Application of melatonin in artemisinin biosynthesis and method for increasing artemisinin content in artemisia apiacea

Abstract

The invention belongs to the field of plant biotechnology and medicinal plant cultivation, and relates to application of melatonin in artemisinin biosynthesis and a method for increasing the content of artemisinin in artemisia apiacea. The melatonin is applied to the artemisia apiacea plants, the content of artemisinin in the artemisia apiacea is increased, and compared with an existing method, the method is more economical and efficient.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology and medicinal plant cultivation, and relates to the application of melatonin in artemisinin biosynthesis and methods for increasing the artemisinin content in Artemisia annua. Background Technology

[0002] Artemisinin has gained worldwide recognition as an effective antimalarial drug and has also shown great potential in treating other diseases such as lupus and diabetes, thus creating a huge market demand. However, countries and regions in Africa and Southeast Asia, with their large populations and relatively underdeveloped economies, have long faced a shortage of artemisinin resources. Currently, the main source of artemisinin is extracted from the above-ground parts of the Artemisia annua plant.

[0003] Artemisia annua L., also known as yellow wormwood, is an annual herbaceous medicinal plant. The very low content of artemisinin in Artemisia annua (0.01%-1% of dry weight) limits its large-scale commercial production. Although artemisinin can now be synthesized artificially, the high difficulty, low yield, and high cost prevent mass production. Using tissue culture and cell engineering to produce artemisinin is an attractive method. However, the content of artemisinin in callus tissue is less than 0.1% of dry weight, and in buds it is only a maximum of 0.16% of dry weight. Most studies have not detected artemisinin in roots, therefore, the feasibility of using tissue culture and cell engineering to produce artemisinin is low. Another feasible method is to use genetic engineering to obtain new varieties of Artemisia annua with higher artemisinin content, but the commercial cultivation of genetically modified Artemisia annua plants is currently restricted by GMO regulations, has a long development cycle, and is impractical for developing countries and regions such as Africa. Therefore, how to rapidly, economically, and efficiently increase the artemisinin content in Artemisia annua remains a major issue for the future and for a long time to come. Summary of the Invention

[0004] In a first aspect, the present invention provides the application of melatonin in the biosynthesis of artemisinin, specifically in the application of improving the biosynthesis of artemisinin.

[0005] Since its discovery in higher plants in 1995, melatonin has been widely recognized as an important plant hormone, playing a significant role in regulating plant growth and development (such as seed germination and seedling growth) and enhancing stress resistance (such as drought and salt stress). In recent years, with the in-depth exploration of the regulatory mechanisms of plant secondary metabolites, the potential regulatory role of melatonin in the synthesis pathways of plant bioactive components has begun to attract attention.

[0006] Existing research has revealed that artemisinin biosynthesis is precisely regulated by various endogenous hormones and external environmental factors. While hormones such as jasmonic acid, abscisic acid, and gibberellin are known to significantly affect the expression of artemisinin synthesis-related genes (e.g., ADS, CYP71AV1, DBR2, ALDH1) and the activity of key enzymes, thereby regulating artemisinin accumulation, research on whether and how melatonin participates in regulating the artemisinin biosynthetic pathway is scarce. Direct evidence is lacking that exogenous application of melatonin can effectively increase artemisinin yield in Artemisia annua plants. Elucidating the specific action sites, signal transduction mechanisms, and interactions of melatonin with known regulatory factors within the artemisinin biosynthetic metabolic network, particularly clarifying its impact on the expression of key enzyme genes involved in artemisinin synthesis, is a crucial scientific issue that urgently needs to be addressed to fill knowledge gaps in this field and explore the use of melatonin, a novel plant hormone, to improve artemisinin production efficiency. Furthermore, it is of great significance for rapidly, economically, and efficiently increasing artemisinin content and achieving large-scale production.

[0007] This invention discovers and verifies that the density of secretory glandular trichomes is significantly increased in Artemisia annua plants treated with melatonin, which can effectively increase the artemisinin content in Artemisia annua.

[0008] The principle is that treating Artemisia annua plants with melatonin solution can increase the expression of some key enzyme genes for artemisinin synthesis within a short period of time (24-72 hours), breaking some rate-limiting steps in the artemisinin synthesis pathway, thereby accumulating a large amount of artemisinin in a short time. At the same time, it activates the glandular trichome development regulation gene, promotes the increase of secretory glandular trichome density, and thus increases artemisinin production. The aforementioned key enzyme genes for artemisinin synthesis refer to Amorpha fruticosa-4,11-diene synthase gene (ADS), Amorpha fruticosa-4,11-diene oxidase gene (CYP71AV1), artemisinin aldehyde Δ11(13) reductase gene (DBR2), and artemisinin aldehyde dehydrogenase gene (ALDH1).

[0009] A second aspect of the present invention provides a method for increasing the artemisinin content in Artemisia annua, which is more economical and efficient than existing methods. This method involves applying melatonin to the Artemisia annua plant.

[0010] The form of melatonin application is not limited, but it is preferred to apply it as a melatonin solution by spraying.

[0011] As a preferred method, the leaves of Artemisia annua plants are sprayed with a melatonin solution at a concentration of 80–120 μM. The treatment time is 3–4 days before harvest or periodically from the seedling stage to the pre-flowering stage.

[0012] Preferably, the concentration of the melatonin solution is 100 μM.

[0013] Preferably, the periodic spraying is performed once a week until the plant enters the pre-flowering stage.

[0014] By implementing the above technical solution, compared with the prior art, the present invention has the following beneficial effects:

[0015] This invention provides a new use for melatonin and expands the scope of its application.

[0016] This invention increases the artemisinin content in Artemisia annua by applying melatonin to the plant, which is more economical and efficient than existing methods. Attached Figure Description

[0017] Figure 1 This shows the relative expression levels of key enzyme genes involved in artemisinin biosynthesis in Artemisia annua leaves treated with melatonin for 3 hours.

[0018] Figure 2 The results of fluorescence microscopy observation of glandular trichomes in Artemisia annua leaves are shown. A is the control group and B is the melatonin treatment group.

[0019] Figure 3 The statistical results of glandular trichome density are shown (fluorescence microscope);

[0020] Figure 4 The scanning electron microscope shows the results of the glandular hairs on Artemisia annua leaves. A is the control group and B is the melatonin treatment group.

[0021] Figure 5 The results of glandular trichome density statistics are shown (scanning electron microscopy);

[0022] Figure 6 This shows the changes in artemisinin content in Artemisia annua plants under long-term melatonin treatment;

[0023] Figure 7 This shows the changes in artemisinin content in Artemisia annua plants under short-term melatonin treatment. Detailed Implementation

[0024] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. 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.

[0025] This invention is achieved through the following technical solution: First, Artemisia annua seeds are planted in a seedling culture medium to germinate. Then, the Artemisia annua seedlings are transplanted into a perlite culture medium. The Artemisia annua seedlings are treated using either of the following two methods: The first method is a long-term treatment, in which the leaves of the Artemisia annua seedlings are sprayed with a 100 μM melatonin solution every week, from the seedling stage to the pre-flowering stage, and then harvested. The second method is a short-term treatment, in which the Artemisia annua seedlings are irrigated with ordinary nutrient solution until the Artemisia annua grows to the pre-flowering stage. 3-4 days before harvest, the leaves of the Artemisia annua seedlings are sprayed with a 100 μM melatonin solution, and then harvested. This method can significantly increase the artemisinin content in Artemisia annua.

[0026] The aforementioned seedling culture medium was prepared by adding 30 g / L sucrose and 2.6 g / L plant gel to MS culture medium. The MS culture medium was published by Murashige and Skoog in 1962.

[0027] The plant-based gel refers to Phytagel, a product purchased from Sigma.

[0028] The transplanting of Artemisia annua seedlings refers to transplanting them into a peat perlite culture medium when they grow to about 5cm.

[0029] The peat-perlite culture medium has a peat and perlite ratio of 1:1.

[0030] The treatment of Artemisia annua seedlings with melatonin solution refers to spraying the entire plant with melatonin solution when the plant reaches the pre-flowering stage. Harvesting can be done in 3-4 days to obtain plants with high artemisinin content.

[0031] The Artemisia annua seedlings were transplanted into a peat perlite culture medium and watered with ordinary nutrient solution once a week.

[0032] The aforementioned ordinary nutrient solution refers to the product 20-20-20+TE, a water-soluble fertilizer containing macro-elements, purchased from Shanghai Yongtong Ecological Engineering Co., Ltd., diluted with water at a ratio of 600-1000.

[0033] Example 1: Long-term treatment of Artemisia annua plants with melatonin

[0034] Artemisia annua seeds (derived from a high-artemisinin-content Artemisia annua variety from Youyang, Chongqing) were planted on a seedling culture medium formulated with MS medium containing 30 g / L sucrose and 2.6 g / L plant gel. Once the seedlings reached approximately 5 cm in height, they were transplanted into a peat-perlite culture medium (1:1 ratio of peat to perlite). The leaves were sprayed weekly with a 100 μM melatonin solution until moist. Water-treated Artemisia annua plants served as a control. The plants were irrigated weekly with standard nutrient solution. Before harvest, the leaves of the entire Artemisia annua plant were harvested, and the artemisinin content was determined by high-performance liquid chromatography (HPLC).

[0035] Example 2: Short-term treatment of Artemisia annua plants with melatonin

[0036] Artemisia seeds (using Artemisia varieties with high artemisinin content from Youyang, Chongqing) were planted in a seedling culture medium for germination. When the seedlings grew to about 5cm, they were transplanted into a perlite culture medium and irrigated with ordinary nutrient solution every two days to keep the perlite culture medium moist. Before harvest, the leaves were sprayed with 100μM melatonin solution. The treated Artemisia leaves were harvested 3 days later, and the artemisinin content was determined by high performance liquid chromatography.

[0037] Example 3: Detection of artemisinin-related gene expression in melatonin-treated plants by quantitative real-time PCR

[0038] 1. Primer design and synthesis: Primers were designed based on the full sequences of key enzyme genes (such as ADS, CYP71AV1, DBR2, ALDH1) and the internal reference gene actin in the artemisinin synthesis pathway. The primers used were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0039] 2. RNA extraction and reverse transcription: Small amounts of tender Artemisia annua leaves from the control group and melatonin-treated group were taken, flash-frozen in liquid nitrogen, and then quickly crushed in a mortar and pestle. 1 mL of TRIzol was added to the mixture. In a 1.5 mL centrifuge tube (Reagents, GIBCOBRL, USA), the RNA was thoroughly shaken and incubated at room temperature for 5 min. 200 μL of chloroform was added, and the mixture was vigorously shaken for 15 sec. After incubating at room temperature for 2-3 min, it was centrifuged at 12,000 g for 15 min at 4 °C. Approximately 600 μL of the supernatant was transferred to a clean 1.5 mL centrifuge tube, and an equal volume of isopropanol was added. The mixture was inverted and mixed thoroughly. After incubating at room temperature for 10 min, it was centrifuged at 12,000 g for 10 min at 4 °C. The supernatant was discarded, and the mixture was washed with 1 mL of 75% ethanol. After shaking, it was centrifuged at 7,500 g for 5 min at 4 °C. After drying at room temperature for 15-20 min, it was dissolved in an appropriate amount (30-50 μL) of RNAase-free water. The RNA content was measured using a Nanodrop spectrophotometer. Genomic DNA was removed from the RNA using DNase I, and the RNA was then processed using qPCR reverse transcription premixed buffer (…). First-strand cDNA was synthesized using qRT Master Mix 2.0, and the cDNA template was diluted 20-fold after reverse transcription before use.

[0040] 3. RT-PCR detection of artemisinin synthesis-related genes and the internal reference gene actin: To adjust for differences in reaction efficiency among samples during RNA extraction and reverse transcription, the expression of the internal reference gene actin was detected simultaneously with the detection of the target gene expression level. SYBR Green quantitative PCR premix solution was used (… The expression levels of the target gene and internal reference gene were measured using 5G qPCR Premix 2.0. The qPCR reaction system is shown in Table 1.

[0041] Table 1. qPCR reaction system

[0042] reaction system volume qPCR Premix 2.0 10μL 10 μM forward primer 0.5μL 10 μM reverse primer 0.5μL template cDNA 3μL ddH2O 6μL Total volume 20μL

[0043] PCR reaction conditions: hot start and denaturation 94℃ for 5 min, 1 cycle; 94℃ for 30 s, 54℃ for 30 s, 72℃ for 10 s, fluorescence acquisition, 40 cycles; melting curve analysis.

[0044] In this embodiment, RT-PCR technology was used to determine the expression of artemisinin biosynthesis-related genes ADS, CYP71AV1, DBR2, and ALDH1 in transgenic Artemisia annua. The detection results are as follows: Figure 1 As shown, after 3 hours of melatonin treatment on Artemisia annua leaves, the expression of ADS, CYP71AV1, and ALDH1 increased significantly.

[0045] Example 4: Observation and statistical analysis of changes in artemisia glandular trichome density after long-term melatonin treatment using fluorescence microscopy

[0046] The leaves of wild-type Artemisia annua and melatonin-treated Artemisia annua were observed using an Olympus BX51 microscope under excitation light with wavelengths of 450nm-480nm. Young leaves of equal size were randomly sampled at five different locations, and the density of glandular trichomes was counted. Figure 2 In the figure, A represents the fluorescence microscopy observation results of the glandular trichomes on the leaves of the control group, Artemisia annua. Figure 2 B in the figure represents the fluorescence microscopy observation results of the glandular trichomes on the leaves of Artemisia annua in the melatonin-treated group. Figure 3 The results are statistical results of glandular hair density, based on Figure 2 , Figure 3 It can be seen that when the glandular trichome density of Artemisia annua in the control group was 17±4.7 per square millimeter, the glandular trichome density of Artemisia annua in the melatonin-treated group increased to more than 29.9±2.1 per square millimeter**, P<0.01 (T test).

[0047] Example 5: Scanning electron microscopy observation and statistical analysis of changes in artemisia glandular trichome density after long-term melatonin treatment

[0048] Mature leaves (5±0.2 mm in diameter) from the control group and the long-term melatonin-treated Artemisia annua were taken at the same size. Using a critical point desiccator, the samples were immersed in anhydrous ethanol, the drying chamber was sealed, the liquid CO2 cylinder valve was opened, and the critical point drying program was started. The dried samples were fixed to a Φ51 mm sample stage with conductive adhesive, and gold was sputtered onto the samples using vacuum sputtering for conductivity. The samples were observed and photographed using a HITACHI S3400N scanning electron microscope. Five different fields of view were randomly selected, and the density of glandular trichomes was statistically analyzed. Figure 4 In the figure, A represents the scanning electron microscopy observation results of glandular trichomes on the leaves of the control group, Artemisia annua. Figure 4 In the diagram, B represents the scanning electron microscopy observation results of the glandular trichomes on the leaves of Artemisia annua in the melatonin-treated group. Figure 5 The results are statistical results of glandular hair density, based on Figure 4 , Figure 5 It can be seen that when the glandular trichome density of Artemisia annua in the control group was 15.8±1.8 per square millimeter, the glandular trichome density of Artemisia annua treated with melatonin increased to more than 22.7±1.9 per square millimeter*, P<0.01 (T test).

[0049] Example 6: Determination of artemisinin content in Artemisia annua after melatonin treatment using HPLC-ELSD

[0050] 1. HPLC-ELSD conditions, system suitability, and preparation of standard solutions

[0051] HPLC: A Water Alliance 2695 system was used with a C-18 reversed-phase silica column (SymmetryShield™ C18, 5 μm, 250 × 4.6 mm, Waters). The mobile phase was methanol:water with a volume ratio of 70:30. The column temperature was 30℃, the flow rate was 1.0 mL / min, the injection volume was 10 μl, the sensitivity (AUFS = 1.0), and the theoretical plate number calculated based on the artemisinin peak was not less than 2000.

[0052] ELSD: Water Alliance 2420 system was used, with an evaporative light scattering detector drift tube temperature of 40°C, a gain of 7, and a carrier gas pressure of 5 bar.

[0053] Accurately weigh 2.0 mg of artemisinin standard (Sigma) and dissolve it completely in 1 mL of methanol to obtain a 2 mg / mL artemisinin standard solution, which is stored at -20°C for later use.

[0054] In this invention, when the mobile phase is methanol:water in a ratio of 70%:30%, the retention time of artemisinin is 5.1 min, with good peak shape. The theoretical plate number, calculated based on artemisinin, is not less than 2000.

[0055] 2. Construction of Standard Curve

[0056] The reference solution was injected at concentrations of 2 μl, 4 μl, 6 μl, 8 μl, and 10 μl under the corresponding chromatographic conditions, and the chromatograms and chromatographic parameters were recorded. Regression analysis was performed using peak area (Y) against the standard content (X, g). The study showed that artemisinin in this invention exhibited a good log-log linear relationship in the range of 4-20 μg. The log-log linear regression equation for the artemisinin reference standard was: Y = 1.02e+000X + 5.23e-001, R = 0.991983.

[0057] 3. Sample preparation and determination of artemisinin content

[0058] Take 2g of fresh Artemisia annua leaves from the upper, middle, and lower parts of the plant and dry them in an oven at 55℃ until constant weight. Then, knock the leaves off the dried branches and grind them into powder. Weigh about 0.1g of the dry powder into a 2mL centrifuge tube, add 2mL of methanol, sonicate at 40W for 30min, centrifuge at 5000rpm for 10min, and filter the supernatant through a 0.22μm filter membrane. This solution can then be used for HPLC-ELSD determination of artemisinin content.

[0059] Artemisinin content was determined by HPLC-ELSD with a sample injection volume of 20 μl. The artemisinin content (mg) in the sample was calculated by substituting the peak area into the linear regression equation, and then divided by the dry weight of Artemisia annua leaves (g) to calculate the artemisinin content in the Artemisia annua plant.

[0060] Figure 6 The changes in artemisinin content in Artemisia annua plants under long-term melatonin treatment, such as... Figure 6 As shown, when the artemisinin content in the control group of Artemisia annua was 20.8±0.9 mg / g DW, the artemisinin content in Artemisia annua treated with long-term melatonin was above 25.4±1.6 mg / g DW, which is about 20% higher, *, P<0.05 (T test); Figure 7 The changes in artemisinin content in Artemisia annua plants under short-term melatonin treatment, such as... Figure 7 As shown, when the artemisinin content in the control group of Artemisia annua was 22.7±1.2 mg / g DW, the artemisinin content in Artemisia annua treated with short-term melatonin was above 31.3±1.6 mg / g DW, an increase of about 40%, **, P<0.01 (T test).

Claims

1. The application of melatonin in enhancing artemisinin biosynthesis.

2. The application according to claim 1, characterized in that, Artemisinin biosynthesis is enhanced by activating the expression of key enzyme genes involved in artemisinin synthesis, wherein the genes include: At least one of the following: Amorpha fruticosa-4,11-diene synthase gene (ADS), Amorpha fruticosa-4,11-diene oxidase gene (CYP71AV1), Artemisinin Δ11(13) reductase gene (DBR2), and Artemisinin dehydrogenase gene (ALDH1).

3. A method for increasing the artemisinin content in Artemisia annua, characterized in that, By applying melatonin to Artemisia annua plants.

4. The method according to claim 3, characterized in that, Spray the leaves of Artemisia annua plants with a melatonin solution at a concentration of 80–120 μM for 3–4 days before harvest.

5. The method according to claim 3, characterized in that, Apply a melatonin solution with a concentration of 80–120 μM periodically from the seedling stage to the pre-flowering stage.

6. The method according to claim 4 or 5, characterized in that, The concentration of the melatonin solution is 100 μM.

7. The method according to claim 5, characterized in that, The periodic spraying is to spray once a week until the plant enters the pre-flowering stage.

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