Preparation method and application of cyclic (isoleucine-isoleucine) dipeptide

Abstract

The invention discloses a preparation method and application of cyclic (isoleucine-isoleucine) dipeptide, and belongs to the technical field of biology. The invention discloses a structure of a synthetic compound through nuclear magnetic identification, and discloses a detection method of the compound through liquid phase detection. Practical application finds that the compound has the functions of promoting plant growth, increasing crop yield, improving the resistance of crops to plant viruses and improving the antibacterial function of the crops, and field experiments find that the compound has the effect of regulating plant growth and has a good prevention effect on viruses.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a preparation method of a cyclic (isoleucine-isoleucine) dipeptide and an application thereof. Background Art

[0002] Plants live in a complex environment and face a variety of potential pathogens and beneficial microorganisms. Plant pathogens directly threaten the growth and yield of crops, resulting in reduced or even total crop failure. Traditional chemical pesticides are widely used worldwide to prevent or control crop diseases. However, the extensive use of chemical pesticides has caused harmful pesticide residues and environmental pollution, reduced crop quality, and threatened human health. Therefore, there is an urgent need to develop new green, efficient, and safe strategies to increase crop yields and improve the current situation.

[0003] The article "Research Progress on Brassinolide and ZNC Immunoinducers" points out that ZNC immunoinducers are secondary metabolites derived from plant endophytic fermentation, specifically secondary metabolites extracted from the mycelium of the endophytic fungus Paecilomyces variotii isolated and purified from wild seabuckthorn plants. Patent CN109824757 discloses a cyclic (isoleucine-isoleucine) dipeptide and its preparation method. A strain of Paecilomyces variotii SJ1 isolated from wild seabuckthorn is fermented, separated and extracted to obtain an extract, which is ZNC. After the extract is concentrated by rotary evaporation, it is chromatographed on a silica gel column, eluted with different solvent gradients, and finally separated and purified by HPLC. Finally, the sample is collected and purified, and the single compound product cyclic (isoleucine-isoleucine) dipeptide is obtained after freeze-drying.

[0004] The patent also provides the application of cyclic (isoleucine-isoleucine) dipeptide in promoting crop seed germination, improving crop salt resistance, inducing crop flowering, resisting cucumber downy mildew, and enhancing crop frost resistance.

[0005] However, there is no further research on the source and application of this substance. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention provides a method for preparing a cyclic (isoleucine-isoleucine) dipeptide and its application. The technical solution implemented by the present invention is:

[0007] As the first aspect of the present invention, a method for preparing a cyclic (isoleucine-isoleucine) dipeptide is provided. The cyclic (isoleucine-isoleucine) dipeptide is obtained by fermentation, separation and purification of Purpureocillium lilacinum. The Purpureocillium lilacinum strain has been deposited in the General Microbiology Center of China National Committee for the Preservation and Administration of Microbiological Cultures on May 27, 2024, with the address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 41240.

[0008] Furthermore, the preparation method of the cyclo(isoleucine-isoleucine) dipeptide comprises: after culturing the Pseudomonas lilacinus on a PDA plate, adding sterile water, scraping spore threads and spores to prepare a bacterial suspension, filtering to prepare a spore suspension, culturing the spore suspension in a culture medium at a culture temperature of 25° C. to 35° C. and a pH value of 5 to 7, centrifuging the culture solution, filtering to collect mycelium, and drying to a constant weight;

[0009] The mycelium is mixed with ethanol, ultrasonically vibrated, and the broken bacterial liquid is vacuum filtered, and the filtrate is collected to obtain the extract; the extract is concentrated by rotary evaporation and freeze-dried to obtain freeze-dried powder.

[0010] As a second aspect of the present invention, it is to provide the use of cyclic (isoleucine-isoleucine) dipeptide in promoting the growth of rice.

[0011] Furthermore, the application in promoting the growth of rice includes promoting the growth of the aboveground part of rice and promoting the growth of the roots of rice.

[0012] Furthermore, the concentration of the cyclic (isoleucine-isoleucine) dipeptide sprayed is 5 to 20 nm / mL.

[0013] As the third aspect of the present invention, it provides the use of cyclic (isoleucine-isoleucine) dipeptide in promoting the synthesis of rice organic matter.

[0014] Furthermore, the application of promoting the synthesis of rice organic matter includes promoting the increase of the synthesis of soluble protein, dry matter, nitrogen accumulation, starch, and free amino acids.

[0015] As a fourth aspect of the present invention, it is to provide the use of cyclic (isoleucine-isoleucine) dipeptide in promoting tobacco resistance to PVX infection.

[0016] Furthermore, in the application of the cyclic (isoleucine-isoleucine) dipeptide in promoting tobacco resistance to PVX infection, the concentration of the cyclic (isoleucine-isoleucine) dipeptide is 5 to 20 ng / mL.

[0017] As a fifth aspect of the present invention, it is to provide the use of cyclic (isoleucine-isoleucine) dipeptide in Arabidopsis thaliana resisting infection by pstDC3000 bacteria.

[0018] Furthermore, the cyclic (isoleucine-isoleucine) dipeptide is used in Arabidopsis thaliana to resist infection by pstDC3000 bacteria. The cyclic (isoleucine-isoleucine) dipeptide is dissolved in an organic solvent and then diluted with water to a use concentration for spraying. The use concentration is 5 to 20 ng / mL.

[0019] As a sixth aspect of the present invention, it is to provide the use of cyclic (isoleucine-isoleucine) dipeptide in increasing the yield of corn, and the use of cyclic (isoleucine-isoleucine) dipeptide in increasing the protein and starch content of corn.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The present invention provides a microbial-based preparation method of cyclic (isoleucine-isoleucine) dipeptide, which is prepared by fermentation, separation and purification using Purpureocillium lilacinum, providing a new approach for the preparation of cyclic dipeptide;

[0022] (2) The present invention has found that the compound has the function of promoting plant growth, increasing crop yield, improving crop resistance to plant viruses, and improving crop antibacterial function. In field experiments, it has been found that the compound has the function of regulating plant growth and has a good preventive effect on viruses.

[0023] (3) The present invention provides the use of cyclic (isoleucine-isoleucine) dipeptide in promoting rice growth and increasing corn yield, especially promoting the growth of the aboveground part of rice and the growth of rice roots, promoting the synthesis of rice organic matter, and increasing the protein and starch content of corn; in addition, cyclic (isoleucine-isoleucine) dipeptide can promote tobacco resistance to PVX infection and Arabidopsis resistance to pstDC3000 infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0025] Figure 1 It is the mass spectrometry negative ion chromatogram in the structure identification experiment of the compound cyclo(isoleucine-isoleucine) dipeptide in Example 3;

[0026] Figure 2 It is the mass spectrometry positive ion chromatogram in the structure identification experiment of the compound cyclo(isoleucine-isoleucine) dipeptide in Example 3;

[0027] Figure 3This is a liquid chromatogram from the structural identification experiment of the compound cyclo(isoleucine-isoleucine) dipeptide in Example 3;

[0028] Figure 4 This is the H NMR spectrum of the compound cyclo(isoleucine-isoleucine) dipeptide structure identification experiment in Example 3;

[0029] Figure 5 This is the liquid chromatogram of the synthetic sample in Example 4;

[0030] Figure 6 is the mass spectrum of b in Example 4;

[0031] Figure 7 is the mass spectrum of c in Example 4;

[0032] Figure 8 The effect of cyclo(isoleucine-isoleucine) dipeptide on the growth of rice seedlings under different nitrogen nutrition conditions in Example 5 (28 days);

[0033] Fig. 9 The effect of cyclo(isoleucine-isoleucine) dipeptide on the growth of rice seedlings under different nitrogen nutrition conditions in Example 5 (35 days);

[0034] Fig.10 The effect of cyclic (isoleucine-isoleucine) dipeptide on underground roots of rice under different nitrogen nutrition in Example 5 (35 days);

[0035] Fig.11 The fluorescence of tobacco leaves under 365nm ultraviolet light in Example 6;

[0036] Fig.12 The relative expression level OD450 of the virus in tobacco leaves under different agent treatments in Example 6;

[0037] Fig.13 The pstDC3000 bacteria infect Arabidopsis leaves after spraying different concentrations of the compound in Example 7;

[0038] Fig.14 This is a diagram showing that the compounds in Example 7 showed a certain resistance to infection by pstDC3000 bacteria 5 days after spraying Arabidopsis leaves. DETAILED DESCRIPTION

[0039] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0040] Pseudomonas lilacinus is an endoparasitic fungus widely distributed around the world. It has the advantages of high efficacy, wide host range, and easy cultivation. Its metabolites can produce physiologically active substances such as indoleacetic acid, cytokinins, and various enzymes, which stimulate crop growth and promote the growth of plant vegetative organs. It also promotes seed germination and growth.

[0041] The present invention ferments, separates and purifies Purpureocillium lilacinum (the strain has been deposited in the General Microbiological Center of China Microorganism Culture Collection Committee on May 27, 2024, the address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 41240), and then performs mass spectrometry and nuclear magnetic resonance analysis to identify it as cyclic (isoleucine-isoleucine) dipeptide. Purpureocillium lilacinum has low requirements for nutritional conditions and can grow not only on a variety of conventional culture media, but also in a variety of natural matrices, such as agricultural and sideline product waste, waste residues and plant leaves or plant extracts.

[0042] Natural cyclic dipeptides are mainly derived from mycelium extracts.

[0043] Example 1, Isolation of the Natural Compound Cyclic (Isoleucine-Isoleucine) Dipeptide

[0044] The components of the culture medium used in the embodiment are as follows:

[0045] PDA culture medium: 150-250 g potato, 20-25 g agar powder, 15-25 g glucose, 1000 mL distilled water; sterilize at 121°C for 20 min before use.

[0046] After the test strain was cultured on the PDA plate for 7 days, 10 mL of sterile water was added, and the spores and spores were scraped to make a bacterial suspension, which was filtered through double-layer gauze to make a spore suspension. The concentration of the spore suspension was adjusted to 1×10 9 pcs / mL for future use.

[0047] The spore suspension with adjusted concentration was transferred to PDA medium at 25°C-35°C and pH 5-7. After culturing for 5 days, the culture solution was centrifuged at 8000 r / min for 20 min, the mycelium was collected by filtration, and dried at 60°C to constant weight.

[0048] The mycelium was mixed with 30% ethanol at a mass ratio of 3:7, and ultrasonically vibrated for 2 hours at an ultrasonic power of 680w and an ultrasonic frequency of 4s working and 6s resting. The broken bacterial liquid was vacuum filtered and the filtrate was collected as the extract. The extract was concentrated by rotary evaporation and freeze-dried to obtain freeze-dried powder.

[0049] The lyophilized powder was dissolved in 30% ethanol and separated by Shimadzu high performance liquid chromatography. The chromatographic column was an Inertsil ODS-3 column (4.6×250 mm, 5 μm). An acetonitrile-water system was used for elution. The flow rate was 1 mL / min. The UV detection wavelength was 200 nm. The mobile phase ratio was: water: acetonitrile = 80:20. 500 μg of the sample was dissolved in 300 μl of 30% ethanol for injection detection. The eluted product was collected, freeze-dried, purified by HPLC and analyzed by NMR. The cyclodipeptide compound was determined to be: cyclo(Ile-Ile), and its structure was:

[0050]

[0051] Example 2: Synthesis of compound cyclo(isoleucine-isoleucine) dipeptide

[0052] If a large number of compounds are needed, chemical synthesis methods are required. The present invention provides a method for synthesizing a cyclic (isoleucine-isoleucine) dipeptide compound.

[0053] Take L-isoleucine (10 g, 0.0762 mol, Mw: 131.17) in toluene at 20 V and heat to reflux, about 110° C., preset a water separator to perform water separation operation every 1 hour, and maintain the reflux reaction for 6 to 10 hours.

[0054] After the reaction, the temperature was lowered to about 75°C, 10V of anhydrous ethanol was added, stirred and dispersed, and then the temperature was lowered to 0-10°C for crystallization for 1-4h, filtered, and dried to obtain 8.96g of the product as a white solid, which was a cyclo(isoleucine-isoleucine) dipeptide compound with a yield of about 50%.

[0055] Screening and optimization of suitable reaction solvents ensures that the reaction temperature is suitable for operation; increasing the amount of solvent will make the reaction system more conducive to the intermolecular cyclization reaction; water separation operation can accelerate the reaction, shorten the reaction time, and reduce side reactions and impurity generation; thanks to the optimization of the previous reaction degree, higher yield products can be obtained after simple post-treatment.

[0056] Example 3: Identification of the structure of the compound cyclo(isoleucine-isoleucine) dipeptide

[0057] Instrument model: Waters SQ-D-2;

[0058] Testing conditions:

[0059] Liquid phase:

[0060] Injection volume: 2 μL;

[0061] Column temperature: 30°C;

[0062] Mobile phase: A-0.1% formic acid water, B-acetonitrile, 0min A: 90%, 10min A: 10%, 12-15min A: 90%;

[0063] Running time: 15min;

[0064] Detection wavelength: 210nm;

[0065] Flow rate: 1 mL / min;

[0066] Chromatographic column: ZORBAX SB-C18 4.6*150mm 3.5μm;

[0067] Mass spectrometry part:

[0068] Ion source: API-ES, positive and negative ion detection mode;

[0069] Capillary voltage: 3.0kV;

[0070] Cone voltage: 40V;

[0071] Desolventizing temperature: 450°C;

[0072] Flow rate: 650L / Hr.

[0073] After mass spectrometry detection, the mass spectrum of the synthetic compound cyclo(isoleucine-isoleucine) dipeptide was determined, and the mass spectrum negative ion chromatogram was as follows: Figure 1 The positive ion chromatogram is shown in Figure 2 As shown in the liquid chromatogram Figure 3 shown.

[0074] HNMR test conditions:

[0075] NMR spectrometer model: Bruker400M

[0076] 5 mg of sample was dissolved in 0.6 mL of DMSO, and the hydrogen spectrum was detected by 400 M nuclear magnetic resonance.

[0077] According to the mass spectrometry results, the isomer spectrum of the cyclodipeptide compound is obtained as follows: Figure 4 shown.

[0078] Example 4: Detection of synthetic samples of compound cyclo(isoleucine-isoleucine) dipeptide

[0079] Weigh 5 mg of the synthetic compound sample, dissolve it in ethanol and make it up to 5 mL. After mixing, take 1 mL and pass it through a 0.22 μm organic filter membrane and measure it on the liquid chromatography machine according to the following reference conditions.

[0080] Liquid chromatography reference conditions:

[0081] Chromatographic column: Inertsil ODS-3, 5μm 4.6×250mm

[0082] Mobile phase: water: acetonitrile = 80:20

[0083] UV detector wavelength: 200nm

[0084] Column temperature: 40°C

[0085] Flow rate: 1mL / min

[0086] Chromatogram Figure 5 As shown, the synthetic sample was detected and analyzed using the area normalization method: the content of cyclo(isoleucine-isoleucine) dipeptide was in the range of 90-95%.

[0087] Further mass spectrometry analysis of the samples revealed that b and c were isomers of a.

[0088] bMass spectrometry results are as follows Figure 6 As shown, the mass spectrum of c Figure 7 shown.

[0089] The invention provides a compound cyclic (isoleucine-isoleucine) dipeptide which has the effects of resisting drought, resisting bacteria, promoting growth and resisting viruses.

[0090] Example 5: Growth-promoting effect of compound cyclo(isoleucine-isoleucine) dipeptide on rice

[0091] 5.1 Effects of compounds on rice growth under different nitrogen nutrition conditions

[0092] This experiment was designed with 12 treatments, each of which was repeated 9 times. The results are shown in Tables 1, 2, and 3.

[0093] Table 1 Seedling stage test treatment design table

[0094]

[0095] Table 2 Effects of compounds on the growth of rice seedlings under different nitrogen nutrition conditions (aerial part) (14-35 days)

[0096]

[0097]

[0098] The results showed that the compound could promote the growth of the aboveground part of rice under different nitrogen concentrations. Through related growth indicators, it was found that after the compound was dissolved in a small amount of dimethyl sulfoxide and then diluted with water to the use concentration for spraying, the rice plant height, leaf area, stem diameter and SPAD were significantly higher than those of the CK group at 35 days. The growth-promoting effect on rice seedlings increased with the increase of compound concentration. Under the nitrogen nutrition condition of 80 mg / L, the growth-promoting effect on the aboveground part of rice was most significant when the compound concentration was 20 ng / mL (P < 0.05), followed by 5 ng / mL and 10 ng / mL ( Figure 8 , 9).

[0099] Table 3 Effects of cyclo(isoleucine-isoleucine) dipeptide on underground roots of rice under different nitrogen nutrition conditions (underground part)

[0100] deal with Fresh weight(g) Dry weight (g) Root length (cm) Number of roots (bars) N20-H0 1.7675 0.16 9.9125 16.06 N20-H5 2.9994 0.22 10.8813 19.81 N20-H10 3.0763 0.22 12.5313 19.88 N20-H20 3.2069 0.25 12.7438 22.06 N40-H0 2.1088 0.17 11.7500 17.63 N40-H5 3.1819 0.20 12.4436 23.19 N40-H10 3.6044 0.25 13.3250 25.19 N40-H20 4.4125 0.31 13.6563 24.56 N80-H0 2.8319 0.23 13.5000 16.69 N80-H5 5.2963 0.31 13.4438 18.81 N80-H10 4.7713 0.42 13.6625 20.88 N80-H20 5.4963 0.43 14.3063 21.13

[0101] The results showed that the compounds could promote the growth of rice roots under different nitrogen nutrition conditions, and each treatment group was significantly better than the CK group (P < 0.05). The best root growth promoting effect was achieved when the nitrogen nutrition was 80 mg / L and the compound was 20 ng / mL ( Fig.10 ).

[0102] 5.2 Effects of compounds on organic matter synthesis in rice plants under different nitrogen nutrition conditions

[0103] The results showed that the organic matter synthesis compounds in each treatment can effectively promote the synthesis of rice organic matter. Under different nitrogen nutrition conditions (20, 40, 80 mg / L), after spraying the compounds, the overall trend of soluble protein, dry matter, total nitrogen, total nitrogen accumulation, and free amino acid synthesis was significantly higher than that of the CK treatment group (P < 0.05). Among them, when the nitrogen nutrition concentration was 80 mg / L, the compound had a more significant effect on the soluble protein, dry matter, total nitrogen, total nitrogen accumulation, and free amino acid synthesis of rice plants, which promoted the increase of soluble protein, dry matter, nitrogen accumulation, starch, and free amino acid synthesis.

[0104] Table 4 Effects of compounds on organic matter synthesis in rice plants under different nitrogen nutrition conditions

[0105]

[0106] As shown in Table 4, the results showed that the compounds in each treatment of organic matter synthesis can effectively promote the synthesis of rice organic matter. Under different nitrogen nutrition conditions (20, 40, 80 mg / L), after spraying the compounds, the overall trend of soluble protein, dry matter, total nitrogen, total nitrogen accumulation, and free amino acid synthesis was significantly higher than that of the CK treatment group (P < 0.05). Among them, when the nitrogen nutrition concentration was 80 mg / L, the compound had a more significant effect on the soluble protein, dry matter, total nitrogen, total nitrogen accumulation, and free amino acid synthesis of rice plants, which promoted the increase of soluble protein, dry matter, nitrogen accumulation, starch, and free amino acid synthesis.

[0107] This experiment preliminarily explored the effects of compounds on the growth of rice seedlings under different nitrogen nutrition conditions, and determined relevant growth indicators and physical and chemical indicators. The results showed that the compounds had a good growth-promoting effect on rice.

[0108] Example 6: Effects of Compounds on Tobacco Viruses

[0109] Test on the effective concentration of compounds against PVX in tobacco

[0110] There were 8 treatment groups in this experiment, each treatment was repeated 5 times, and the liquid medicine was sprayed on the leaves by foliar spraying. The treatment methods are shown in Table 5.

[0111] Table 5 Drug treatment table

[0112]

[0113] The virus was inoculated into three true leaves below the tobacco systemic leaves by friction inoculation. On the 5th day after inoculation, the fluorescence of the leaves was observed under 365nm ultraviolet light. The diseased leaves were sampled and stored in a -80℃ refrigerator.

[0114] Potato virus X enzyme-linked immunosorbent assay kit was used to detect the virus content in the leaves of each treatment group. The results showed that after spraying the compound, the fluorescence level on the leaves of each treatment group was significantly reduced compared with CK ( Fig.11 ).

[0115] Different concentrations of compounds have a certain effect on tobacco's ability to resist PVX. As the concentration of the compound increases, tobacco's resistance to PVX first increases and then decreases slightly. The best effect is achieved when the concentration is 5-20 ng / mL ( Fig.12 ), the inhibition rates were 68.74%, 69.93%, 70.62% and 71.53% respectively. When the concentration of the compound was 50 ng / mL, the antiviral infection ability decreased, and the inhibition rate was 53.13%. When the concentration of the compound was 100 ng / mL, the inhibition rate was 29.56% (Table 6).

[0116] Table 6 Inhibition rate of PVX by different agents

[0117]

[0118] Note: Lowercase letters in the table indicate significant differences at 5%.

[0119] The results showed that the compound had a good promoting effect on tobacco resistance to PVX infection, and the effect was most obvious when the concentration was 5-20 ng / mL.

[0120] Example 7: Antibacterial Function of Compounds

[0121] Effects of compounds on Arabidopsis thaliana resistance to pstDC3000 infection

[0122] pstDC3000 vaccination and disease investigation

[0123] Each expanded leaf of Arabidopsis thaliana was inoculated with pstDC3000 bacterial solution by infiltration inoculation method, and 4 Arabidopsis thaliana were inoculated in each replicate. After culturing for 3-5 days in a 24℃ artificial climate chamber for 16 / 8h, samples were taken for determination according to the disease situation. Calculation formula: cfu / g = colony count × 10 6 ×100 / g

[0124] The infection of Arabidopsis leaves by pstDC3000 after spraying different concentrations of compounds (5 days) Fig.13 shown.

[0125] Table 7 Effect of compounds on Arabidopsis thaliana resistance to pstDC3000 (protective effect / %)

[0126]

[0127]

[0128] The results are shown in Table 7. After spraying the compounds on Arabidopsis leaves for 5 days, the leaves showed a certain resistance to the infection of pstDC3000 bacteria ( Fig.14 ), but different concentrations of compounds had different effects on Arabidopsis thaliana's resistance to pstDC3000 infection. Among them, the concentration of 20ng / mL had the best effect, the lowest leaf fungus content, and a protection efficiency of 66.68%.

[0129] Example 8: Field trials of compounds

[0130] The experiment was conducted in Tianbao Village 4, Tianbao Town, Tai'an City, Shandong Province (117°21′20″E, 35°56′36″N), which belongs to the warm temperate semi-humid continental monsoon climate. The test soil is brown loam, which is a common simple moist eluvial soil in the Chinese soil system classification. The test corn variety is Zhengdan 958, with a growth period of 96 to 125 days and a 100-grain weight of about 33.0g. The traditional fertilizers tested were urea (N 46%), superphosphate (P2O5 16%), and potassium sulfate (K2O51%). The experiment had a total of 5 treatments, each with 3 replicates. The area of ​​the plot was 20m 2 (5m×4m), 3 repetitions. The corn field experiment adopted a randomized block design, as shown in the table. The corn planting method was direct seeding, with a row spacing of 60cm and a plant spacing of 20cm. The experimental design and results are shown in Tables 8 and 9.

[0131] Table 8 Experimental design

[0132]

[0133] Table 9 Effect of soluble solution of compound on corn plant height

[0134]

[0135] The data analysis results show that when the dosage of the cyclodipeptide soluble solution is between 0.02 and 0.033 mg / kg, the weight of 100 grains is between 25.41 and 27.20 g, which shows a significant increase trend compared with the blank control.

[0136] When the dosage of the cyclodipeptide soluble solution is between 0.02 and 0.033 mg / kg, the per-acre yield of corn plots is between 560 and 610 kg, and the yield increase rate is between 18.6% and 29.2%. Compared with the blank, spraying the cyclodipeptide soluble solution can significantly increase the per-acre yield of corn.

[0137] Table 10 Corn quality analysis

[0138]

[0139] In terms of quality, protein and starch content are important indicators for evaluating the nutritional value of corn. The test data in Table 10 show that the addition of cyclodipeptide soluble solution improves the protein and starch content of corn, with the starch content increasing by 0.98% to 1.05% and the protein content increasing by 4.52% to 5.85%. It effectively improves the nutritional value of corn and meets the market demand for high-nutrition corn.

[0140] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a cyclic (isoleucine-isoleucine) dipeptide, characterized in that: The cyclic (isoleucine-isoleucine) dipeptide is obtained by fermentation, separation and purification of Pseudomonas lilacinus. The Pseudomonas lilacinus strain was deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee on May 27, 2024, with the address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 41240.

2. The method for preparing the cyclic (isoleucine-isoleucine) dipeptide according to claim 1, characterized in that: The preparation method of the cyclo(isoleucine-isoleucine) dipeptide comprises: culturing the Pseudomonas lilacinus on a PDA plate, adding sterile water, scraping spores and spores to prepare a bacterial suspension, filtering to prepare a spore suspension, culturing the spore suspension in a culture medium at a temperature of 25° C. to 35° C. and a pH value of 5 to 7, centrifuging the culture solution, filtering to collect mycelium, and drying to a constant weight; The mycelium is mixed with ethanol, ultrasonically vibrated, and the broken bacterial liquid is vacuum filtered, and the filtrate is collected to obtain the extract; the extract is concentrated by rotary evaporation and freeze-dried to obtain freeze-dried powder.

3. Application of cyclic (isoleucine-isoleucine) dipeptide in promoting rice growth.

4. The use of the cyclic (isoleucine-isoleucine) dipeptide in promoting rice growth according to claim 3, characterized in that: It includes promoting the growth of the aboveground part of rice and promoting the growth of rice roots.

5. Application of cyclo(isoleucine-isoleucine) dipeptide in promoting the synthesis of organic matter in rice.

6. The use of the cyclic (isoleucine-isoleucine) dipeptide according to claim 5 in promoting the synthesis of rice organic matter, characterized in that: Including promoting the increase of soluble protein, dry matter, nitrogen accumulation, starch and free amino acid synthesis.

7. Application of cyclic (isoleucine-isoleucine) dipeptide in promoting tobacco resistance to PVX infection.

8. Application of cyclic (isoleucine-isoleucine) dipeptide in Arabidopsis thaliana's resistance to infection by pstDC3000 bacteria.

9. Application of cyclic (isoleucine-isoleucine) dipeptide in increasing corn yield, and in increasing the protein and starch content of corn.

10. The use according to any one of claims 3 to 9, characterized in that: Cyclic (isoleucine-isoleucine) dipeptide is dissolved in an organic solvent and then diluted with water to a use concentration for spraying, and the use concentration is 5 to 20 ng / mL.

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