core funnel for vacuum filtration to obtain a solid product;

[0010] S4. Wash the solid product alternately with pre-cooled anhydrous ethanol and pre-cooled deionized water, then transfer the washed solid product to a petri dish and dry it in a vacuum drying oven for 8 hours to obtain p-hydroxymethylpeptide.

[0011] This invention also discloses a method for preparing p-hydroxymethylpeptide, comprising the following steps:

[0012] A1. Inoculate *C. 42391* of *C. 42391* into a fermentation medium and ferment at 25–30°C and 150–220 rpm for 4–7 days to obtain the fermentation broth.

[0013] A2. Centrifuge the fermentation broth to collect the mycelium. Then wash the mycelium with distilled water and freeze-dry it to obtain mycelium powder. Then crush the mycelium powder with methanol using ultrasound. After centrifugation, vacuum concentration, rotary evaporation and freeze-drying, the crude extract is obtained.

[0014] A3. Using macroporous resin for initial separation, the crude extract was dissolved in distilled water, and then purified by reversed-phase chromatography to elute and separate it, obtaining a collection solution. The collection solution was then concentrated and freeze-dried to obtain p-hydroxymethylpeptide.

[0015] The present invention also discloses the application of p-hydroxymethyl peptide prepared by the above preparation method in agriculture.

[0016] Furthermore, the application is the use of p-hydroxymethylpeptide in the preparation of compositions that promote potassium absorption by crops.

[0017] Furthermore, the potassium absorption-promoting composition for crops contains p-hydroxymethylpeptide and an agriculturally acceptable carrier; the concentration of p-hydroxymethylpeptide in the potassium absorption-promoting composition for crops is 5–200 ng / mL.

[0018] Furthermore, the agriculturally acceptable carrier is at least one of deionized water, N,N-dimethylformamide, ethanol, kaolin, diatomaceous earth, and bentonite.

[0019] Furthermore, the potassium-enhancing composition for crops is applied by foliar spraying or root irrigation.

[0020] Furthermore, the crop is at least one of Arabidopsis thaliana, wheat, corn, rice, tomato, pepper, and peanut.

[0021] Compared with existing technologies, the method for preparing p-hydroxymethylpeptide and its application in agriculture provided by this invention have the following beneficial effects:

[0022] (1) The p-hydroxymethyl peptide synthesis process provided by the present invention is simple, the reaction conditions are mild, the product has high purity, and it is suitable for large-scale preparation.

[0023] (2) The p-hydroxymethyl peptide provided by the present invention can significantly promote the absorption and utilization of potassium by crops at extremely low concentrations, and its effect is better than that of traditional plant growth regulators.

[0024] (3) The potassium-enhancing composition based on p-hydroxymethyl peptide provided by the present invention has specific effects on enhancing the potassium absorption of crops, exhibits good stability on crops such as wheat, and has good effects and has good prospects for crop promotion and application.

[0025] (4) The potassium-absorbing composition based on p-hydroxymethyl peptide provided by the present invention is suitable for various application methods, such as foliar spraying and root irrigation. It is convenient to use and environmentally friendly. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0027] Figure 1 This is a process route diagram for the synthesis of p-hydroxymethylpeptide provided in Embodiment 1 of the present invention. Detailed Implementation

[0028] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0029] Example 1:

[0030] Please see Figure 1 A method for preparing p-hydroxymethylpeptide specifically includes the following steps:

[0031] S1. Place L-serine and ethylene glycol in a molar ratio of 1:1.5-2 in a single-necked round-bottom flask, then add anhydrous N,N-dimethylformamide as a solvent, and stir magnetically until the solid is completely dissolved to obtain a mixed solution.

[0032] S2. The mixed solution was reacted in an oil bath at 130°C under reflux for 10 hours. After the reaction was completed, it was cooled to room temperature to obtain the reactants.

[0033] S3. Transfer the reactants to a sand core funnel for vacuum filtration to obtain a solid product;

[0034] S4. Wash the solid product alternately with pre-cooled anhydrous ethanol and pre-cooled deionized water, then transfer the washed solid product to a petri dish and dry it in a vacuum drying oven for 8 hours to obtain p-hydroxymethylpeptide.

[0035] The specific implementation method is as follows: accurately weigh 10.0 mmol (1.05 g) of L-serine and 15.0 mmol (0.93 mL) of ethylene glycol, and add them sequentially to a dry 250 mL single-necked round-bottom flask equipped with a reflux condenser and a magnetic stirrer. Then add 50 mL of anhydrous N,N-dimethylformamide (DMF) as the reaction solvent, turn on the magnetic stirrer, and set the speed to 100-200 rpm. After the solid is completely dissolved and a homogeneous reaction system is formed, a mixed solution is obtained.

[0036] The reaction oil bath temperature was set to 130℃ to keep the mixed solution under mild reflux. Under these conditions, the reaction was stirred continuously for 10 hours to promote the esterification reaction between the serine carboxyl group and the ethylene glycol hydroxyl group and the subsequent intramolecular cyclization process. After the reaction was completed, the heat source was removed and the reaction system was allowed to cool naturally to room temperature to obtain the reactants. At this time, a white solid was observed to precipitate in the reactants.

[0037] The reactants were transferred to a sand core funnel for vacuum filtration, and the solid substances were collected to obtain the solid product.

[0038] The solid product was washed alternately with pre-cooled anhydrous ethanol (10 mL three times) and pre-cooled deionized water (10 mL twice) to thoroughly remove residual solvent, unreacted raw materials, and linear byproducts. The resulting wet solid product was then transferred to a petri dish and placed in a vacuum drying oven at 40 °C and -0.095 MPa for 8 hours to obtain a white crystalline powder, namely p-hydroxymethylpeptide, with a mass of approximately 0.92 g. The synthetic route is as follows: Figure 1 As shown. Preliminary proton nuclear magnetic resonance (NMR) spectrum (… 1 H NMR confirmed that its characteristic structure was consistent with expectations, and its structure is as follows:

[0039]

[0040] Example 2:

[0041] A method for preparing p-hydroxymethylpeptide specifically includes the following steps:

[0042] A1. Scytalidium circinatum with accession number CGMCC No.42391 was inoculated onto PDA slant for activation, and then transferred to PDB liquid fermentation medium for fermentation culture. The culture was carried out at 28℃ and 160r / min for 7 days to obtain a fermentation broth rich in p-hydroxymethylpeptide.

[0043] A2. Centrifuge the fermentation broth at 8000×g for 15 min to collect the mycelium. Then wash the mycelium with distilled water and freeze-dry it to obtain mycelium powder. Then, ultrasonically break up the mycelium powder with 70%wt methanol, centrifuge to collect the supernatant, and remove the organic solvent by rotary evaporation of the supernatant through vacuum concentration. Freeze-dry to obtain crude extract.

[0044] A3. Using macroporous resin for initial separation, the crude extract was dissolved in distilled water and loaded onto an HP-20 resin column. Impurities were first eluted with water, followed by a gradient elution of the target component with 30% ethanol. Reversed-phase chromatography was used for purification. The eluent was separated by HPLC, and the collected solution was concentrated and lyophilized. 1 H NMR identification confirmed that the cyclic dipeptide compound was p-hydroxymethylpeptide.

[0045] Example 3:

[0046] A composition for promoting potassium absorption in crops, comprising p-hydroxymethylpeptide and deionized water, is prepared by the following method:

[0047] Under stirring conditions, 1.0 g of the p-hydroxymethylpeptide prepared in Example 1 or Example 2 was slowly added to 1 L of deionized water and stirred continuously until completely dissolved to obtain a stock solution with a concentration of 1 mg / mL (1,000,000 ng / mL). The prepared stock solution should be stored in a cool, dark place.

[0048] The mother liquor is further diluted with deionized water to prepare a solution with a concentration of 5-200 ng / mL, which is the composition for promoting potassium absorption by crops. The concentration of p-hydroxymethyl peptide in the composition for promoting potassium absorption by crops can be 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL, or 200 ng / mL.

[0049] Example 4:

[0050] Screening of effective concentrations of p-hydroxymethylpeptide in compositions that promote potassium absorption in crops:

[0051] (1) Experimental design:

[0052] This experiment was conducted indoors in pots, using Arabidopsis thaliana, wheat, corn, rice, tomato, pepper, and peanut as experimental materials. A substrate consisting of peat moss, vermiculite, and perlite in a volume ratio of 2:1:1 was used, with 350g of soil placed in each pot (10×10×8cm). Eight treatments were set up for each crop: water control (CK treatment group), treatment 1: 5 ng / mL of p-hydroxymethylpeptide, treatment 2: 10 ng / mL of p-hydroxymethylpeptide, treatment 3: 20 ng / mL of p-hydroxymethylpeptide, treatment 4: 50 ng / mL of p-hydroxymethylpeptide, treatment 5: 100 ng / mL of p-hydroxymethylpeptide, treatment 6: 150 ng / mL of p-hydroxymethylpeptide, and treatment 7: 200 ng / mL of p-hydroxymethylpeptide. All treatment groups were uniformly basal applied with urea (0.058 g / pot) and superphosphate (0.035 g / pot). The available potassium content of the substrate was less than 30 mg / kg. No potassium fertilizer was applied to any treatment group to create a stable and consistent low potassium stress environment. At the three-leaf stage of the crop, 100 mL of the corresponding concentration of p-hydroxymethylpeptide was applied once. After 21 days, the aboveground dry weight and total potassium content of the plants were measured, and the potassium accumulation was calculated. The optimal effective concentration of p-hydroxymethylpeptide in each crop was determined by analysis of variance and multiple comparisons.

[0053] (2) Experimental results:

[0054] Table 1. Effects of different concentrations of hydroxymethylpeptide on the dry weight of different plants.

[0055] Dry weight of plant material (g) Arabidopsis wheat corn rice tomato chili peanut CK 0.02 0.1 0.32 0.15 0.5 0.4 0.35 p-hydroxymethyl 5 ng / mL 0.026 0.115 0.35 0.165 0.55 0.45 0.385 p-hydroxymethyl 10 ng / mL 0.035 0.145 0.42 0.195 0.625 0.51 0.435 p-hydroxymethyl 20 ng / mL 0.032 0.17 0.48 0.225 0.7 0.565 0.485 p-hydroxymethyl 50 ng / mL 0.028 0.155 0.57 0.21 0.775 0.645 0.55 p-hydroxymethyl 100 ng / mL 0.024 0.14 0.535 0.19 0.84 0.605 0.615 p-hydroxymethyl 150 ng / mL 0.022 0.125 0.48 0.175 0.79 0.565 0.575 p-hydroxymethyl 200 ng / mL 0.02 0.11 0.43 0.165 0.73 0.525 0.54

[0056] Table 2. Effects of different concentrations of hydroxymethylpeptide on total potassium content in different plants.

[0057] Total potassium content of plants (% DW) Arabidopsis wheat corn rice tomato chili peanut CK 0.95 1.15 1.25 1.1 1.45 1.55 1.2 p-hydroxymethyl 5 ng / mL 1.25 1.35 1.45 1.3 1.75 1.85 1.45 p-hydroxymethyl 10 ng / mL 1.65 1.58 1.68 1.52 2.08 2.18 1.68 p-hydroxymethyl 20 ng / mL 1.55 1.85 1.95 1.78 2.35 2.45 1.88 p-hydroxymethyl 50 ng / mL 1.35 1.65 2.25 1.62 2.65 2.75 2.05 p-hydroxymethyl 100 ng / mL 1.2 1.52 2.05 1.48 2.85 2.58 2.25 p-hydroxymethyl 150 ng / mL 1.05 1.42 1.88 1.38 2.65 2.45 2.08 p-hydroxymethyl 200 ng / mL 0.98 1.35 1.75 1.32 2.48 2.35 1.95

[0058] As shown in Tables 1 and 2, p-hydroxymethylpeptide has a certain promoting effect on crop growth and potassium absorption under low potassium stress. The dry weight and total potassium content showed an overall trend of first increasing and then decreasing with increasing concentration. The 20-50 ng / mL range was the broad-spectrum effective concentration range, with 20 ng / mL being the optimal concentration, which is suitable for crops such as wheat, corn, rice, tomato, pepper, and peanut. The effect of p-hydroxymethylpeptide has a concentration threshold effect, with the effect being weaker below 5 ng / mL and decreasing above 100 ng / mL.

[0059] Example 5:

[0060] Synergistic effect of p-hydroxymethylpeptide on wheat at different potassium levels:

[0061] (1) Experimental design:

[0062] This example is an indoor pot experiment using spring wheat 'Kenpi Mai 12' as the experimental material. The substrate used was a mixture of peat moss, vermiculite, and perlite in a 2:1:1 volume ratio. This substrate had an available potassium content below 30 mg / kg, thus creating a stable and consistent low-potassium stress environment. 10×10×8cm plastic pots were used, each precisely filled with 350g of potassium. Six treatment groups were set up, containing three potassium fertilizer gradients: Treatment Group 1: Low potassium (K1, 0 kg / hm²) 2 Group 2: Low potassium (K1, 0 kg / hm) + clean water, treatment group 2: 2 Treatment 3: 20 ng / mL p-hydroxymethylpeptide (K1-CO), treatment 3: normal potassium (K2, 90 kg / hm) 2 + clean water, treatment 4: normal potassium (K2, 90 kg / hm) 2 Treatment 5: High potassium (K3, 270 kg / hm²) + 20 ng / mL p-hydroxymethylpeptide (K2-CO), Treatment 5: High potassium (K3, 270 kg / hm²) 2 + clean water, treatment 6: high potassium (K3, 270 kg / hm) 2 ) + 20 ng / mL p-hydroxymethylpeptide (K3-CO), wherein the amount of water used for irrigation in treatment groups 1, 3, and 5 was equal to the amount of p-hydroxymethylpeptide solution used for irrigation in treatment groups 2, 4, and 6.

[0063] Each treatment group was treated with urea at a uniform standard (150 kg / hm²). 2 ) and superphosphate (90 kg / hm 2 As a base fertilizer, all fertilizers should be thoroughly mixed with the soil. Sow 10 seeds per pot, and after germination, thin to 5 seedlings per pot. Cultivate in an artificial climate chamber (day / night temperature 25 / 18℃, photoperiod 14h / d), maintaining soil moisture content at 70% field capacity using a weighing method. Apply as a fertigation treatment at the three-leaf stage of wheat, using a 20 ng / mL p-hydroxymethylpeptide solution or an equal volume of water at a concentration of 500 mL / m³. 2 The dosage was evenly applied to the soil; after 21 days of treatment, samples were taken to measure the aboveground dry weight of the plants, the dry weight of the roots, the total potassium content, and the SPAD of the leaves. By calculating the total potassium content and potassium accumulation, and combining with physiological data, the synergistic effect of hydroxymethyl peptide under different potassium environments was analyzed.

[0064] (2) Experimental results:

[0065] Table 3 Effects of p-hydroxymethylpeptide on wheat seedling growth at different potassium levels

[0066] deal with Plant height (cm) Root length (cm) Fresh weight on the ground (g) Fresh weight underground (g) Dry weight on the ground (g) Root dry weight (g) K1-CK 18.52±0.85c 10.28±0.67c 0.15±0.012c 0.14±0.011c 0.030±0.0024c 0.025±0.0020c K1-C0 22.18±0.73b 15.42±0.89b 0.25±0.018b 0.25±0.016b 0.050±0.0036b 0.045±0.0029b K2-CK 24.35±0.71a 22.81±1.15a 0.38±0.022a 0.35±0.019a 0.076±0.0044a 0.063±0.0034a K2-C0 26.07±0.68a 24.53±1.08a 0.46±0.025a 0.42±0.021a 0.092±0.0050a 0.076±0.0038a K3-CK 22.94±0.78b 20.15±1.02ab 0.32±0.020b 0.30±0.018ab 0.064±0.0040b 0.054±0.0032ab K3-C0 24.88±0.75a 22.38±1.12a 0.40±0.023a 0.38±0.020a 0.080±0.0046a 0.068±0.0036a

[0067] Note: K1-CK: Low potassium treatment, K1-CO: Low potassium + p-hydroxymethylpeptide treatment, K2-CK: Normal potassium treatment, K2-CO: Normal potassium + p-hydroxymethylpeptide treatment, K3-CK: High potassium treatment, K3-CO: High potassium + p-hydroxymethylpeptide treatment

[0068] Table 4. Effects of p-hydroxymethylpeptide on potassium uptake and accumulation in wheat plants under different potassium levels.

[0069] deal with Total potassium content (%) Potassium accumulation (mg / plant) K1-CK 1.35±0.06d 4.05±0.38e K1-C0 2.05±0.07c 10.25±0.85d K2-CK 3.20±0.07b 24.32±1.65c K2-C0 3.55±0.08ab 32.66±2.10b K3-CK 3.75±0.08a 24.00±1.72c K3-C0 4.10±0.08a 32.80±2.15b

[0070] Note: K1-CK: Low potassium treatment, K1-CO: Low potassium + p-hydroxymethylpeptide treatment, K2-CK: Normal potassium treatment, K2-CO: Normal potassium + p-hydroxymethylpeptide treatment, K3-CK: High potassium treatment, K3-CO: High potassium + p-hydroxymethylpeptide treatment

[0071] In Tables 3 and 4, different lowercase letters indicate significant differences at the 0.05 level. Combining the data from Tables 3 and 4, the indoor pot experiment using spring wheat 'Kenpi Mai 12' as the experimental material shows that 20 ng / mL of p-hydroxymethylpeptide can produce a positive synergistic effect on wheat seedling growth and potassium absorption and accumulation under three different potassium levels: low potassium, normal potassium, and high potassium. The synergistic effect is particularly significant under low potassium stress.

[0072] Under low potassium conditions (K1, 0 kg / hm) 2 Compared with the water control (K1-CK), the wheat plant height in the treatment group treated with p-hydroxymethylpeptide (K1-CO) increased from 18.52 cm to 22.18 cm, root length increased significantly from 10.28 cm to 15.42 cm, aboveground fresh weight and underground fresh weight increased from 0.15 g and 0.14 g to 0.25 g and 0.25 g, respectively, and aboveground dry weight and root dry weight also increased from 0.030 g and 0.025 g to 0.050 g and 0.045 g, respectively, with comprehensive improvement in growth indicators. At the same time, the total potassium content increased from 1.35% to 2.05%, and the potassium accumulation increased significantly from 4.05 mg / plant to 10.25 mg / plant, effectively alleviating the inhibition of wheat growth and potassium absorption by low potassium stress.

[0073] Under normal potassium conditions (K2, 90 kg / hm) 2 Under normal potassium levels, the plant height of the p-hydroxymethylpeptide treatment group (K2-C0) increased slightly from 24.35 cm to 26.07 cm, the root length increased from 22.81 cm to 24.53 cm, the aboveground fresh weight, underground fresh weight and dry weight all increased significantly, the total potassium content increased from 3.20% to 3.55%, and the potassium accumulation increased from 24.32 mg / plant to 32.66 mg / plant, further enhancing the growth of wheat and its potassium absorption and accumulation capacity under normal potassium levels.

[0074] Under high potassium conditions (K3, 270 kg / hm) 2Under the condition of hydroxymethylpeptide treatment (K3-C0), the group still showed a significant improvement over the control (K3-CK). The plant height increased from 22.94 cm to 24.88 cm, the root length increased from 20.15 cm to 22.38 cm, and all fresh and dry weight indicators were improved. The total potassium content increased from 3.75% to 4.10%, and the potassium accumulation increased from 24.00 mg / plant to 32.80 mg / plant. Moreover, its potassium accumulation was close to that of the normal potassium + hydroxymethylpeptide treatment group.

[0075] In summary, p-hydroxymethylpeptide can not only effectively alleviate the adverse effects of low potassium stress on wheat, but also synergistically promote wheat growth and potassium absorption and accumulation under normal and high potassium conditions. This indicates that the promoting effect of p-hydroxymethylpeptide on potassium absorption in wheat is not limited by potassium level and has broad applicability.

[0076] Example 6:

[0077] Effects of p-hydroxymethylpeptide on potassium reduction and synergistic effects in potted wheat:

[0078] (1) Experimental design: This embodiment uses the pot method, with spring wheat 'Kenpi Mai 12' as the material, and is carried out in an artificial mixed substrate of peat moss, vermiculite and perlite in a volume ratio of 2:1:1. The available potassium content of this substrate is less than 30 mg / kg, which can create a stable and consistent low potassium environment; 10×10×8cm flower pots are used, and each pot is filled with 350g of soil. Five treatment groups are set up, including treatment group 1: normal potassium (T1, K2O 90kg / hm) 2 +Clean water), Treatment Group 2: Potassium reduction (T2, K2O 63kg / hm) 2 +Clean water), Treatment Group 3: Potassium reduction (T3, K2O 63kg / hm) 2 Treatment group 4: Potassium reduction (T4, K2O 63 kg / hm) with 20 ng / mL p-hydroxymethylpeptide. 2 Treatment group 5: Potassium reduction (T5, K2O 63 kg / hm) with 0.05 mg / L brassinolide. 2 +3 mg / L sodium nitrophenolate), with treatment group 1 serving as the control; all treatment groups were uniformly treated with urea (150 kg N / hm). 2 ) and superphosphate (90kg P2O5 / hm 2 As a base fertilizer, all fertilizers should be thoroughly mixed with the soil; apply at 500 mL / m² when the wheat has three leaves and one heart stage. 2 Equal amounts of p-hydroxymethyl peptide, brassinolide, sodium nitrophenolate or water were applied by irrigation. After 21 days of cultivation, the wheat growth indicators, dry weight, total potassium content, potassium accumulation and superoxide dismutase activity and other stress resistance indicators of each treatment group were measured.

[0079] (2) Experimental results:

[0080] Table 5 Effects of different treatments on wheat seedling growth phenotypes

[0081] deal with Plant height (cm) Root length (cm) Fresh weight on the ground (g) Fresh weight underground (g) T1 24.8±1.2a 23.5±1.8a 0.39±0.03a 0.36±0.03a T2 18.2±1.5d 13.8±1.5d 0.24±0.02c 0.22±0.02d T3 23.5±1.4a 20.9±1.6a 0.35±0.03ab 0.32±0.03ab T4 20.5±1.3c 17.2±1.4c 0.30±0.03b 0.27±0.02c T5 21.8±1.6b 18.8±1.7b 0.29±0.03b 0.26±0.03c

[0082] Note: T1: Normal potassium control, T2: Reduced potassium control, T3: Reduced potassium + p-hydroxymethylpeptide, T4: Reduced potassium + brassinolide, T5: Reduced potassium + sodium nitrophenolate

[0083] Table 6. Effects of different treatments on dry matter, potassium absorption, and antioxidant physiology in wheat plants.

[0084] deal with Dry weight on the ground (g) Dry weight of underground soil (g) Total potassium content (%DW) <![CDATA[SOD activity (U·g -1 ·min -1 )]]> Potassium accumulation (mg / plant) T1 0.078±0.006a 0.065±0.005a 3.25±0.12a 1140±80d 25.4±2.5a T2 0.048±0.005d 0.040±0.004d 2.55±0.15d 1530±100b 12.2±1.8d T3 0.070±0.006b 0.058±0.005b 3.45±0.14a 1650±110a 24.2±2.8a T4 0.060±0.005c 0.050±0.004c 3.00±0.13c 1480±95bc 18.0±2.0b T5 0.058±0.006c 0.048±0.005c 2.90±0.14c 1380±90c 16.8±2.2c

[0085] Note: T1: Normal potassium control, T2: Reduced potassium control, T3: Reduced potassium + p-hydroxymethylpeptide, T4: Reduced potassium + brassinolide, T5: Reduced potassium + sodium nitrophenolate

[0086] In Tables 5 and 6, different lowercase letters indicate significant differences at the 0.05 level. Combining the data from Tables 5 and 6, it can be seen that compared with the normal potassium control (T1), the wheat plant height, root length, and other growth phenotypic indicators (above-ground and below-ground dry weight, total potassium content, potassium accumulation, etc.) of the water control (30% potassium reduction) were significantly decreased, while the SOD activity was increased. The T3 treatment (30% potassium reduction + 20 ng / mL p-hydroxymethylpeptide) was significantly better than T2 in terms of plant height, root length, and above-ground and below-ground fresh and dry weight, and showed no significant difference from T1. Meanwhile, its total potassium content (3.45% DW) and potassium accumulation (24.2 mg / plant) were similar to T1, and its SOD activity (1650 U·g) was also similar. -1 ·min -1 The potassium content of the T3 treatment was significantly higher than that of the T2 treatment and other regulator treatments (T4, T5). Furthermore, all core indicators of the T3 treatment were superior to those of the T4 (potassium reduction + brassinolide) and T5 (potassium reduction + sodium nitrophenolate) treatments. This indicates that under 30% potassium reduction conditions, p-hydroxymethylpeptide can not only effectively alleviate the inhibitory effect of potassium reduction stress on wheat growth and maintain normal potassium nutrition levels, but also enhance wheat's stress resistance by increasing antioxidant enzyme activity. Its potassium reduction-enhancing effect is significant and superior to commonly used plant regulators.

[0087] Example 7:

[0088] Effects of hydroxymethylpeptide on potassium reduction and synergistic effects in field wheat:

[0089] (1) Experimental design:

[0090] This embodiment was conducted in a field trial on a mildly potassium-deficient plot with a baseline available potassium content of 90 mg / kg. The test crop was spring wheat 'Kenpi Mai 12'. The trial included three core treatment groups: Treatment Group 1: Conventional potassium fertilizer control CK (K2O 60 kg / hm²). 2+Clean water), Treatment Group 2: Conventional potassium fertilizer with reduced potassium content control, K- (K2O 30kg / hm) 2 +Clean water) and treatment group 3: conventional potassium fertilizer reduction control + p-hydroxymethyl peptide, K-+CO (K2O 30kg / hm 2 +20 ng / mL p-hydroxymethylpeptide), nitrogen in all plots (255 kg / hm²) 2 ), phosphorus (67.5 kg / hm) 2 The fertilizer application rate was consistent and applied as base fertilizer and top dressing according to local standards. At the three-leaf and one-heart stage of wheat, p-hydroxymethyl peptide (15L / mu, 20ng / mL) or an equal amount of water was applied by foliar spraying. Yields were calculated by region at wheat maturity, and yield per unit area was measured. Plant samples were collected to determine the total potassium content of grains and straw to calculate potassium fertilizer utilization rate. The formula for potassium fertilizer agronomic efficiency (kg / kg) is:

[0091]

[0092] Where K represents the yield in the potassium-applied area and K0 represents the yield in the non-potassium-applied area; in this example, the yield (K0) in the non-potassium-applied area was 6460 kg / hm². 2 The protein content is on a dry basis.

[0093] (2) Experimental results:

[0094] Table 7 Wheat Yield Composition and Grain Yield under Different Treatments

[0095] deal with <![CDATA[Effective panicle number (×10 4 hm -2） > Number of grains per ear (grains) 1000-grain weight (g) <![CDATA[Grain yield (kg ha -2） > CK 633.7±19.8a 37.1±1.2a 45.2±0.8a 7915±168a K- 587.4±23.1b 31.8±1.4b 40.6±1.0b 6392±191c K-+C0 648.3±18.5a 35.9±1.1a 43.9±0.7a 7783±162a

[0096] Note: CK: Control group for conventional potassium fertilizer; K-: Control group for conventional potassium fertilizer with reduced potassium content; K-+CO: Control group for conventional potassium fertilizer with reduced potassium content plus p-hydroxymethylpeptide.

[0097] Table 8. Potassium uptake and utilization efficiency and grain protein content in wheat under different treatments.

[0098] deal with <![CDATA[Total potassium uptake of plants (kg / hm 2） > Potassium fertilizer partial productivity (kg / kg) Potash fertilizer agronomic efficiency (kg / kg) Protein content (%) CK 134.8±6.9b 86.0±2.1c 7.8±0.9b 13.35±0.26b K- 111.2±7.4c 99.9±3.2b 5.1±1.1c 12.63±0.29c K-+C0 141.6±6.4a 121.6±2.8a 10.7±0.8a 13.96±0.24a

[0099] Note: CK: Control group for conventional potassium fertilizer; K-: Control group for conventional potassium fertilizer with reduced potassium content; K-+CO: Control group for conventional potassium fertilizer with reduced potassium content plus p-hydroxymethylpeptide.

[0100] In Tables 7 and 8, different lowercase letters indicate significant differences at the 0.05 level. Combining the data from Tables 7 and 8, it can be seen that the K-+C0 treatment (conventional potassium fertilizer reduced by 50% + 20 ng / mL p-hydroxymethylpeptide) is significantly superior to the conventional potassium fertilizer reduced potassium control K- (conventional potassium fertilizer reduced by 50% + water) in terms of yield components such as effective panicle number, grain number per panicle, thousand-grain weight, and grain yield. Specifically, the effective panicle number (648.3 × 10⁻⁶) is significantly higher. 4 hm -2), number of grains per ear (35.9 grains), thousand-grain weight (43.9g), and grain yield (7783kg / hm). 2 The total potassium uptake of plants treated with K-+C0 was not significantly different from that of the conventional potassium fertilizer control (CK); meanwhile, the total potassium uptake of plants treated with K-+C0 was 141.6 kg / hm². 2 The partial productivity (121.6 kg / kg), agronomic efficiency (10.7 kg / kg), and grain protein content (13.96%) of p-hydroxymethylpeptide were all significantly higher than those of the conventional potassium fertilizer reduced-potassium control K-, and the partial productivity, agronomic efficiency, and protein content of p-hydroxymethylpeptide were better than those of the conventional potassium fertilizer control CK. This indicates that under mild potassium-deficient field conditions, p-hydroxymethylpeptide can achieve half the application of potassium fertilizer without reducing wheat yield, and can also significantly improve potassium fertilizer utilization efficiency and grain quality.

[0101] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A method for preparing p-hydroxymethylpeptide, characterized in that, Includes the following steps: S1. Place L-serine and ethylene glycol in a molar ratio of 1:1.5-2 in a single-necked round-bottom flask, then add anhydrous N,N-dimethylformamide as a solvent, and stir magnetically until the solid is completely dissolved to obtain a mixed solution. S2. The mixed solution was reacted in an oil bath at 130°C under reflux for 10 hours. After the reaction was completed, it was cooled to room temperature to obtain the reactants. S3. Transfer the reactants to a sand core funnel for vacuum filtration to obtain a solid product; S4. Wash the solid product alternately with pre-cooled anhydrous ethanol and pre-cooled deionized water, then transfer the washed solid product to a petri dish and dry it in a vacuum drying oven for 8 hours to obtain p-hydroxymethylpeptide.

2. A method for preparing p-hydroxymethylpeptide, characterized in that, Includes the following steps: A1. Inoculate *C. 42391* of *C. 42391* into a fermentation medium and ferment at 25–30°C and 150–220 rpm for 4–7 days to obtain the fermentation broth. A2. Centrifuge the fermentation broth to collect the mycelium. Then wash the mycelium with distilled water and freeze-dry it to obtain mycelium powder. Then crush the mycelium powder with methanol using ultrasound. After centrifugation, vacuum concentration, rotary evaporation and freeze-drying, the crude extract is obtained. A3. Using macroporous resin for initial separation, the crude extract was dissolved in distilled water, and then purified by reversed-phase chromatography to elute and separate it, obtaining a collection solution. The collection solution was then concentrated and freeze-dried to obtain p-hydroxymethylpeptide.

3. The application of p-hydroxymethylpeptide prepared by the method described in claim 1 or 2 in agriculture.

4. The application of p-hydroxymethylpeptide in agriculture according to claim 3, characterized in that, The application described is the use of p-hydroxymethylpeptide in the preparation of compositions that promote potassium absorption by crops.

5. The application of p-hydroxymethylpeptide in agriculture according to claim 4, characterized in that, The composition for promoting potassium absorption in crops contains p-hydroxymethylpeptide and an agriculturally acceptable carrier; the concentration of p-hydroxymethylpeptide in the composition for promoting potassium absorption in crops is 5–200 ng / mL.

6. The application of p-hydroxymethylpeptide in agriculture according to claim 4, characterized in that, The agriculturally acceptable carrier is at least one of deionized water, N,N-dimethylformamide, ethanol, kaolin, diatomaceous earth, and bentonite.

7. The application of p-hydroxymethylpeptide in agriculture according to claim 4, characterized in that, The potassium-absorbing composition for crops is applied by foliar spraying or root irrigation.

8. The application of p-hydroxymethylpeptide in agriculture according to claim 4, characterized in that, The crop is at least one of Arabidopsis thaliana, wheat, corn, rice, tomato, pepper, and peanut.

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