Preparation method of p-hydroxyethyl peptide and application of p-hydroxyethyl peptide in agriculture

Abstract

The invention discloses a preparation method of p-hydroxyethyl peptide and application of p-hydroxyethyl peptide in agriculture, and relates to the technical field of agricultural biologication.The p-hydroxyethyl peptide is prepared through an intermolecular dehydration condensation reaction of L-threonine and ethylene glycol under the heating reflux condition; the p-hydroxyethyl peptide can also be prepared by the following steps: carrying out fermentation culture on the Helabeam scrollata, 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 on the crude extract; the preparation process provided by the invention is simple, and the product is easy to purify and has the characteristic of environmental friendliness; the p-hydroxyethyl peptide, the diethyl aminoethyl hexanoate, the brassinolide, the indoleacetic acid and the compound sodium nitrophenolate are compounded for use under a specific concentration, so that the growth and development of crops can be remarkably promoted, and the yield of the crops can be increased; the p-hydroxyethyl peptide-based plant growth regulation composition is widely applicable to crops, is effective to monocotyledonous and dicotyledonous plants, and has broad spectrum.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, specifically to a method for preparing p-hydroxyethyl peptide and its application in agriculture. Background Technology

[0002] Plant growth regulators are widely used in agricultural production. They promote crop growth, improve stress resistance, and increase yield by regulating the plant's endogenous hormone system. Commonly used growth regulators include brassinolide, indoleacetic acid, aminoethyl esters, and sodium nitrophenolate, which have shown certain growth-promoting effects on different crops.

[0003] However, existing plant growth regulators still have the following problems: First, their effects are limited, with most regulators only effective for a certain type of crop or a specific growth stage; second, improper concentrations can easily lead to phytotoxicity or insignificant effects; third, their scope of action is limited, lacking broad-spectrum activity; and fourth, some chemically synthesized regulators pose residue risks, failing to meet the needs of green agriculture development. Therefore, developing a novel, highly efficient, safe, broad-spectrum, and easily prepared plant growth promoter is of significant practical importance. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing p-hydroxyethyl peptide and its application in agriculture, in order to solve the problems of existing plant growth regulators such as single effect, difficulty in controlling concentration, limited range of action and risk of residue.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing p-hydroxyethyl peptide, specifically comprising the following steps:

[0006] S1. Using L-threonine and ethylene glycol as reactants, accurately weigh L-threonine and ethylene glycol using a balance, transfer them to a 250mL single-necked flask, and react under reflux for 10 hours. After the reaction, cool the resulting mixed solution to room temperature to obtain the reaction solution.

[0007] S2. Transfer the reaction solution to a Buchner funnel, filter it to obtain a solid product;

[0008] S3. Wash the solid product 2-3 times with an organic solvent, and then dry the washed solid product in a desiccator or vacuum drying oven to obtain p-hydroxyethyl peptide.

[0009] Furthermore, the organic solvent in S3 is at least one of ethylene glycol, ethanol, methanol, and isopropanol.

[0010] This invention also discloses a method for preparing p-hydroxyethyl peptide, specifically including the following steps:

[0011] A1. The *C. 42394* species of *C. 42394* was inoculated into a fermentation medium and fermented to obtain the fermentation broth.

[0012] 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.

[0013] 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-hydroxyethyl peptide.

[0014] Furthermore, the fermentation conditions in A1 are as follows: the culture temperature is set to 25-30℃, the shaking speed is set to 150-220 rpm, and the culture time is set to 4-7 days.

[0015] This invention also discloses the application of p-hydroxyethyl peptide in agriculture. The p-hydroxyethyl peptide prepared by the above preparation method, together with at least one plant growth regulator component selected from amino acid esters, brassinolide, indoleacetic acid, and sodium nitrophenolate, is used to prepare a plant growth regulator composition. The plant growth regulator composition is applied to promote crop growth.

[0016] Further, the concentration of the p-hydroxyethoxypeptide is 5-100 ng / mL; the concentration of the amino ester is 15-20 mg / L; the concentration of the brassinolide is 0.1-1 mg / L; the concentration of the indoleacetic acid is 10-20 mg / L; and the concentration of the sodium nitrophenolate is 8-20 mg / L.

[0017] Furthermore, the composition is applied to the crop by fertigation, spraying, or seed soaking.

[0018] Furthermore, the crop is a monocotyledonous plant or a dicotyledonous plant.

[0019] Furthermore, the monocotyledonous plant is maize.

[0020] Furthermore, the dicotyledonous plant is at least one of tomato, Arabidopsis thaliana, and lettuce.

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

[0022] (1) The p-hydroxyetheptide prepared by this invention has a clear and wide range of application concentrations, with 20 ng / mL being the optimal concentration, and is unlikely to cause phytotoxicity to plants.

[0023] (2) The preparation process of p-hydroxyethyl peptide provided by the present invention is simple, the product is easy to purify, and it has environmentally friendly characteristics.

[0024] (3) The plant growth regulator composition based on p-hydroxyethyl peptide provided by the present invention significantly promotes the growth of multiple indicators of crops, such as plant height, root length, fresh weight, and chlorophyll content, and its effect is better than that of commonly used plant growth regulators.

[0025] (4) The plant growth regulating composition based on p-hydroxyethyl peptide provided by the present invention is applicable to a wide range of crops and is effective for both monocotyledonous and dicotyledonous plants, thus having a broad spectrum of applicability.

[0026] (5) The plant growth regulating composition based on p-hydroxyethyl peptide provided by the present invention can be used in combination with other agronomic measures, and its application is flexible. Attached Figure Description

[0027] 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.

[0028] Figure 1 This is a schematic diagram of a method for preparing p-hydroxyethyl peptide provided by the present invention. Detailed Implementation

[0029] 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.

[0030] Example 1:

[0031] Please see Figure 1 A method for preparing p-hydroxyetheptide specifically includes the following steps:

[0032] S1. Using L-threonine and ethylene glycol as reactants, accurately weigh L-threonine and ethylene glycol using a balance, transfer them to a 250mL single-necked flask, and react under reflux for 10 hours. After the reaction, cool the resulting mixed solution to room temperature to obtain the reaction solution.

[0033] S2. Transfer the reaction solution to a Buchner funnel, filter it to obtain a solid product;

[0034] S3. Wash the solid product 2-3 times with an organic solvent, and then dry the washed solid product in a desiccator or vacuum drying oven to obtain p-hydroxyethyl peptide.

[0035] The specific implementation method is as follows: raw material preparation and feeding:

[0036] Raw material selection: L-threonine (L-Thr) and ethylene glycol were selected as reactants;

[0037] L-Threonine is a chiral amino acid containing an amino group (-NH2), a carboxyl group (-COOH), and a hydroxyl group (-OH); ethylene glycol is a diol that combines reactivity and solvent properties.

[0038] Weighing and Transfer: Accurately weigh a fixed amount of L-threonine and ethylene glycol (molar ratio of 1:20-25 or according to the experimentally optimized ratio) using an analytical balance, and transfer the two to a clean, single-necked flask containing 250 mL of uncontaminated material.

[0039] Setup and execution of the reflux reaction apparatus:

[0040] Apparatus assembly: Install a spherical condenser tube on the single-necked flask to enhance condensation efficiency and reduce the loss of volatile substances; connect the lower end of the condenser tube to a constant temperature water bath to control the cooling water to flow from bottom to top and ensure continuous condensation; place the bottom of the flask on a heating device that can precisely control the temperature, such as an oil bath or an electric heating mantle.

[0041] Reaction conditions control: Heat to 197-198℃ and maintain a stable temperature, so that the mixed solution continues to boil and the steam is refluxed to the single-necked flask through the condenser; maintain the reflux reaction for 10 hours, during which the temperature and time need to be monitored to ensure that the reaction proceeds fully; in this stage, the amino / carboxyl groups of L-threonine undergo intermolecular dehydration condensation reaction with the hydroxyl groups of ethylene glycol, forming cyclic / chain structures connected by functional groups such as amide bonds and ester bonds by removing water molecules.

[0042] Separation and purification:

[0043] Cooling to terminate the reaction: After the reaction is complete, turn off the heating device and allow it to cool naturally to room temperature to avoid sudden cooling that could cause product decomposition or container rupture, thus obtaining the reaction solution.

[0044] Solid-liquid separation (filtration): Transfer the cooled reaction liquid to a Buchner funnel and assemble it into a vacuum filtration device with a vacuum filtration flask; turn on the vacuum pump or water pump to filter the liquid quickly through the filter paper, and retain the solid product (crude p-hydroxyethyl peptide) on the filter paper. The filtrate (containing a small amount of unreacted raw materials, by-products, etc.) is collected and properly disposed of.

[0045] Washing and drying: Wash the solid product 2-3 times with a small amount of cold solvent, such as ethylene glycol, ethanol, methanol, isopropanol, or other organic solvents that match the solubility of the product, to remove residual reactants / byproducts on the solid surface; transfer the washed solid product to a desiccator or vacuum drying oven and dry it at 40-60℃ (avoiding thermal decomposition of the product) to constant weight. The final product was identified by NMR, confirming that the cyclic dipeptide compound was a p-hydroxyetheptide, with the following structure:

[0046]

[0047] Example 2:

[0048] A method for preparing p-hydroxyetheptide specifically includes the following steps:

[0049] A1. Cephalotrichum heliciforme with accession number CGMCC No.42394 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-hydroxyethyl peptide. The depositary of this strain is China General Microbiological Culture Collection Center, and the deposit date is November 27, 2025.

[0050] 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.

[0051] A3. Using macroporous resin initial separation method, the crude extract was dissolved in distilled water and loaded onto an HP-20 resin column. Impurities were first eluted with water, and then the target component was eluted with a gradient of 30% ethanol. The eluent was purified by reversed-phase chromatography and separated by HPLC. The collected solution was concentrated and lyophilized. The NMR identification confirmed that the cyclic dipeptide compound was p-hydroxyetheptide.

[0052] Example 3:

[0053] Growth-promoting effects of different concentrations of p-hydroxyethinopeptide on maize:

[0054] (1) Experimental design

[0055] Select uniform, plump corn seeds and soak them in water for 5-8 hours (enough to submerge the seeds). After soaking, evenly place the seeds in a petri dish lined with moist nutrient soil, cover with an equal amount of moist nutrient soil, and germinate in the dark. Once the seeds show white sprouts, select those that germinate evenly for later use. Fill transplanting baskets 3 / 4 full with moist nutrient soil, sow 4 seeds per basket (lay them flat without overlapping), and cover with a thin layer of soil. Set up 6 treatment solutions: Treatment 1: CK (water); Treatment 2: 5 ng / mL of p-hydroxyethyl peptide; Treatment 3: 10 ng / mL of p-hydroxyethyl peptide; Treatment 4: 20 ng / mL of p-hydroxyethyl peptide; Treatment 5: 50 ng / mL of p-hydroxyethyl peptide; Treatment 6: 100 ng / mL of p-hydroxyethyl peptide; each group was replicated 6 times, with 100 mL of the corresponding treatment solution applied per basket (the control group was watered with water). The planting baskets were placed in a climate chamber at 23℃, humidity of 50%–60%, and light intensity of 6000–7000 lux. The treatment solution was applied every 7 days, and the plants were regularly watered and kept moist. The growth was also recorded by taking photos.

[0056] (2) Indicator Measurement

[0057] The corn plant height, root length, fresh weight, and stem diameter of each group were statistically analyzed.

[0058] Measure the length from the base of the stem to the tip of the top leaf and record it as plant height; measure the length from the base of the root to the tip of the root and record it as root length; wash the plant with tap water, dry it, and weigh it fresh; measure the diameter at the base of the stem and record it as stem diameter.

[0059] Table 1 Experimental Design

[0060] (3) Analysis of experimental results

[0061] The experimental data in Tables 1 and 2 show that the promoting effect of p-hydroxyethoxypeptide on maize growth exhibits a significant concentration-dependent effect. Different lowercase letters in Table 2 indicate significant differences at the 0.05 level. The 20 ng / mL concentration significantly outperformed other concentrations in five growth indicators: plant height (21.55 cm), root length (10.12 cm), stem diameter (3.52 mm), aboveground fresh weight (1.327 g), and underground fresh weight (1.205 g), and was significantly higher than the control group (CK) and other treatment groups. While low concentrations (5–10 ng / mL) improved some growth indicators (such as plant height and root length), the effect was less than that of 20 ng / mL. High concentrations (50–100 ng / mL) had no significant effect on maize growth indicators. The experiment demonstrates that p-hydroxyethoxypeptide application promotes maize growth in a concentration-dependent manner, with 20 ng / mL being the optimal concentration for promoting maize growth, significantly improving plant height, root length, stem diameter, and aboveground and underground fresh weight.

[0062] Table 2 Effects of different treatments on growth indicators of corn seedlings

[0063] Example 4:

[0064] Effects of different concentrations of hydroxyethyl peptide on tomato growth:

[0065] (1) Experimental design:

[0066] The effects of different concentrations of p-hydroxyethyl peptide (p-hydroxyethyl peptide) on tomato growth were verified through an indoor pot experiment. Danish Pinsch peat moss was used as the seedling substrate. After sterilization, water was added and thoroughly mixed, then the substrate was poured into seedling trays and the surface was leveled. Tomato seeds were then sown at a depth of 0.5 cm. The seedling trays were placed in a climate-controlled environment with the following conditions: temperature 23℃, humidity 50%–60%, and light intensity 6000–7000 lx. Once the tomato seedlings reached the four-leaf stage, disease-free seedlings with uniform growth were selected and transplanted into flowerpots. The experiment included six treatment groups, with the control group (CK): Treatment 1: CK (water); Treatment 2: p-hydroxyethyl peptide 5 ng / mL; Treatment 3: p-hydroxyethyl peptide 10 ng / mL; Treatment 4: p-hydroxyethyl peptide 20 ng / mL; Treatment 5: p-hydroxyethyl peptide 50 ng / mL; Treatment 6: p-hydroxyethyl peptide 100 ng / mL. Each treatment group was replicated nine times. After transplanting the seedlings, apply the treatment solution once; after the seedlings have recovered and during the flowering period, apply the solution again to the samples. Throughout the growing season, water and fertilizer are continuously supplied to maintain the normal growth of the tomatoes. In the later stages of the experiment, relevant indicators of tomato root growth are recorded and photographed.

[0067] Table 3 Experimental Design

[0068] (2) Experimental results:

[0069] In Table 4, different lowercase letters indicate significant differences at the 0.05 level. Combining the data from Tables 3 and 4, it can be seen that, in terms of plant height, treatment group 4 (16.4±0.3acm) was the highest, followed by treatment groups 3 and 5 (15.6±0.3bcm, 14.2±0.3bcm), while treatment group 1 (11.1±0.2dcm) was the lowest. Regarding root length, treatment group 4 (14.7±0.3acm) was the longest, followed by treatment groups 3 and 5 (13.1±0.2bcm, 12.5±0.2bcm). Group 1 (9.7±0.1 dcm) had the shortest root length. Regarding underground fresh weight, treatment group 4 (2.18±0.09 ag) had the highest, followed by treatment groups 5 and 6 (1.55±0.06 bg, 1.62±0.07 bg), with treatment group 1 (1.16±0.05 dg) having the lowest. In terms of chlorophyll / SPAD ratio, treatment group 4 (41.6±0.5 a) had the highest, followed by treatment groups 3 and 6 (40.1±0.6 b, 39.2±0.6 b), with treatment group 1 (34.3±0.4 d) having the lowest. The experimental results indicate that fertigation with hydroxyethyl peptide can promote the increase of tomato plant height, root length, underground fresh weight, and chlorophyll content, with the best overall performance across all growth indicators at a concentration of 20 ng / mL. Its promoting effect on tomato growth is concentration-dependent.

[0070] Table 4 Effects of different treatments on tomato growth indicators

[0071] Example 5:

[0072] Effects of different concentrations of hydroxyethoxypeptide on the intact growth period of Arabidopsis thaliana:

[0073] (1) Experimental design

[0074] First, soak an appropriate amount of Arabidopsis thaliana seeds in sterile water for 1 minute, then disinfect them with 5% sodium hypochlorite for 5 minutes, shake them thoroughly, rinse them three times with clean water, then disinfect them with 75% ethanol for 1 minute, rinse them three to five times with sterile water, protect them from light, and place them in a refrigerator at 4°C for vernalization for 2 days.

[0075] Vermiculite, earthworm soil, and potting soil were all sterilized and thoroughly mixed in a specific ratio. The substrate was then poured into flowerpots, the surface of which was leveled. These pots were placed in white trays, with 12 small pots in each tray. The treatment solution was slowly added to the bottom of the trays. Three Arabidopsis seeds were placed in each flowerpot. The plants were covered with a film and allowed to grow until they had four rosette leaves. The film was then removed, and the seedlings were thinned out, leaving one Arabidopsis seed of uniform size in each pot. After thinning, 600 mL of the treatment solution was applied to each tray weekly, and the plant growth was observed daily during this period.

[0076] The seedling trays were placed in an artificial climate incubation chamber with the following environmental parameters: temperature 23℃, air humidity 50%–60%, and light intensity 6000–7000 lux. The substrate was kept moist to ensure normal seed germination. Six different treatment groups were set up: Treatment 1: CK (water); Treatment 2: p-hydroxyethylpeptide 5 ng / mL; Treatment 3: p-hydroxyethylpeptide 10 ng / mL; Treatment 4: p-hydroxyethylpeptide 20 ng / mL; Treatment 5: p-hydroxyethylpeptide 50 ng / mL; Treatment 6: p-hydroxyethylpeptide 100 ng / mL. To ensure repeatability and data reliability, each treatment group was replicated 12 times. During the experiment, the growth status of Arabidopsis seedlings was observed and photographed regularly. At designated growth stages, growth indicators such as leaf disc diameter, bolting height, number of flowers, and number of pods were measured to analyze the effects of different concentrations on Arabidopsis growth.

[0077] Table 5 Effects of different treatments on Arabidopsis thaliana growth

[0078] (2) Test results

[0079] In Table 6, different lowercase letters indicate significant differences at the 0.05 level. Based on Tables 5 and 6 combined with the experimental data, it can be seen that, in terms of leaf disc diameter, treatment group 4 (20 ng / mL) had the largest leaf disc diameter, followed by treatment group 5 (50 ng / mL), while treatment groups 1 (water) and 6 (100 ng / mL) had relatively smaller diameters. In terms of bolting height, treatment group 4 (5 ng / mL) had a significantly higher bolting height than the other groups, followed by treatment group 3 (10 ng / mL), while treatment group 5 had the lowest bolting height. In terms of the number of flowers, treatment group 4 had the most flowers (14.7 ± 0.1 a), followed by treatment group 3, while treatment groups 1 and 5 had fewer flowers. In terms of the number of pods, treatment group 4 had the most pods, followed by treatment group 5, while treatment groups 2 and 6 had relatively fewer pods. The experimental results showed that the effect of p-hydroxyethoxyphenide on Arabidopsis thaliana growth indicators was concentration-dependent. The 20 ng / mL treatment had the best effect on improving leaf disc, bolting, flowering and pod formation, while the high concentration (100 ng / mL) had an effect close to that of the water control.

[0080] Table 6 Effects of different treatments on Arabidopsis thaliana growth

[0081] Example 6:

[0082] The promoting effect of p-hydroxyethyl peptide on the growth of lettuce seedlings:

[0083] (1) Experimental design

[0084] First, sterilize the seedling substrate. After the substrate cools, add an appropriate amount of water and stir thoroughly until the moisture content is suitable. Fill the seedling trays with the treated substrate, level and compact the surface, then sow the lettuce seeds. After sowing, lightly cover with a thin layer of substrate.

[0085] The seedling trays were placed in an artificial climate incubation chamber with the following environmental parameters: temperature 23℃, air humidity 50%–60%, and light intensity 6000–7000 lux. The substrate was kept moist to ensure normal seed germination. When the lettuce seedlings reached the two-leaf-one-heart stage, seedlings free from pests and diseases and with uniform growth were selected for subsequent transplanting.

[0086] The experiment included six different treatment groups: Treatment 1: CK (water); Treatment 2: p-hydroxyethyl peptide 20 ng / mL; Treatment 3: aminoethyl ester 15 mg / L; Treatment 4: brassinolide 0.1 mg / L; Treatment 5: indoleacetic acid 10 mg / L; Treatment 6: sodium nitrophenolate 8 mg / L. To ensure repeatability and data reliability, each treatment group was replicated five times. During the experiment, the growth status of lettuce seedlings was observed and photographed regularly. At designated growth stages, leaf length, leaf width, number of leaves, and fresh weight were measured to analyze the effects of different treatments on lettuce seedling growth.

[0087] Table 7 Effects of different treatments on lettuce growth

[0088] (2) Test results

[0089] In Table 8, different lowercase letters indicate significant differences at the 0.05 level. Based on Tables 7 and 8 and the experimental data, it can be seen that under the irrigation treatment, p-hydroxyethyl peptide (treatment 2) had the most prominent promoting effect on various growth indicators of lettuce. Its leaf length (19.9±0.2a), leaf width (12.1±0.2a), SPAD value (29.8±0.3a), number of leaves (7.8±0.1a), and whole plant fresh weight (12.6±0.1a) were all significantly higher than other treatments. Brassinolide (treatment 4), indoleacetic acid (treatment 5), and sodium nitrophenolate (treatment 6) had certain promoting effects on leaf length, SPAD value, and number of leaves, and the effects were better than the water control (treatment 1). However, aminoethyl ester (treatment 3) showed no significant difference in various indicators compared with the water control and did not show a significant growth promoting effect. Experiments have shown that hydroxyethyl peptide has the most significant effect on promoting lettuce growth, while brassinolide, indoleacetic acid, and sodium nitrophenolate have some effect, and aminoethyl esters did not show a significant growth-promoting effect.

[0090] Table 8 Effects of different treatments on lettuce growth

[0091] Example 7:

[0092] The growth-promoting and yield-increasing effects of p-hydroxyethoxypeptide on Arabidopsis thaliana:

[0093] (1) Experimental design

[0094] First, soak an appropriate amount of Arabidopsis thaliana seeds in sterile water for 1 minute, then disinfect them with 5% sodium hypochlorite for 5 minutes, shake them thoroughly, rinse them three times with clean water, then disinfect them with 75% ethanol for 1 minute, rinse them three to five times with sterile water, protect them from light, and place them in a refrigerator at 4°C for vernalization for 2 days.

[0095] Vermiculite, earthworm soil, and potting soil were all sterilized and thoroughly mixed in a specific ratio. The substrate was then poured into flowerpots, the surface of which was leveled. These pots were placed in white trays, with 12 small pots in each tray. The treatment solution was slowly added to the bottom of the trays. Three Arabidopsis seeds were placed in each flowerpot. The plants were covered with a film and allowed to grow until they had four rosette leaves. The film was then removed, and the seedlings were thinned out, leaving one Arabidopsis seed of uniform size in each pot. After thinning, 600 mL of the treatment solution was applied to each tray weekly, and the plant growth was observed daily during this period.

[0096] The seedling trays were placed in an artificial climate incubation chamber with the following environmental parameters: temperature 23℃, air humidity 50%–60%, and light intensity 6000–7000 lux. The substrate was kept moist to ensure normal seed germination. Six different treatment groups were set up: Treatment 1: CK (water); Treatment 2: p-hydroxyethoxyphenide 20 ng / mL; Treatment 3: aminoethyl ester 15 mg / L; Treatment 4: brassinolide 0.1 mg / L; Treatment 5: indoleacetic acid 10 mg / L; Treatment 6: sodium nitrophenolate 8 mg / L. To ensure repeatability and data reliability, each treatment group was replicated 12 times. During the experiment, the growth status of Arabidopsis seedlings was observed and photographed regularly. At designated growth stages, growth indicators such as leaf disc diameter, bolting height, number of flowers, and number of pods were measured to analyze the effects of different treatments on Arabidopsis growth.

[0097] Table 9 Effects of different treatments on Arabidopsis thaliana growth

[0098] (2) Test results

[0099] In Table 10, different lowercase letters indicate significant differences at the 0.05 level. Based on Tables 9 and 10 and the experimental data, it can be seen that p-hydroxyethoxylate (treatment 2) has the most prominent promoting effect on Arabidopsis thaliana, with leaf disc diameter (10.0±0.07a), bolting height (22.1±0.2a), number of flowers (15.0±0.1a), and number of pods (235.2±0.1a) all at the best level among the treatments. Brassinolide (treatment 4) showed good performance in leaf disc diameter, bolting height, and number of pods, but there was no significant difference in the number of flowers compared with the water control (treatment 1). Aminoethyl ester (treatment 3), indoleacetic acid (treatment 5), and sodium nitrophenolate (treatment 6) have a certain promoting effect on the growth indicators of leaf disc diameter and number of flowers, but the effect is weaker than that of p-hydroxyethoxylate. The water control (treatment 1) has the lowest growth indicators in all aspects. The experimental results showed that p-hydroxyethyl peptide had the most significant promoting effect on both the vegetative and reproductive growth of Arabidopsis thaliana, while the growth-promoting effects of other reagents were relatively limited or exhibited index specificity.

[0100] Table 10 Effects of different treatments on Arabidopsis thaliana growth

[0101] Example 8:

[0102] The promoting effect of p-hydroxyethyl peptide on maize growth:

[0103] (1) Experimental design

[0104] Select uniform, plump corn seeds and soak them in water for 5-8 hours (enough to submerge the seeds). After soaking, evenly place the seeds in a petri dish lined with moist nutrient soil, cover with an equal amount of moist nutrient soil, and germinate in the dark. Once the seeds show white sprouts, select those that germinate evenly for later use. Fill transplanting baskets 3 / 4 full with moist nutrient soil, sow 4 seeds per basket (lay them flat without overlapping), and cover with a thin layer of soil. Six treatments were administered: CK (water); Treatment 2: p-hydroxyethyl peptide 20 ng / mL; Treatment 3: aminoethyl ester 15 mg / L; Treatment 4: brassinolide 0.1 mg / L; Treatment 5: indoleacetic acid 10 mg / L; Treatment 6: sodium nitrophenolate 8 mg / L. Each group was replicated 6 times, with 100 mL of the corresponding treatment solution applied per basket (control group watered with water). The planting baskets were placed in a climate chamber at 23℃, humidity of 50%–60%, and light intensity of 6000–7000 lux. The treatment solution was applied every 7 days, and the plants were regularly watered and kept moist. The growth was also recorded by taking photos.

[0105] (2) Indicator Measurement

[0106] Statistics were collected on indicators such as corn plant height, root length, fresh weight, and stem diameter.

[0107] Measure the length from the base of the stem to the tip of the top leaf and record it as plant height; measure the length from the base of the root to the tip of the root and record it as root length; wash the plant with tap water, dry it, and weigh it fresh; measure the diameter at the base of the stem and record it as stem diameter.

[0108] Table 11 Experimental Design

[0109] (3) Analysis of experimental results

[0110] In Table 12, different lowercase letters indicate significant differences at the 0.05 level. As shown in Tables 11 and 12, combined with the experimental data, p-hydroxyethyl peptide (treatment 2) had the most prominent promoting effect on various growth indicators of maize seedlings. Its plant height (28.52±0.49a), root length (28.73±1.26a), stem diameter (4.62±0.07a), aboveground fresh weight (2.31±0.07a), and underground fresh weight (1.85±0.04a) were all at the best level among the treatments. Brassinolide (treatment 4) showed good performance in plant height, root length, and aboveground fresh weight, second only to p-hydroxyethyl peptide. Aminoethyl ester (treatment 3), indoleacetic acid (treatment 5), and sodium nitrophenolate (treatment 6) had certain promoting effects on some indicators, but the effects were weaker than those of p-hydroxyethyl peptide and brassinolide treatment groups. The water control (treatment 1) had the lowest growth indicators. The experimental results showed that p-hydroxyethyl peptide had the most significant promoting effect on the vegetative growth (plant height, root length, stem diameter) and biomass accumulation (above-ground and underground fresh weight) of maize seedlings, while the growth-promoting effects of other reagents were relatively limited.

[0111] Table 12 Effects of different treatments on growth indicators of corn seedlings

[0112] 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-hydroxyethyl peptide, characterized in that, Specifically, the following steps are included: S1. Using L-threonine and ethylene glycol as reactants, accurately weigh L-threonine and ethylene glycol using a balance, transfer them to a 250mL single-necked flask, and react under reflux for 10h. After the reaction, cool the resulting mixed solution to room temperature to obtain the reaction solution. S2. Transfer the reaction solution to a Buchner funnel, filter it to obtain a solid product; S3. Wash the solid product 2-3 times with an organic solvent, and then dry the washed solid product in a desiccator or vacuum drying oven to obtain p-hydroxyethyl peptide.

2. The method for preparing p-hydroxyethyl peptide according to claim 1, characterized in that, The organic solvent in S3 is at least one of ethylene glycol, ethanol, methanol, and isopropanol.

3. A method for preparing p-hydroxyethyl peptide, characterized in that, Specifically, the following steps are included: A1. The *C. 42394* species of *C. 42394* was inoculated into a fermentation medium and fermented 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-hydroxyethyl peptide.

4. The method for preparing p-hydroxyethyl peptide according to claim 3, characterized in that, The fermentation conditions in A1 are as follows: the culture temperature is set to 25-30℃, the shaking speed is set to 150-220 rpm, and the culture time is set to 4-7 days.

5. The application of p-hydroxyethyl peptide prepared by the method for preparing p-hydroxyethyl peptide according to claim 1 or 3 in agriculture, characterized in that, A plant growth regulating composition is prepared by using the p-hydroxyethyl peptide prepared by the method described in claim 1 or 3, and at least one plant growth regulator component selected from amino acid esters, brassinolide, indoleacetic acid, and sodium nitrophenolate. The plant growth regulating composition is used to promote crop growth.

6. The application of p-hydroxyethyl peptide in agriculture according to claim 5, characterized in that, The concentration of the p-hydroxyethoxypeptide is 5–100 ng / mL; the concentration of the amino ester is 15–20 mg / L; the concentration of the brassinolide is 0.1–1 mg / L; the concentration of the indoleacetic acid is 10–20 mg / L; and the concentration of the sodium nitrophenolate is 8–20 mg / L.

7. The application of p-hydroxyethyl peptide in agriculture according to claim 5, characterized in that, The composition is applied to crops by irrigation, spraying, or seed soaking.

8. The application of p-hydroxyethyl peptide in agriculture according to claim 5, characterized in that, The crop is a monocotyledonous plant or a dicotyledonous plant.

9. The application of p-hydroxyethyl peptide in agriculture according to claim 8, characterized in that, The monocotyledonous plant mentioned is maize.

10. The application of p-hydroxyethyl peptide in agriculture according to claim 8, characterized in that, The dicotyledonous plant is at least one of tomato, Arabidopsis thaliana, and lettuce.

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