Preparation method of simplest peptide and application of simplest peptide in agriculture

Abstract

The invention discloses a preparation method of simplest peptide and application of the simplest peptide in agriculture, and relates to the field of agricultural science and technology and bio-fertilizer.The preparation method comprises the following steps that L-glycine and ethylene glycol serve as raw materials, under catalysis of p-toluenesulfonic acid and protection of nitrogen, a reflux reaction is conducted for 8 hours at the temperature of 120-150 DEG C, after the reaction is completed, cooling crystallization and suction filtration are conducted, and a product is collected; washing the product with precooled absolute ethyl alcohol and diethyl ether in sequence to obtain a washed product; recrystallizing the washed product by using an ethanol-water mixed solvent, and carrying out vacuum drying at 40-50 DEG C for 12 hours until the weight is constant to obtain cyclo (glycine-glycine) dipeptide, namely the simplest peptide; according to the preparation method of the simplest peptide and the application of the simplest peptide in agriculture, elongation and branching of roots of various crops can be effectively promoted through the simplest peptide, the root surface area and the capillary root number can be increased, the root activity can be remarkably improved, and the viability and growth recovery of plants under drought stress can be enhanced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of agricultural science and bio-fertilizer technology, in particular to a preparation method of a minimal peptide and application thereof in agriculture. BACKGROUND

[0002] Plant root system is the core organ for plant fixation, water absorption and mineral nutrient acquisition, and its development degree (including main root length, lateral root and capillary root density, total root surface area, etc.) directly determines the utilization efficiency of water and nutrients by plants, and further directly affects the growth and development, stress resistance and final yield of crops. Therefore, promoting root development is one of the key links to improve the efficiency of agricultural production.

[0003] At present, the substances commonly used in agricultural production to promote plant rooting and regulate growth are mostly chemical synthetic plant growth regulators, such as indole butyric acid (IBA), naphthalene acetic acid (NAA), prohexadione-calcium (PAC), amine fresh ester (DA-6), etc. However, these traditional regulators have several obvious limitations in practical application: first, their effects are often unstable and easily affected by environmental conditions, application time and crop species, making it difficult to achieve stable and predictable rooting promotion effects in different scenarios; second, their use concentration is generally high, usually reaching micrograms per milliliter (μg / mL) or even higher, which not only increases production cost but also brings difficulties to precision application; third, some chemical regulators have a long residual time and slow degradation in soil, posing a certain risk to the quality safety of agricultural products, and their long-term use also has potential negative impacts on the soil ecological environment, which is contrary to the urgent need for developing green and sustainable agriculture. SUMMARY

[0004] The purpose of the present application is to provide a preparation method of a minimal peptide and application thereof in agriculture, so as to solve the problems of unstable effect, high use concentration and residue or environmental risk of existing regulators.

[0005] In order to achieve the above purpose, the present application provides the following technical scheme: a preparation method of a minimal peptide, comprising the following steps:

[0006] A1, inoculate the Tuberolysipha fungus into PDA solid culture medium, and incubate at 25℃ for 5 days; after the strain is activated, transfer it to a triangular flask containing 200mL PDA liquid seed culture medium by agar block method, and shake culture for 5-7 days to obtain a first-stage seed liquid;

[0007] A2, sterilize and cool a 5L fermenter, then inoculate 6% of the first-stage seed liquid, and culture for 5 days to obtain a fermentation liquid;

[0008] A3. Centrifuge the fermentation broth to separate mycelium and filtrate. After washing the mycelium, dry it at 60°C, weigh it and crush it. Extract it three times with an equal volume of ethanol. Mix the extracts three times with a magnetic stirrer, sonicate for 1 hour, vacuum filter, and collect the filtrate, which is the extract.

[0009] A4. After the extract is concentrated by rotary evaporation, it is extracted three times with an equal volume of ethyl acetate containing 5% acetone. The supernatant is collected, and the extract is evaporated by rotary evaporation and dried under nitrogen to obtain the crude extract.

[0010] A5. The crude extract was initially separated by silica gel column chromatography (200-300 mesh) and ODS pressurized chromatography. After TLC merging, it was purified by HPLC and analyzed by NMR to obtain the cyclic (glycine-glycine) dipeptide, which is the simplest peptide.

[0011] Furthermore, the *Pterocarya nodosa* described in A1 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.42392.

[0012] A simple method for preparing peptides includes the following steps:

[0013] S1. Using L-glycine and ethylene glycol as raw materials, the reaction was carried out under reflux at 120-150℃ for 8 hours under the catalysis of p-toluenesulfonic acid and nitrogen protection. The reaction progress was monitored by TLC during the reaction. After the reaction was completed, the product was collected by cooling and crystallization and by filtration.

[0014] S2. Wash the product with pre-cooled anhydrous ethanol and diethyl ether in sequence, washing each three times with pre-cooled anhydrous ethanol and diethyl ether to obtain the washed product.

[0015] S3. The washed product was recrystallized using an ethanol-water mixed solvent and dried under vacuum at 40-50°C for 12 hours to constant weight to obtain a cyclic (glycine-glycine) dipeptide, which is the simplest peptide.

[0016] Furthermore, the developing solvent for the TLC in S1 is a mixed solvent of ethyl acetate and methanol, wherein the volume ratio of ethyl acetate to methanol is 5:1.

[0017] Furthermore, the cooling and crystallization temperature described in S1 is 0-5°C.

[0018] Furthermore, in the ethanol-water mixed solvent described in S3, the volume ratio of ethanol to water is 8:2.

[0019] Application of a simple peptide prepared by the aforementioned method in agriculture.

[0020] Furthermore, the application in agriculture is an application in promoting the development of plant roots, wherein the plant is a dicotyledonous plant or a monocotyledonous plant.

[0021] Further, the dicotyledonous plants include Arabidopsis thaliana and tomato; the monocotyledonous plants include rice, wheat and corn.

[0022] Further, the use concentration of the minimal peptide is 5-100 ng / mL.

[0023] Compared with the prior art, the present application provides a preparation method of a minimal peptide and its application in agriculture. The minimal peptide shows a significant root promoting effect at a concentration of nanogram per milliliter (ng / mL), which is much lower than the use concentration (usually microgram per milliliter level) of traditional plant growth regulators. The minimal peptide can not only effectively promote the elongation and branching of the root system of various crops, increase the root surface area and the number of capillary roots, but also significantly improve the root activity and the survival ability and growth recovery of plants under drought stress. Through promoting the development of the root system and nutrient absorption, the minimal peptide can ultimately significantly improve the yield per plant of crops (such as tomato). Due to its natural amino acid composition and extremely low use amount, the minimal peptide has a small potential risk to the environment and non-target organisms.

[0024] Further, the preparation method of the minimal peptide has easily available raw materials, simple synthesis route and convenient purification, and is easy to scale up. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced. Obviously, the drawings in the following description are only some embodiments described in the present application, and other drawings can also be obtained by those skilled in the art according to these drawings.

[0026] Figure 1 The preparation method of the minimal peptide provided in the embodiments of the present application is shown in the schematic diagram of the principle flow.

[0027] Figure 2 The root surface area and capillary root phenotype of Arabidopsis thaliana after using different concentrations of the minimal peptide are shown in the schematic diagram.

[0028] Figure 3 The root surface area and capillary root phenotype of rice after using different concentrations of the minimal peptide are shown in the schematic diagram.

[0029] Figure 4 The root surface area and capillary root phenotype of tomato after using the minimal peptide and other regulators are shown in the schematic diagram.

[0030] Figure 5 The yield of tomato after using the minimal peptide and other regulators is shown in the schematic diagram.

[0031] Figure 6A schematic diagram of the root phenotype of wheat under drought stress after using the minimal peptide and other modulators provided by the embodiments of the present application;

[0032] Figure 7 A schematic diagram of the root phenotype of corn under drought stress after using the minimal peptide and other modulators provided by the embodiments of the present application. DETAILED DESCRIPTION

[0033] In order for those skilled in the art to better understand the technical solutions of the present application, the present application will be further described in detail below with reference to the accompanying drawings.

[0034] Example 1:

[0035] A preparation method of a minimal peptide, comprising the following steps:

[0036] A1, inoculate Scytalidium tuberculatum into PDA solid medium, and incubate at 25°C for 5 days. After the strain is activated, transfer it to a 200 mL PDA liquid seed culture medium in a triangular flask by agar block method, and incubate at 25°C and 180 r / min for 5-7 days to prepare a first seed liquid. Scytalidium tuberculatum is preserved in the General Microbiological Center of the Chinese Microbial Culture Collection Committee, No. 3, Institute of Microbiology, Chinese Academy of Sciences, No. 1, Beichen West Road, Chaoyang District, Beijing, with a preservation date of November 27, 2025, and a preservation number of CGMCC NO. 42392.

[0037] A2, then transfer the first seed liquid to a 5 L fermenter for secondary fermentation. First, sterilize the fermenter at 121°C for 20 minutes, then inoculate 6% of the first seed liquid after cooling, control the temperature at 25.8°C, the initial pH at 7.2, and the stirring speed at 160 r / min, and incubate for 5 days. Take samples daily for detection, and finally obtain the fermentation broth. The fermentation medium in the 5 L fermenter consists of 1.0 L of potato extract, 1.0 g of yeast extract, 3.0 g of peptone, and 15.0 g of glucose.

[0038] A3, centrifuge the fermentation broth into mycelium and filtrate, wash the mycelium, dry it at 60°C, weigh and crush it, extract it with an equal volume of ethanol for 3 times, mix each extraction liquid with a magnetic stirrer, ultrasonic oscillation for 1 h, vacuum filtration, and collect the filtrate, which is the extraction liquid;

[0039] A4, after the extraction liquid is concentrated by rotary evaporation, extract it with an equal volume of ethyl acetate containing 5% acetone for 3 times, collect the supernatant, rotary evaporate it with a rotary evaporator, and dry it with nitrogen blowing to obtain the crude extract;

[0040] A5, the crude extract was separated by 200-300 mesh silica gel column chromatography under reduced pressure, and eluted with dichloromethane, a mixture of dichloromethane and methanol with a volume ratio of 70:1, a mixture of dichloromethane and methanol with a volume ratio of 30:1, a mixture of dichloromethane and methanol with a volume ratio of 8:1, a mixture of dichloromethane and methanol with a volume ratio of 2:1, acetone and methanol in seven polarity systems, and then collected each fraction. The preliminary separation was performed by ODS pressure chromatography (gradient elution of methanol / water 12:88-100:0), and then the similar components were combined by TLC. Finally, the preparation purification was performed by HPLC on a C18 column with methanol-water gradient (50%-100%, 40 minutes), and the simplest peptide was obtained by NMR identification .

[0041] Example Two

[0042] Referring to Figure 1 , a preparation method of the simplest peptide comprises the following steps:

[0043] S1, L-glycine and ethylene glycol were used as raw materials, and a toluenesulfonic acid was used as catalyst under nitrogen protection, and a reflux reaction was performed at 120-150°C for 8 hours. The reaction process was monitored by TLC. After the reaction was completed, the product was collected by cooling and crystallization and filtration.

[0044] S2, the product was washed with pre-cooled anhydrous ethanol and ether in sequence, and each was washed for 3 times to obtain a washed product.

[0045] S3, the washed product was recrystallized by using an ethanol-water mixed solvent, and vacuum dried at 40-50°C for 12 hours to constant weight to obtain a cyclic (glycine-glycine) dipeptide, i.e. the simplest peptide .

[0046] Specifically, 250 mL single-mouth round-bottom flask was taken, L-glycine, ethylene glycol and toluenesulfonic acid (5-10 mol%) were added, a reflux condenser was installed, nitrogen protection was performed, and oil bath heating was performed to 120-150°C, and maintained for 8 hours of reflux. The reaction process was monitored by taking samples in time and using TLC (developing agent: ethyl acetate / methanol=5:1). After the reaction was completed, the product was collected by slowly cooling to room temperature, cooling to 0-5°C in an ice water bath, and filtering by a Buchner funnel.

[0047] The product was washed with 10 mL of pre-cooled anhydrous ethanol and ether in sequence, each for 3 times. The washed product was recrystallized by using an ethanol-water (8:2) mixed solvent, and vacuum dried at 40-50°C for 12 hours to constant weight. The white powder product was determined by melting point, HPLC purity analysis and NMR identification, and the cyclic dipeptide compound was determined as cyclic (glycine-glycine) dipeptide, i.e. the simplest peptide, and the structure was as follows: .

[0048] Example Three:

[0049] Please refer to Figure 2 and Figure 3 , this embodiment provides a technical scheme on the basis of example one or example two: the regulation and influence experiment of different concentrations of minimal peptides on crop rooting and growth.

[0050] 1. Influence on the growth and development of Arabidopsis thaliana root system in culture medium:

[0051] Weigh 35g of 1 / 2MS medium and add it to 1L of pure water to make a solid culture medium for sterilization. After sterilization, add different concentrations of minimal peptides when the medium is cooled to 50-55℃. The concentrations are 5ng / mL, 20ng / mL, 50ng / mL, and 100ng / mL. The control group CK is treated with the same volume of sterile water. Then pour them into sterilized culture dishes for standby.

[0052] Take the sealed and still viable Arabidopsis thaliana seeds, sterilize them in a super-clean bench with 75% alcohol for 30S, 10% sodium hypochlorite for 1min, and then wash them with sterile water for 3-4 times for standby. Choose uniform and full Arabidopsis thaliana seeds, 20 per plate, and place them evenly on the same horizontal line above the plate. Place them on the super-clean bench until the water around the seeds dries, the seeds can be fixed on the culture plate with the culture cover, and the plate is sealed with a sealing film. Place it in the plate holder and culture it in a culture room at a temperature of 22℃ with a light duration of 16h.

[0053] Observe and record the seed germination time, seedling stage root length, lateral root number, and Arabidopsis thaliana fresh weight every day. Calculate the root surface area using a root scanning instrument and take photos for record.

[0054] The test results are shown in Table 1 and Figure 2 , where treatment 1 is minimal peptide 5ng / mL, treatment 2 is minimal peptide 20ng / mL, treatment 3 is minimal peptide 50ng / mL, and treatment 4 is minimal peptide 100ng / mL. Different lowercase letters in Table 1 indicate significant differences at the 0.05 level. Through the analysis of the related data of Arabidopsis thaliana root system, it is shown that the concentration of minimal peptide in the range of 5-100ng / mL can promote the growth and development of root system to different degrees. Among them, the comprehensive performance is the most outstanding when the concentration of minimal peptide is 20ng / mL, the root length (5.78cm), fresh weight (1.0669g), and lateral root number (15.73) are increased by 23.2%, 2.4%, and 155% respectively compared with the CK control. Especially in the root surface area (24.64cm 2 ), which is only (12.57cm 2The root length increased by 96.02% compared with CK. It is speculated that the simplest peptide at a concentration of 20 ng / mL may simultaneously activate the synergistic regulation pathway of root elongation and branching, thereby promoting the increase of root surface area and capillary roots. The root length increased by 11.7% and 6.8% at the simplest peptide concentrations of 50 ng / mL and 100 ng / mL, respectively, compared with CK. However, the root surface area still increased by 53.94% and 68.50%, respectively. In summary, different concentrations of the simplest peptide all played a positive role in promoting the root development of Arabidopsis thaliana. Among them, the simplest peptide concentration of 20 ng / mL (treatment 2) had the best effect on promoting the increase of root surface area and capillary roots of Arabidopsis thaliana.

[0055] Table 1 Effect of different concentrations of the simplest peptide on the root system indicators of Arabidopsis thaliana.

[0056] Treatment Root length (cm) Fresh weight (g) Capillary root number (stripes) Root surface area (cm 2 )]]> CK 4.69 ± 0.21 c ]] 1.0415 ± 0.0065 c ]] 6.18 ± 1.87 c ]] 12.57 ± 1.29 d ]] Treatment 1 5.19 ± 0.12 b ]] 1.053 ± 0.0026 bc ]] 9.91 ± 2.42 bc ]] 16.71 ± 1.85 c ]] Treatment 2 5.78 ± 0.18 a ]] 1.0669 ± 0.0063 a ]] 15.73 ± 1.09 a ]] 24.64 ± 3.14 a ]] Treatment 3 5.24 ± 0.12 ab ]] 1.0566 ± 0.0028 ab ]] 11.18 ± 1.9 abc ]] 19.35 ± 2.40 b ]] Treatment 4 5.01 ± 0.24 bc ]] 1.0547 ± 0.0031 abc ]] 11.64 ± 1.89 ab ]] 21.18 ± 2.71 ab ]]

[0057] 2. Effect on the root growth and development of water-cultured rice:

[0058] The rice variety "Nipponbare" was selected, and the full, uniform, and consistent rice seeds were disinfected with 5% sodium hypochlorite solution for 5 min, 75% alcohol for 1 min, and then washed with sterile water for 5-6 times. The disinfected and dried rice seeds were used for germination until they were white. Then, the white rice seeds were transplanted into 96-hole small water culture boxes and treated with nutrient solution (Yoshida nutrient solution, pH 5.8±0.2). Five groups of treatments were set: the simplest peptide was 5 ng / mL, 20 ng / mL, 50 ng / mL, and 100 ng / mL, respectively, and the control group CK was treated with the same volume of sterile water. The water culture boxes were placed in a climate chamber at a temperature of 28°C, a humidity of 50%-60%, and a light intensity of 12000-14000 lux for 12 h. The growth of rice aboveground and root system was observed. After 14 days of growth, the capillary roots with a diameter of <0.5 mm were counted by stereomicroscopy, and the total root surface area was quantified by Epson root scanner combined with ImageJ.

[0059] The test results are shown in Table 2 and Figure 3Table 2 Effects of different concentrations of minimal peptides on rice root indexes, wherein treatment 1 is 5 ng / mL of minimal peptides, treatment 2 is 20 ng / mL of minimal peptides, treatment 3 is 50 ng / mL of minimal peptides, and treatment 4 is 100 ng / mL of minimal peptides; different lowercase letters in Table 2 represent significant differences at the level of 0.05. The effects of different concentrations of minimal peptides on rice root growth show significant concentration effects and parameter differences. Minimal peptides at 5 ng / mL (9.15 cm) and 100 ng / mL (9.07 cm) show the best effects on the root length index, which is increased by 13.80% and 14.81% respectively compared with CK, and is significantly better than other treatments; the minimal peptides at 20 ng / mL show the highest values in fresh weight (0.326 g), capillary root number (56.5) and root surface area (72.9 cm 2 ), which are increased by 168.65%, 80.50% and 66.86% respectively compared with CK, and show comprehensive root promoting effects. The minimal peptides at 50 ng / mL show sub-optimal effects on fresh weight and root surface area, but the capillary root number is not significantly different from that of treatment 2, which may increase the absorption efficiency by increasing the root branching rather than lengthening the main root. The comprehensive analysis of the test results shows that the treatment 2, i.e., 20 ng / mL of minimal peptides, is the optimal treatment concentration, which promotes the elongation, weight increase and branching of rice root, and makes rice show comprehensive growth advantages; and the higher concentration of 100 ng / mL has a slight inhibitory effect on the development of rice root.

[0060] Table 2 Effects of different concentrations of minimal peptides on rice root indexes.

[0061] Treatment Root length (cm) Fresh weight (g) Capillary root number (stripes) Root surface area (cm 2 )]> CK 7.97 ± 0.25 c ]] 0.1212 ± 0.008 d ]] 31.28 ± 3.85 c ]] 43.66 ± 3.13 d ]] Treatment 1 9.07 ± 0.23 a ]] 0.1502 ± 0.009 c ]] 42.57 ± 2.18 bc ]]> 59.67 ± 3.19 bc ]] Treatment 2 8.62 ± 0.28 b ]] 0.3256 ± 0.021 a ]] 56.46 ± 4.11 a ]] 72.85 ± 5.16 a ]] Treatment 3 8.49 ± 0.22 b ]] 0.2128 ± 0.011 b ]] 50.14 ± 3.24 ab ]] 61.38 ± 4.21 b ]] Treatment 4 9.15 ± 0.27 a ]] 0.1618 ± 0.013 c ]] 41.71 ± 3.54 bc ]] 55.28 ± 2.95 c ]]

[0062] Example Four

[0063] Please refer to Figure 4 and Figure 5 , this embodiment provides a technical solution based on example one: the promoting effect of minimal peptides on tomato root growth and yield.

[0064] After the matrix is sterilized, water is added and mixed thoroughly, then it is loaded into the seedling tray, the surface is flattened, and tomato seeds (dwarf tomato "Tommy") are spotted. The tray is placed in a climate chamber for cultivation. When the tomato grows to the four-leaf stage, the seedlings that are disease-free, pest-free, and have uniform growth are selected and transplanted into flowerpots. The soil in the flowerpots is a mixture of peat soil, earthworm soil, and vermiculite at a ratio of 4:4:6, and is sterilized. The flowerpots are placed in a climate chamber at a temperature of 23°C, a humidity of 50%-60%, and an illumination of 12000-14000lx. The experiment includes 6 groups: 20 ng / mL of the simplest peptide; 50 ug / mL of indole-3-butyric acid (IBA); 10 ug / mL of amaranthine (DA-6); 5 ug / mL of sodium nitrophenol; 10 ug / mL of naphthalene acetic acid (NAA); and a control group CK: the same volume of water. After the seedlings are acclimated for 3 days, the above-mentioned groups are treated by spraying. After the tomato grows for one month, some samples are taken from each group for root washing, and the root development of the tomato is observed. The root surface area and root activity are calculated, and the aboveground and root tissues of the tomato are collected for analysis of the tissue nutrient content (total nitrogen, total phosphorus, and total potassium). The rest of the samples are normally cultivated, and after harvesting, the fruits of each group are weighed for yield determination.

[0065] The root activity is determined according to the following formula:

[0066] ;

[0067] The root development-related indexes are shown in Table 3 and Figure 4 Table 3. Root development-related indexes of dwarf tomato. 2 The simplest peptide has a significant effect on promoting the root development of the tomato. The effect of the simplest peptide on improving the root morphology and enhancing the root activity is equivalent to or even better than that of the commonly used plant growth regulators (indole-3-butyric acid and amaranthine). The total root length (27.24 cm), root surface area (228.13 cm 2 ), and number of capillary roots (142.85) of the tomato treated with the simplest peptide are significantly higher than those of the CK group (p<0.05), and are significantly increased by 37.51%, 56.67%, and 83.02%, respectively, compared with the CK. The root surface area and the number of capillary roots are significantly better than those of the sodium nitrophenol and naphthalene acetic acid treatments. In terms of root activity, the simplest peptide > amaranthine > sodium nitrophenol > indole-3-butyric acid > naphthalene acetic acid > CK. The root activity of the simplest peptide treatment is significantly higher than that of the CK, and is higher than that of the indole-3-butyric acid and naphthalene acetic acid treatments, and is equivalent to that of amaranthine, with an increase of 48.53%, 18.64%, 7.75%, 9.90%, and 33.84%, respectively.

[0068] Table 3. Root development-related indexes of dwarf tomato.

[0069] Treatment Total root length (cm) Root surface area (cm 2 )]> Capillary root number (stripes) Root activity (μg TTF / g·h) CK 19.81 ± 1.18 bc ]] 145.61 ± 11.22 d ]] 78.05 ± 7.52 e ]] 125.02 ± 10.05 d ]]> Minimal peptide 27.24 ± 2.53 a ]] 228.13 ± 18.61 a ]] 142.85 ± 13.62 a ]] 185.69 ± 15.33 a ]] Indole-3-butyric acid 26.40 ± 2.44 a ]] 208.55 ± 16.37 ab ]] 135.2 ± 12.74 ab ]] 156.52 ± 13.82 b ]] Prohydrojasmon 28.79 ± 2.67 a ]] 215.43 ± 17.54 ab ]] 128.74 ± 11.05 b ]] 172.34 ± 14.04 ab ]] Sodium nitrophenolate 23.65 ± 2.10 ab ]] 192.72 ± 15.18 bc ]] 92.89 ±8.45 d ]]> 168.97 ± 14.17 ab ]]> Naphthalene acetic acid 16.34 ± 1.55 c ]] 168.34 ± 13.25 cd ]] 115.85 ± 10.12 c ]] 138.74 ± 11.31 c ]]

[0070] Table 4. Nitrogen, phosphorus, and potassium absorption efficiency of tomato. Figure 5, different lower case letters in Table 4 indicate significant difference at 0.05 level. The minimal peptide showed the best performance in improving nutrient absorption and fruit yield of tomato plants, and the comprehensive effect was significantly better than that of traditional growth regulators. Compared with the CK control group, the absorption efficiency of nitrogen (N), phosphorus (P), and potassium (K) of the plant treated with the minimal peptide increased by 79.6%, 123.5%, and 110.3%, respectively, and significantly promoted the yield of single plant color-changing fruit (+24.0%) and total yield (+29.8%), all reaching a significant level (p < 0.05). Indole-3-butyric acid and amino acid also showed good performance in nitrogen, phosphorus, and potassium absorption and yield, but were still slightly lower than the minimal peptide. The yield-increasing effect of indole-3-butyric acid was similar to that of the minimal peptide, while amino acid was more prominent in potassium absorption. In contrast, the promoting effect of sodium nitrophenolate was weak, and naphthalene acetic acid promoted nutrient absorption to some extent, but the total yield increase was unstable, and the promoting effect on potassium absorption was the weakest.

[0071] From the above index statistics, the minimal peptide treatment not only showed excellent performance in promoting root nutrient absorption, but also significantly improved tomato yield, showing more potential application value.

[0072] Table 4 Nutrient absorption efficiency of nitrogen, phosphorus, and potassium and yield per plant of dwarf tomato.

[0073] Treatment Total nitrogen (N) absorption (mg / plant) Total phosphorus (P) absorption (mg / plant) Total potassium (K) absorption (mg / plant) Single plant color fruit yield (g) Single plant total yield (g) CK 6.22 ± 0.65 c ]] 0.98 ± 0.16 c ]] 7.51 ± 0.72 d ]] 84.51 ± 6.55 c ]] 114.32 ± 12.03 d ]] Minimal peptide 11.17 ± 1.31 a ]] 2.19 ± 0.28 a ]] 15.79 ± 1.29 a ]] 104.76 ± 7.01 a ]] 148.39 ± 11.05 a ]]> Indole-3-butyric acid 10.21 ± 1.08 ab ]] 1.83 ± 0.20 ab ]] 13.82 ± 1.32 ab ]] 98.46 ± 6.23 ab ]]> 136.63 ± 10.78 b ]]> Prohydrojasmon 10.94 ± 1.02 a ]] 1.92 ± 0.24 ab ]] 14.14 ± 1.18 ab ]]> 97.92 ± 6.35 ab ]] 131.23 ± 10.09 b ]]> Sodium nitrophenolate 7.84 ± 0.76 bc ]] 1.26 ± 0.13 bc ]] 11.27 ± 1.10 bc ]] 95.5 ± 7.00 b ]] 123.99 ± 10.21 c ]] Naphthalene acetic acid 9.41 ± 0.94 b ]] 1.54 ± 0.15 b ]] 10.36 ± 0.93 cd ]] 91.48 ± 6.25 bc ]]> 138.63 ± 11.52 b ]]

[0074] Example Five

[0075] For reference Figure 6 and Figure 7 , this embodiment provides a technical scheme based on example one: the effect of minimal peptide on root growth of crops under drought stress.

[0076] 1. Promote the development of wheat roots under drought stress

[0077] The test wheat variety is Jimei 22. Select full, uniform, and consistent test wheat seeds, disinfect with 5% sodium hypochlorite solution for 5 minutes, disinfect by soaking in 75% alcohol for 1 minute, rinse with sterile water for 5-6 times, and prepare for use. The PEG6000 concentration for drought stress of wheat seeds is determined to be 20% in the preliminary experiment. Six groups of CK0, CK1, 20 ng / mL minimal peptide, 200 ug / mL paclobutrazol, 500 ng / mL methyl jasmonate, and 10 ug / mL S-elicitor are set up, wherein CK0 and CK1 are treated with water soaking, and the remaining four groups are treated with the above-mentioned agents, with a soaking time of 20 hours. Add 6 mL of 20% PEG6000 solution in a culture dish with 2 layers of sterile filter paper, and treat CK0 with water; then supplement the solution regularly; repeat each treatment 3 times, with 25 seeds after soaking in each repeat. The culture temperature is 22°C (16 / 8h), the relative humidity is about 70%, and the culture is in the dark. On the 7th day, measure the wheat sprout length, root length, root surface area, number of capillary roots, and root activity.

[0078] The test results are shown in Table 5 and Figure 6 Table 5. Influence of different treatments on the growth of wheat under drought stress.

[0079] Table 5. Influence of different treatments on the growth of wheat under drought stress.

[0080] Treatment Root length (cm) Bud length (cm) Root dry weight (mg) Root activity (μg TTF / g·h) Proline (μg / g FW) CK0 11.89 ± 0.70 a ]] 9.78 ± 0.51 a ]] 28.52 ± 2.10 a ]] 68.52 ± 3.21 a ]] 25.4 ± 3.81 e ]] CK1 5.23 ± 0.46 d ]] 2.41 ± 0.39 d ]] 12.64 ± 1.24 d ]] 26.39 ± 2.42 d ]]> 98.25 ± 5.12 a ]]> Minimal peptide 7.16 ± 0.58 bc ]] 4.30 ± 0.42 b ]] 20.09 ± 1.06 b ]] 48.02 ± 4.06 b ]] 65.79 ± 5.25 d ]] PP333 7.84 ± 0.65 b ]] 4.57 ± 0.44 b ]] 19.31 ± 1.51 b ]] 42.84 ± 3.75 b ]] 69.85 ± 5.86 d ]] Methyl jasmonate 6.09 ± 0.51 cd ]] 3.92 ± 0.31 bc ]] 15.84 ± 1.37 c ]] 35.85 ± 3.08 c ]] 82.02 ± 6.01 b ]]> S-elicitor 6.82 ± 0.64 c ]] 3.22 ± 0.33 cd ]] 17.52 ± 1.45 bc ]] 38.29 ± 3.14 c ]]> 75.24 ± 5.25 c ]]>

[0081] 2. Promote the development of corn root system under drought stress:

[0082] The tested corn variety is Zhengdan 958. The field soil and nutrient medium are sterilized respectively, mixed uniformly at a ratio of 1:1, and water is added to 80% of the soil saturated water holding capacity. Each pot contains 5 seeds of the same size and fullness. The test has 6 groups of treatments: CK0, CK1, 20 ng / mL minimal peptide, 200 ug / mL paclobutrazol, 500 ng / mL methyl jasmonate, and 10 ug / mL S-antibiotic, with 4 repeats for each treatment. The treatments are applied by flushing. The pots are cultured in an artificial climate room at 23°C, with light (16 / 8h) and humidity (60%-70%). The pot mass is measured at regular intervals after stopping watering, and the total weight of the pots is adjusted to maintain the soil water content at 30%-40% of the soil saturated water holding capacity. The growth of the corn is observed regularly, and photographs are taken for record. The corn is harvested when the differences are significant. The morphological indicators such as root length, fresh weight, and root surface area, and the physiological indicators such as antioxidant enzyme activity are measured.

[0083] The corn root development indicators are shown in Table 6 and Figure 7 Under drought stress, the root length, root fresh weight, root surface area, capillary root number, and root activity of the corn are significantly reduced. After the addition of exogenous regulatory substances, the minimal peptide shows the best drought relief effect. Its root length, root surface area, and root activity are significantly increased by 32.16%, 41.05%, and 78.80% respectively compared with CK1. It is significantly better than the paclobutrazol, methyl jasmonate, and S-antibiotic treatments, especially in promoting capillary root development, which is increased by 106.73%, 58.47%, 35.65%, and 13.21% respectively compared with CK1, paclobutrazol, methyl jasmonate, and S-antibiotic. S-antibiotic is the second best in restoring root fresh weight and root activity, while methyl jasmonate has a relatively limited effect on improving the corn root indicators. In summary, the minimal peptide can promote the development of corn roots, increase the root surface area, and increase the number of capillary roots under drought stress.

[0084] Table 6 Corn root development indicators under drought stress of different treatments.

[0085] Treatment Root length (cm) Root fresh weight (g) Root surface area (cm 2 )]> Capillary root number Root activity (μg TTF / g·h) CK0 41.52 ± 1.82 a ]]> 1.98 ± 0.15 a ]] 325.85 ± 12.02 a ]] 105.25 ± 4.25 a ]] 72.29 ± 3.52 a ]]> CK1 29.29 ± 2.14 d ]]> 1.15 ± 0.10 c ]] 205.25 ± 10.25 d ]]> 42.79 ± 2.75 d ]] 21.56 ± 1.57 c ]] Minimal peptide 38.71 ± 1.92 b ]] 1.59 ± 0.14 ab ]] 289.51 ± 13.52 b ]] 88.46 ± 4.41 ab ]] 38.55 ± 3.06 b ]]> PP333 36.49 ± 2.34 bc ]]> 1.42 ± 0.10 b ]] 245.36 ± 15.13 c ]]> 55.82 ± 2.98 c ]]> 30.85 ± 2.45 bc ]] Methyl jasmonate 33.65 ± 2.08 c ]] 1.30 ± 0.09 b ]] 227.85 ± 14.21 cd ]] 65.21 ± 3.08 bc ]] 28.19 ± 1.95 bc ]] S-elicitor 34.84 ± 1.74 c ]] 1.48 ± 0.11 ab ]] 260.47 ± 13.21 bc ]] 78.14 ± 3.14 b ]] 35.84 ± 2.75 b ]]

[0086] The corn-related physiological indexes are shown in Table 7, wherein different lowercase letters represent significant differences at the 0.05 level. Under the drought stress condition, the antioxidant system of corn is unbalanced and the membrane lipid peroxidation is intensified, which is manifested by the significant decrease of SOD activity and CAT activity, the increase of POD activity, and the dramatic increase of MDA and proline contents, reflecting serious oxidative damage and osmotic regulation response. Among different treatments, the minimal peptide shows the strongest antioxidant repair capacity, and the SOD activity thereof is restored to 88.5% of that of CK0, which is significantly higher than that of other treatments; meanwhile, the minimal peptide effectively inhibits the excessive activation of POD, and makes the CAT activity and proline tend to be normalized, and the MDA content close to the level of the control group. In comparison, although the methyl jasmonate can induce the further increase of POD and proline, the repair effect on the SOD / CAT system is weak; and the S-antibiotic and the paclobutrazol show an intermediate phenotype. This shows that the minimal peptide can more efficiently relieve the oxidative damage caused by drought stress by synergistically improving the efficiency of the SOD / CAT primary antioxidant defense and reducing the demand for POD compensation.

[0087] Table 7. Physiological indexes of corn under drought stress conditions of different treatments.

[0088] Treatment SOD (U / g FW) POD (U / g FW / min) CAT (pmol H202 / g FW / min) MDA (nmol / g FW) Proline (μg / g FW) CK0 70.34 ± 4.28 a ]] 25.74 ± 2.16 d ]] 15.42 ± 1.08 a ]] 3.85 ± 0.45 d ]] 26.56 ± 2.34 e ]] CK1 30.27 ± 2.85 e ]] 65.63 ± 5.22 a ]] 5.67 ± 0.64 c ]] 14.45 ± 1.22 a ]] 96.23 ± 10.65 a ]] Minimal peptide 62.26 ± 4.04 b ]] 32.64 ± 2.61 cd ]] 10.05 ± 0.91 b ]] 5.32 ± 0.50 cd ]] 45.23 ± 3.94 d ]] PP333 49.85 ± 3.64 d ]] 38.52 ± 3.09 c ]] 9.76 ± 0.85 b ]] 6.26 ± 0.55 c ]] 68.45 ± 5.81 c ]] Methyl jasmonate 41.52 ± 3.29 d ]] 52.85 ± 4.18 b ]] 7.44 ± 0.72b c ]] 9.73 ± 0.86 b ]] 80.36 ± 7.58 b ]] S-elicitor 52.06 ± 3.82 c ]] 46.46 ± 3.73 bc ]] 7.29 ± 0.43 bc ]] 7.56 ± 0.61 bc ]] 58.41 ± 4.72 cd ]]

[0089] The above merely describes certain exemplary embodiments of the present application in a descriptive manner, and it is needless to say that the described embodiments can be modified in various ways without departing from the spirit and scope of the present application for those skilled in the art. Therefore, the above figures and descriptions are illustrative in nature and should not be understood as limiting the scope of protection of the claims of the present application.

Claims

1. A simple method for preparing peptides, characterized in that, Includes the following steps: A1. Inoculate *Pseudomonas nodosa* onto PDA solid medium and incubate at 25°C for 5 days. After the strain is activated, transfer it to an Erlenmeyer flask containing 200 mL of PDA liquid seed medium using the agar block method. Shake and incubate for 5-7 days to obtain the first-stage seed culture. A2. After sterilizing and cooling the 5L fermenter, add 6% primary seed liquid and culture for 5 days to obtain fermentation broth; A3. Centrifuge the fermentation broth to separate mycelium and filtrate. After washing the mycelium, dry it at 60°C, weigh it and crush it. Extract it three times with an equal volume of ethanol. Mix the extracts three times with a magnetic stirrer, sonicate for 1 hour, vacuum filter, and collect the filtrate, which is the extract. A4. After the extract is concentrated by rotary evaporation, it is extracted three times with an equal volume of ethyl acetate containing 5% acetone. The supernatant is collected, and the extract is evaporated by rotary evaporation and dried under nitrogen to obtain the crude extract. A5. The crude extract was initially separated by silica gel column chromatography (200-300 mesh) and ODS pressurized chromatography. After TLC merging, it was purified by HPLC and analyzed by NMR to obtain the cyclic (glycine-glycine) dipeptide, which is the simplest peptide.

2. The method for preparing a simple peptide according to claim 1, characterized in that, The tuberculate fungus described in A1 is deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC NO.42392.

3. A simple method for preparing peptides, characterized in that, Includes the following steps: S1. Using L-glycine and ethylene glycol as raw materials, the reaction was carried out under reflux at 120-150℃ for 8 hours under the catalysis of p-toluenesulfonic acid and nitrogen protection. The reaction progress was monitored by TLC during the process. After the reaction is complete, the product is collected by cooling and crystallization. S2. Wash the product with pre-cooled anhydrous ethanol and diethyl ether in sequence, washing each three times with pre-cooled anhydrous ethanol and diethyl ether to obtain the washed product. S3. The washed product was recrystallized using an ethanol-water mixed solvent and dried under vacuum at 40-50°C for 12 hours to constant weight to obtain a cyclic (glycine-glycine) dipeptide, which is the simplest peptide.

4. The method for preparing a simple peptide according to claim 3, characterized in that, The developing solvent for the TLC described in S1 is a mixture of ethyl acetate and methanol, with a volume ratio of 5:1 between the two.

5. The method for preparing a simple peptide according to claim 3, characterized in that, The cooling and crystallization temperature described in S1 is 0-5℃.

6. The method for preparing a simple peptide according to claim 3, characterized in that, The volume ratio of ethanol to water in the ethanol-water mixed solvent described in S3 is 8:

2.

7. The application of a simple peptide prepared by the method of any one of claims 1-6 in agriculture.

8. The application according to claim 7, characterized in that, The application in agriculture is for promoting the development of plant roots, where the plant is a dicotyledonous or monocotyledonous plant.

9. The application according to claim 8, characterized in that, The dicotyledonous plants include Arabidopsis thaliana and tomato; the monocotyledonous plants include rice, wheat and corn.

10. The application according to claim 7, characterized in that, The concentration of the simplest peptide used is 5-100 ng / mL.

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