A small peptide and its use

Abstract

The application belongs to the technical field of bioengineering, and discloses a small peptide and application thereof. The small peptide sequence is VITD, and the salt tolerance of crops can be obviously improved under salt stress. The small peptide can be developed as an agricultural composition, and is especially a growth regulator which has a significant effect on crops under salt stress.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology and relates to a small peptide and its applications. Background Technology

[0002] Salt stress has become one of the important abiotic factors restricting crop growth. In-depth research into exogenous regulatory measures against salt stress in crops is of great significance for improving crop salt tolerance, increasing crop yield, and improving crop quality. Salt stress reduces the ability of crop roots to absorb water and nitrogen, and accelerates the accumulation of reactive oxygen species within the crop, leading to cytotoxicity. Simultaneously, low osmotic potential and high ion concentration also reduce the ability of cells to absorb water from the external environment, restricting seed germination and affecting the growth and development of mature plants.

[0003] Recent studies have shown that exogenous plant growth regulators can enhance plant stress resistance by inducing gene expression and altering metabolite content. For example, melatonin participates in plant growth and development, helping to improve plant resistance to various abiotic and biotic stresses. Salicylic acid can act as a signaling regulator to regulate membrane lipid peroxidation and increase the content of soluble sugars and proteins to maintain the physiological and ecological balance of the intracellular environment and alleviate salt stress. Traditional plant hormones such as auxin (IAA), gibberellin (GA), cytokinin (CTK), abscisic acid (ABA), and ethylene enable plants to respond to abiotic stresses through signaling pathways. Polyamines play an important role in plant biotic and abiotic stress signaling networks. Steroid hormones can enhance root water uptake and stabilize membrane system structure to enhance plant stress resistance. Jasmonic acid can act as an endogenous signaling molecule under salt stress to regulate plant growth and development and participate in regulating plant responses to abiotic stresses.

[0004] Polypeptides are a new class of plant growth regulators, following traditional plant hormones, amine hormones such as melatonin, polyamines, and jasmonic acid. In addition to possessing the characteristics of protein molecules, they play a crucial regulatory role in plant growth, development, reproduction, and responses to environmental stresses. However, current research on the role of single small peptides / oligopeptides in promoting plant salt tolerance is limited. Summary of the Invention

[0005] To enrich the exogenous regulatory measures for salt stress in crops, this invention provides a small peptide and its application. The exogenous addition of this small peptide under salt stress conditions has a significant impact on seed germination and seedling growth in crops such as pak choi, Arabidopsis thaliana, soybean, and corn.

[0006] This invention uses maize as experimental material and exogenously adds small peptide VITD (valine-isoleucine-threonine-aspartic acid) under 100 mmol / L NaCl (salt stress) conditions. Based on metabolomics, the effects of small peptide VITD are explored, the molecular mechanism of maize seedling response to salt stress is elucidated as a whole, differentially expressed metabolites under salt stress are identified, and the metabolic pathways of differentially expressed metabolites are analyzed. The effects of small peptide VITD on maize seedlings under salt stress conditions are investigated, and the mechanism of maize seedling response to salt stress is revealed.

[0007] The small peptides described in this invention, also known as small molecule active peptides, oligopeptides, or oligopeptides, generally have a molecular weight of less than 1000 Daltons.

[0008] Specifically, the present invention provides a small peptide, the sequence of which is VITD.

[0009] Furthermore, the small peptide can improve the salt tolerance of plants including Chinese cabbage, Arabidopsis thaliana, soybean, and corn under salt stress conditions.

[0010] Furthermore, the salt stress condition is a NaCl solution with a concentration of 100 mmol / L.

[0011] Furthermore, the concentration of the small peptide is 1×10⁻⁶. -6 mol / L~1×10 -11 mol / L.

[0012] Furthermore, the concentration of the small peptide is 1×10⁻⁶. -9 mol / L.

[0013] Furthermore, the small peptide can reduce the accumulation of malondialdehyde;

[0014] The small peptide can increase the content of at least one of the following substances: proline and soluble sugars.

[0015] Furthermore, the small peptide can increase the expression level of at least one enzyme, including peroxidase and catalase.

[0016] Furthermore, the small peptide can increase the expression levels of auxin and gibberellin, and the small peptide can decrease the expression level of abscisic acid.

[0017] Furthermore, the small peptides can increase the biosynthesis of flavonoids, including but not limited to flavones, flavonols, and flavonoids.

[0018] On the other hand, the present invention relates to the application of the above-mentioned small peptides in the salt stress resistance of crops.

[0019] Compared with the prior art, the small peptides and their applications provided by the present invention have the following beneficial effects or advantages.

[0020] (1) The small peptide VITD described in this invention has the ability to regulate the expression of a variety of metabolites. These metabolites include, but are not limited to, malondialdehyde, proline, soluble sugars, peroxidase, catalase, auxin, gibberellin, and abscisic acid.

[0021] (2) The small peptide VITD described in this invention can improve the salt tolerance of crops by regulating the expression of metabolites in crops under salt stress conditions. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a graph showing the growth-promoting effect of the small peptide VITD described in this invention at different salt concentrations. Figure 1 CK0-4 represents the control group; T1-4 represents the experimental group with added small peptide VITD.

[0024] Figure 2 This is a graph showing the effect of the small peptide VITD described in this invention on the root system of maize seedlings under salt stress. Figure 2 In this context, A represents the blank control group CK0, which is salt-free and does not contain the small peptide VITD. Figure 2 In this context, B represents the stress control group CK1, which contains salt and does not contain the small peptide VITD. Figure 2 In this context, C represents the salt-free experimental group T0, which contains the small peptide VITD. Figure 2 The "D" in the figure represents the experimental group T1, which contains salt and the small peptide VITD.

[0025] Figure 3 This is a graph showing the measurement results of the small peptide VITD described in this invention on the osmotic regulation substances of maize seedlings under salt stress. Figure 3 CK0 represents the blank control group, which is salt-free and does not contain the small peptide VITD. Figure 3 CK1 in the figure represents the stress control group with salt and without the addition of the small peptide VITD; Figure 3 T0 in the figure represents the salt-free experimental group with added small peptide VITD; Figure 3 T1 in the figure represents the experimental group with salt and added small peptide VITD.

[0026] Figure 4 This is a graph showing the measurement results of antioxidant enzyme activity in maize seedlings treated with the small peptide VITD described in this invention under salt stress. Figure 4CK0 represents the blank control group, which is salt-free and does not contain the small peptide VITD. Figure 4 CK1 in the figure represents the stress control group with salt and without the addition of the small peptide VITD; Figure 4 T0 in the figure represents the salt-free experimental group with added small peptide VITD; Figure 4 T1 in the figure represents the experimental group with salt and added small peptide VITD.

[0027] Figure 5 This is a graph showing the results of hormone content measurement in maize seedlings treated with the small peptide VITD described in this invention under salt stress. Figure 5 CK0 represents the blank control group, which is salt-free and does not contain the small peptide VITD. Figure 5 CK1 in the figure represents the stress control group with salt and without the addition of the small peptide VITD; Figure 5 T0 in the figure represents the salt-free experimental group with added small peptide VITD; Figure 5 T1 in the figure represents the experimental group with salt and added small peptide VITD.

[0028] Figure 6 This is a PCA score chart for all samples (including QC samples).

[0029] Figure 7 This is a graph showing the OPLS-DA scores for each experimental group.

[0030] Figure 8 This is a graph showing the cluster analysis results of maize seedling metabolites under different treatment conditions.

[0031] Figure 9 This is a volcano diagram of metabolic differences between the VITD group and the CK1 group of the small peptide described in this invention under salt stress. Figure 9 The horizontal axis represents the fold change in metabolite expression between the two groups, and the vertical axis represents the statistical test value (p-value) of the difference in metabolite expression levels. Figure 9 Each dot in the diagram represents a specific metabolite; blue dots indicate metabolites with differentially downregulated expression, while red dots indicate metabolites with differentially upregulated expression.

[0032] Figure 10 This is a map showing the enrichment of differentially metabolized substances via the KEGG pathway. Detailed Implementation

[0033] The technical solution of the present invention will be described below with reference to the embodiments. However, the present invention is not limited to the following embodiments.

[0034] Unless otherwise specified, the experimental and detection methods described in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0035] The small peptide VITD involved in the following examples was synthesized by Sangon Biotech (Shanghai) Co., Ltd., with a purity of 95%.

[0036] Example 1

[0037] This embodiment describes the determination of the optimal concentration of the small peptide VITD for promoting salt tolerance in maize seedlings.

[0038] 1. Corn Seed Pretreatment

[0039] Select plump, undamaged, and uniformly sized corn seeds (Ningdan 33 variety). Soak the seeds in 95% alcohol for 10 seconds to remove surface tension, then soak them in 0.1% mercuric chloride solution for 30 seconds to disinfect the surface. Finally, rinse the seeds thoroughly with plenty of sterile water. Place them in an Erlenmeyer flask and soak them in sterile water for later use.

[0040] 2. Germination and cultivation of corn seeds

[0041] CK control group: corn seeds were soaked in sterile water for 12 hours.

[0042] Experimental group: Using concentrations of 1×10⁻⁶ -4 mol / L, 1×10 -5 mol / L, 1×10 -6 mol / L, 1×10 -7 mol / L, 1×10 -8 mol / L, 1×10 -9 mol / L, 1×10 -10 mol / L, 1×10 -11 Corn seeds were soaked in 1 mol / L VITD (a small peptide) for 12 hours.

[0043] Germination: Place the soaked corn seeds in water agar medium and incubate at 28°C in the dark for 48 hours to germinate and prepare for use.

[0044] 3. Determination of the optimal concentration of small peptide VITD for promoting salt tolerance in maize seedlings

[0045] CK0 control group: No salt solution was added, and no small peptide VITD was added, serving as a blank control.

[0046] CK1 control group: salt solution with a concentration of 100 mmol / L was added, and the small peptide VITD was not added as a salt stress control.

[0047] The salt solution is a NaCl solution.

[0048] Small peptide VITD experimental group: Corn seedlings soaked and germinated at various concentration gradients were planted in a plant nutrient solution containing 100 mmol / L NaCl, and small peptide VITD at corresponding concentration gradients were added exogenously to the corn roots.

[0049] The plant nutrient solution consists of the following components:

[0050] Mother liquor 1: 0.004g / LCoCl2·6H2O, 2.86g / LH3BO3, 1.9g / LMnCl2·4H2O, 0.22g / LZnSO4·7H2O, 0.102g / LCuSO4·5H2O, 0.122g / LNa2MnO4·2H2O;

[0051] Mother liquor 2: 24.084 g / L MgSO4;

[0052] Mother liquor 3: 228.168 g / L K2HPO4·3H2O, 136.886 g / L KH2PO4;

[0053] Mother liquor 4: 83.806 g / L CaCl2;

[0054] Mother liquor 5: 5.6 g / LFeC6H5O7·5H2O;

[0055] Dilute 1 mL of each of the five stock solutions in 1000 mL of distilled water before use.

[0056] Four corn seedlings were planted in each bottle, and five biological replicates were set up. All replicates were cultured in a light incubator (25℃, 16h light, 10000Lx light intensity, 18℃, 8h darkness).

[0057] When the corn seedlings grew to a point where there were significant differences, the plant height and root length of the corn in the CK0 group, CK1 group, and experimental group were measured. The corn plants were rinsed with distilled water several times until clean, and the water was absorbed. The fresh weight of the plants was then measured.

[0058] Table 1. Effects of different concentrations of VITD on maize seedlings under salt stress.

[0059]

[0060] As shown in Table 1, compared with the blank control, the growth of maize seedlings was significantly inhibited under salt stress. Treatment with different concentrations of the small peptide VITD exogenously alleviated the inhibition caused by salt stress to varying degrees. The difference between CK0 and CK1 treatments was significant. Under salt stress conditions, a concentration of 1×10⁻⁶ was used. -6 mol / L, 1×10 -8 mol / L, 1×10 -9 mol / L, 1×10 -10 mol / L, 1×10 -11The root length increased by 2.67%, 6.66%, 1.99%, 9.80%, and 5.43% respectively compared with CK1 after treatment with 1×10 mol / L small peptide VITD. -10 The mol / L VITD treatment showed the most significant difference from CK1; in terms of plant height, except for 1×10⁻⁶, the difference was most significant. -10 Except for the mol / L VITD treatment, which was lower than CK1, all other treatments were higher than CK1, increasing by 8.75%, 3.17%, 13.41%, 2.98%, 9.58%, 24.02%, and 8.12%, respectively. Among these, the 1×10⁻⁶ mol / L VITD treatment showed the highest VITD concentration. -9 The mol / L VITD treatment showed the most significant difference from CK1; in terms of fresh weight, each treatment increased by 4.64%, 9.13%, 9.29%, 7.26%, 10.52%, 16.29%, 3.52%, and 1.60% compared to CK1, respectively. Among these, 1×10⁻⁶ VITD treatment... -9 The most significant difference was observed between mol / LVITD treatment and CK1. Comprehensive analysis showed that the small peptide VITD at 1×10⁻⁶ mol / L was significantly different from CK1. -9 The salt concentration of mol / L is most effective in promoting salt tolerance in maize seedlings.

[0061] Example 2

[0062] This embodiment describes the optimal conditions for the small peptide VITD to promote salt tolerance in maize seedlings.

[0063] The corn seed pretreatment was the same as in Example 1.

[0064] Germination: Place the pretreated corn seeds in water agar medium and incubate them in the dark at 28°C for 48 hours to germinate and prepare for use.

[0065] Control group: Germinated corn seeds were planted in plant nutrient solutions with salt concentration gradients of 0, 100 mmol / L, 150 mmol / L, 200 mmol / L, and 250 mmol / L, respectively, as control groups CK0-4.

[0066] Experimental group: The concentration was 1.0 × 10⁻⁶ -9 The small peptide VITD at a concentration of mol / L was added to plant nutrient solutions with salt concentration gradients of 100 mmol / L, 150 mmol / L, 200 mmol / L, and 250 mmol / L, respectively, and germinated corn seeds were planted, which were used as experimental groups T1 to T4.

[0067] The composition of the plant nutrient solution is the same as in Example 1.

[0068] The salt concentration is the NaCl concentration.

[0069] Four corn seedlings were planted in each bottle, and five biological replicates were set up. All replicates were cultured in a light incubator (25℃, 16h light, 10000Lx light intensity, 18℃, 8h darkness).

[0070] When the corn seedlings grew to a point where there were significant differences, the plant height, stem diameter, chlorophyll content, and root length of the corn in the control group and the experimental group were measured. The corn plants were rinsed multiple times with distilled water until clean, and the water was dried. The fresh weight of the plants was measured, and the germination rate was calculated.

[0071] Germination rate = (Number of normally germinated seeds at the end of the experiment / Number of tested seeds) × 100%

[0072] Table 2. Effects of VITD on maize seedlings at different salt concentrations.

[0073]

[0074] like Figure 1 As shown in Table 2, the growth of maize seedlings was inhibited with increasing salt concentration, indicating that the small peptide VITD had a regulatory effect on maize growth under salt stress. Table 2 shows that various indicators of maize seedlings in each treatment generally decreased gradually with increasing salt concentration. When the salt concentration was 100 and 150 mmol / L, the small peptide VITD treatment increased the germination rate of maize seedlings compared to the control (CK). When the salt concentration rose to 200 and 250 mmol / L, the small peptide VITD treatment decreased the germination rate of maize seedlings compared to the CK. Under different salt stress conditions, the root length of maize seedlings treated with the small peptide VITD treatment and the CK group did not differ significantly, both decreasing with increasing salt concentration. When the salt concentration was 100, 150, and 200 mmol / L, the plant height, fresh weight, and chlorophyll content of the small peptide VITD treatment were all higher than those of the control group, with plant height increasing by 16.8% with increasing salt concentration. The fresh weight increased by 4.3%, 0%, and 16.3% respectively with increasing salt concentration, and the chlorophyll content increased by 7.9%, 1.6%, and 36.1% respectively with increasing salt concentration. When the salt concentration increased to 250 mmol / L, the plant height and fresh weight of the VITD treatment were lower than the control, decreasing by 29.1% and 16.4% respectively, while the chlorophyll content was slightly higher than the control, but not significantly. When the salt concentration was 100 and 250 mmol / L, the stem diameter ratio of the VITD treatment decreased by 12.8% and 6.4% respectively, while when the salt concentration was 150 and 200 mmol / L, the stem diameter ratio of the VITD treatment increased by 10.7% and 16.5% respectively.

[0075] Seed germination is the most vulnerable and crucial stage in a plant's entire life cycle, and also the period most sensitive to salt stress. Salt stress leads to osmotic stress and ion toxicity, inhibiting root and above-ground growth, affecting seedling height and dry matter accumulation. Different plants can absorb exogenous substances from roots, leaves, and seeds to enhance their stress resistance, and the effectiveness of these exogenous substances in enhancing plant stress resistance depends on their concentration.

[0076] In experiments on the promotion of salt tolerance in maize seedlings by the small peptide VITD, root length, plant height, and fresh weight generally showed a trend of increasing and then decreasing with increasing VITD concentration. Among different VITD concentration treatments, 1x10-1... - 9 Treatment with 100 mmol / L VITD peptide showed the best effect in promoting salt tolerance in maize seedlings, increasing root length, plant height, and fresh weight by 1.99%, 24.02%, and 16.29%, respectively. This indicates that an appropriate concentration of VITD peptide can promote the salt tolerance of maize seedlings, and further demonstrates that peptides can act as plant hormone analogs to regulate plant growth under stress. In this example, the growth-promoting effect of VITD peptide treatment varied under different salt stresses, with the best effect observed at a salt concentration of 100 mmol / L. This may suggest that the environment in which the substance is located affects its effectiveness.

[0077] Example 3

[0078] This embodiment describes the effect of the small peptide VITD on the root growth of maize seedlings.

[0079] The corn seed pretreatment, germination, and plant nutrient solution components are the same as in Example 2.

[0080] Control group: Germinated corn seeds were planted in a salt-free plant nutrient solution without added VITD, as CK0; germinated corn seeds were planted in a plant nutrient solution containing 100 mmol / L NaCl solution without added VITD, as CK1.

[0081] Experimental group: Germinated corn seeds were planted in a salt-free environment with a concentration of 1x10⁻⁶. -9 Plant nutrient solution containing the small peptide VITD at a concentration of 1 mol / L was used as T0; germinated corn seeds were planted in a solution containing 100 mmol / L NaCl and 1 x 10 mol / L VITD. -9 In a plant nutrient solution containing mol / L of the small peptide VITD, it is used as T1.

[0082] The salt is NaCl.

[0083] Four corn seedlings were planted in each group, and five biological replicates were set up. All replicates were cultured in a light incubator (25℃, 16h light, 10000Lx light intensity, 18℃, 8h darkness).

[0084] When the seedlings were cultured for 14 days, the roots of corn seedlings in groups T1, T0, CK1, and CK0 were cut and analyzed using a root system analyzer.

[0085] Under salt stress, treatment with the small peptide VITD significantly promoted root growth in maize seedlings, such as... Figure 2 As shown.

[0086] Table 3. Effects of small peptide VITD on the root system of maize seedlings under salt stress.

[0087] Total length <![CDATA[4190±476.2 a ]]> <![CDATA[4374±698.8 a ]]> <![CDATA[1981±391.3 c ]]> <![CDATA[2261±286.9 b ]]> Total projected area <![CDATA[3286±304.0 a ]]> <![CDATA[3426±342.3 a ]]> <![CDATA[1676±263.4 c ]]> <![CDATA[1962±272.7 b ]]> Total surface area <![CDATA[10318±954.6 a ]]> <![CDATA[10758±1191 a ]]> <![CDATA[5259±827.1 c ]]> <![CDATA[6162±856.4 b <!-- 6 -->]]> Total volume <![CDATA[4323±737.0 a ]]> <![CDATA[4537±779.0 a ]]> <![CDATA[2792±620.6 c ]]> <![CDATA[3348±563.9 b ]]> average diameter <![CDATA[8.444±0.5413 b ]]> <![CDATA[8.223±0.5469 b ]]> <![CDATA[8.617±0.8780 ab ]]> <![CDATA[9.150±0.4050 a ]]> Number of connections <![CDATA[2696±439.4 a ]]> <![CDATA[3084±562.6 a ]]> <![CDATA[1209±263.7 b ]]> <![CDATA[1316±117.0 b ]]> Number of nodes <![CDATA[1858±287.5 a ]]> <![CDATA[2138±383.1 a ]]> <![CDATA[861±177.2 b ]]> <![CDATA[931±114.2 b ]]> Root tip number <![CDATA[210±32.95 a ]]> <![CDATA[252±48.80 a ]]> <![CDATA[124±15.85 b ]]> <![CDATA[131±20.79 b ]]> Number of branches <![CDATA[1561±254.7 a ]]> <![CDATA[1764±324.2 a ]]> <![CDATA[690±156.7 b ]]> <![CDATA[752±92.73 b ]]>

[0088] Table 3 shows that under salt stress, treatment with the small peptide VITD promoted the growth of maize seedling roots. The total length, total projected area, total surface area, total volume, average diameter, number of connections, number of nodes, number of root tips, and number of branches increased by 14.13%, 17.06%, 17.17%, 19.91%, 6.18%, 8.85%, 8.13%, 5.64%, and 8.98%, respectively. Significant differences were observed between the small peptide VITD treatment and the salt control in total length, total projected area, total surface area, and total volume. Under no salt stress, the small peptide VITD treatment had no significant effect on maize seedling roots, and the T0 value was not significantly different from the control, indicating that salt stress is an inducing factor for the regulation of maize seedling growth and development by the small peptide VITD treatment.

[0089] Salt stress damages the cellular structure and physiological systems of plant seedling roots, while exogenous substances can alleviate root damage and promote root growth under salt stress. In this example, the total length, total projected area, total surface area, and total volume of maize seedling roots showed significant differences compared to the salt control, increasing by 14.13%, 17.06%, 17.17%, and 19.91%, respectively. This indicates that soaking in the exogenous peptide VITD under salt stress can promote root development in maize seedlings. However, VITD treatment under salt stress could not restore the root growth of maize seedlings to normal levels.

[0090] Example 4

[0091] This embodiment describes the effects of the small peptide VITD on the biochemical indicators of maize seedlings.

[0092] The pretreatment, germination, control group and experimental group setup, culture process and conditions of corn seeds are the same as in Example 3.

[0093] When the seedlings were cultured for 14 days, corn seedlings from groups T1, T0, CK1, and CK0 were taken, rinsed with distilled water, dried with sterile filter paper, and then cut off the corn seedlings for biochemical index determination.

[0094] Superoxide dismutase (SOD) test kit, catalase (CAT) test kit, peroxidase (POD) test kit, malondialdehyde (MDA) assay kit, proline Pro assay kit (colorimetric method), and plant soluble sugar content test kit (colorimetric method) (all kits were purchased from Nanjing Jiancheng Bioengineering Institute) to test the corresponding indicators of maize seedlings.

[0095] The difference significance was analyzed using Graphpad 2018 software (Tukey method, p < 0.05) and plotted.

[0096] 1. Effects of small peptide VITD on MDA, Pro, and soluble sugars in maize seedlings under salt stress

[0097] from Figure 3 As shown in Figure A, the MDA mass fractions of both the aboveground and underground parts of the salt control CK1 group were significantly higher than those of the salt-free control CK0 group (P < 0.05). Under salt stress, the MDA mass fractions of both the aboveground and underground parts of the VITD-treated T1 group were lower than those of the CK1 group, and the difference between the underground part and the salt control CK1 group was significant, with a mean value of 516.0 nmol / g (DW), which was 0.625 times that of CK1 and close to that of the salt-free control CK0 group. Under salt-free stress, there was little difference between the VITD-treated T0 group and the CK0 group.

[0098] from Figure 3 According to the results in B, the Pro mass fraction in both the aboveground and underground parts of the salt control CK1 group was slightly higher than that of the salt-free control CK0 group, but the difference was not significant. Under salt stress, the Pro mass fraction in the underground part of the small peptide VITD treatment T1 group was significantly higher than that of the CK1 group, with a mean value of 692.946 μg / g (DW), which was 4.64 times that of the CK1 group, while the difference in the aboveground parts was not significant. Under salt-free stress, the Pro mass fraction in the T0 group was slightly higher than that in the CK0 group, but the difference was not significant.

[0099] from Figure 3 The results showed that the soluble sugar content in the underground part of the salt control group CK1 was significantly lower than that in the salt-free control group CK0, while there was no significant difference in the aboveground part between the CK1 and CK0 groups. Under salt stress, the soluble sugar content in the underground part of the small peptide VITD-treated group T1 was significantly higher than that of the CK1 group, with a mean of 3.51 mg / g (DW), which was 1.17 times that of the CK1 group. Under salt-free stress, the soluble sugar content in both the aboveground and underground parts of the T0 group was significantly higher than that of the salt-free control group CK0.

[0100] Salt stress leads to the accumulation of harmful peroxides such as malondialdehyde (MDA) in maize seedlings, causing membrane lipid peroxidation and ultimately affecting the dynamic balance between the production and scavenging of reactive oxygen species. To adapt to saline-alkali environments, plants synthesize large amounts of osmotic regulators such as proline (Pro) and soluble sugars to prevent water loss. In this study, under salt stress conditions, the MDA content in the underground parts of the VITD-treated group T1 was significantly different from that of the salt control group CK1, indicating that VITD treatment can reduce MDA accumulation. Furthermore, under salt stress conditions, the Pro mass fraction and soluble sugar content in the underground parts of the VITD-treated group T1 were significantly higher than those of the CK1 group. This may be because VITD treatment promotes the synthesis of osmotic regulators in the maize seedling roots, reduces MDA accumulation, and thus improves the salt tolerance of maize seedlings.

[0101] 2. Effects of small peptide VITD on POD, CAT, and SOD levels in maize seedlings under salt stress

[0102] Depend on Figure 4 As shown in Figure A, the POD enzyme activity in the underground part of the salt control CK1 group was significantly lower than that in the salt-free stress group (P < 0.05). Under salt stress, the POD enzyme activities in both the aboveground and underground parts of the T1 group were significantly higher than those of CK1, with mean values ​​of 842 U / g (DW) and 934 U / g (DW), respectively, which were 1.27 and 1.05 times that of CK1. Under salt-free stress, there was no significant difference between the T0 group and CK0.

[0103] from Figure 4 According to the results in B, the CAT enzyme activity in the underground part of the salt control CK1 group was significantly lower than that in the salt-free stress group (P<0.05); under salt stress, the CAT enzyme activity in the underground part of the T1 group was higher than that of the CK1 group, at 211.637 U / g (DW), which was 1.17 times that of CK1, but the difference was not statistically significant; under salt-free stress, there was no significant difference between the T0 group and the CK0 group.

[0104] from Figure 4 According to the results, the aboveground and underground parts of the salt control CK1 group showed slightly higher SOD enzyme activity than the CK0 group, but the difference was not significant. Under salt stress, there was no significant difference between the T1 group and the CK0 group. Under no salt stress, there was no significant difference between the T0 group and the CK0 group.

[0105] Under normal conditions, the production and scavenging of reactive oxygen species (ROS) in plants are always in dynamic equilibrium. When plants are under abiotic stress (including salinity), ROS production increases, and these ROS act as toxic and signaling molecules in the plant stress response. Peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD), as antioxidant enzymes, can act as ROS scavengers. Under adverse conditions, they maintain high activity to regulate ROS levels within the plant, mitigating stress damage and serving as markers of plant resistance. In this example, under 100 mmol / L salt stress, the CAT enzyme activity in maize seedlings was significantly higher than that in the salt-free control group, while the POD enzyme activity was significantly lower. Simultaneously, the MDA mass fraction was significantly higher in maize seedlings than in the salt-free control group. This may be because salt stress treatment increases CAT enzyme activity in maize seedlings to regulate ROS levels, while the 14-day salt accumulation leads to cell membrane damage, resulting in the accumulation of harmful peroxides such as MDA and a decrease in POD enzyme activity. After exogenous addition of the small peptide VITD, under 100 mmol / L salt stress, the MDA content of maize seedlings treated with VITD was lower than that of the salt control, while the CAT enzyme activity was significantly lower, and the POD enzyme activity was slightly higher. This may be because VITD treatment reduced the peroxidation and osmotic damage caused by salt stress to maize seedlings, resulting in a decrease in MDA content and POD enzyme activity, thus alleviating the stress on the seedlings. Therefore, the CAT enzyme activity was only slightly higher than that of the salt-free control. Furthermore, under salt-free conditions, there were no significant differences in CAT enzyme activity, POD enzyme activity, and MDA content between the VITD treatment and the control group (CK0), indicating that salt stress is an inducing factor for VITD to regulate the growth and development of maize seedlings. Under normal conditions and salt stress, the SOD enzyme activity of the VITD treatment was not significantly different from that of the control group, which may be because VITD has no regulatory ability on SOD enzyme activity.

[0106] The levels of auxin (IAA), gibberellin (GA), and abscisic acid (ABA) were determined using the Plant hormone ELISA Kit (purchased from USCNLIFETM).

[0107] First, prepare the extraction and lysis buffers; then follow the kit instructions for the remaining steps.

[0108] The extract is prepared by dissolving 100g of trichloroacetic acid (C2HCl3O2, TCA) in water and bringing the volume to 1000mL.

[0109] The lysis buffer is prepared as follows: Weigh 54g of urea [CO(NH2)2] and 4g of 3-[(3-cholesterol aminopropyl)dimethylamino]-1-propanesulfonic acid (CHAPS), dissolve and dilute to 100mL. Prepare immediately before use.

[0110] Result Calculation and Processing

[0111] If duplicate wells are used, the average value is taken for calculation. Using the concentration of the standard and the OD value as the ordinate, analysis is performed using professional curve creation software Curve Expert 1.3. The regression equation of the standard curve is fitted, and the OD value of the sample is substituted into the equation to calculate the sample concentration. This concentration is then multiplied by the dilution factor to obtain the actual concentration of the sample. The actual concentration of the sample multiplied by the volume and divided by the sample mass gives the endogenous hormone content of the corn shoot tip tissue.

[0112] The difference significance was analyzed using Graphpad 2018 software (Tukey method, p < 0.05) and plotted.

[0113] Depend on Figure 5 As shown in Figure A, the auxin content in the aboveground parts of the salt control group CK1 was significantly higher than that in the CK0 group, while the auxin content in the underground parts was significantly lower than that in the CK0 group. Under salt stress, the auxin content in the underground parts of the T1 group was significantly higher than that of the CK1 group, with a mean of 4.409 μmol / ml (DW), which was 1.38 times that of the CK1 group. Under no salt stress, the auxin content in the aboveground parts of the T0 group was significantly higher than that of the CK0 group, while there was no significant difference in the underground parts.

[0114] Depend on Figure 5 According to the results in B, the gibberellin content in both the aboveground and underground parts of the salt control group CK1 was significantly higher than that in the CK0 group. Under salt stress, the gibberellin content in the underground part of the T1 group was significantly higher than that of the CK1 group, with a mean value of 97.63 pmol / ml (DW), which was 1.43 times that of the CK1 group, while there was no significant difference in the aboveground part. Under no salt stress, the gibberellin content in both the aboveground and underground parts of the T0 group was significantly higher than that of the CK0 group.

[0115] Depend on Figure 5 According to the data from the study, the abscisic acid content in both the aboveground and underground parts of the salt control group CK1 was significantly higher than that of the CK0 group. Under salt stress, the abscisic acid content in the aboveground parts of the T1 group was significantly lower than that of the CK1 group, with a mean value of 5.875 ng / ml (DW), which was 0.83 times that of the CK1 group, while there was no significant difference in the underground parts. Under no salt stress, the abscisic acid content in both the aboveground and underground parts of the T0 group was significantly higher than that of the CK0 group, while there was no significant difference from the T1 group.

[0116] Plant hormones are endogenous molecules essential for regulating plant development and can modulate tolerance or sensitivity to various stresses, including salt stress. Appropriate concentrations of IAA, GA, and ABA can alleviate the damage caused by salt stress. In this study, under salt stress, the IAA and GA contents in the underground parts of group T1 were significantly higher than those in group CK1, being 1.38 times and 1.43 times higher, respectively. This indicates that treatment with the small peptide VITD may promote IAA and GA synthesis in maize seedlings to alleviate the damage caused by salt stress. Under salt stress, the abscisic acid content in the aboveground parts of group T1 was significantly lower than that of group CK1, suggesting that treatment with the small peptide VITD may reduce the ABA content in maize seedlings, thereby alleviating the damage caused by salt stress.

[0117] Example 5

[0118] This embodiment describes an investigation based on metabolomics into the effect of the small peptide VITD on improving salt tolerance in maize.

[0119] The pretreatment, germination, control group and experimental group setup, culture process and conditions of corn seeds are the same as in Example 3.

[0120] After 14 days of cultivation, when the growth of maize plants showed obvious differences, the above-ground parts (stems and leaves) of maize in groups T1, T0, CK1, and CK0 were cut off, with six replicates in each group. The samples were then frozen in liquid nitrogen for 30 minutes and stored at -80℃ for later use.

[0121] Metabolites were extracted and separated from maize seedlings and analyzed by GC-TOF / MS (extraction and detection of metabolites were handled by Shanghai Meiji Biomedical Technology Co., Ltd.). Graphpad 2018 software was used for significance analysis (Tukey method, p < 0.05) and plotting. VIP values ​​(VIP > 1) and t-test p-values ​​(p < 0.05) were used to screen differentially expressed metabolites among different treatments using the OPLS-DA model. Key metabolic pathways enriched by differentially expressed metabolites were then identified using relevant databases such as KEGG and PubChem.

[0122] 1. Principal component analysis (PCA) of metabolites in maize seedling leaves

[0123] Depend on Figure 6As shown, the QC samples (purple triangles) clustered tightly, indicating good stability of the experimental instrument analysis system and stable and reliable experimental data. The remaining samples were generally within the 95% confidence interval, indicating high reliability and statistical significance of the experimental sample data. Significant principal component separation was observed between CK0 and T04 in the salt-free group and CK1 and T14 in the salt-stressed group, indicating that salt stress affected maize seedlings. Simultaneously, the separation trend between the VITD treatment and control samples in both the salt-free and salt-stressed groups also largely demonstrates the inter-group differences between the VITD treatment and control under salt stress. Subsequent orthogonal partial least squares discriminant analysis (OPLS-DA) of each treatment group yielded consistent conclusions. Figure 7 ).

[0124] 2. Analysis and cluster analysis of metabolites in maize seedling leaves

[0125] To observe the functional correlation of metabolites from different treatments, hierarchical cluster analysis was performed on the metabolites, and a cluster heatmap was generated. Figure 8 The results showed that the differentially metabolites were broadly classified into peptides (amino acids, organic amines), lipids, carbohydrates (monosaccharides, oligosaccharides), and hormones and neurotransmitters (steroid hormones, neurotransmitters). Significant changes in metabolites were also observed between salt stress and no-salt stress conditions. This indicates that maize seedlings begin to cope with salt stress. Furthermore, the changes in metabolites observed in the VITD treatment compared to the control suggest that VITD treatment can regulate the response of maize seedlings to salt stress.

[0126] 3. Differential Metabolite Analysis under Salt Stress

[0127] Depend on Figure 9 It can be seen that there are significant differences in the expression of metabolites between the experimental group's small peptide VITD and the control group's CK1. Analysis of these differentially expressed metabolites revealed 1571 differentially expressed metabolites in both groups under salt stress. Of these, 641 were significantly upregulated (p<0.05, VITD / CK1, FC>1), with a maximum upregulation of 30.5633-fold, while 930 were significantly downregulated (p<0.05, P / CK1, FC<1), with a maximum downregulation of 0.0001-fold. Table 7.2 below shows 20 differentially expressed metabolites with searchable names, mainly including flavonoids and organic acids.

[0128] Table 3. 20 metabolites showing significant differences between the VITD small peptide group and the control group.

[0129]

[0130]

[0131] Note: (1) VIP: The fold difference in the model VIP value of the metabolite between the two groups; (2) FC(Y / X): The fold change in the expression of the metabolite between the two groups; (3) p value: The result of the significance test of the difference between the two samples; The default screening criteria are VIP>1, p<0.05.

[0132] 4. Enrichment of differentially metabolized substances via the KEGG pathway

[0133] The selected differential metabolites were subjected to KEGG pathway enrichment and classification analysis (see Table 4).

[0134] Table 4. Enrichment of differentially metabolites via the KEGG pathway

[0135]

[0136]

[0137] It is mainly involved in phosphonate and phosphonite metabolism, anthocyanin biosynthesis, purine metabolism, flavonoid biosynthesis, and flavonoid and flavonol biosynthesis. The KEGG pathway enrichment of the top 20 differentially regulated metabolites between the small peptide VITD group and the control group under salt stress is shown below. Figure 10 Among them, the differences between flavonoid and flavonol biosynthesis and flavonoid biosynthesis in the metabolite pathway were extremely significant and significant, respectively.

[0138] Flavonoids are a class of polyphenolic antioxidants widely distributed in nature. They possess antioxidant properties, scavenge free radicals, and participate extensively in plant physiological metabolism, stress resistance, disease resistance, and growth and development. When plants are under stress, the production of reactive oxygen species (ROS) increases, and flavonoids can act as antioxidants to eliminate or mitigate ROS damage caused by abiotic stresses such as drought, cold, salinity, and heavy metals. Flavonols can enhance the role of enzymatic and non-enzymatic antioxidants in reducing excessive ROS production under salt stress, thus improving the tolerance of tomato seedlings to high salt levels. Flavonoids can also regulate the sodium content in plants. + / K + Ion balance mitigates the effects of salinity on plants. In this study, the levels of four flavonoid and flavonol metabolites—astragalin, isoquercitrin, quercetin, and rutin—were significantly upregulated (P<0.05) in maize seedlings treated with the small peptide VITD under salt stress, and these metabolites were significantly enriched (P<0.05, VIP>1) along the flavonoid and flavonol biosynthetic pathways. Therefore, it is hypothesized that the small peptide VITD can induce the production of flavonoids and flavonols in maize seedlings under salt stress, thereby enhancing antioxidant capacity and maintaining the dynamic balance of reactive oxygen species in the plant. Quercetin and rutin may be important metabolites that the small peptide VITD regulates in maize seedlings' response to salt stress.

[0139] In summary, this invention proposes a small peptide VITD concentration of 1x10⁻¹ under 100 mmol / L NaCl stress. -9 The optimal concentration of sodium 60 mol / L significantly improved the salt tolerance of maize seedlings. Under the optimal conditions, VITD reduced the peroxidation and osmotic damage caused by salt stress in maize seedlings, enhancing their salt tolerance. VITD also regulated the inducing factors of maize seedling growth and development under salt stress. Furthermore, VITD induced changes in maize seedling metabolites under salt stress, upregulating 641 differentially expressed metabolites, primarily through the regulation of Na+. + / K + Ion balance enhances antioxidant capacity to alleviate the damage of salt stress to maize seedlings and improve their salt tolerance.

[0140] As described above, the present invention can be well implemented. The above embodiments are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, all changes and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the present invention.

Claims

1. The application of small peptides in crop salt stress resistance, characterized in that, The amino acid sequence of the small peptide is VITD; The crop in question is corn.

2. The application according to claim 1, characterized in that, The small peptides can improve the salt tolerance of crops under salt stress conditions.

3. The application according to claim 2, characterized in that, The salt stress condition is a NaCl solution with a concentration of 100 mmol / L.

4. The application according to claim 2, characterized in that, The concentration of the small peptide is 1×10⁻⁶. -6 mol / L ~ 1×10 - 11 mol / L.

5. The application according to claim 2, characterized in that, The concentration of the small peptide is 1×10⁻⁶. -9 mol / L.

6. The application according to claim 2, characterized in that, The small peptide can reduce the accumulation of malondialdehyde, and the small peptide can increase the content of at least one substance among proline and soluble sugars.

7. The application according to claim 2, characterized in that, The small peptide can increase the expression level of at least one of the enzymes, namely peroxidase and catalase.

8. The application according to claim 2, characterized in that, The small peptide can increase the content of auxin and gibberellin, and the small peptide can decrease the content of abscisic acid.

9. The application according to claim 2, characterized in that, The small peptides can increase the biosynthesis of flavonoids and flavonols.

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