Salvia miltiorrhiza SmCLE45-3 polypeptide as well as preparation method and application thereof

Abstract

The invention relates to the field of preparation and application of biological peptides, in particular to a salvia miltiorrhiza SmCLE45-3 polypeptide as well as a preparation method and application thereof. The amino acid sequence of the polypeptide is as shown in SEQ ID NO. 1; by utilizing the system regulation function of the plant endogenous signal peptide, the salvia miltiorrhiza terpene and phenylpropane metabolic pathway can be efficiently and synergistically activated, and the salvia miltiorrhiza root biomass and the content and accumulation of tanshinone IIA, cryptotanshinone and salvianolic acid B are synchronously and remarkably improved on the premise of not inhibiting plant growth. The technology has the advantages of being accurate in effect, safe, environmentally friendly, low in dosage, easy and convenient to operate and the like, and is suitable for high-quality and high-yield standardized cultivation of salvia miltiorrhiza.

Description

Technical Field

[0001] This invention relates to the field of preparation and application of bioactive peptides, and particularly to a Danshen SmCLE45-3 polypeptide, its preparation method, and its application. Background Technology

[0002] Danshen, a traditional and widely used medicinal herb in my country, primarily relies on two main categories of active ingredients for its medicinal value: fat-soluble tanshinones (such as tanshinone IIA and cryptotanshinone) and water-soluble salvianolic acids (such as salvianolic acid B). These two types of components exhibit clear pharmacological activity in treating cardiovascular and cerebrovascular diseases and are core indicators for evaluating the quality of Danshen. However, in actual production, the content of these active ingredients in Danshen is significantly affected by various factors such as genetic background, growth environment, cultivation years, and harvesting timing, resulting in considerable fluctuations and poor stability. This has become a key bottleneck restricting the high-quality development of the Danshen industry.

[0003] Currently, strategies for enhancing the active components of Salvia miltiorrhiza mainly include genetic improvement, cultivation optimization, and regulation by exogenous substances. Among these, the application of exogenous plant hormones (such as methyl jasmonate and salicylic acid) or chemical inducers can stimulate secondary metabolism to some extent, but they have problems such as non-specificity of action, narrow concentration window, potential growth inhibition, or residual risks, making it difficult to apply on a large scale in the green and sustainable production of Chinese medicinal materials.

[0004] Tanshinone biosynthesis belongs to the plant terpene metabolic pathway, originating from the mevalonate (MVA) and methyl erythritol phosphate (MEP) pathways. It is synthesized via geranyl-geranyl pyrophosphate (GGPP) and forms the tanshinone skeleton under the catalysis of key enzymes such as copacpite pyrophosphate synthase (CPS) and kauriene synthase (KSL). Tanshinone, on the other hand, belongs to the phenylpropane metabolic pathway, and its synthesis is regulated by key enzymes such as phenylalanine ammonia-lyase (PAL) and 4-coumaroyl-CoA ligase (4CL). Both pathways are precisely regulated by complex transcriptional networks and signaling pathways.

[0005] In recent years, research on endogenous small peptide signaling molecules in plants has provided new insights into the precise regulation of crop traits. CLE (CLAVATA3 / EMBRYO SURROUNDING REGION-RELATED) family peptides are important intercellular signaling molecules involved in regulating various biological processes such as plant stem cell maintenance, organ development, nutrient allocation, and stress responses. Previous studies have shown that certain CLE peptides can act as long-range signals to systematically regulate root-shoot structure, nitrogen and phosphorus uptake, and secondary metabolic flux. Chinese patent CN121293312A discloses a mature SmCLE peptide and its application in plant stress resistance. The technical solution includes a mature SmCLE peptide, the amino acid sequence of which is SEQ ID NO:1. This invention addresses the problem that existing CLE peptides cannot be applied to stress resistance in plants, especially medicinal plants or non-model crops. It possesses high biocompatibility and targeting capabilities, and can significantly improve crop biomass and yield under stress conditions. Chinese patent CN121227797A discloses the application of a mature SmCLE peptide in inhibiting plant senescence. The amino acid sequence of the mature SmCLE peptide is SEQ ID NO:1. This invention addresses the problem that existing CLE peptides cannot be used in anti-senescence applications in plants, especially medicinal plants or non-model crops. It features small molecular weight, easy synthesis, low concentration, good biocompatibility, and the ability to precisely regulate leaf senescence through exogenous application, avoiding the safety issues associated with genetically modified organisms. However, whether the SmCLE peptide has a positive effect on enhancing plant bioactive components has not been reported; whether the application of SmCLE peptide can promote the biosynthesis of tanshinone / tanshinone acid compounds also requires further investigation. Summary of the Invention

[0006] The technical problem this invention aims to solve is the current pain point in the cultivation of Salvia miltiorrhiza, namely, the low and unstable content of active ingredients and the lack of efficient, green, and specialized regulatory products. Current research on SmCLE peptides mainly focuses on improving plant stress resistance and inhibiting plant senescence; however, there are no reports on whether SmCLE peptides have a positive effect on enhancing plant active ingredients. Furthermore, whether applying SmCLE peptides can promote the biosynthesis of tanshinone / tanshinone acid compounds also requires further investigation.

[0007] To address the aforementioned problems, this invention provides a biological agent with the novel signal peptide SmCLE45-3 derived from Tanshinone as its core active ingredient, along with its corresponding application method. This agent effectively promotes the healthy growth of Tanshinone plants and specifically and significantly increases the content and total accumulation of tanshinone IIA, cryptotanshinone, and salvianolic acid B in their roots. Furthermore, it provides a standardized and quantifiable application method for this agent. By optimizing the spraying concentration, timing, frequency, and auxiliary management measures, a set of agronomic schemes for improving the quality and yield of Tanshinone suitable for different production models is established, achieving a synergistic leap in both the "yield" and "quality" of the medicinal material.

[0008] To achieve the above objectives, the present invention is implemented through the following technical means: a Danshen SmCLE45-3 polypeptide, the amino acid sequence of which is shown in SEQ ID NO.1.

[0009] SEQ ID NO.1: RRVPKGSDPIHN.

[0010] SmCLE45-3 is a novel gene identified and cloned from the transcriptome of *Salvia miltiorrhiza* by our team. It encodes a 12-amino acid secretory peptide, and its precursor protein contains a typical N-terminal signal peptide and a conserved CLE domain. Preliminary bioinformatics analysis and tissue expression profiling showed that the SmCLE45-3 gene was highly expressed in *Salvia miltiorrhiza* roots, suggesting its potential involvement in root development or rhizosphere signaling. However, there are currently no publicly available reports, either domestically or internationally, on its development into a biopharmaceutical for exogenous application to target and enhance specific active components of medicinal plants.

[0011] Based on the core role of CLE peptides in plant systemic signal transduction and the specific expression pattern of SmCLE45-3 in Salvia miltiorrhiza roots, we hypothesize that exogenous application of SmCLE45-3 peptides may act as a highly efficient "biological trigger," systematically coordinating the growth-defense resource allocation of Salvia miltiorrhiza by mimicking or enhancing endogenous signals, thereby simultaneously promoting the accumulation of root biomass and the biosynthesis of tanshinone / tanshinone acid derivatives. Therefore, developing biological agents with SmCLE45-3 as the core active ingredient has significant theoretical innovation value and broad industrial application prospects, and is expected to provide a novel technological solution for the high-quality, stable, and efficient production of Salvia miltiorrhiza.

[0012] A method for preparing the above-mentioned Tanshinone SmCLE45-3 polypeptide, wherein it is synthesized artificially. A solid-phase synthesis method, such as the Fmoc method, is preferred.

[0013] The above-mentioned SmCLE45-3 polypeptide from Danshen is used in the preparation of formulations that enhance the content of active ingredients in Danshen.

[0014] Furthermore, the active ingredients of tanshinone are tanshinone IIA, cryptotanshinone, and salvianolic acid B.

[0015] Furthermore, the effective working concentration of the SmCLE45-3 peptide in the formulation is 0.5 nM to 500 nM. Preferably, it is 5 nM to 100 nM; the most preferred concentration is 25 nM.

[0016] Furthermore, the solvent for the formulation is sterile deionized water, or a buffer system containing the following auxiliary components:

[0017] Ionic strength modifier: 0.1-1.0 mM calcium chloride (CaCl2), which helps maintain peptide conformational stability and participates in signal transduction; Wetting and penetrating agents: nonionic surfactants, such as Tween-20, at a volume ratio of 0.005%-0.02% (v / v) to promote the spreading and absorption of the pesticide solution on the leaf surface; Buffer system: 2-10 mM MES or phosphate buffer, adjust pH to 5.5-6.5 to maintain peptide stability during administration.

[0018] A method for preparing the above-mentioned formulation includes the following steps: (1) The SmCLE45-3 peptide shown in SEQ ID NO: 1 was synthesized by solid-phase synthesis. (2) The purified peptide fragments were freeze-dried into powder and stored at -20℃ or -80℃. (3) When using, take an appropriate amount of freeze-dried powder, dissolve it in a solvent, and prepare a high-concentration stock solution; (4) Before application, dilute the stock solution with a solvent to the required working concentration, prepare and use immediately, or store at 4°C for a short period of time (not exceeding 72 hours).

[0019] A method for enhancing the content of active ingredients in Salvia miltiorrhiza using the above-mentioned formulation involves spraying the formulation solution evenly onto the leaves of the plant during the vegetative growth stage and the period of effective ingredient accumulation in the roots. This method precisely regulates the growth, development, and secondary metabolic network of Salvia miltiorrhiza, achieving a synergistic improvement in the yield and quality of the medicinal material. It is suitable for standardized field planting and facility cultivation of Salvia miltiorrhiza.

[0020] Furthermore, the specific steps include: (1) First stage: Spraying is carried out 30-50 days after transplanting when the plants have 4-6 true leaves; the purpose of spraying at this stage is to systematically activate the overall metabolic potential of the plants and lay the foundation for the efficient synthesis of subsequent secondary metabolites.

[0021] (2) Second stage: Spraying is carried out 80-110 days after transplanting the Salvia miltiorrhiza plants, at the early stage of budding; the plants are in the transition period from vigorous vegetative growth to reproductive growth (early stage of budding). This stage is the key period for the rapid accumulation of dry matter and secondary metabolites (tanshinone, salvianolic acid) in the roots of Salvia miltiorrhiza.

[0022] After all spraying treatments are completed, Salvia miltiorrhiza can continue to grow normally for 50-70 days. Compared with traditional cultivation, Salvia miltiorrhiza grown using this method can achieve better yields and significantly improved quality within a cycle that is not extended or is even slightly shortened, thus optimizing the harvest period.

[0023] Furthermore, within each spraying stage, spray once every 10-15 days, and spray twice consecutively for each stage.

[0024] Furthermore, the first stage involves spraying once on the 40th and 55th days after transplanting; the second stage involves spraying once on the 90th and 105th days after transplanting.

[0025] Furthermore, when spraying, use conventional spraying equipment and spray the solution evenly and meticulously on both sides of the leaves of the Salvia miltiorrhiza plant in the afternoon or evening on a sunny day, until the leaf surface is completely wet but no droplets roll off.

[0026] Furthermore, during and before spraying, maintain the relative soil moisture content at 65%-75%; create moderate and mild moisture conditions that ensure normal growth while slightly activating the plant's secondary metabolic defense response. Implement balanced fertilization, paying attention to the supply of phosphorus, potassium, and micronutrients such as calcium, magnesium, zinc, and boron. Appropriately control nitrogen fertilizer, especially excessive application of ammonium nitrogen, to avoid excessive vegetative growth that dilutes secondary metabolites.

[0027] Furthermore, in facility cultivation, day / night temperatures are controlled at 22-28℃ / 16-20℃, and light intensity is maintained at 300-500 µmol / m² / s to optimize the signal performance of SmCLE45-3.

[0028] A type of Salvia miltiorrhiza medicinal material cultivated using the above method has significantly higher contents of tanshinone IIA, cryptotanshinone, and salvianolic acid B in its roots than conventionally cultivated Salvia miltiorrhiza that did not use this preparation.

[0029] The beneficial effects of this invention are as follows: (1) SmCLE45-3 is an endogenous CLE family signal peptide of Tanshinone. In this invention, it is applied as an exogenous inducer, which can be specifically recognized by the plant. By binding to membrane receptor kinases, it initiates downstream specific signal cascade reactions, precisely and efficiently regulating the gene network related to root development and tanshinone / tanshinone synthesis, thus achieving "signal-guided" metabolic engineering. This preparation can not only significantly increase the fresh weight and dry weight of the root system (increase yield) by optimizing root architecture (increasing the number and length of lateral roots) and promoting the distribution of photosynthetic products to the roots, but also systematically guide carbon flow and reducing power to the tanshinone and tanshinone synthesis pathways, thereby significantly increasing the percentage content of the target active ingredients and the total yield per plant (improving quality), solving the problem of component dilution that may be caused by general growth-promoting measures.

[0030] (2) SmCLE45-3 is composed of natural amino acids and is easily degraded by proteases in plants and the environment, leaving no residues and being non-toxic, making it safe for non-target organisms. Its action is based on the plant's own signaling system and will not cause the growth malformations or phytotoxicity commonly seen with chemical hormones, fully meeting the strict requirements for inputs in organic agriculture and GAP production of Chinese medicinal herbs. The active ingredients can exert significant effects at nanomolar (nM) concentrations, resulting in low cost per unit area. The chemically synthesized peptides are stable and easy to prepare. The foliar spraying method is easy to integrate into existing agricultural operations, has high farmer acceptance, and faces little resistance to promotion. Activated secondary metabolic pathways are usually accompanied by an increase in the synthesis of phytoalexins, lignin, and other disease-resistant and stress-resistant substances. Therefore, the application of SmCLE45-3 may indirectly enhance the tolerance of Salvia miltiorrhiza plants to drought, diseases, and other stresses while improving quality, thus improving the stability of field production. Attached Figure Description

[0031] Figure 1 Comparison of Salvia miltiorrhiza roots after harvest: 25 nM and 100 nM MSmCLE45-3 preparations have a significant growth-promoting effect on Salvia miltiorrhiza roots.

[0032] Figure 2 The relative expression levels of the SmCLE45-3 gene in different tissues of Salvia miltiorrhiza.

[0033] Figure 3 Effects of different concentrations of SmCLE45-3 on the root biomass of Salvia miltiorrhiza. * indicates significant difference; ** indicates extremely significant difference.

[0034] Figure 4 Content of tanshinone IIA, cryptotanshinone, and salvianolic acid B in Salvia miltiorrhiza roots under different treatments. * indicates significant difference; ** indicates extremely significant difference.

[0035] Figure 5Comparison of the effects of SmCLE45-3 treatment and methyl jasmonate (MeJA) treatment. * indicates significant difference; ** indicates extremely significant difference.

[0036] Figure 6 qRT-PCR analysis of the effect of SmCLE45-3 treatment on the expression of key genes in the secondary metabolism of Salvia miltiorrhiza. * indicates significant difference; ** indicates extremely significant difference. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0038] Furthermore, unless otherwise specified, all methods used in the embodiments of this invention are common processing methods; and all materials used, unless otherwise specified, are obtained by purchasing from the market.

[0039] Example 1: Synthesis of SmCLE45-3 polypeptide and preparation of formulation stock solution.

[0040] The SmCLE45-3 peptide with the sequence RRRVPKGSDPIHN (SEQ ID NO: 1) was synthesized using a standard Fmoc solid-phase synthesis strategy. After synthesis, the peptide was purified by preparative reversed-phase high-performance liquid chromatography (RP-HPLC, C18 column), the main peak was collected, and the sample was lyophilized. A portion of the sample was analyzed by mass spectrometry (MALDI-TOF MS), and the chromatographic purity was ≥96%.

[0041] Preparation of stock solution: Accurately weigh 1.38 mg of the above-mentioned lyophilized peptide powder, dissolve it in 10.0 mL of sterile, enzyme-free ultrapure water, and vortex thoroughly to obtain a 100 µM SmCLE45-3 stock solution. Aliquot 100 µL into sterile centrifuge tubes and store at -20°C for later use.

[0042] Preparation of working solution (using 25 nM as an example): Take 25 µL of the 100 µM stock solution and add it to 99.975 mL of the "spraying solvent," gently inverting to mix. The "spraying solvent" is a 5 mM MES buffer solution (pH 6.0) containing 0.5 mM CaCl2 and 0.01% (v / v) Tween-20. It is recommended to use the working solution within 4 hours of preparation.

[0043] Example 2: Pot experiment to verify the optimal concentration window of SmCLE45-3.

[0044] Materials and Methods: Uniformly grown tissue culture seedlings of "Ji Danshen No. 1" were selected and transplanted into plastic pots (18 cm in diameter) containing a uniform substrate. These pots were then placed in an intelligent artificial climate chamber (25 / 18℃, 14h light, 60% humidity). After 30 days of acclimatization, plants with uniform growth were selected for the experiment.

[0045] Set up 5 processes: CK: Apply an equal amount of "spraying solvent".

[0046] T1: Spray with 5 nM SmCLE45-3 working solution.

[0047] T2: Spray with 25 nM SmCLE45-3 working solution.

[0048] T3: Spray with 100 nM SmCLE45-3 working solution.

[0049] T4: Spray with 500 nM SmCLE45-3 working solution.

[0050] Each treatment consisted of 15 pots, arranged completely randomly. Foliar spraying was applied on days 35 and 50 after transplanting (two stages, one application each time), with each application ensuring the leaves were moistened. Harvesting took place 45 days after the last application.

[0051] Measurement indicators: Growth indicators: Carefully dig up the complete root system, wash and dry it, and weigh the fresh weight of the root. Dry the root system at 65℃ until it reaches a constant weight, and weigh the dry weight of the root.

[0052] Content of active ingredients: The contents of tanshinone IIA, cryptotanshinone, and salvianolic acid B were simultaneously determined by HPLC using dried root powder, following the methods described in the Chinese Pharmacopoeia (2020 edition). Chromatographic conditions: Agilent Eclipse Plus C18 column; mobile phase: acetonitrile-0.1% formic acid-water gradient elution; detection wavelengths: 270 nm (tanshinone) and 286 nm (salvianolic acid B).

[0053] Results statistics:

[0054] Conclusions are as follows Figure 1 As shown, the 25 nM and 100 nM MSmCLE45-3 preparations have significant growth-promoting effects on Salvia miltiorrhiza roots; for example... Figure 3 As shown in the bar chart, the average root dry weight of *Salvia miltiorrhiza* treated with different concentrations (5 nM, 25 nM, 100 nM, 500 nM) of SmCLE45-3 is displayed. The results show that the root dry weight of the 25 nM treatment group (T2) reached its peak value, significantly higher than that of the CK and other treatment groups (P<0.05). Figure 4As shown, different concentrations of treatment on the accumulation of the main active components of *Salvia miltiorrhiza* exhibited a clear dose-response effect. The content trends of tanshinone IIA, cryptotanshinone, and salvianolic acid B were basically consistent, all reaching peak values ​​in the T2 treatment group (25 nM), at 3.05 mg / g, 1.38 mg / g, and 49.8 mg / g, respectively, significantly higher than the CK control group. When the treatment concentration was further increased to T3 (100 nM) and T4 (500 nM), the contents of all three components decreased. Although the content in the T4 group was higher than that in the CK group, it was significantly lower than the peak level. The results indicate that an appropriate concentration (25 nM in this experiment) can most effectively promote the synthesis and accumulation of active components of *Salvia miltiorrhiza*, while excessively high concentrations weaken the promoting effect.

[0055] As shown in the table above, the 25 nM SmCLE45-3 treatment was the most effective in promoting root growth and increasing the content of the three target active ingredients, significantly outperforming other concentration treatments and establishing its optimal concentration position.

[0056] Example 3: The expression profile of the SmCLE45-3 gene in different tissues of Salvia miltiorrhiza was analyzed using real-time quantitative PCR.

[0057] First, root, stem, leaf, and flower tissue samples were collected from healthy *Salvia miltiorrhiza* plants, flash-frozen in liquid nitrogen, and stored at -80°C for RNA extraction. Then, total RNA was extracted from each tissue using a plant total RNA extraction kit, and its concentration and purity were determined using Nanodrop. RNA integrity was verified using agarose gel electrophoresis. Next, equal volumes of high-quality total RNA were used to synthesize first-strand cDNA using a reverse transcription kit and Oligo(dT) primers. Based on this, specific primers were designed according to the SmCLE45-3 gene sequence, with the *Salvia miltiorrhiza* Actin gene selected as an internal control. The specificity and amplification efficiency of the primers were verified. A qRT-PCR reaction system was prepared using SYBR Green Premix Pro Taq HS premixed solution, with three technical replicates for each sample and a template-free negative control. A standard procedure including pre-denaturation, amplification cycles, and melting curve analysis was run on a real-time quantitative PCR instrument. Finally, based on the obtained Ct values, root tissue was used as the calibration sample, and 2... -ΔΔCt The relative expression level of the gene in each tissue was calculated using the relative quantification method.

[0058] The results are as follows Figure 2As shown, qRT-PCR analysis revealed that the expression of the SmCLE45-3 gene exhibited significant tissue specificity, with the highest expression level in roots (set as the baseline of 1), followed by stems with approximately half the expression level of roots (0.53), while expression further decreased to 0.31 in leaves and was lowest in flowers at only 0.21. These results indicate that this gene primarily functions in the roots.

[0059] Example 4: The effects of different treatments on the growth and accumulation of active ingredients of Salvia miltiorrhiza were evaluated through exogenous treatment experiments.

[0060] The experiment used *Salvia miltiorrhiza* seedlings of uniform growth stage, cultivated under greenhouse hydroponic or soil-based conditions. Three treatment groups were set up: a control group (no treatment), a 25 nM SmCLE45-3 treatment group, and a conventional methyl jasmonate (MeJA) treatment group, with three biological replicates in each group. Treatments were applied via foliar spraying, periodically during the mid-to-late stages of *Salvia miltiorrhiza* growth. After treatment, intact plants were harvested, roots were separated, washed, and dried in an oven to constant weight. Root dry weight data were accurately obtained. Subsequently, the dried roots were pulverized and sieved, and the contents of the main active ingredients, tanshinone IIA and salvianolic acid B, were determined using high-performance liquid chromatography (HPLC). All data were statistically analyzed to compare the significant differences among groups in root dry weight, component content, and calculated yield per plant, in order to evaluate the actual effects of different treatments.

[0061] The results are as follows Figure 5 As shown, among different treatments, the SmCLE45-3 treatment had the most significant promoting effect on the growth and accumulation of active ingredients in *Salvia miltiorrhiza*. Compared with the control group, it significantly increased root dry weight (18.5 g vs 15.2 g) and simultaneously increased the contents of tanshinone IIA and salvianolic acid B to 3.20 mg / g and 46.5 mg / g, respectively, thereby achieving a highly significant yield per plant (59.2 mg / plant and 860.3 mg / plant, respectively). In contrast, while methyl jasmonate treatment also significantly increased the contents of the two active ingredients and the yield per plant, it had no significant effect on root dry weight. Therefore, under the same cultivation conditions, the 25 nM SmCLE45-3 treatment of this invention is significantly superior to the conventionally used MeJA treatment in terms of comprehensive indicators such as increasing root dry weight, tanshinone IIA content, and salvianolic acid B content. Moreover, the plants in the MeJA treatment group showed a slight growth inhibition phenotype, while the plants in the SmCLE45-3 treatment group grew vigorously. This indicates that SmCLE45-3 is a more effective strategy that can synergistically promote the synthesis of biomass and active ingredients in Salvia miltiorrhiza.

[0062] Example 5: Verification of the synergistic effect of the "two-stage four-application method" under field conditions.

[0063] Field trials were conducted at the Danshen GAP base in Linyi, Shandong. The tested variety was one-year-old seedlings of "Lu Danshen No. 2," which were transplanted in spring.

[0064] Experimental design: Set up 3 treatments: CK: Routine management, spray with clean water.

[0065] T_M: Conventional exogenous inducer control, sprayed with 100 µM methyl jasmonate (MeJA) at the same time as T_S.

[0066] T_S: The treatment of this invention involves spraying 25 nM SmCLE45-3 formulation using a "two-stage, four-application method": the first stage is spraying on the 45th and 60th days after transplanting; the second stage is spraying on the 95th and 110th days after transplanting.

[0067] Each treatment consists of 4 duplicate cells arranged in a randomized block layout.

[0068] Field management: Unified fertilization, weeding, and green pest and disease control. During the critical spraying period, control the soil moisture content to around 70%.

[0069] Sample collection and analysis: Harvest approximately 165 days after transplanting (55 days after the last spray). Ten plants were randomly sampled from each plot, and the fresh / dry weight of the roots was determined. The root samples were then dried, pulverized, and subjected to HPLC component analysis.

[0070] Results Display:

[0071] Conclusion: The SmCLE45-3 formulation and its matching spraying method provided by this invention can achieve a better overall effect than conventional chemical inducers (MeJA) under field conditions. While significantly increasing yield, it also greatly improves the content of core active ingredients and the yield per plant, and the plants are in better growth condition.

[0072] Example 6: Preliminary exploration of mechanism of action - key gene expression analysis.

[0073] To elucidate the mechanism of action of the SmCLE45-3 peptide, this study conducted a systematic temporal analysis of gene expression in a controlled hydroponic system. Using four-leaf stage *Salvia miltiorrhiza* seedlings as material, the experimental group was foliar-sprayed with 25 nM SmCLE45-3, while the control group was sprayed with an equal amount of solvent. Leaf and root tip samples were collected at 6, 24, and 48 hours after treatment and preserved after flash freezing in liquid nitrogen. Subsequently, total RNA was extracted from each sample and reverse transcribed into cDNA. Real-time quantitative PCR (qRT-PCR) was used to dynamically detect the expression changes of key genes in terpene backbone synthesis (SmDXR, SmCPS1, SmKSL1) and key genes in the phenylpropanone metabolic pathway (SmPAL, Sm4CL, SmRAS), thereby elucidating the regulatory pattern and temporal characteristics of this peptide on the secondary metabolic pathway of *Salvia miltiorrhiza* at the transcriptional level.

[0074] The results are as follows Figure 6 As shown, the relative expression levels of a series of key genes in *Salvia miltiorrhiza* (SmDXR, SmPAL, Sm4CL, and SmRAS) changed at 6h, 24h, and 72h after spraying with 25 nM SmCLE45-3. qRT-PCR analysis revealed a significant and temporally induced pattern in the expression of key genes in *Salvia miltiorrhiza* secondary metabolism after spraying with 25 nM SmCLE45-3. The expression levels of all genes were upregulated after treatment, but the kinetic characteristics differed: the expression of SmPAL and SmRAS peaked at 24h (3.77-fold and 3.42-fold higher than the control, respectively), and then declined at 72h; while the expression of Sm4CL continued to rise, reaching its highest level (2.87-fold) at 72h. The expression of SmDXR, involved in the terpene pathway, changed relatively gradually, also peaking at 24h (2.78-fold). Overall, this treatment can rapidly and strongly activate core genes in the biosynthetic pathways of tanshinone and tanshinone compounds, with their expression peaks occurring mostly within 24 hours. This provides a direct transcriptional explanation for the accumulation of downstream active ingredients.

[0075] In conclusion, the biological agent provided by this invention, with 25 nM concentration SmCLE45-3 polypeptide as the core active ingredient, combined with the "two-stage four-application method," can serve as a highly efficient, safe, and specific bioregulatory tool. Without altering conventional agricultural operations, it synergistically and significantly increases the yield of Salvia miltiorrhiza and the content and accumulation of its core fat-soluble and water-soluble active ingredients, providing an innovative technical path for the standardized and high-quality production of Salvia miltiorrhiza.

[0076] Finally, it should be noted that although the above embodiments describe specific implementations of the present invention, they are not intended to limit the invention. Those skilled in the art should understand that these are merely illustrative examples, and the scope of protection of the present invention is defined by the appended claims. All modifications or equivalent substitutions should be included within the scope of protection of the present invention.

Claims

1. A tanshinone SmCLE45-3 polypeptide, characterized in that: The amino acid sequence is shown in SEQ ID NO.

1.

2. The method for preparing the Tanshinone SmCLE45-3 polypeptide according to claim 1, characterized in that: It is artificially synthesized.

3. The application of the tanshinone SmCLE45-3 polypeptide of claim 1 in the preparation of formulations that enhance the content of active ingredients in tanshinone.

4. The application as described in claim 3, characterized in that: The active ingredients of tanshinone are tanshinone IIA, cryptotanshinone, and salvianolic acid B.

5. The application as described in claim 3, characterized in that: The effective working concentration of the tanshinone SmCLE45-3 polypeptide in the formulation is 0.5 nM ~ 500 nM.

6. The application as described in claim 3, characterized in that: The solvent for the formulation is sterile deionized water, or a buffer system containing the following auxiliary components: ionic strength regulator: 0.1-1.0 mM calcium chloride; wetting and penetrating agent: 0.005%-0.02% by volume of nonionic surfactant; buffer system: 2-10 mM MES or phosphate buffer, adjusting the pH to 5.5-6.

5.

7. The method for preparing the formulation according to claim 3, characterized in that... Includes the following steps: (1) The SmCLE45-3 peptide of tanshinone shown in SEQ ID NO: 1 was synthesized by solid-phase synthesis. (2) The purified peptide fragments were freeze-dried into powder and stored at -20℃ or -80℃. (3) When using, take an appropriate amount of freeze-dried powder, dissolve it in a solvent, and prepare a high-concentration stock solution; (4) Before application, dilute the stock solution with a solvent to the required working concentration, prepare and use immediately, or store at 4°C for a short period of time.

8. A method for increasing the content of active ingredients in Danshen using the formulation of claim 3, characterized in that: The preparation solution is sprayed evenly onto the leaves of the plant during the vegetative growth period and the period of accumulation of effective ingredients in the roots of Salvia miltiorrhiza by spraying.

9. The method as described in claim 8, characterized in that... Includes the following steps: (1) First stage: Spray the plants 30-50 days after transplanting and when they have 4-6 true leaves; (2) Second stage: Spraying is carried out 80-110 days after the transplanting of Salvia miltiorrhiza plants, at the early stage of budding.

10. A type of Salvia miltiorrhiza medicinal material obtained by cultivation using the method described in claim 8 or 9.

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