Application of triterpene saponins in promoting synthesis of plant scopoletin

Abstract

This invention discloses the application of triterpenoid saponins in promoting hyoscyamine synthesis in plants, belonging to the field of biotechnology. Through screening and verification, this invention has determined that small molecule compounds with the structural characteristics of triterpenoid saponins can significantly regulate hyoscyamine synthesis in plants. By applying these triterpenoid saponin compounds to plants, they can significantly promote the synthesis and accumulation of hyoscyamine, and further reveal that these small molecule compounds competitively bind with the negative regulator WRKY70. F6'H1 Gene promoter segments promote F6'H1 The mechanism of action of gene expression and hyoscyamine synthesis. Therefore, this invention can provide a new strategy for the regulation and application of plant secondary metabolites, and also provide new ideas for the research on the regulation of hyoscyamine synthesis, with good application prospects.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to the application of triterpenoid saponins in promoting the synthesis of hyoscyamine in plants. Background Technology

[0002] Scopolettein is a phenolic coumarin and an important member of the phytoalexin group isolated from many plants. It is a natural compound with broad pharmacological activities, and its medicinal value has attracted attention in the treatment of various diseases. In traditional applications, scopolettein and its plant-derived forms have been used to relieve symptoms of convulsions, inflammation, rheumatic pain, and leprosy. Modern research shows that this compound exhibits antihypertensive effects in the cardiovascular system, while also possessing non-specific antispasmodic (neuromuscular blockade) activity at the neuromuscular level and participating in anticonvulsant processes. Furthermore, scopolettein can enhance the activation of eosinophils, which play a key role in the pathogenesis of asthma, suggesting its potential regulatory potential for asthma. At a broader pharmacological level, scopolettein also exhibits various biological activities, including hepatoprotection, antioxidation, anti-inflammation, and anticancer effects, providing a scientific basis for its development as a multi-target medicinal resource.

[0003] Hyoscyamine is one of the main polyphenols in tobacco leaves. This type of substance has a significant impact on the color of tobacco leaves, the aroma of cigarettes, and the smoke itself, and is one of the important indicators for evaluating tobacco quality. Therefore, research on the regulation of hyoscyamine synthesis has important medicinal and application value.

[0004] The biosynthesis of hyoscyamine originates from the phenylpropane metabolic pathway. Its direct precursor, phenylalanine, is generated via the shikimic acid pathway, which uses phosphoenolpyruvate from glycolysis and erythrose-4-phosphate from the pentose phosphate pathway. Phenylalanine is converted to 4-coumaroyl-CoA via a cascade of phenylalanine ammonia-lyase (PAL), cinnamate hydroxylase (C4H), and 4-coumaroyl-CoA ligase (4CL). Subsequently, under the action of shikimic acid O-hydroxycinnamoyltransferase (HCT) and coumarate-3-hydroxylase (C3H), coumaroyl-shikimic acid and caffeoyl-shikimic acid are generated sequentially. The latter is then catalyzed by HCT to release caffeoyl-CoA, which is then methylated to feruloyl-CoA by caffeoyl-CoA O-methyltransferase (CCoAOMT). Finally, feruloyl-CoA is methylated in feruloyl-CoA-6- 6-hydroxyferuloyl-CoA is generated under the action of hydroxylase 1 (F6'H1), and then hyoscyamine is synthesized through cis-trans isomerization and intramolecular lactone formation. The activity of F6'H1 protein is crucial for the synthesis and accumulation of hyoscyamine in plants, and the content of hyoscyamine can be regulated by controlling its activity.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide the application of triterpenoid saponin compounds in promoting the synthesis of hyoscyamine in plants. These compounds can promote the synthesis of hyoscyamine by regulating the expression of the key gene F6'H1 in hyoscyamine synthesis.

[0007] This invention is implemented as follows: In a first aspect, the present invention provides the application of triterpenoid saponins in the preparation of products that promote the synthesis of hyoscyamine in plants.

[0008] To further investigate the effective regulation of hyoscyamine synthesis in tobacco, this invention utilizes AlphaFold to predict key genes involved in hyoscyamine synthesis. F6 H1 The conserved structural domains of the promoter were modeled using AI deep learning technology. Candidate lead compounds were screened using a small molecule compound library and a highly automated drug screening platform, and their biological activity was validated. Experimental results show that triterpenoid saponins can regulate key genes involved in hyoscyamine synthesis. F6'H1 It promotes the synthesis of hyoscyamine through expression.

[0009] In some embodiments, triterpenoid saponins include Fulvotomentoside A.

[0010] Fulvotomentoside A (Flavorotomentoside A) is derived from the plant *Lonicera japonica* (Flavorotomentoside A) of the Caprifoliaceae family. Lonicera fulvotomentosa Fulvotomentoside A is a triterpenoid saponin compound isolated from flower buds, also known as Decaisoside E. Current research indicates that Fulvotomentoside A has anti-inflammatory and immunomodulatory effects, but there is currently no publicly available information regarding its gene regulation.

[0011] In some embodiments, the aforementioned plants include, but are not limited to, tobacco. Since the core mechanisms by which plants regulate the synthesis of secondary metabolites are conserved across different species, theoretically, other plants that synthesize hyoscyamine through the same metabolic pathway can also promote hyoscyamine synthesis through the aforementioned triterpenoid saponins.

[0012] Furthermore, this invention verifies that the above-mentioned triterpenoid saponins can regulate... F6'H1 Gene expression further promotes the synthesis of hyoscyamine in plants.

[0013] In a second aspect, the present invention provides a method for promoting the synthesis of hyoscyamine in plants, comprising: applying a triterpenoid saponin compound to the plant; wherein the triterpenoid saponin compound includes Fulvotomentoside A.

[0014] In some embodiments, the application of terpene saponin compounds is selected from at least one of basal application, drip irrigation, spraying, or hydroponics. Due to limitations in experimental time and conditions, this invention cannot exhaust all application methods. During the verification process, the effects were verified using representative application methods (spraying and hydroponics). However, this does not mean that the above-mentioned applications of this invention can only be achieved through spraying and hydroponics. Those skilled in the art can treat plants based on the research of this invention and conventional techniques in the field. Therefore, any method that promotes the synthesis of hyoscyamine in plants using the treatment ideas of this invention falls within the protection scope of this invention.

[0015] In some embodiments, the triterpenoid saponins are applied via hydroponics; wherein the concentration of the triterpenoid saponins in the hydroponic solution is ≥5 μM, and the hydroponic time is ≥5 days. In the hydroponic experiment, the inventors found that when the concentration of the triterpenoid saponins is below 5 μM, its effect on hyoscyamine content is not significant; however, when the concentration of the triterpenoid saponins is ≥5 μM and the treatment time is ≥5 days, it can significantly increase hyoscyamine content.

[0016] In some embodiments, the triterpenoid saponins are applied via foliar spraying; the concentration of the triterpenoid saponins in the foliar spray is ≥0.01 μM. In the foliar spraying experiment, when the concentration of the triterpenoid saponins is ≥0.01 μM, the hyoscyamine content can be increased, but the increase is small, lower than that of the 5 μM hydroponic treatment. Therefore, different treatment methods may have different effects on the hyoscyamine content, and those skilled in the art can select and set the treatment method according to the actual situation.

[0017] Thirdly, the present invention provides a product for promoting the synthesis of hyoscyamine in plants, comprising a triterpenoid saponin compound and an agriculturally acceptable carrier or adjuvant; the triterpenoid saponin compound includes Fulvotomentoside A.

[0018] In this invention, agriculturally acceptable carriers or adjuvants refer to auxiliary substances that do not possess biological activity themselves but are added to formulations to improve their physical or chemical properties, enhance application effects, or facilitate processing and use, including but not limited to solid carriers / fillers, liquid carriers / solvents, surfactants, and functional enhancement adjuvants.

[0019] The solid carrier / filler can be mineral materials such as kaolin, clay, diatomaceous earth, talc, silica, bentonite, and attapulgite; or plant materials such as corn cob powder and peanut shell powder. The liquid carrier / solvent can be xylene, toluene, diesel oil, petroleum ether, etc. The surfactant can be a wetting agent (such as alkylbenzene sulfonate, alkylnaphthalene sulfonate, sodium dodecyl sulfate, soapberry powder, etc.), an emulsifier (such as calcium alkylbenzene sulfonate, polyoxyethylene nonionic surfactants, etc.), or a dispersant (such as lignin sulfonate, alkylaryl sulfonate formaldehyde condensate, etc.). The functional enhancing agent can be an adhesive (such as mineral oil, gelatin, polyvinyl alcohol, starch paste, etc.), a stabilizer (such as anti-photolysis agent, antifreeze agent, preservative, etc.), or a synergist or defoamer, etc. Those skilled in the art can select and combine these agents according to the required dosage form, and this invention does not limit this.

[0020] The present invention has the following beneficial effects: This invention identifies small molecule compounds with the structural characteristics of triterpenoid saponins, particularly Fulvotomentoside A, through screening and verification, which can significantly regulate hyoscyamine synthesis in plants. Applying these triterpenoid saponin compounds to plants significantly promotes the synthesis and accumulation of hyoscyamine, and further reveals that these small molecule compounds competitively bind to the negative regulator NtWRKY70. F6'H1 Gene promoter segments promote F6'H1 The mechanism of action of gene expression and hyoscyamine synthesis. Therefore, this invention can provide a new strategy for the regulation and application of plant secondary metabolites, and also provide new ideas for the research on the regulation of hyoscyamine synthesis, with good application prospects. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 Key genes for the synthesis of hyoscyamine NtF6'H1 Promoter fragment modeling and compound library screening; A is NtF6'H1 DNA modeling of key gene fragments; B is a set of candidate small molecule compounds obtained based on two algorithms; Figure 2 This is a schematic diagram of the co-injection of small molecule compounds with the LUC fluorescence system; Figure 3The effect of small molecule compounds on the fluorescence signals of pGreenII 0800-LUC-Wbox and pGreenII 62SK-NtWRKY70; Figure 4 Fulvotomentoside A promotes tobacco NtF6'H1 Gene expression verification experiment; A is the result of identifying positive plants by PCR amplification vector specific fragment; B is the expression level of LUC gene in 5 positive lines; C is the LUC signal after Fulvotomentoside A treatment of positive lines; D is the quantitative detection of LUC signal after Fulvotomentoside A treatment of positive lines. Figure 5 Experiment A is a validation experiment to show the inhibitory activity of Fulvotomentoside A on NtWRKY70 protein binding; Experiment B is a Western blot analysis of the expression and purification of tobacco NtWRKY70 protein; Experiment C is the binding of Fulvotomentoside A to NtWRKY70 protein under 10 μM treatment. NtF6'H1 Effect of Fulvotomentoside A on the W-box activity of NtWRKY70 binding to NtF6'H1 gene at different concentrations; C represents the inhibitory effect of Fulvotomentoside A on the W-box activity of NtWRKY70 binding to NtF6'H1 gene at different concentrations. Figure 6 The content of hyoscyamine in tobacco leaves under hydroponic treatment with Fulvotomentoside A; Figure 7 The content of hyoscyamine in tobacco leaves after foliar spraying with Fulvotomentoside A. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0024] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0025] Example 1 This example demonstrates the screening and validation of candidate small molecule compounds. 1. Identification and screening of candidate small molecule compounds against NtF6'H1Key segments in gene promoters were analyzed, and their potential binding regions were predicted using the Grid Box tool in software such as COACH, SiteMap, and AutoDockTools. Figure 1 As shown in Part A of the diagram), candidate small molecules in SDF or MOL2 format were downloaded from databases such as PubChem, ZINC, and TCMSP. Based on the predicted potential binding regions and the conformational files of the small molecule compounds, molecular simulation docking was performed using docking software such as dock and glide, resulting in a set of 35 potential candidate compounds. Figure 1 (As shown in Part B of the document). Further verification through compound library queries and standard searches resulted in a final list of 18 candidate small molecule compounds for subsequent activity validation.

[0026] 2. Preliminary validation of small molecule compounds using dual-luciferase reporter gene assays. To preliminarily verify whether the 18 small molecule compounds obtained from the screening affect NtWRKY70's effect on... NtF6'H1 To regulate gene expression, a reporter plasmid, pGreenII 0800-LUC-Wbox, was successfully constructed. Co-injection of pGreenII 62SK-NtWRKY70 with the effector plasmid in *Tobacco Benedict* leaves created a dual-luciferase reporter gene experimental system. In this system, a mixed infection of pGreenII 0800-LUC-Wbox and pGreenII 62SK-NtWRKY70 served as a negative control. Simultaneously, 18 small molecule compounds (initial concentration 50 μM) obtained through screening were added to the mixed infection solution of pGreenII 0800-LUC-Wbox and pGreenII 62SK-WRKY70 to achieve a final concentration of 20 μM. Figure 2 After inoculation, all plants recovered under the same growth conditions for 3 days, and then fluorescence signals were observed using the CDD plant in vivo imaging system.

[0027] like Figure 3 As shown, compared with the negative control without the addition of Fulvotomentoside A, the fluorescence signal generated by the mixed infection of pGreenII 0800-LUC-Wbox and pGreenII 62SK-NtWRKY70 was significantly enhanced after the addition of Fulvotomentoside A, indicating that these three small molecule compounds may interfere with the synergistic effect of the negatively regulating transcription factor NtWRKY70. NtF6'H1 The gene's W-box cis-acting element binds, thereby promoting the expression of the gene.

[0028] Among the candidate compounds mentioned above, Fulvotomentoside A (see the red dashed circle) showed the strongest fluorescence signal, so Fulvotomentoside A was selected for further investigation.

[0029] 3. Candidate compounds promote stable transformation lines NtF6'H1 promoter activity To further determine the effect of Fulvotomentoside A on tobacco products NtF6'H1 Regarding the impact on gene expression, this embodiment utilizes the Pro507:LUC expression vector to carry out leaf disc genetic transformation.

[0030] After obtaining positive seedlings, genomic DNA samples were extracted from each line, and PCR identification was performed using vector-specific primers, resulting in 5 positive lines ( Figure 4 (As shown in Part A of the diagram). RNA samples were then extracted from each positive line, and the expression level of the LUC gene was detected. It was found that the expression level of the LUC gene in all five lines was significantly higher than that in the wild-type control. Three lines were selected for subsequent experimental analysis. Figure 4 (As shown in Part B of the document). Using positive seedlings with uniform growth under normal conditions, 10 μM Fulvotomentoside A was sprayed on each seedling. After 3 days of treatment, the LUC signal in each sample was detected.

[0031] The test results showed that the LUC signal in the leaves of tobacco seedlings treated with Fulvotomentoside A was significantly stronger than that in the untreated control. Figure 4 As shown in section C), quantitative results also indicate that the LUC intensity after Fulvotomentoside A treatment was significantly higher than that of the control ( Figure 4 (as shown in section D of the diagram). These results further confirm that Fulvotomentoside A can promote the metabolism of tobacco products. NtF6'H1 Gene expression.

[0032] Example 2 In vitro experiments in this embodiment confirmed that the candidate compound affects NtWRKY70. NF6'H1 Expression inhibition To further investigate the effect of the candidate small molecule compounds obtained from screening on the binding activity of NtWRKY70, this example uses E. coli to express and purify tobacco NtWRKY70-MBP fusion protein of approximately 80 kD size. Figure 5 (As shown in Part A of the diagram). Subsequently, EMSA spotting analysis was performed using the expressed and purified NtWRKY70-MBP fusion protein and a biotin-labeled W-box probe to determine the effect of the target small molecule compound on the binding activity of NtWRKY70.

[0033] The results are as follows Figure 5 As shown in Part B, the NtWRKY70-MBP fusion protein and NtF6'H1 The W-box probe of the gene formed a significant binding after co-incubation in vitro. Adding 10 μM Fulvotomentoside A to the incubation system further enhanced the binding of the NtWRKY70-MBP fusion protein to the probe. NtF6'H1 The binding of the gene W-box probe was completely eliminated, indicating that Fulvotomentoside A can inhibit the binding activity of NtWRKY70 in vitro.

[0034] The binding activity was further examined after treatment with 1 μM and 2 μM concentrations. The results are as follows: Figure 5 As shown in section C, adding 1x or 2x of unlabeled competitive probes can completely eliminate the NtWRKY70-MBP fusion protein and... NtF6'H1 The binding of the gene W-box probe. Meanwhile, the addition of 1 μM Fulvotomentoside A almost completely prevented the NtWRKY70-MBP fusion protein from binding with... NtF6'H1 The binding of the gene W-box probe was detected, but no binding was detected after treatment with 2 μM Fulvotomentoside A. These results further confirm the inhibitory effect of Fulvotomentoside A on NtWRKY70 binding activity.

[0035] Example 3 This embodiment verifies the regulatory effect of Fulvotomentoside A on the content of hyoscyamine in tobacco. To further clarify the effect of the screened Fulvotomentoside A on the content of hyoscyamine in tobacco leaves and to explore the establishment of an effective treatment system, hydroponic and foliar spraying treatments of the compound were carried out.

[0036] When conducting hydroponic treatment with Fulvotomentoside A, five concentration gradients (0 μM, 0.01 μM, 0.1 μM, 1 μM, and 5 μM) and four time gradients (1 day, 3 days, 5 days, and 7 days) were established. After treatment, fresh tobacco leaf samples were collected for hyoscyamine content determination.

[0037] like Figure 6As shown, the hyoscyamine content in the control tobacco leaves did not change significantly at the four time points. In contrast, the hyoscyamine content did not change significantly after 1, 3, 5, and 7 days of hydroponic treatment with 0.01 μM, 0.1 μM, and 1 μM Fulvotomentoside A. Notably, the hyoscyamine content did not change significantly after 1 and 3 days of treatment with 5 μM Fulvotomentoside A, but after 5 and 7 days of treatment, the hyoscyamine content in the tobacco leaves increased by 2.2 times and 2.8 times, respectively, compared to the untreated control.

[0038] Subsequently, foliar spraying treatment was carried out using Fulvotomentoside A, with five concentration gradients of 0 (Mock), 0.01, 0.1, 1 and 5 μM, and four time gradients of 1 day (D), 3D, 5D and 7D. After treatment, the hyoscyamine content in each sample of tobacco leaves was measured.

[0039] The results are as follows Figure 7 As shown, the hyoscyamine content in tobacco leaves treated with 0.01 μM Fulvotomentoside A for 1, 3, 5, and 7 days was higher than that in the untreated control. The hyoscyamine content in tobacco leaves treated with 0.1 μM Fulvotomentoside A for 3, 5, and 7 days was slightly higher than that in the untreated control. However, with 5 μM Fulvotomentoside A treatment, the hyoscyamine content was only higher on day 7 than in the untreated control. Although treatments with 0.01 μM, 0.1 μM, and 5 μM Fulvotomentoside A all increased the hyoscyamine content in tobacco leaves, the increase ranged from 0.22 to 1.4 times, lower than the increase observed with the 5 μM hydroponic treatment. These results indicate that exogenous application of Fulvotomentoside A can promote the synthesis of hyoscyamine in tobacco leaves. Although multiple concentrations of foliar spraying with Fulvotomentoside A can promote the synthesis of hyoscyamine in tobacco leaves, the 5 μM hydroponic treatment induces a more dramatic effect.

[0040] In summary, this invention has found that the saponin compound Fulvotomentoside A can significantly promote the synthesis and accumulation of hyoscyamine in tobacco, and further provides evidence that Fulvotomentoside A binds competitively with the negative regulator NtWRKY70. NtF6'H1 The promoter fragment of the gene promotes the latter's expression and the mechanism of hyoscyamine synthesis. Therefore, the results of this study can provide new insights into the regulation of hyoscyamine synthesis.

[0041] 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. The application of triterpenoid saponins in the preparation of products that promote the synthesis of hyoscyamine in plants, characterized in that, The triterpenoid saponins include Fulvotomentoside A.

2. The application according to claim 1, characterized in that, The plant mentioned includes tobacco.

3. The application according to claim 1, characterized in that, The triterpenoid saponins are regulated by F6'H1 Gene expression promotes the synthesis of hyoscyamine in plants.

4. A method for promoting the synthesis of hyoscyamine in plants, characterized in that, include: Applying triterpenoid saponins to plants; The triterpenoid saponins include Fulvotomentoside A.

5. The method according to claim 4, characterized in that, The triterpenoid saponin compounds are applied by at least one of the following methods: basal application, drip irrigation, spraying, or hydroponics.

6. The method according to claim 5, characterized in that, The triterpenoid saponins were applied via hydroponics; the concentration of the triterpenoid saponins in the hydroponic solution was ≥5 μM, and the hydroponic time was ≥5 days.

7. The method according to claim 5, characterized in that, The triterpenoid saponin compound is applied by foliar spraying; the concentration of the triterpenoid saponin compound applied by foliar spraying is ≥0.01μM.

8. The method according to any one of claims 4 to 7, characterized in that, The plant mentioned includes tobacco.

9. A product for promoting the synthesis of hyoscyamine in plants, characterized in that, It contains triterpenoid saponins, as well as agriculturally acceptable carriers or adjuvants; The triterpenoid saponins include Fulvotomentoside A.

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