Eelgrass-sourced polypeptide and application thereof in increasing fucose content

Abstract

The invention belongs to the technical field of active polypeptides, and particularly relates to an eelgrass-derived polypeptide and application thereof in increasing fucose content. The invention discloses an exocrine polypeptide ZosmaRALF2 capable of responding to a high-salt seawater environment and increasing the content of fucose serving as an effective component in eelgrass and application of the exocrine polypeptide ZosmaRALF2. The expression of the polypeptide ZosmaRALF2 is remarkably accumulated in a high-salt seawater environment. Through exogenous application of the polypeptide, accumulation of fucose in eelgrass can be effectively improved. The invention provides a new technical approach and product support for improving the medicinal material quality of eelgrass through a molecular biotechnological means, especially for improving the content of fucose serving as an effective component of the eelgrass.

Description

Technical Field

[0001] This invention belongs to the field of active polypeptide technology, specifically relating to a polypeptide derived from eelgrass and its application in promoting fucose content. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Eelgrass ( Zostera Marina L. (also known as seaweed or kelp), mainly distributed in the temperate shallow seas of the Northern Hemisphere, is adapted to high-salinity submerged environments. It uses its roots and rhizomes to anchor itself to the seabed, reducing the risk of wind, waves, and currents eroding the coastline. Its leaves absorb suspended matter, improving seawater transparency. L. is also a traditional Chinese medicine plant native to the Shandong and Liaoning peninsulas. According to the *Compendium of Materia Medica*, the whole L. plant is used medicinally; it is salty and cold in nature. Decoctions, pills, or powders can clear heat and resolve phlegm, soften hardened masses, and dissipate nodules. It has the effects of resolving phlegm and softening hardened masses, purging heat and promoting diuresis, reducing lipids and lowering blood pressure, relieving cough and asthma, dissipating nodules, and fighting cancer. Fucoido, one of the main active ingredients of L., is a natural six-carbon deoxyglucose widely found in various biomolecules. Recent studies have shown that fucose and its derivatives have shown promising medicinal prospects in anti-tumor, anti-inflammatory, immunomodulatory, antibacterial, and gut health maintenance. With increased human activity and global warming, seawater salinity continues to rise, seriously threatening the survival of L. seagrass beds. This not only jeopardizes coastal ecological security but may also lead to unstable accumulation or reduced content of fucose, the core active ingredient in *Erigeron breviscapus*, when used as a medicinal resource, affecting the sustainable supply of medicinal materials and their clinical efficacy. Currently, methods to increase the content of effective components in medicinal plants mainly include genetic improvement, cultivation optimization, and exogenous induction. In particular, there is a lack of efficient biotechnological methods that can specifically respond to salt signals and activate the fucose synthesis pathway in *Erigeron breviscapus*. Therefore, a new technical solution is urgently needed in this field to effectively increase the fucose content of *Erigeron breviscapus* in high-salinity seawater environments to ensure its medicinal quality and sustainable resource utilization. Summary of the Invention

[0004] The present invention aims to overcome the shortcomings of the prior art and provide an exocrine polypeptide that can respond to high-salinity seawater environments and increase the fucose content of eelgrass, and its application.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, a polypeptide derived from eelgrass is provided, the polypeptide satisfying at least one of the following conditions: (1) Its amino acid sequence is shown in SEQ ID NO:1: (2) A derivative polypeptide formed by adding, substituting or deleting one or more amino acids of the sequence shown in SEQ ID NO:1, wherein the derivative polypeptide has the same or substantially the same function as the polypeptide of the sequence shown in SEQ ID NO:1. (3) A modified polypeptide after chemical or genetic modification of the polypeptide described in (1) or the derivative polypeptide described in (2).

[0006] This invention analyzed transcriptome data from *Erigeron annuus* under salt stress and found that ZosmaRALF2 is one of the genes most highly expressed under salt stress in *Erigeron annuus*. It exhibits typical characteristics of an exocrine protein, and the polypeptide shown in SEQ ID NO:1 is the exocrine polypeptide of ZosmaRALF2. Hereinafter, ZosmaRALF2 will be used to refer to this polypeptide. Verification showed that ZosmaRALF2 has higher stability under salt stress, and exogenous application of ZosmaRALF2 can effectively increase the content of fucose, glucose, fructose, galactose, sucrose, rhamnose, mannose, xylose, and arabinose in *Erigeron annuus*, especially showing a significant effect on fucose, glucose, and fructose content. Among these, the effect on fucose is the most significant. Of the aforementioned monosaccharides or disaccharides, fucose has high medicinal value. Its proven medicinal activities include anti-inflammatory, anticancer, anti-infective, and metabolic regulation. It can be used as an adjunct treatment for inflammatory bowel disease, arthritis, etc., to inhibit related expression on the surface of liver cancer cells, lower blood lipids, and assist in metabolic regulation, etc.

[0007] In some embodiments verified by this invention, exogenous application of ZosmaRALF2 alone can increase the fucose content in Eriocaulon buergerianum by 8.95%, and exogenous application of ZosmaRALF2 combined with high-salinity seawater cultivation can further increase the fucose content by 18.64%. The preparation method of this polypeptide is selected from solid-phase synthesis, liquid-phase synthesis, or recombinant expression methods.

[0008] The recombinant expression method described above includes transferring nucleic acid material encoding the polypeptide described in the first aspect into an expression vector and expressing it through engineered microorganisms. Further, the nucleic acid material includes a coding nucleic acid capable of being translated to obtain the polypeptide due to codon degeneracy, including DNA or RNA forms; wherein the DNA form includes cDNA, genomic DNA, or artificially synthesized DNA, including single-stranded or double-stranded, coding or non-coding strands. In one embodiment verified by this invention, the sequence of the coding nucleic acid is shown in SEQ ID NO:2.

[0009] Furthermore, the expression vector is a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, or mammalian cell virus, wherein the mammalian cell virus is an adenovirus or a retrovirus.

[0010] In (2) above, the "same or substantially the same function" includes physical properties, chemical properties, and biological properties. The physical properties are selected from hydrophobicity / hydrophilicity, solubility, spatial conformation, optical properties, thermal stability, melting point, and fluidity, etc.; the chemical properties are selected from acidity / alkalinity, functional group reactivity, degradation stability, redox properties, and binding ability with metal ions, etc. The biological properties indicate the enhancing effect of the polypeptide on the monosaccharide or disaccharide components in eelgrass. The derived polypeptide has 90% similarity to the sequence shown in SEQ ID NO:1, further, more than 93%; and even more than 95%.

[0011] In (3) above, the chemical or genetic modifiers include, but are not limited to, polyethylene glycol, streptavidin, biotin, radioactive isotopes, fluorescent agents, enzymes, cytotoxic substances, and antitumor agents.

[0012] In a second aspect, a composition is provided, wherein the composition comprises the polypeptide described in the first aspect, and further comprises a pharmaceutically necessary carrier.

[0013] Preferably, the carrier includes, but is not limited to, buffers, antioxidants, preservatives, bactericides, and anti-degradation agents. In one embodiment, the composition is a formulation or an immobilized product, including, but not limited to, products that use physical adsorbents, carriers, or cross-linking agents to block or immobilize the polypeptide described in the first aspect.

[0014] Thirdly, the application of the polypeptides described in the first aspect in increasing the fucose content in eelgrass is provided.

[0015] As mentioned in the background section, the content of fucose in eelgrass, as a medicinal plant, is of great significance for exerting its medicinal activity. The polypeptide isolated by the present invention can effectively increase the fucose content in eelgrass, which is of great significance for enhancing the medicinal activity of eelgrass.

[0016] In one embodiment, the above-mentioned polypeptide is applied to all stages of *Erigeron annuus* cultivation, with a concentration of 1-5 μM, and applied to all parts of the plant. Further, the concentration of the above-mentioned polypeptide is 2-4 μM, and even more specifically, 3 μM.

[0017] In another embodiment, the above-mentioned polypeptide is applied to the entire stage of eel grass cultivation, wherein the eel grass is cultivated in high-salinity seawater with a salinity not exceeding 30.

[0018] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention provides for the first time an exocrine polypeptide ZosmaRALF2 derived from eelgrass that can specifically respond to high-salinity seawater signals.

[0019] 2. The polypeptides or their compositions of the present invention can effectively increase the fucose content of eelgrass in normal seawater and high-salinity seawater environments through exogenous application, providing a new tool for improving the quality of marine medicinal plants through molecular biotechnology. Attached Figure Description

[0020] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0021] Figure 1 The phenotypic changes and physiological and biochemical indicators of *Eriocheir sinensis* under simulated high-salinity seawater stress as described in Example 1 were observed after treatment. in, Figure 1 In Figure A, the phenotypic results of *Eriocheir sinensis* after 14 days of treatment with high-salinity seawater are shown. Figure 1 Figure B shows the results of photosynthetic pigment content in Eriocaulon buergerianum leaves after 0, 3, and 6 days of high-salinity seawater treatment. Figure 1 C represents the SOD content determination results in Eriocaulon buergerianum after 0, 3, and 6 days of high-salinity seawater treatment; Figure 1 D represents the CAT content determination results in Eriocaulon buergerianum after 0, 3, and 6 days of high-salinity seawater treatment; Figure 1 E represents the results of MDA activity determination in Eriocheir sinensis after 0, 3, and 6 days of high-salinity seawater treatment; Figure 1 The results of proline activity determination in Eriocaulon buergerianum after 0, 3, and 6 days of high-salt seawater treatment are shown in Figure F. Figure 1 G represents the Na content in the root and leaf tissues of *Eriocaulon buergerianum* after 0 and 6 days of high-salinity seawater treatment. + / K + The results of the ratio determination; Figure 1 H represents the results of flavonoid content determination in Eriocaulon buergerianum after 0 and 6 days of high-salinity seawater treatment; Figure 1 Figure I shows the results of the determination of the content of 11 monosaccharides in Eriocaulon buergerianum after 0 days and 14 days of high-salt seawater treatment.

[0022] Figure 2 This study describes the analysis and heterologous expression verification of salt-responsive exocrine peptides and ZosmaRALF2 in Eriocaulon buergerianum based on whole-genome data screening as described in Example 1. Figure 2 In Figure A, the results of the screening of salt-responsive exocrine peptides from Eriocaulon buergerianum are shown. Figure 2 B represents the gene structure analysis results of ZosmaRALF2; Figure 2 C represents the analysis results of the signal peptide, cleavage site, domain, and conserved motif of ZosmaRALF2; Figure 2 In Figure D, the growth results of the yeast mutant YTK12 through heterologous expression of the exocrine signal peptide ZosmaRALF2 are shown. Figure 2E represents the TTC colorimetric reaction result: YTK12 represents a mutant yeast lacking invertase and tryptophan genes, pSUC2 represents an empty plasmid, pSUC2-Avr1b represents the Avr1b signal peptide positive control, and pSUC2-SP represents the ZosmaRALF2 exocrine signal peptide heterologous expression group.

[0023] Figure 3 The results are the expression response and stability analysis of the polypeptide ZosmaRALF2 described in Example 1 under high salinity seawater. in, Figure 3 In the middle, A represents the predicted cis-acting element of the ZosmaRALF2 promoter region; Figure 3 Figure B shows the expression analysis results of ZosmaRALF2 in root and leaf tissues after 12 hours of high-salt seawater treatment; Figure 3 In the middle C, the results of Coomassie brilliant blue staining and Western blot identification of prokaryotic-induced ZosmaRALF2 protein are shown. Figure 3 D represents the relative protein expression levels under each induction condition; Figure 3 E represents the retention of ZosmaRALF2 protein under normal conditions at different time points (0, 1, 2, 4 h). Figure 3 F represents the retention of ZosmaRALF2 protein under salt stress conditions at different times (0, 1, 2, 4 h).

[0024] Figure 4 Verification of the application effect of the exogenous peptide ZosmaRALF2 in increasing the fucose content in Eriocaulon buergerianum; in, Figure 4 Figure A shows the phenotypic results of *Erigeron angustifolia* after 14 days of culture under different treatments; Figure 4 Figure B shows the results of the determination of the content of 11 monosaccharides in eelgrass after 14 days under different treatments; Figure 4 In the middle, C represents the three-dimensional scoring results of principal component analysis (PCA) of sugar components under different treatments; Figure 4 In the diagram, D represents the two-dimensional PCA plane formed by PC1 and PC2; Figure 4 E in the figure represents the two-dimensional PCA plane formed by PC1 and PC3. Detailed Implementation

[0025] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0026] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0027] In the context of this specification, the word "comprising" is considered to mean "especially including". It should not be interpreted as "consisting of only".

[0028] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0029] Example 1 (I) The impact of high-salinity marine environment on the growth of eelgrass 1.1 Effects of high-salinity seawater environment on the phenotype of eelgrass The *Erigeron annuus* samples used in this embodiment were collected from the Rongcheng sea area of ​​Weihai City, Shandong Province (122°08' E, 36°45' N). The *Erigeron annuus* was cultured under a light intensity of 10,000 lux, with a photoperiod of 14 hours of light and 10 hours of darkness, and the temperature was maintained at 15±1℃. After 3 days of acclimatization, the *Erigeron annuus* was placed in normal seawater (salinity 30) and high-salinity seawater (salinity 60) for two more weeks. The results showed that under normal seawater conditions, the *Erigeron annuus* grew well, and no significant changes in phenotype were observed. However, the high-salinity seawater treatment caused obvious stress phenotypes, manifested as darkening of leaf color, accompanied by wilting of leaves and stem tissues. Figure 1 (A)

[0030] 1.2 Determination of physiological indicators of eelgrass under different treatments In high-salinity seawater environments, the chlorophyll content in *Erigeron eelgrass* significantly decreases, leading to the accumulation of malondialdehyde (MDA), a product of membrane lipid peroxidation. To cope with this stress, *Erigeron eelgrass* activates its antioxidant enzyme system in the short term, with increased activities of superoxide dismutase (SOD) and catalase (CAT). It also accumulates free proline to maintain cellular homeostasis. Under long-term high-salinity seawater stress, the Na+ content in the roots and leaves of *Erigeron eelgrass* decreases. + / K + The ratios increased by 5.57-fold and 4.60-fold, respectively (P<0.001), indicating an imbalance in ion homeostasis. Simultaneously, flavonoids and monosaccharides accumulated significantly, potentially acting as antioxidants in the stress response. Figure 1 in BI).

[0031] based on Figure 1It can be seen that high-salinity seawater environments lead to phenotypic changes in *Erigeron eelgrass*, such as darkening of leaf color, softening of texture, and wilting of rhizomes. Simultaneously, it significantly affects its physiological and biochemical processes: reducing photosynthetic pigment content and weakening photosynthetic capacity; activating the activity of antioxidant enzymes (SOD, CAT) to combat oxidative damage; inducing MDA accumulation and exacerbating cell membrane damage; promoting the accumulation of monosaccharides and flavonoids to regulate osmotic balance and enhance stress resistance; and specifically increasing the content of monosaccharides such as fucose. These results indicate that high-salinity seawater environments affect the photosynthetic, antioxidant, osmotic regulation, and secondary metabolic processes of *Erigeron eelgrass*, thereby significantly regulating its growth status and the accumulation of the active ingredient fucose. These results suggest that *Erigeron eelgrass* adapts to high-salinity seawater environments through multiple mechanisms, including synergistic regulation of antioxidant defense and osmotic regulation, providing a physiological basis for further elucidating its response mechanism to salt signals.

[0032] (II) Bioinformatics screening of peptide ZosmaRALF2 Based on the eelgrass whole proteome database, proteins were initially classified according to the length of their encoded amino acid sequences. Potential extracellular secretory proteins were screened by analyzing the presence of signal peptides, subcellular localization, and GO functional annotation. The analysis workflow is as follows: Figure 2 As shown in section A: (1) represents the identification of secretory proteins, including the division of the target protein group into secretory and non-secretory proteins based on signal peptide prediction; (2) represents the subcellular localization prediction, including the main predicted subcellular localization of gene products; (3) represents GO enrichment analysis, including listing the gene ontology (GO) entries that are significantly enriched in the target gene group; (4) represents the differential expression analysis, including the display of gene expression differences between the salt stress group and the control group based on the transcriptome data of *Zosma arachidica* under salt stress. The values ​​in the "Expression Difference" column are log2 fold differences (log2FC); "↑" indicates upregulation, and "↓" indicates downregulation. Gene abbreviation annotation: RALF (rapid alkalization factor), GASA (gibberellin regulatory protein), PLA2 (phospholipase A2), GH28 (glycosylated enzyme family 28). Combined with the analysis of *Zosma arachidica* under salt stress transcriptome data, it was found that ZosmaRALF2 had the highest fold upregulation under salt stress ( Figure 2 (A)

[0033] Bioinformatics analysis was used to analyze the physicochemical properties of ZosmaRALF2 (as shown in Table 1 below), gene structure, conserved domains, signal peptide cleavage sites, and to verify its exocrine function. Figure 2 (BE).

[0034] Table 1 Physicochemical Properties of ZosmaRALF2 The results showed that ZosmaRALF2 is one of the genes with the highest expression levels in eelgrass under salt stress. It has typical characteristics of exocrine proteins, stable physicochemical properties, and can be successfully secreted in yeast through heterologous expression. This provides an important gene for further research on its role in regulating fucose accumulation in eelgrass.

[0035] (III) Expression response and stability of peptide ZosmaRALF2 in high-salinity seawater 3.1 ZosmaRALF2 Regulatory Elements and Gene Expression Analysis Bioinformatics analysis of the promoter sequence 1500 bp upstream of the transcription start site of ZosmaRALF2 predicted several cis-regulatory elements associated with abiotic stress responses. Figure 3 (A). After treating *Zosma RALF2* in normal seawater and high-salinity seawater for 12 hours, RNA was extracted from root and leaf tissues and analyzed by real-time quantitative PCR. The results showed that under high-salinity stress, the expression of *Zosma RALF2* was significantly upregulated by 1.64-fold in roots and 5-fold in leaves. Figure 3 (B)

[0036] 3.2 Gene Cloning and Vector Construction Specific primers were designed using SnapGene software to amplify the CDS region of the signal peptide coding sequence (forward primer: 5'-gctgatatcggatccgaattcATGGGAGGAGGAAGAACGAGA-3', SEQ ID NO:4; reverse primer: 5'-tgcggccgcaagcttgtcgacTTAAGACCTGCACCTGGCGA-3', SEQ ID NO:5). Amplification was performed using *Eriocaulon buergerianum* cDNA as a template. The purified fragment was ligated into the prokaryotic expression vector pET-30a, transformed into *E. coli* competent cells, and sequencing confirmed the correctness of the obtained recombinant plasmid sequence.

[0037] 3.3 Induction of expression and protein purification The validated recombinant strain was induced to express the protein at 30°C for 8 hours using 1 mmol / L isopropyl-β-D-thiogalactopyranoside (IPTG). After cell collection, the cells were sonicated and the His-tagged recombinant ZosmaRALF2 protein was purified by nickel column affinity chromatography. SDS-PAGE electrophoresis showed that the purified product exhibited a single, high-purity band at the expected molecular weight, indicating successful acquisition of the recombinant ZosmaRALF2 peptide (…). Figure 3 Medium CD).

[0038] 3.4 Stability of peptide ZosmaRALF2 under salt stress In a semi-in vitro reaction system simulating normal and salt stress conditions, exogenous ZosmaRALF2 protein was added and its stability was tested. The results showed that after 4 hours of incubation under salt stress, approximately 26.2% of ZosmaRALF2 remained intact; while under normal conditions, the protein was almost completely degraded after the same treatment time, with a residual rate of only 12.5%. These results demonstrate that the peptide exhibits higher stability under salt stress. Figure 3 Medium EF).

[0039] Figure 3 The results indicate that ZosmaRALF2 responds to high-salinity seawater stress through a complex transcriptional regulatory network. Its promoter region's cis-regulatory elements, along with bound transcription factors, jointly regulate its expression in root and leaf tissues. This provides important theoretical basis for a deeper understanding of the molecular mechanism of ZosmaRALF2's response to high-salinity seawater in *Erigeron brevis* and for subsequent applications in regulating fucose accumulation in *Erigeron brevis* using this gene. Furthermore, under salt stress conditions, the ZosmaRALF2 protein exhibits high stability and good retention. These results provide crucial experimental evidence for elucidating the molecular mechanism by which ZosmaRALF2 mediates plant salt stress responses.

[0040] (iv) Verification of the effect of exogenous application of peptide ZosmaRALF2 in increasing fucose content in eelgrass 4.1 Growth settings of eelgrass under different seawater treatments Eelgrass seedlings with consistent growth were randomly divided into four groups for hydroponic treatment: (1) normal seawater (salinity 15, prepared using artificial sea salt crystals); (2) high-salinity seawater (salinity 30, adjusted by supplementing normal seawater with sea salt); (3) normal seawater + 3 μM ZosmaRALF2 peptide (peptide directly added to normal seawater); (4) high-salinity seawater + 3 μM ZosmaRALF2 peptide (peptide directly added to high-salinity seawater). The ZosmaRALF2 peptide was derived from the purified product of Example 1. Three biological replicates were set up for each group, and the treatment lasted for 14 consecutive days. After exogenous application of ZosmaRALF2 peptide, eelgrass showed a phenotype of leaf tip wilting. Figure 4 (A)

[0041] The above Figure 4 The results showed that exogenous application of ZosmaRALF2 peptide resulted in leaf tip wilting in *Erigeron angustifolia*, indicating possible localized water stress and metabolic redistribution. Simultaneously, it specifically promoted the accumulation of fucose in *Erigeron angustifolia*, altering its monosaccharide metabolism pattern and shifting the fucose content towards a direction beneficial to improving medicinal quality. This provides important experimental evidence for utilizing ZosmaRALF2 to regulate the content of effective components in *Erigeron angustifolia* and enhance its application value in traditional Chinese medicine resources and marine drug development.

[0042] 4.2 Determination of fucose content in eelgrass under different seawater environments Collect eelgrass samples, dry and pulverize them, and weigh 0.1 g (60 mesh) of the dried sample into a 50 mL centrifuge tube. Add 10 mL of ultrapure water and extract ultrasonically at 50℃ for 60 min, followed by centrifugation at 8000 rpm and 4℃ for 10 min. Collect the supernatant, and repeat the extraction once with 10 mL of ultrapure water on the residue. Combine the two supernatants and dilute to volume in a 100 mL volumetric flask. Take 1 mL of the extract and add 5 mL of Sevage reagent (n-butanol:chloroform = 1:5). v / v The polysaccharide extract was subjected to deproteinization treatment until no flocculent matter was found at the interface. After standing, the supernatant was collected as the polysaccharide extract. 1 mL of the polysaccharide extract was added to 1 mL of 2 mol / L trifluoroacetic acid (TFA), and hydrolyzed at 100℃ for 2 h in a sealed container. The hydrolysate was dried under nitrogen, reconstituted with methanol, and then subjected to nitrogen evaporation again. This process was repeated three times to completely remove TFA. The residue was reconstituted with 1 mL of ultrapure water, diluted 50 times, and filtered through a 0.22 μM nylon membrane for HPAEC-Q-Orbitrap HRMS analysis. Chromatographic and mass spectrometric conditions: Chromatographic separation was performed using a Thermo Scientific CarboPac PA20 analytical column (3.0 mm × 150 mm) and a corresponding guard column (3.0 mm × 30 mm), at a column temperature of 35℃. Isocratic elution was used (A:B = 97.5:2.5). v / v Separation was performed at a flow rate of 0.4 mL / min, with an injection volume of 5 μL and a total analysis time of 20 minutes. Mass spectrometry detection was performed using a HESI ion source in positive ion mode with full scan data acquisition and a resolution of 70,000. Key ion source parameters were as follows: ion source temperature 350℃, capillary temperature 320℃, sheath gas pressure 45 psi, auxiliary gas pressure 10 arb, and spray voltage 3.0 kV.

[0043] The content of monosaccharides was determined by HPLC and analyzed by PCA. Figure 4 (BE), among which, Figure 4 The fucose content of each treatment group in treatment B is shown in Table 2 below: Table 2 Effects of different treatments on fucose content Note: Values ​​are mean ± SD (n=3). Different lowercase letters indicate significant differences between treatments (p<0.05).

[0044] according to Figure 4The results showed that high-salinity seawater induced a core shift in the monosaccharide metabolite profile of *Erigeron annuus* (PC1, 88.8%), transforming it from a state of energy storage dominated by sucrose and fructose to a state of synthesizing large amounts of xylose, rhamnose, mannitol, and other key substances involved in cell wall remodeling and osmotic regulation. Meanwhile, the accumulation pattern of fucose was independent of the aforementioned core shift (mainly contributing to PC2, 6.1%), and it significantly accumulated under both high-salinity seawater and ZosmaRALF2 peptide treatment, exhibiting a synergistic effect when treated together. These results indicate that exogenous application of ZosmaRALF2 peptide can significantly promote fucose accumulation in *Erigeron annuus*, demonstrating its potential application as a tool for regulating the content of this active ingredient.

[0045] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A polypeptide derived from eelgrass, characterized in that, The polypeptide derived from eelgrass has the amino acid sequence shown in SEQ ID NO:

1.

2. The polypeptide derived from eelgrass as described in claim 1, characterized in that, The preparation method of the polypeptide is selected from solid-phase synthesis, liquid-phase synthesis or recombinant expression method; the recombinant expression method includes transferring the coding nucleic acid of the polypeptide shown in SEQ ID NO:1 into an expression vector and expressing it through engineered microorganisms, wherein the sequence of the coding nucleic acid is shown in SEQ ID NO:

2.

3. A composition, characterized in that, The composition includes the polypeptide of claim 1 or 2, and also includes a pharmaceutically necessary carrier.

4. The composition according to claim 3, characterized in that, The carrier is selected from buffers, antioxidants, preservatives, bactericides, or anti-degradation agents.

5. The composition according to claim 3, characterized in that, The composition is in the form of a formulation or an immobilized product, including products in which the polypeptide of claim 1 or 2 is blocked or immobilized using physical adsorbents, carriers, or cross-linking agents.

6. The application of the polypeptide described in claim 1 or 2 in increasing the fucose content in eelgrass.

7. The application of the polypeptide as described in claim 6 to enhance the fucose content in eelgrass, characterized in that, The polypeptide is applied to all stages of Eriocaulon buergerianum cultivation at a concentration of 1-5 μM, and is applied to all parts of the plant.

8. The application of the polypeptide as described in claim 6 to enhance the fucose content in eelgrass, characterized in that, The polypeptide is applied to all stages of eelgrass cultivation, and the eelgrass is cultivated in high-salinity seawater with a salinity not exceeding 30.

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