Application of plant elicitor peptide MsPep1 in improving salt tolerance of medicago sativa
By binding the plant elicitor peptide MsPep1 to the cell membrane PEPR receptor, the systemic immune resistance of alfalfa is activated, solving the problem of germination and growth inhibition of alfalfa under salt stress and achieving efficient and safe improvement of salt tolerance.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
- Filing Date
- 2026-02-27
- Publication Date
- 2026-05-12
AI Technical Summary
Current technologies lack efficient and safe salt-resistant biological agents for alfalfa. Under salt stress, the seed germination rate, root development and growth of alfalfa are inhibited, affecting its application in saline-alkali land.
The plant elicitor peptide MsPep1 is used to activate systemic immune resistance and enhance the salt stress adaptability of alfalfa by binding to the PEPR receptor on the cell membrane. The synthesis methods include microbial fermentation or Fmoc solid-phase chemical synthesis.
It significantly improved the seed germination rate, root development and photosynthetic capacity of alfalfa under salt stress, alleviated the growth inhibition caused by salt stress, and enhanced the growth potential of alfalfa in saline-alkali land.
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Figure CN122012591A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, and in particular to a plant elicitor peptide MsPep1 and its application in improving the salt tolerance of alfalfa. Background Technology
[0002] Alfalfa (Medicago sativa) is rich in protein and highly palatable, making it one of the most nutritious forage crops among leguminous plants. It is known as the "King of Forage" and is extremely important for livestock development. Given the high proportion of saline-alkali land in these regions, improving alfalfa's salt tolerance is crucial for the sustainable development of my country's forage industry and livestock farming.
[0003] Plant elicitor peptides (Pep) are a class of endogenous damage-associated molecular pattern (DAMP) signaling molecules generated by the cleavage of precursor proteins (PROPEPs) by proteases. They can activate systemic immune resistance by binding to PEPR receptors on cell membranes. Currently, research on Pep peptides' disease resistance is relatively advanced. Previous research in this invention (CN202511092830.6) has isolated and identified MsPep1 from alfalfa and confirmed that MsPep1 can effectively improve the genetic transformation efficiency of alfalfa. However, its mechanism of action and application value under abiotic stress, especially salt stress, remain unclear, and research on the salt-resistance function of endogenous Pep peptides in alfalfa is even more lacking.
[0004] Based on this, the purpose of this invention is to study the effects of different concentrations of alfalfa elicitor peptide MsPep1 on seed germination rate, seedling growth phenotype and physiological indicators under salt stress, in order to develop a novel, efficient and safe small peptide to improve alfalfa salt tolerance, which will have important application value for improving alfalfa growth in saline-alkali land. Summary of the Invention
[0005] To address the lack of efficient and safe salt-tolerant biological agents for alfalfa in existing technologies, this invention provides an application of the plant elicitor peptide MsPep1 in improving the salt tolerance of alfalfa. This invention is the first to demonstrate that the elicitor peptide MsPep1 can enhance seed germination, root development, and photosynthetic capacity under salt stress, providing a research foundation for the growth and development of alfalfa and having significant implications for the sustainable development of my country's forage industry and animal husbandry.
[0006] This invention provides for the first time the application of plant elicitor peptide MsPep1 in positively regulating the salt stress resistance of alfalfa, wherein the amino acid sequence of MsPep1 is shown in SEQ ID NO.1.
[0007] Preferably, the amino acid sequence of MsPep1 is a derived protein sequence that has the same activity as the amino acid sequence shown in SEQ ID NO.1, obtained by substituting, deleting or adding one or more amino acid residues.
[0008] Preferably, the method for synthesizing MsPep1 includes a microbial synthesis method or a chemical synthesis method.
[0009] Preferably, the microbial synthesis includes fermentation synthesis by prokaryotic Escherichia coli or fungal yeast.
[0010] Preferably, the chemical synthesis method employs Fmoc solid-phase chemical synthesis technology.
[0011] Another object of the present invention is to provide the application of plant elicitor peptide MsPep1 in improving the salt tolerance of alfalfa.
[0012] Another objective of this invention is to provide the application of plant elicitor peptide MsPep1 in improving seed germination rate of alfalfa under salt stress.
[0013] Another objective of this invention is to provide the application of plant elicitor peptide MsPep1 in improving seedling growth traits of alfalfa under salt stress.
[0014] Another object of the present invention is to provide the application of the plant elicitor peptide MsPep1 in alleviating the inhibition of root growth in alfalfa caused by salt stress.
[0015] Another objective of this invention is to provide the application of plant elicitor peptide MsPep1 in alleviating leaf wilting in alfalfa under salt stress.
[0016] Another object of the present invention is to provide the application of plant elicitor peptide MsPep1 in reducing the degree of damage to alfalfa leaves under salt stress.
[0017] Furthermore, the present invention also provides a product that resists salt stress in alfalfa, positively regulates salt stress in alfalfa, improves salt tolerance of alfalfa, increases seed germination rate of alfalfa under salt stress, improves seedling growth traits of alfalfa under salt stress, alleviates the inhibition of root length of alfalfa by salt stress, alleviates wilting of alfalfa leaves under salt stress, or reduces the degree of damage to alfalfa leaves under salt stress, wherein the product comprises the above-mentioned plant elicitor peptide MsPep1 and its excipients.
[0018] Preferably, the excipients are selected from one or more of surfactants, osmosis regulators, stabilizers, or pH buffers.
[0019] The present invention also provides the use of the above-mentioned plant elicitor peptide MsPep1 in the preparation of products that resist salt stress in alfalfa, positively regulate salt stress in alfalfa, improve salt tolerance of alfalfa, improve seed germination rate of alfalfa under salt stress, improve seedling growth traits of alfalfa under salt stress, alleviate the inhibition of root length of alfalfa by salt stress, alleviate wilting of alfalfa leaves under salt stress, or reduce the degree of damage to alfalfa leaves under salt stress.
[0020] The advantages of this invention are as follows: This invention is the first to demonstrate that the elicitor peptide MsPep1 can enhance seed germination, root development and photosynthetic capacity under salt stress, providing a research basis for the growth and development of alfalfa, and is of great significance to the sustainable development of my country's forage industry and animal husbandry. Attached Figure Description
[0021] Figure 1 Effect of MsPep1 on the germination of alfalfa seed 'M1' under 200 mM NaCl stress: where a is a seed germination phenotypic diagram and b is a statistical graph of seed germination rate. Figure 2 Effects of MsPep1 on root length of alfalfa seedlings No. 1 under 200 mM NaCl stress: where a is the root length phenotype and b is the root length statistical graph. Figure 3 Effect of MsPep1 on the germination of alfalfa seed 'M1' under 250 mM NaCl stress: where a is a seed germination phenogram and b is a statistical graph of seed germination rate. Figure 4 Effects of MsPep1 on the growth of alfalfa seedlings No. 1 under 200 mM NaCl stress: where a is the phenotypic diagram of alfalfa after 28 days of salt stress, b is the chlorophyll content statistical diagram, and c is the trypan blue staining results. Figure 5 Effects of MsPep1 on seed germination of Xinjiang large-leaf alfalfa under 100 mM NaCl stress. Detailed Implementation
[0022] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.
[0023] The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art based on the specific embodiments of the invention without inventive effort are within the protection scope of the invention.
[0024] In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the embodiments of the present invention, unless specifically specified, the technical means used are conventional means well known to those skilled in the art.
[0025] MsPep1 is released from its precursor proteins (PROPEPs) after cleavage by a protease, and its amino acid sequence is shown in SEQ ID NO. 1. MsPep1 can activate a series of defense responses by binding to receptors on the cell membrane (such as PEPR), thereby enhancing the systemic immune resistance of plants. MsPep1 can be synthesized by microorganisms such as prokaryotic Escherichia coli and fungi / yeasts, or by Fmoc solid-phase chemical synthesis technology.
[0026] Example 1 Effects of MsPep1 on seed germination of alfalfa variety Zhongmu 1 under 200 mM salt stress Using plump alfalfa seeds (Zhongmu 1), four treatment groups were established: ① Salt stress control group (200 mM NaCl); ② Treatment group 1 (200 mM NaCl + 1 nM MsPep1); ③ Treatment group 2 (200 mM NaCl + 5 nM MsPep1); ④ Treatment group 3 (200 mM NaCl + 10 nM MsPep1). The control group used salt-free distilled water, while the other groups were prepared using salt-free distilled water as the solvent. The amino acid sequence of MsPep1 is shown in SEQ ID NO.1.
[0027] Fifty plump seeds were randomly selected from each group and placed in a petri dish lined with 2-4 layers of sterile filter paper. 3-5 mL of the corresponding treatment solution was added to each dish. The edges of the petri dishes were sealed with sealing film to prevent moisture evaporation and contamination. Each group was repeated three times. The sterilized seeds were placed in a light incubator for cultivation. Germination rate was recorded on days 5-6, and seedling root length was measured.
[0028] Germination rate results as follows Figure 1 As shown: Treatment with 200 mM NaCl resulted in a germination rate of 34.0%, indicating that salt stress severely inhibited seed germination. Adding 1 nM MsPep1 resulted in a germination rate of 12.0%, and adding 5 nM MsPep1 resulted in a germination rate of 53.3%, an increase of 19.3% compared to the NaCl control, significantly alleviating salt stress. Adding 10 nM MsPep1 resulted in a germination rate of 52.0%, an increase of 18% compared to the NaCl control, also showing a mitigating effect. Therefore, adding 5-10 nM MsPep1 can significantly increase the seed germination rate of alfalfa seeds under 200 mM NaCl stress by 18-19%.
[0029] Seedling roots as long as Figure 2 As shown: under 200 mM NaCl treatment, the root length was 0.94 cm, indicating that salt stress significantly inhibited root elongation; with the addition of 1 nM MsPep1, the root length was 0.73 cm; with the addition of 5 nM MsPep1, the root length was 2.02 cm, showing a significant alleviating effect; with the addition of 10 nM MsPep1, the root length was 0.57 cm. Therefore, 5 nM MsPep1 can significantly alleviate the inhibitory effect of salt stress on root length.
[0030] Example 2 Effects of MsPep1 on seed germination of alfalfa cultivar Zhongmu 1 under 250 mM salt stress Using alfalfa seeds (Zhongmu 1) as material, four treatment groups were established: ① Salt stress control group (250 mM NaCl); ② Treatment group 1 (250 mM NaCl + 1 nM MsPep1); ③ Treatment group 2 (250 mM NaCl + 5 nM MsPep1); ④ Treatment group 3 (250 mM NaCl + 10 nM MsPep1). The control group used salt-free distilled water, while the other groups used salt-free distilled water as the solvent. The amino acid sequence of MsPep1 is shown in SEQ ID NO.1.
[0031] Fifty plump seeds were randomly selected from each group and placed in a petri dish lined with 2-4 layers of sterile filter paper. 3-5 mL of the corresponding treatment solution was added to each dish. The edges of the petri dishes were sealed with sealing film to prevent moisture evaporation and contamination. Each treatment was repeated three times. The sterilized seeds were placed in a light incubator for cultivation. Germination rate was recorded on day 7, and seedling root length was measured.
[0032] Germination rate results as follows Figure 3 As shown: Treatment with 250 mM NaCl resulted in a sharp drop in germination rate to 27.3%, indicating that salt stress severely inhibited seed germination; addition of 1 nM MsPep1 resulted in a germination rate of 36.7%, an increase of 9.4% compared to the NaCl control; addition of 5 nM MsPep1 resulted in a germination rate of 34.7%, an increase of 7.4% compared to the NaCl control; and addition of 10 nM MsPep1 resulted in a germination rate of 53.3%, a significant increase of 26.0% compared to the NaCl control, effectively alleviating salt stress. Therefore, adding 1-10 nM MsPep1 can significantly improve the seed germination rate of alfalfa variety Zhongmu 1. Under high salt stress, 10 nM MsPep1 has a more significant effect, increasing the seed germination rate by up to 26%.
[0033] Example 3 Effects of MsPep1 on the seedling stage of alfalfa variety Zhongmu 1 under salt stress One-month-old alfalfa seedlings of the variety Zhongmu 1 were used as experimental subjects. Four treatment groups were set up: ① Salt stress control group (200 mM NaCl); ② Treatment group 1 (200 mM NaCl + 1 nM MsPep1); ③ Treatment group 2 (200 mM NaCl + 5 nM MsPep1); ④ Treatment group 3 (200 mM NaCl + 10 nM MsPep1). The seedlings of each group were watered with the above treatments in sequence, once a day for 28 consecutive days. Phenotypic results were observed and physiological indicators were measured.
[0034] The results are as follows Figure 4 As shown, under 200 mM NaCl stress, the wilting rate of leaves without MsPep1 was 80%; while the wilting rate of leaves with 1 nM, 5 nM, and 10 nM MsPep1 was only 30%. Simultaneously, physiological indicators of stress resistance showed that the chlorophyll content of seedlings in the treatment groups was significantly higher than that in the salt-stressed control group, and the trypan blue staining color of the seedling leaves was also significantly lighter than that in the salt-stressed control group. These results fully demonstrate that the degree of leaf damage in the treatment groups was significantly reduced compared to the control. Therefore, MsPep1 improves the salt tolerance of alfalfa seedlings.
[0035] Example 4 To further verify the universality of MsPep1 in improving salt tolerance of alfalfa, Ganong No. 4 alfalfa was used as an example to further analyze its universality.
[0036] Four treatment groups were set up: ① Salt stress control group (200 mM NaCl); ② Treatment group 1 (200 mM NaCl + 1 nM MsPep1); ③ Treatment group 2 (200 mM NaCl + 5 nM MsPep1); ④ Treatment group 3 (200 mM NaCl + 10 nM MsPep1).
[0037] Fifty plump seeds were randomly selected from each group and placed in a petri dish lined with 2-4 layers of sterile filter paper. 3-5 mL of the corresponding treatment solution was added to each dish. The edges of the petri dishes were sealed with sealing film to prevent moisture evaporation and contamination. Each treatment was repeated three times. The sterilized seeds were placed in a light incubator for cultivation, and the germination rate was recorded on day 5.
[0038] The experimental results showed that: 200 mM NaCl treatment resulted in a sharp decrease in germination rate to 10.7%, indicating that salt stress severely inhibited seed germination; the addition of 1 nM MsPep1 resulted in a germination rate of 5.3%, 5 nM MsPep1 resulted in a germination rate of 10.7%, and 10 nM MsPep1 resulted in a germination rate of 26.0%, significantly higher than the NaCl control by 15.3%, effectively alleviating salt stress. Therefore, the addition of 10 nM MsPep1 can significantly improve the seed germination rate of alfalfa variety Gannong 4 under 200 mM NaCl stress. In conclusion, MsPep1 has varietal applicability and is suitable for multiple major alfalfa varieties.
[0039] Example 5 To further verify the universality of MsPep1 in improving salt tolerance of alfalfa, Xinjiang large-leaf alfalfa was used as an example to further analyze its universality.
[0040] Four treatment groups were set up: ① Salt stress control group (100 mM NaCl); ② Treatment group 1 (100 mM NaCl + 1 nM MsPep1); ③ Treatment group 2 (100 mM NaCl + 5 nM MsPep1); ④ Treatment group 3 (100 mM NaCl + 10 nM MsPep1).
[0041] Fifty plump seeds were randomly selected from each group and placed in a petri dish lined with 2-4 layers of sterile filter paper. 3-5 mL of the corresponding treatment solution was added to each dish. The edges of the petri dishes were sealed with sealing film to prevent moisture evaporation and contamination. Each treatment was repeated three times. The sterilized seeds were placed in a light incubator for cultivation, and the germination rate was recorded on day 5.
[0042] The experimental results show that ( Figure 5 Treatment with 100 mM NaCl resulted in a germination rate of 61.3%, indicating that salt stress inhibited seed germination. Adding 1 nM MsPep1 resulted in a germination rate of 71.3%, adding 5 nM MsPep1 resulted in a germination rate of 67.3%, and adding 10 nM MsPep1 resulted in a germination rate of 70.0%, significantly higher than the NaCl control by approximately 10%, effectively alleviating salt stress. Therefore, adding MsPep1 can significantly improve the seed germination rate of Xinjiang large-leaf alfalfa seeds under 100 mM NaCl stress. In conclusion, MsPep1 has varietal applicability and is suitable for multiple major alfalfa varieties.
[0043] The above embodiments are intended to illustrate the essential content of the present invention, but are not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of protection of the present invention.
Claims
1. The application of plant elicitor peptide MsPep1 in positively regulating the salt stress resistance of alfalfa, characterized in that, The amino acid sequence of MsPep1 is shown in SEQ ID NO.
1.
2. The application of plant elicitor peptide MsPep1 in improving salt tolerance in alfalfa, characterized in that, The amino acid sequence of MsPep1 is shown in SEQ ID NO.
1.
3. The application of plant elicitor peptide MsPep1 in improving seed germination rate of alfalfa under salt stress, characterized in that, The amino acid sequence of MsPep1 is shown in SEQ ID NO.
1.
4. The application of plant elicitor peptide MsPep1 in improving seedling growth traits of alfalfa under salt stress, characterized in that, The amino acid sequence of MsPep1 is shown in SEQ ID NO.
1.
5. The application of plant elicitor peptide MsPep1 in alleviating the inhibition of alfalfa root length by salt stress, characterized in that... The amino acid sequence of MsPep1 is shown in SEQ ID NO.
1.
6. The application of plant elicitor peptide MsPep1 in alleviating leaf wilting in alfalfa under salt stress, characterized in that... The amino acid sequence of MsPep1 is shown in SEQ ID NO.
1.
7. The application of plant elicitor peptide MsPep1 in reducing the degree of damage to alfalfa leaves under salt stress, characterized in that... The amino acid sequence of MsPep1 is shown in SEQ ID NO.
1.
8. The use of plant elicitor peptide MsPep1 in the preparation of products that resist alfalfa salt stress, positively regulate alfalfa salt stress, improve alfalfa salt tolerance, improve seed germination rate of alfalfa under salt stress, improve seedling growth traits of alfalfa under salt stress, alleviate the inhibition of alfalfa root length by salt stress, alleviate alfalfa leaf wilting under salt stress, or reduce the degree of damage to alfalfa leaves under salt stress, characterized in that... The amino acid sequence of MsPep1 is shown in SEQ ID NO.
1.
9. The use as described in claim 8, characterized in that, The product includes plant elicitor peptide MsPep1 and its excipients, wherein the excipients are selected from one or more of surfactants, osmotic regulators, stabilizers or pH buffers.
10. The application as described in any one of claims 1-9, characterized in that, The amino acid sequence of MsPep1 is a derived protein sequence that has the same activity as the amino acid sequence shown in SEQ ID NO.1, by substituting, deleting or adding one or more amino acid residues.