Screening method of a cyclic dipeptide cyclo(l-phenylalanine-l-proline) and its application in promoting germination of peanut seeds

Abstract

The present application belongs to the field of agricultural biotechnology, and relates to a screening method of a cyclic dipeptide Cyclo(L-Phe-L-Pro) and application thereof in promoting germination of peanut seeds. The cyclic dipeptide Cyclo(L-Phe-L-Pro) is isolated from a biocontrol Bacillus velezensis, and application thereof in promoting germination of aged peanut seeds and in preparing a preparation for promoting germination of aged peanut seeds and other products is provided. In the present application, a concentration and seed soaking time double-factor test design is adopted to perform seed soaking treatment on natural aged peanut varieties Huayu 25 and Nongda R271. The results show that each treatment group can significantly improve the germination performance of the aged peanut seeds, including germination energy, germination uniformity, germination rate and germination index. The present application is suitable for seeds of different peanut varieties such as Huayu 25 and Nongda R271, and provides a general solution to the problems of low emergence rate and uneven emergence caused by seed aging.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically relating to a screening method for cyclic dipeptide Cyclo (L-Phe-L-Pro) and its application in promoting peanut seed germination. Background Technology

[0002] Seed viability is a common problem in agricultural production. Seed aging leads to reduced viability, resulting in decreased germination rate, reduced germination potential, and weak seedling growth, ultimately severely impacting crop yield and quality. Aged peanut seeds refer to peanut seeds that have undergone natural physiological deterioration after long-term conventional drying and storage following harvest, exhibiting decreased seed viability, reduced germination potential, uneven emergence, and a significantly reduced germination rate. Traditional methods to address seed aging generally employ physical and chemical treatments. However, physical treatments have limited effectiveness, and chemical treatments easily cause environmental pollution and pesticide residues, failing to meet the needs of green agriculture. In recent years, biocontrol bacteria and their metabolites have gained widespread attention in agricultural production due to their environmental friendliness and high safety, and have been applied to remediate seed aging. For example, Bacillus belye (… Bacillus velezensis Biological control bacteria (Bacillus belychnophora) have been reported to possess growth-promoting and disease-resistant activities. However, current technologies that directly use fermentation broth or crude extracts of biocontrol bacteria suffer from problems such as complex composition, unclear active substances, ambiguous mechanisms of action, and unstable effects, making precise application and standardized production difficult. Therefore, systematically analyzing and identifying key active monomers from active biocontrol bacteria to develop seed germination promoters with clearly defined components and stable effects is a pressing technical problem to be solved in this field. Currently, there are no reports of isolating Cyclo(L-Phe-L-Pro) from Bacillus belychnophora and using it to promote germination in aged peanut seeds.

[0003] To address the problems of low germination rate, uneven emergence, residual pollution from chemical regulators, and unclear and unstable effects of microbial agents in aged peanut seeds, this invention, through experimental research, screened and identified a cyclic dipeptide Cyclo (L-Phe-L-Pro) from Bacillus belyeis. Verification showed that it plays a key role in regulating the germination of aged seeds. Cyclo (L-Phe-L-Pro) is a naturally occurring small-molecule metabolite with stable structure, strong biological activity, and is safe and non-toxic. This provides an important approach for developing efficient and environmentally friendly seed germination promoters. Summary of the Invention

[0004] The purpose of this invention is to address the aforementioned problems in the existing technology by proposing a screening method for the cyclic dipeptide Cyclo (L-Phe-L-Pro) and its application in promoting peanut seed germination. This method is based on metabolomics technology to screen the cyclic dipeptide Cyclo (L-Phe-L-Pro) from Bacillus belye, and further provides a novel application of this cyclic dipeptide in promoting the germination of aged peanut seeds. This invention provides a highly efficient, environmentally friendly, residue-free, and precisely controllable technology for restoring the viability of aged peanut seeds, which can significantly improve the germination quality of naturally aged peanut seeds and is suitable for agricultural production applications.

[0005] The technical solution of this invention includes two aspects: This invention provides the application of the cyclic dipeptide Cyclo (L-Phe-L-Pro) in promoting peanut seed germination. In particular, it provides the application of the cyclic dipeptide Cyclo (L-Phe-L-Pro) in promoting the germination of aged peanut seeds.

[0006] This invention also provides the application of the cyclic dipeptide Cyclo (L-Phe-L-Pro) in the preparation of products that promote the germination of peanut seeds. Preferably, this invention provides the application of the cyclic dipeptide Cyclo (L-Phe-L-Pro) in the preparation of products that promote the germination of aged peanut seeds.

[0007] Furthermore, the cyclic dipeptide Cyclo(L-Phe-L-Pro) was screened from Bacillus belye ( Bacillus velezensis) BVZX-1.

[0008] Furthermore, the aged peanut seeds are peanut seeds that spontaneously undergo physiological deterioration under natural storage conditions as the storage time is extended; the aged peanut seed varieties include, but are not limited to, Huayu 25 and Nongda R271.

[0009] Furthermore, the product is an agriculturally acceptable seed treatment agent, including formulations, soaking solutions, or seed coating agents.

[0010] Furthermore, the cyclic dipeptide Cyclo (L-Phe-L-Pro) is used to improve germination potential or uniformity of germination, and to increase germination rate and germination index.

[0011] This invention provides a method for preparing the cyclic dipeptide Cyclo (L-Phe-L-Pro), comprising the following steps: (1) Inoculate Bacillus belye into the fermentation medium for fermentation culture, centrifuge the fermentation broth and collect the supernatant; (2) The metabolites in the fermentation broth were analyzed by non-targeted metabolomics using ultra-high performance liquid chromatography-mass spectrometry. The chromatographic column used was a C18 column, and the mobile phase was 0.1% formic acid water and 0.1% formic acid acetonitrile. (3) Principal component analysis and orthogonal partial least squares discriminant analysis were used to screen differential metabolites with variable projection importance >1 and P value <0.05; (4) By Bayes correlation analysis, the metabolite that was present at all three soaking times related to the germination index was identified as Cyclo (L-Phe-L-Pro).

[0012] This invention also provides the application of Cyclo (L-Phe-L-Pro), a cyclic dipeptide prepared according to the above-described preparation method, in promoting the germination of aged peanut seeds.

[0013] This invention provides a formulation for promoting the germination of aged peanut seeds, wherein the active ingredient of the formulation is Cyclo (L-Phe-L-Pro) obtained by the preparation method described above.

[0014] Furthermore, the concentration of Cyclo (L-Phe-L-Pro) in the formulation is 333.33~2000 μg / mL.

[0015] This invention also provides a method for promoting the germination of aged peanut seeds. A certain mass of Cyclo (L-Phe-L-Pro) is weighed and dissolved in analytical grade DMSO to prepare a stock solution with a concentration of 10 mg / mL. Then, the stock solution is diluted with DMSO solvent to a working solution with a concentration of 333.33~2000 μg / mL. DMSO of the same concentration is used as a control. Aged peanut seeds are soaked in the working solution for 60~180 min at a temperature of 20℃.

[0016] Furthermore, when it is necessary to improve the germination potential or germination uniformity of aged peanut seeds, the concentration of the working solution used is 444.44 μg / mL, and the soaking time is 180 min. When it is necessary to improve the germination rate and germination index of aged peanut seeds, the concentration of the working solution should be 2000 μg / mL, and the soaking time should be any time within the range of 91.5 to 177 min.

[0017] The beneficial effects of this invention are: (1) This invention establishes for the first time a method for screening cyclic dipeptide Cyclo (L-Phe-L-Pro) from Bacillus belye using metabolomics, providing a technical demonstration for the precise discovery of microbial active monomers.

[0018] (2) Significant germination promotion effect: All five treatment groups of the present invention can significantly improve the germination performance of aged peanut seeds. Compared with the water control group, Huayu 25 has a germination potential of at least 10 percentage points, a germination rate of at least 15 percentage points, and a germination index of at least 8.23; Nongda R271 has a germination potential of at least 13.33 percentage points, a germination rate of at least 18.33 percentage points, and a germination index of at least 6.08.

[0019] (3) Precise and controllable parameters: Combinations of different concentrations and soaking times can specifically improve different germination indicators. Therefore, they can be flexibly selected and optimized according to production needs. Among them, the 444.44 μg / mL + 180 min treatment has the best effect on improving germination potential or uniformity (difference of 0.2160), and the 2000 μg / mL soaking treatment for 91.5~177 min has the best effect on improving germination rate (increase of 0.2451) and germination index (increase of 13.2330).

[0020] (4) Environmentally friendly and safe: Cyclo (L-Phe-L-Pro) is a natural metabolite of the biocontrol bacterium Bacillus belye, with no chemical residues, and is safe for seeds, seedlings and the environment, which is in line with the concept of green agricultural development.

[0021] (5) Wide range of applications: It is applicable to seeds of different peanut varieties such as Huayu 25 and Nongda R271, and provides a general technical solution to solve the problem of low germination rate caused by seed aging. Attached Figure Description

[0022] Figure 1 This is the total ion chromatogram from the metabolomics analysis results of Bacillus belyssus.

[0023] Figure 2 This is a graph showing the OPLS-DA score from the metabolomics analysis results of Bacillus belyssus.

[0024] Figure 3 Volcano plot of differentially metabolites in the metabolomics analysis results of Bacillus belyssus.

[0025] Figure 4 This is a mass spectrometry image of the cyclic dipeptide Cyclo (L-Phe-L-Pro).

[0026] Figure 5 This is the ion current chromatography (EIC) chromatogram of the cyclic dipeptide Cyclo (L-Phe-L-Pro).

[0027] Figure 6 The results of the experiment on the effect of different soaking times / concentrations of cyclic dipeptide Cyclo (L-Phe-L-Pro) on the germination of aged peanut seeds of Huayu 25.

[0028] Figure 7 The results of the experiment on the effect of different soaking times / concentrations of cyclic dipeptide Cyclo (L-Phe-L-Pro) on the germination of aged peanut seeds of Agricultural University R271. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Unless otherwise specified, any techniques or conditions not specifically described in this invention can be performed according to the conventional techniques or conditions described in literature, books, papers, etc. in this field, or according to the product instructions. Unless otherwise stated, the raw materials, reagents, or instruments used in the following examples that do not specify the manufacturer are all conventional products that can be purchased from legitimate channels or prepared by known methods.

[0031] The aged peanut seeds used in this invention are naturally aged peanut seeds harvested in September 2024 and stored at room temperature until March 2026.

[0032] The Cyclo (L-Phe-L-Pro) used in this invention is a commercial standard purchased from Shanghai Taoshu Biotechnology Co., Ltd. (TargetMol), batch number 226979 (5 mg, purity 97.08%).

[0033] Reagent: DMSO (Solepro).

[0034] The Bacillus velezensis used in this invention was isolated from peanut kernel HY25, with accession number CGMCC No. 36546. The depository institution is the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is November 7, 2025, and the classification name is Bacillus velezensis.

[0035] Example 1

[0036] Metabolomics screening and identification of cyclic dipeptide Cyclo (L-Phe-L-Pro)

[0037] 1. Bacillus vesiculosus fermentation The preserved Bacillus belye strain BVZX-1 was inoculated into LB liquid medium and cultured at 30℃ and 180 rpm for 24 h with shaking to obtain the seed culture. The seed culture was then transferred at a 1% inoculum to fermentation medium (soluble starch 20 g / L, soybean peptone 10 g / L, NaCl 5 g / L, K₂HPO₄ 1 g / L, MgSO₄·7H₂O 0.5 g / L, pH 7.0) and fermented at 30℃ and 180 rpm for 72 h. The fermentation broth was centrifuged at 8000 rpm for 10 min, and the supernatant was collected.

[0038] 2. Metabolite extraction Weigh 60 mg of sample and place it in a 1.5 mL EP tube; add two small steel balls and 600 μL of methanol-water (V:V=4:1 including mixed internal standard); pre-cool in a -40℃ freezer for 2 min, then grind in a grinder (45 Hz, 2 min); extract by sonication in an ice-water bath for 30 min, and let stand overnight at -40℃; centrifuge for 20 min (12000 rpm, 4℃), and take 150 μL of supernatant into an LC-MS vial with a foot liner for analysis; the quality control sample (QC) is prepared by mixing equal volumes of the extracts from all samples.

[0039] 3. LC-MS / MS analysis Untargeted metabolomics analysis was performed using an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) system. Chromatographic column: ACQUITY UPLC HSS T3 (100 mm × 2.1 mm, 1.8 μm); column temperature: 45℃; mobile phase: A-water (containing 0.1% formic acid), B-acetonitrile; flow rate: 0.35 mL / min; injection volume: 5 μL. Ion source: HESI; sample mass spectrometry signals were acquired using both positive and negative ion scanning modes. Data acquisition mode: DDA; scan mode: Full MS / dd-MS2 (TOP 10).

[0040] The total ion current chromatogram results are as follows: Figure 1 As shown.

[0041] from Figure 1 It can be seen that the chromatographic separation performance is stable and reliable: the metabolic components of different polarities in the sample were effectively separated within a 15-minute elution cycle, with symmetrical and sharp peaks, stable baselines without obvious drift, and no severe tailing or co-elution phenomenon, indicating that the chromatographic conditions in this study can meet the requirements for the separation and detection of multiple metabolites in the sample.

[0042] The component distribution characteristics are clear: strongly polar components preferentially elute in the 0-2 min range, moderately polar components concentrate in the 3-6 min range, and weakly polar components gradually elute in the 7-15 min range; the signal intensity is positively correlated with the relative abundance of the components, with the components in the 3-6 min range showing higher signal responses, indicating that the relative content of metabolites is higher in this range.

[0043] The target compound can be effectively separated and detected: the characteristic elution peak of the target cyclic dipeptide Cyclo (L-Phe-L-Pro) is located at approximately 5.0 min, with good peak shape and no obvious interference, which is consistent with the subsequent extractive ion chromatography (EIC) and mass spectrometry fragment identification results (see...). Figure 4 and Figure 5 This matches the results, providing a reliable basis for the qualitative and subsequent quantitative analysis of the compound.

[0044] 4. Data analysis and differential metabolite screening LC-MS data were processed using Progenesis QI V2.3 (Nonlinear, Dynamics, Newcastle, UK) software, including baseline filtering, peak identification, integration, retention time correction, peak alignment, and normalization. The main parameters were a 5 ppm precursor tolerance, a 10 ppm product tolerance, and a 5% product ion threshold.

[0045] Compounds were identified using the Human Metabolome Database (HMDB), Lipidmaps (V2.3), Metlin, and a self-built database, based on precise mass-to-charge ratio (M / z), secondary fragments, and isotopic distribution. The extracted data were then further processed, removing any peaks with more than 50% missing values ​​(ionic intensity = 0), replacing zero values ​​with half the minimum value, and filtering based on the qualitative results. Compounds with a database matching score below 36 (out of 80) were also considered inaccurate and deleted. The positive and negative ion data were combined into a data matrix.

[0046] The data matrix was imported into the R package for principal component analysis (PCA). To clarify the overall differences in the metabolome of samples treated with Bacillus belysse and under different time conditions, this embodiment performed principal component analysis (PCA) on the metabolome data obtained by LC-MS detection. The results are as follows: Figure 2 As shown, PC1 and PC2 explained 24.0% and 9.86% of the total sample variance, respectively, with a combined contribution of 33.86%, effectively reflecting the overall metabolic differences among samples in a two-dimensional plane. The biological replicates in each group clustered within the 95% confidence ellipse, indicating low intra-group dispersion and reliable experimental repeatability. The data stability meets the requirements for subsequent analysis.

[0047] from Figure 2 It can be seen that the control group CK3H sample showed a clear separation trend from other groups in both PC1 and PC2 directions, with particularly significant differences in metabolic characteristics compared to the CK1H and ZX-1-1H groups. The ZX-1 treatment groups (ZX-1-1H, ZX-1-2H, ZX-1-3H) partially overlapped with the control group CK1H and CK2H samples on the PC1 axis. Among them, the metabolic characteristics of the ZX-1-3H group and the CK2H group were relatively similar, suggesting that the metabolic differences between the treatment group and the control group were not significant at the early time point, but as time went on, the metabolic characteristics of the control group CK3H became significantly different from those of the treatment group.

[0048] The overall distribution of samples and the stability of the entire analysis process were observed. Orthogonal partial least squares discriminant analysis (OPLS-DA) and partial least squares discriminant analysis (PLS-DA) were used to distinguish metabolite differences between groups. In this embodiment, p<0.05 and |log2FC|>1 were used as screening criteria to perform differential analysis on the expression data of the two groups of samples and to draw volcano plots. Figure 3 This method visually demonstrates the distribution characteristics of differentially expressed substances. The results showed that 1134 significantly differentially expressed substances were identified, including 492 significantly upregulated substances and 642 significantly downregulated substances, with the number of significantly downregulated substances slightly exceeding the number of upregulated substances.

[0049] from Figure 3 It can be seen that the log2 fold change of the differential substances is mainly distributed in the range of -4 to 4. Some substances have a high fold change (|log2FC|>4) and strong statistical significance, suggesting that these substances may play a key role in the biological differences between the two groups of samples.

[0050] Representative difference substances: Figure 3 The study highlighted some representative substances with significant differences, including significantly upregulated HOPX, CD36, AKR1C2, ADAMTSL1, etc., and significantly downregulated NLRP12, FGF21, TMEM156, FUT3, etc. These substances can serve as potential targets for subsequent functional verification and mechanism research.

[0051] To prevent overfitting, 7-fold cross-validation and the 200-response permutation test (RPT) were used to assess model quality. The projected importance (VIP) values ​​of the V variables obtained from the OPLS-DA model were used to rank the overall contribution of each variable to group discrimination. A two-tailed Student's t-test was further used to verify whether the differences in metabolites between groups were significant. Metabolites with VIP values ​​greater than 1.0 and p-values ​​less than 0.05 were selected. Finally, Bayesian correlation analysis was used to screen for metabolites significantly associated with three different soaking times and germination indicators (see Tables 1 to 3).

[0052] Table 1. Bayesian correlation analysis to screen metabolites related to germination index over 1 hour.

[0053] Table 2. Bayesian correlation analysis screens metabolites associated with germination index at 2 h.

[0054] Table 3. Bayesian correlation analysis to screen metabolites related to the 3-hour germination index.

[0055] 5. Structural identification The secondary mass spectrum of this metabolite was compared with natural product databases (such as HMDB and METLIN), and it was initially identified as the cyclic dipeptide Cyclo (L-Phe-L-Pro). Further comparison with a standard (Shanghai Taoshu Biotechnology, purity 97.08%) showed that the retention time and mass spectrometry fragmentation mode were completely consistent, confirming that the metabolite is the cyclic dipeptide Cyclo (L-Phe-L-Pro), with the molecular formula C2. 14 H 16 N2O2, molecular weight 244.12.

[0056] Example 2 Application verification of Cyclo (L-Phe-L-Pro) in promoting the germination of aged peanut seeds 1. Selection of aged peanut seeds Naturally aged peanut seeds of Huayu 25 (HY25) and Nongda R271 were selected. Spring peanuts harvested in September 2024 were dried and stored until March 2026 for use in the experiment.

[0057] 2. Experimental Design Two-factor (dilution concentration and soaking time) experiments were designed using DesignExpert software. The minimum soaking time, maximum soaking time, maximum concentration and minimum concentration were input into the software. The software automatically generated the design of the most suitable soaking time and concentration. The design time and concentration are shown below, and the specific treatment combinations are shown in Table 4.

[0058] Table 4. Treatment concentration and time of cyclic dipeptide Cyclo (L-Phe-L-Pro)

[0059] 3. Experimental Procedure (1) Preparation of cyclic dipeptide solution: Weigh Cyclo (L-Phe-L-Pro) and prepare a standard stock solution with a concentration of 10 mg / mL using DMSO as the solvent.

[0060] According to the concentration gradient in Table 4 above, the stock solution was diluted by 5, 19.3, 22.5 and 30 times with DMSO as solvent to obtain a series of working solutions with concentrations of 2000, 518.13, 444.44 and 333.33 μg / mL, which were prepared and used immediately.

[0061] The dilution factor is determined based on the Design-Expert response surface methodology, which can uniformly cover the optimal germination concentration range and meet the requirements of model fitting and condition optimization.

[0062] (2) Seed soaking treatment: Naturally aged peanut seeds were selected and soaked in working solutions of different concentrations at 20℃ for the corresponding times shown in Table 4. The control group (CK) was soaked in DMSO for the same time (120 min). Each treatment consisted of 30 seeds, and the treatment was repeated twice.

[0063] (3) Germination culture: After soaking, the seeds were placed in petri dishes lined with moistened filter paper and incubated at 25℃. Germination was observed and recorded daily (germination was defined as the radicle breaking through the seed coat by 2 mm). Germination potential was calculated on day 3, and germination rate and germination index were calculated on day 7. Germination potential (%) = (Number of normally germinated seeds on day 3 / Number of tested seeds) × 100% Germination rate (%) = (Number of seeds that germinated normally on day 7 / Number of seeds tested) × 100% Germination index GI = Σ(Gt / Dt), where Gt is the number of germinated seeds on day t and Dt is the corresponding number of germination days.

[0064] (4) Data statistics: Germination potential, germination rate, and germination index were statistically analyzed using Jamovi software. A Beta regression model was used to analyze germination potential and germination rate, and multiple comparisons were performed after Bonferroni correction tests to correct for differences between groups (P < 0.05). A general linear model (GLM) was used to analyze the germination index.

[0065] 4. Test Results (1) Effects on the seeds of Flower 25 like Figure 6 The image shows the germination status of aged peanut seeds of Huayu 25 after soaking in cyclic dipeptide Cyclo (L-Phe-L-Pro) at different times / concentrations.

[0066] As shown in Tables 5 and 6, the germination indices of all treatment groups were significantly better than those of the control group. Among them, treatment 3 (444.44 μg / mL + 180 min) had the largest germination potential difference (0.2160) and the best germination uniformity; treatment 2 (2000 μg / mL + 177 min) had the best germination rate (increased by 23.33%) and germination index (increased by 13.23), and the differences between all treatment groups and the control group were significant or highly significant (P<0.05).

[0067] Table 5 Effects of Cyclo (L-Phe-L-Pro) treatment on germination indices of aged seeds of HY25 var. florida.

[0068] Note: Different lowercase letters in the same column indicate significant differences between treatments (P<0.05); CK is the solvent DMSO control.

[0069] Table 6. Analysis of the differences in germination indices of HY25 seeds under different treatments.

[0070] (2) Impact on the R271 seed of China Agricultural University like Figure 7 The image shows the germination status of aged peanut seeds of Nongda R271 after soaking in cyclic dipeptide Cyclo (L-Phe-L-Pro) at different times / concentrations.

[0071] As shown in Tables 7 and 8, treatment 3 (444.44 μg / mL + 180 min) showed the greatest increase in germination potential (25.00%), treatment 4 (2000 μg / mL + 91.5 min) showed the best increase in germination index (11.83), and treatment 2 (2000 μg / mL + 177 min) showed the highest increase in germination rate (25.00%). The germination indices of all treatment groups were significantly higher than those of the control group, confirming that the cyclic dipeptide has a stable germination-promoting effect on aging peanut seeds of different varieties. There were extremely significant differences among the six treatment groups.

[0072] Table 7 Effects of Cyclo (L-Phe-L-Pro) treatment on germination indices of aged Nongda R271 seeds

[0073] Table 8. Analysis of the differences in germination indices of Nongda R271 seeds under different treatments.

[0074] In summary, this invention successfully screened the cyclic dipeptide Cyclo (L-Phe-L-Pro) using metabolomics technology, and significantly promoted the germination of aged peanut seeds by adjusting its concentration and soaking time parameters. Based on production targets, the following optimal combination is recommended: To improve germination potential (uniformity): the optimal concentration is 444.44 μg / mL + 180 min; To improve germination rate / germination index (germination rate and final germination ratio): the optimal value is 2000 μg / mL + 91.5~177 min.

[0075] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, alterations, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of Cyclo (L-Phe-L-Pro) in promoting peanut seed germination.

2. Application of Cyclo (L-Phe-L-Pro) in the preparation of products that promote peanut seed germination.

3. The application according to any one of claims 1 or 2, characterized in that, The cyclic dipeptide Cyclo (L-Phe-L-Pro) was screened from Bacillus belysinus (… Bacillus velezensis BVZX-1.

4. The application according to any one of claims 1 or 2, characterized in that, The peanut seeds include aged peanut seeds, which are peanut seeds that spontaneously undergo physiological deterioration under natural storage conditions as the storage time is extended; the aged peanut seed varieties include, but are not limited to, Huayu 25 and Nongda R271.

5. The application according to claim 2, characterized in that, The product is an agriculturally acceptable seed treatment agent, including formulations, soaking solutions, or seed coating agents.

6. A method for preparing the cyclic dipeptide Cyclo (L-Phe-L-Pro), characterized in that, Includes the following steps: (1) Inoculate Bacillus belye into the fermentation medium for fermentation culture, centrifuge the fermentation broth and collect the supernatant; (2) The metabolites in the fermentation broth were analyzed by non-targeted metabolomics using ultra-high performance liquid chromatography-mass spectrometry. The chromatographic column used was a C18 column, and the mobile phase was 0.1% formic acid water and 0.1% formic acid acetonitrile. (3) Principal component analysis and orthogonal partial least squares discriminant analysis were used to screen differential metabolites with variable projection importance >1 and P value <0.05; (4) By Bayes correlation analysis, the metabolite that was present at all three soaking times related to the germination index was identified as Cyclo (L-Phe-L-Pro).

7. The application of Cyclo (L-Phe-L-Pro), a cyclic dipeptide prepared by the method described in claim 6, in promoting peanut seed germination.

8. A preparation for promoting the germination of aged peanut seeds, characterized in that, The active ingredient of the formulation is Cyclo (L-Phe-L-Pro) obtained by the preparation method described in claim 6; The concentration of Cyclo (L-Phe-L-Pro) in the formulation is 333.33~2000 μg / mL.

9. A method for promoting the germination of aged peanut seeds, characterized in that, Weigh a certain amount of Cyclo (L-Phe-L-Pro) and dissolve it in DMSO to prepare a stock solution with a concentration of 10 mg / mL. Then, use DMSO solvent to dilute the stock solution to a working solution with a concentration of 333.33~2000 μg / mL. Soak aged peanut seeds in the working solution for 60~180 min at a temperature of 20℃.

10. The method according to claim 9, characterized in that, When it is necessary to improve the germination potential or germination uniformity of aged peanut seeds, the concentration of the working solution should be 444.44 μg / mL, and the soaking time should be 180 min. When it is necessary to improve the germination rate and germination index of aged peanut seeds, the concentration of the working solution should be 2000 μg / mL, and the soaking time should be any time within the range of 91.5 to 177 min.

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