Application of DNA methylation inhibitor in regulation and control of synthesis of ginkgo terpene lactones

Abstract

The invention discloses application of a DNA methylation inhibitor in regulation and control of synthesis of ginkgo terpene lactones in the technical field of plant cultivation, and the epigenetic regulation and control effect of 5-azacytidine on leaf growth and terpene lactones synthesis is researched by taking ginkgo as a material. 5-AzaC with different concentrations is set for treatment, and the dynamic change of leaf growth traits and terpene lactone accumulation is systematically analyzed. Researches show that treatment of 5-AzaC shows a significant dose effect: low concentration (50 [mu] mol / L) promotes increase of fresh weight and increase of terpene lactone content, and high concentration (greater than or equal to 150 [mu] mol / L) inhibits growth but significantly increases accumulation of bilobalide and ginkgolide C. Correlation analysis shows that biomass and terpene lactone content are in significant negative correlation, a growth and defense balance phenomenon is proved, the invention reveals that 5-AzaC affects synthesis and accumulation of different terpene lactones by regulating DNA methylation level difference, and a theoretical basis and a technical path are provided for accurate regulation of ginkgo medicinal components.

Description

Technical Field

[0001] This invention relates to the field of plant cultivation technology, specifically to the application of DNA methylation inhibitors in regulating the synthesis of ginkgolide lactones. Background Technology

[0002] ginkgo( Ginkgo biloba As the only extant species in the Ginkgoaceae family, Ginkgo biloba has been widely introduced to various parts of the world due to its unique medicinal and ornamental value. Modern research shows that Ginkgo biloba leaves contain abundant bioactive substances. To date, more than 10 ginkgolides have been isolated from Ginkgo biloba leaves, including four major ginkgolides: ginkgolide A (GA), ginkgolide B (GB), ginkgolide C (GC), and ginkgolide (BB). Their biosynthetic mechanisms have been studied in depth. The precursors of Ginkgo terpenoids are synthesized via the mevalonic acid (MVA) and 2-C-methyl-D-erythritol-4-phosphate (MEP) metabolic pathways. Ginkgolides are diterpenes synthesized via the MEP pathway, while ginkgolide is a sesquiterpene synthesized via the MVA pathway. Several key enzyme genes involved in the synthesis pathway of Ginkgo terpenoid lactones have been cloned and studied, including those encoding 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), 1-deoxyxylulose-5-phosphate synthase (DXS), 1-deoxy-D-xylulose-5-phosphate reductase (DXR), geraniol-geraniol pyrophosphate synthase (GGPPS), farnesyl pyrophosphate synthase (FPS), and L-pinediene synthase (LPS) (Han et al., 2021). With the development of the health industry, the global market for Ginkgo products is continuously expanding. Therefore, increasing the content of medicinal metabolites in Ginkgo leaves through breeding and cultivation techniques is of great significance for improving the quality and efficiency of Ginkgo leaf cultivation.

[0003] DNA methylation, as an important epigenetic regulatory mechanism, dynamically regulates gene expression activity by adding methyl groups to cytosine bases without altering the DNA sequence. Epigenetic regulation can lead to gene silencing, thereby affecting plant growth, development, and secondary metabolic processes. Studies have shown that DNA methylation plays a crucial regulatory role in the synthesis of secondary metabolites in various medicinal plants through spatiotemporally specific epigenetic modifications. For example, in *Salvia miltiorrhiza* (Danshen),... Salvia miltiorrhiza Key genes regulating the synthesis pathways of tanshinone and phenolic acids in ) SmCPS5 , SmCYP71D464 The expression of ) in Angelica sinensis ( Angelica sinensis (This is achieved by influencing genes related to phenylpropanol metabolism) AsCOMT1 Expression promotes lignin synthesis and thus participates in the root lignification process; in tea plants ( Camellia sinensis Dynamic regulation of catechins during leaf development SCPL1A , LAR) and terpenoids ( NES Expression of synthetic genes. Previous studies have not only revealed the fine regulatory mechanism of DNA methylation on plant secondary metabolic networks, but also provided new ideas for the targeted improvement of medicinal plant quality through epigenetic means.

[0004] 5-Azacytidine (5-AzaC), as a DNA methyltransferase inhibitor, has been shown to irreversibly bind to and inhibit the activity of DNA methyltransferase 1 (DNMT1 / MET1), blocking its function in maintaining DNA methylation. This leads to a decrease in genomic methylation levels, thereby breaking epigenetic silencing of specific gene promoter regions, activating the expression of related genes, and affecting plant growth and secondary metabolic processes. For example, in licorice (… Glycyrrhiza inflata In tomatoes, 5-AzaC significantly increased the accumulation of glycyrrhizin A. Although whole-genome methylation sequencing showed that its direct effect on DNA methylation was limited, it may indirectly regulate flavonoid biosynthesis pathways by activating transcription factors such as MYB, ERF, and WRKY. Furthermore, 5-AzaC in tomatoes (… Solanum lycopersicum In the salt stress response, 5-AzaC works synergistically with trehalose to enhance plant salt tolerance, possibly by regulating the accumulation of osmotic regulators (such as proline and soluble sugars) and antioxidant defense systems to alleviate stress damage. Furthermore, 5-AzaC can not only regulate the synthesis of secondary metabolites through demethylation but also enhance plant resistance to abiotic stresses, providing an important tool for agronomic trait improvement and targeted regulation of active ingredients in medicinal plants.

[0005] However, there are few reports on the effects of DNA methylation modification on the accumulation of secondary metabolites in Ginkgo biloba leaves. To promote quality improvement and targeted regulation in the cultivation industry of Ginkgo biloba leaves, this invention is proposed. Summary of the Invention

[0006] ginkgo( Ginkgo bilobaAs the only extant species in the Ginkgoaceae family, Ginkgo biloba has been widely introduced to various parts of the world due to its unique medicinal and ornamental value. Modern research shows that Ginkgo biloba leaves contain abundant bioactive substances. To date, more than 10 ginkgolides have been isolated from Ginkgo biloba leaves, including four major ginkgolides: ginkgolide A (GA), ginkgolide B (GB), ginkgolide C (GC), and ginkgolide (BB). Their biosynthetic mechanisms have been studied in depth. The precursors of Ginkgo terpenoids are synthesized via the mevalonic acid (MVA) and 2-C-methyl-D-erythritol-4-phosphate (MEP) metabolic pathways. Ginkgolides are diterpenes synthesized via the MEP pathway, while ginkgolide is a sesquiterpene synthesized via the MVA pathway. Several key enzyme genes involved in the synthesis pathway of Ginkgo terpenoid lactones have been cloned and studied, including those encoding 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), 1-deoxyxylulose-5-phosphate synthase (DXS), 1-deoxy-D-xylulose-5-phosphate reductase (DXR), geraniol-geraniol pyrophosphate synthase (GGPPS), farnesyl pyrophosphate synthase (FPS), and L-pinediene synthase (LPS) (Han et al., 2021). With the development of the health industry, the global market for Ginkgo products is continuously expanding. Therefore, increasing the content of medicinal metabolites in Ginkgo leaves through breeding and cultivation techniques is of great significance for improving the quality and efficiency of Ginkgo leaf cultivation.

[0007] DNA methylation, as an important epigenetic regulatory mechanism, dynamically regulates gene expression activity by adding methyl groups to cytosine bases without altering the DNA sequence. Epigenetic regulation can lead to gene silencing, thereby affecting plant growth, development, and secondary metabolic processes. Studies have shown that DNA methylation plays a crucial regulatory role in the synthesis of secondary metabolites in various medicinal plants through spatiotemporally specific epigenetic modifications. For example, in *Salvia miltiorrhiza* (Danshen),... Salvia miltiorrhiza Key genes regulating the synthesis pathways of tanshinone and phenolic acids in ) SmCPS5 , SmCYP71D464 The expression of ) in Angelica sinensis ( Angelica sinensis (This is achieved by influencing genes related to phenylpropanol metabolism) AsCOMT1 Expression promotes lignin synthesis and thus participates in the root lignification process; in tea plants ( Camellia sinensis Dynamic regulation of catechins during leaf development SCPL1A , LAR ) and terpenoids ( NES Expression of synthetic genes. Previous studies have not only revealed the fine regulatory mechanism of DNA methylation on plant secondary metabolic networks, but also provided new ideas for the targeted improvement of medicinal plant quality through epigenetic means.

[0008] 5-Azacytidine (5-AzaC), as a DNA methyltransferase inhibitor, has been shown to irreversibly bind to and inhibit the activity of DNA methyltransferase 1 (DNMT1 / MET1), blocking its function in maintaining DNA methylation. This leads to a decrease in genomic methylation levels, thereby breaking epigenetic silencing of specific gene promoter regions, activating the expression of related genes, and affecting plant growth and secondary metabolic processes. For example, in licorice (… Glycyrrhiza inflata In tomatoes, 5-AzaC significantly increased the accumulation of glycyrrhizin A. Although whole-genome methylation sequencing showed that its direct effect on DNA methylation was limited, it may indirectly regulate flavonoid biosynthesis pathways by activating transcription factors such as MYB, ERF, and WRKY. Furthermore, 5-AzaC in tomatoes (… Solanum lycopersicum In the salt stress response, 5-AzaC works synergistically with trehalose to enhance plant salt tolerance, possibly by regulating the accumulation of osmotic regulators (such as proline and soluble sugars) and antioxidant defense systems to alleviate stress damage. Furthermore, 5-AzaC can not only regulate the synthesis of secondary metabolites through demethylation but also enhance plant resistance to abiotic stresses, providing an important tool for agronomic trait improvement and targeted regulation of active ingredients in medicinal plants.

[0009] However, there are few reports on the effects of DNA methylation modification on the accumulation of secondary metabolites in Ginkgo biloba leaves. To promote quality improvement and targeted regulation in the cultivation industry of Ginkgo biloba leaves, this invention is proposed. Attached Figure Description

[0010] Figure 1 The effects of different concentrations of exogenous 5-AzaC treatment on the biomass accumulation of Ginkgo biloba leaves provided by this invention; Figure 2 The effect of different concentrations of exogenous 5-AzaC on Ginkgo leaf area provided by this invention; Figure 3 The effects of different concentrations of exogenous 5-AzaC treatment on the accumulation of terpene lactone compounds in Ginkgo biloba leaves provided by this invention; Figure 4 This invention provides a correlation analysis of growth and metabolic traits under exogenous 5-AzaC treatment; Figure 5 The comprehensive effects of exogenous 5-AzaC treatment provided by this invention on the yield and quality of Ginkgo biloba leaves. Detailed Implementation

[0011] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0012] 1. Materials and Methods 1.1 Test Materials In April 2023, three healthy mature ginkgo trees were selected on the campus of Nanjing Forestry University, and their newly sprouted branches in early spring were collected as experimental materials. All collected branches were of uniform length (50 cm) and systematically grouped according to the number of buds and the source of the mother tree. The experiment consisted of four treatment groups, each containing nine samples (three from each mother tree) to ensure a balanced genetic background among the treatment groups. The standardized branches were then used for subsequent hydroponic system establishment and 5-AzaC treatment experiments.

[0013] 1.2 Experimental Design The 36 mature Ginkgo branches were divided into four groups (n=9 per group) according to the principle of biological replication to ensure that the number of buds was basically consistent among the groups. A standard hydroponic system was used for treatment. In three treatment groups, appropriate amounts of 5-AzaC were added to the culture medium to concentrations of 50 μmol / L, 150 μmol / L, and 300 μmol / L (referred to as groups T1, T2, and T3, respectively). The other treatment group did not receive any 5-AzaC and was designated as the control group (CK group).

[0014] During the cultivation period, continuous oxygenation was maintained for 24 hours, and the culture medium was changed weekly. After 4 weeks of treatment, morphologically intact new leaves (one leaf per bud) were selected from each bud point for relevant index measurements.

[0015] 1.3 Indicator Measurement (1) Biomass accumulation determination Fresh leaf weight (FW) was immediately determined after sampling using a precision electronic balance (accuracy 0.0001 g); leaf area (LA) was standardized using a LI-3100C leaf area meter; dry weight (DW) was determined by drying fresh leaf samples in a 60℃ constant temperature oven to constant weight (48 h) and then weighing. All measurements were performed in triplicate.

[0016] Leaf samples, after morphological index determination, were pulverized in a grinder, passed through a 50-mesh standard sieve, and aliquoted and stored in a desiccator (relative humidity <30%) for subsequent HPLC-MS / MS analysis of ginkgolide lactones (including ginkgolide A (GA), ginkgolide B (GB), ginkgolide C (GC) and ginkgolide (BB)).

[0017] (2) Determination of terpene lactone accumulation Standardized methods were used for the extraction and detection of terpene lactones from Ginkgo biloba leaves.

[0018] First, the sample was pretreated using Soxhlet extraction: 0.5 g of ginkgo powder was accurately weighed, defatted with petroleum ether (80℃) for 6 h, and then extracted with methanol solution (80℃) for 6 h. The extract was concentrated by rotary evaporation. The sample was purified using a gradient extraction method, which involved acidification with hydrochloric acid, extraction with ethyl acetate (4 times), and washing with sodium acetate solution to finally obtain the purified extract.

[0019] The purified extract was analyzed using ultra-high performance liquid chromatography (UPLC). Chromatographic conditions: reversed-phase column (RPC), mobile phase: methanol-water (31:69, v / v), drift tube temperature: 90 ℃. The sample was filtered through a 0.22 μm organic filter before injection. The contents of GA, GB, GC, and BB were quantitatively analyzed by characteristic peak area, and the concentrations of each component were calculated using the external standard method.

[0020] The total content of terpene lactones (TT) was calculated as the sum of ginkgolide A (GA), ginkgolide B (GB), ginkgolide C (GC), and ginkgolide (BB).

[0021] 1.4 Data Processing The raw data were processed using Excel 2010. For growth phenotypic traits, after removing the maximum and minimum values, seven biological replicates were retained for each treatment, while for metabolic traits, three biological replicates were set for each treatment. Statistical analysis was performed using SPSS 23.0: One-way ANOVA was used to test for significant differences between groups using Duncan's multiple comparisons test, and Pearson correlation analysis was used to assess the association between traits (*P<0.05, **P<0.01). Experimental results are expressed as mean ± standard deviation (Mean ± SD), and differences between groups in ANOVA are indicated with lowercase letters (P<0.05). Data visualization was performed using the ggplot2 package in R (version 4.1.0).

[0022] 1. Test Results 2.1 Effects of exogenous 5-AzaC treatment on Ginkgo biloba leaf biomass accumulation The results are as follows Figure 1 As shown, 5-AzaC treatment significantly altered the phenotypic traits of Ginkgo biloba leaves. With increasing treatment concentration, leaf dry weight showed a dose-dependent decreasing trend; the dry weight of the high-concentration T3 group was 17.3%–43.7% lower than that of the CK group.

[0023] Fresh weight changes showed a trend where low concentrations of 5-AzaC promoted fresh weight accumulation while high concentrations inhibited it: low-concentration T1 treatment induced polarization in fresh weight, with some samples maintaining control levels while a small number of samples showed significant weight gain (9.7%); while treatments with ≥150 μmol / L 5-AzaC (T2 and T3 groups) resulted in a general decrease in fresh weight of 12.5%–28.3%. The dose-response relationship suggests that low concentrations of 5-AzaC may activate some metabolic pathways through epigenetic regulation, while high concentrations inhibit overall growth. The abnormal increase in fresh weight observed in the T1 group indicates that 50 μmol / L 5-AzaC treatment may specifically regulate the expression of certain growth-related genes in Ginkgo biloba leaves through demethylation. This finding provides an important concentration reference for subsequent research on the targeted regulatory role of 5-AzaC in Ginkgo biloba growth.

[0024] 2.2 Effects of exogenous 5-AzaC treatment on Ginkgo leaf morphogenesis The results are as follows Figure 2 As shown, 5-AzaC treatment significantly affected Ginkgo leaf morphogenesis. With increasing treatment concentration, leaf area generally decreased. Medium- and high concentrations of 5-AzaC (T2 and T3 groups) resulted in a significant reduction in leaf area, with decreases ranging from 18.6% to 34.4% compared to the control group. However, the low-concentration T1 group exhibited a dual effect: some samples showed leaf areas comparable to the control, while the maximum leaf area increased by 5.0% compared to the control. This suggests that low-concentration 5-AzaC may promote the expansion and growth of specific leaves through epigenetic regulation, revealing the complex regulatory role of 5-AzaC in Ginkgo leaf development.

[0025] 2.3 Effects of exogenous 5-AzaC treatment on the accumulation of terpene lactones in Ginkgo biloba leaves In this study, standard curves for four major terpene lactone components in Ginkgo biloba leaves were successfully established using high-performance liquid chromatography (HPLC). The results showed that the standard curves for BB, GA, GB, and GC all exhibited good linear relationships, and their linear regression equations are shown in Table 1.

[0026] Table 1. Linear Regression Equations ; The coefficients of determination for all standard curves were greater than 0.99, indicating that each terpene lactone compound exhibited excellent linear response within the range of measured concentrations.

[0027] Test results as follows Figure 3As shown, 5-AzaC treatment significantly altered the accumulation pattern of terpenoid lactones in Ginkgo biloba leaves. With increasing treatment concentration, BB and GC exhibited a clear concentration-dependent accumulation, with the high-concentration T3 group showing the most significant effect, where BB and GC contents reached 2.1 times and 1.8 times that of the CK control group, respectively. Notably, GA accumulation showed a non-linear response; the medium-concentration T2 group induced the highest accumulation (0.75 mg / g), an increase of 15.5% compared to the control group, but this was not statistically significant (P>0.05). For GB, both the T1 and T3 groups significantly promoted its accumulation, with increases of 45.0% and 30.2% compared to the control group, respectively, indicating that different concentrations of 5-AzaC may affect the GB synthesis pathway through differential epigenetic regulatory mechanisms.

[0028] In terms of total terpene lactone content, the 300 μmol / L treatment showed the best effect, mainly attributed to the significant increase in BB and GC. Of particular note is that although the high-concentration treatment (T3 group) significantly increased the total terpene lactone content, the proportions of each monomer compound changed significantly: the proportion of BB increased from 22.5% in the control to 34.4%, while the proportion of GA decreased from 45.5% to 31.5%. These compositional changes may affect the pharmacological activity of the extract.

[0029] 2.4. Comprehensive effects of exogenous 5-AzaC treatment on the yield and quality of Ginkgo leaves Pearson correlation analysis showed that the accumulation of terpenoid lactones in Ginkgo biloba leaves after 5-AzaC treatment was significantly negatively correlated with growth traits. Figure 4 BB content showed a significant negative correlation with leaf dry weight (r=-0.986, P=0.014) and leaf area (r=-0.964, P=0.036), while total terpene lactone content also showed a negative correlation with dry weight (r=-0.849) and leaf area (r=-0.802). Meanwhile, leaf fresh weight showed a highly significant positive correlation with dry weight and leaf area (r>0.97, P<0.05). These results indicate that 5-AzaC may promote terpene lactone biosynthesis through epigenetic regulation while potentially inhibiting leaf biomass accumulation.

[0030] Furthermore, by calculating the total content of terpene lactones in a single leaf (the product of terpene lactone content and single leaf dry weight), it was found that the low concentration of 5-AzaC treatment had the most significant effect, increasing it by 16.25% compared to the control group. Figure 5 However, as the treatment concentration continued to increase to 300 μmol / L, the content of terpene lactones in single leaves decreased, although it was still higher than that in the control group, but the increase was smaller.

[0031] The results showed that low-concentration 5-AzaC treatment effectively promoted the biosynthesis of terpenoids while maintaining leaf biomass, achieving the optimal balance between yield and quality. While high-concentration treatment still increased the terpenoid content per unit mass of leaf, it significantly inhibited leaf growth, leading to a decrease in the increase in the total accumulation of terpenoids per leaf. These findings provide important reference for the rational application of 5-AzaC in Ginkgo leaf cultivation, and a concentration of 50 μmol / L is recommended to achieve the best economic benefits.

[0032] 1. Conclusion The results of the 5-AzaC exogenous treatment experiment of this invention show that: 5-AzaC treatment significantly affected the biomass accumulation and terpene lactone synthesis in Ginkgo biloba leaves, suggesting that the dynamic regulation of DNA methylation may play a key role in the metabolic network of Ginkgo biloba.

[0033] The regulation of Ginkgo biloba growth and metabolism by 5-AzaC exhibits a typical dose-dependent biphasic effect: low concentrations (50 μmol / L) promote fresh weight and terpene lactone accumulation, achieving an optimal balance between leaf biomass and terpene lactone content, while high concentrations (≥150 μmol / L) inhibit growth but enhance terpene lactone accumulation. This dose-response effect may be related to the degree of inhibition of the DNA methyltransferase DNMT. Low concentrations of 5-AzaC may only partially inhibit DNMT activity, leading to selective demethylation of specific genes, while high concentrations may cause a significant decrease in genome-wide methylation levels, triggering a widespread stress response and thus inhibiting overall growth.

[0034] 5-AzaC exhibits significant differences in its regulation of different terpene lactone monomers. BB and GC showed concentration-dependent increases, with increases of 110.9% and 82.6%, respectively, at 300 μmol / L treatment. Ginkgolide A, however, showed a non-linear response, with only a slight increase in GA content after treatment at 150 μmol / L. These differences may stem from varying sensitivities of key enzyme genes in different synthetic pathways to methylation modification. Therefore, 5-AzaC may finely regulate the branching of the terpene lactone metabolic flux through differential epigenetic regulation.

[0035] In addition, it is worth noting that the total terpene lactone content was significantly negatively correlated with the leaf dry weight, indicating that the resource allocation of Ginkgo exhibits a typical trade-off between growth and defense.

[0036] This study elucidates the role of 5-AzaC in regulating the growth, development, and secondary metabolism of Ginkgo biloba leaves through epigenetic pathways, laying a theoretical foundation for the precise regulation of the medicinal active components of Ginkgo biloba.

[0037] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. Application of DNA methylation inhibitors in regulating the synthesis of ginkgolide lactones.

2. The application of the DNA methylation inhibitor according to claim 1 in regulating the synthesis of ginkgolide lactones, characterized in that, The DNA methylation inhibitor is 5-azacytidine.

3. The application of the DNA methylation inhibitor according to claim 1 in regulating the synthesis of ginkgolide lactones, characterized in that, The application method involves using 5-azacytidine for exogenous treatment during the cultivation of Ginkgo biloba.

4. The application of the DNA methylation inhibitor according to claim 1 in regulating the synthesis of ginkgolide lactones, characterized in that, The ginkgolide is at least one of ginkgolide A, ginkgolide B, ginkgolide C, or ginkgolide.

5. The application of the DNA methylation inhibitor according to claim 2 or 3 in regulating the synthesis of ginkgolide lactones, characterized in that, The concentration of 5-azacytidine exogenously treated Ginkgo biloba is 50-300 μmol / L.

6. The application of the DNA methylation inhibitor according to claim 2 or 3 in regulating the synthesis of ginkgolide lactones, characterized in that, When the concentration of 5-azacytidine exogenously treated Ginkgo biloba was 50 μmol / L, the biomass and terpene lactone content of Ginkgo biloba leaves were increased simultaneously.

7. The application of the DNA methylation inhibitor according to claim 6 in regulating the synthesis of ginkgolide lactones, characterized in that, The biomass of ginkgo leaves refers to the fresh weight of ginkgo leaves.

8. The application of the DNA methylation inhibitor according to claim 2 or 3 in regulating the synthesis of ginkgolide lactones, characterized in that, When the concentration of 5-azacytidine exogenously treated Ginkgo biloba is ≥150 μmol / L, the content of ginkgolide C and ginkgolide in Ginkgo biloba leaves is increased.

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