Method for promoting synthesis of walnut callus serotonin by methyl jasmonate and tryptamine

Abstract

This invention relates to a method for promoting serotonin synthesis from walnut callus using methyl jasmonate and tryptophan, belonging to the fields of plant cell engineering and metabolic regulation technology. The invention includes the following steps: (1) selecting the embryo of a mature fresh walnut seed, disinfecting it, and placing it in an induction culture medium. After 14 days of induction culture, white callus tissue is formed; (2) after sterilizing the subculture culture medium and cooling it to 50°C, sterilized methyl jasmonate and tryptophan are added. After cooling, the callus tissue from step (1) is transferred to the subculture culture medium for culture; (3) after 10-20 days of culture, serotonin is extracted from the callus tissue. This invention can efficiently and cost-effectively promote the synthesis of large amounts of serotonin from deep-striped walnut callus tissue, providing an innovative solution for the large-scale production of plant-derived serotonin.

Description

Technical Field

[0001] This invention relates to the fields of plant cell engineering and metabolic regulation technology, and in particular to a method for promoting the synthesis of serotonin from walnut callus using methyl jasmonate and tryptophan. Background Technology

[0002] Serotonin (5-Hydroxytryptamine, 5-HT) is an important bioactive substance that plays a crucial role in plant stress resistance, growth and development, and fruit ripening. Simultaneously, serotonin participates in neural regulation, immune function, and cardiovascular health in the human body, possessing extremely high medicinal value.

[0003] Although serotonin exists in most plants, its synthesis efficiency under natural conditions is low, and its extraction is limited by seasonality and raw material supply. Traditional production methods rely on chemical synthesis, plant extraction, and microbial fermentation. Chemical synthesis requires multiple steps, produces many byproducts, and causes serious environmental pollution; plant extraction is limited by the seasonality of raw materials, has complex extraction processes, and is costly; microbial fermentation is difficult to scale up due to strain domestication challenges and competitive inhibition of metabolites.

[0004] Plant-derived serotonins are healthier and safer due to their natural properties, and plant callus tissue is a plant raw material that can be stably cultured in large quantities and whose metabolites are easy to extract. Therefore, it is necessary to provide a method that can promote the synthesis of high concentrations of 5-HT in deep-striped walnut callus tissue, thereby providing a solution for the large-scale production of plant-derived serotonins. Summary of the Invention

[0005] To overcome the problems existing in the background technology, the present invention provides a method for promoting the synthesis of serotonin from walnut callus using methyl jasmonate and tryptophan. This method can efficiently and cost-effectively promote the synthesis of large amounts of serotonin from deep-striped walnut callus tissue, providing an innovative solution for the large-scale production of plant-derived serotonin.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution: This invention provides a method for promoting serotonin synthesis in walnut callus using methyl jasmonate and tryptophan, specifically including the following steps: (1) Select the embryos of mature fresh walnut seeds, disinfect them and place them in an induction culture medium. After 14 days of induction culture, white callus tissue will form. (2) After the subculture medium is sterilized and cooled to 50°C, sterilized methyl jasmonate and tryptophan are added. After cooling, the callus tissue described in step (1) is transferred to the subculture medium for culture. (3) After culturing for 10-20 days, serotonin is extracted from the callus tissue.

[0007] In the above technical solution, the subculture medium in step (2) is MS solid medium supplemented with 0.5 mg / L NAA and 0.5 mg / L 6-BA.

[0008] In the above technical solution, the induction culture conditions in step (1) are culture at 25±2℃ under dark conditions.

[0009] In the above technical solution, the final concentration of methyl jasmonate in step (2) is 200 μM and the final concentration of tryptophan is 5 µg / mL.

[0010] In the above technical solution, the culture conditions in step (3) are: culture at 25±2℃ in the dark for 10 days.

[0011] In the above technical solution, the extraction method of serotonin in step (3) is as follows: freeze-dry the induced callus tissue and then crush it. Add 2 mL of methanol, shake for 5 min, centrifuge at 5000 rpm for 10 min, remove the supernatant, add 2 mL and 1 mL of methanol to the precipitate and repeat the extraction twice. Centrifuge and combine the supernatants, filter through a 0.22 μm filter membrane to obtain serotonin extract.

[0012] In the above technical solution, the walnut is a deep-striped walnut.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention, through the synergistic induction treatment of 200 μM MeJA and 5 µg / mL tryptophan, significantly increased the serotonin synthesis in deep-striped walnut callus tissue compared to treatments using MeJA, tryptophan, or tryptophan alone. This solves the problem of low synthesis efficiency in existing methods and provides a new technical approach for the regulation of secondary metabolites in plant metabolic engineering.

[0014] 2. The raw material of this invention is deep-striped walnut callus, which has low culture cost and fast proliferation rate; the amount of reagent used in the induction system is small, no complicated equipment is required, and the production cost is low.

[0015] 3. The entire synthesis process of this invention only involves callus culture, induction treatment, extraction and detection steps to obtain plant-derived serotonin. It is simple to operate, easy to scale up and promote, has no environmental pollution, and the product is highly safe. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0017] Figure 1 This invention relates to deep-textured walnut callus tissue; Figure 2 In Figure A, the serotonin content of *Hedysarum sylvestris* callus was obtained after treatment with MeJA, MeJA, and serotonin synthesis precursors (Trp and TAM) according to the present invention. In Figures B and C, the expression of synthase genes was obtained after treatment with MeJA, MeJA, and serotonin synthesis precursors (Trp and TAM) according to the present invention. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of this application will be described in further detail below with reference to the accompanying drawings.

[0019] Where the following description relates to the accompanying drawings, unless otherwise indicated, the same numbers in different drawings denote the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present invention. Rather, they are merely examples of apparatuses and methods consistent with some aspects of the invention as detailed in the appended claims.

[0020] In the description of this invention, it should be understood that the terms "first," "second," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance. Those skilled in the art can understand the specific meaning of these terms in this invention based on the specific circumstances. Furthermore, in the description of this invention, unless otherwise stated, "multiple" refers to two or more. "And / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, and B existing alone. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0022] Example 1: Preparation of deep-striped walnut callus Embryos from mature, fresh, deep-striped walnut seeds were sterilized and placed in induction medium MS + 2 mg / L 2,4-D + 0.25 mg / L 6-BA. After 14 days of culture in the dark (25 ± 2℃), white callus tissue was formed. Then, the callus tissue was transferred to subculture medium (MS + 0.5 mg / L NAA + 0.5 mg / L 6-BA) for amplification. Subculture was performed approximately every 25 days, and stable callus tissue was selected for use.

[0023] Example 2 The stable callus tissue from Example 1 was transferred to a new subculture medium (MS + 0.5 mg / L NAA + 0.5 mg / L 6-BA). The subculture medium was first sterilized and then cooled to approximately 50°C in a clean bench. Sterilized methyl jasmonic acid was added to the medium to a final concentration of 200 μM. The culture was carried out in the dark at 25 ± 2°C for 10–20 days. The cultured callus tissue was harvested at 10, 15, and 20 days.

[0024] Take 0.05-0.1g of induced and cultured callus tissue, grind it with liquid nitrogen, add 2 mL of methanol, sonicate for 5 min, then transfer it to a centrifuge tube, centrifuge at 5000 rpm for 10 min, take the supernatant, add 2 mL and 1 mL of methanol to the precipitate and repeat the above steps twice, centrifuge, combine the supernatants, filter through a 0.22μm filter membrane to obtain crude serotonin extract.

[0025] The crude serotonin extract was detected by high performance liquid chromatography (HPLC). The chromatographic conditions were as follows: C18 column (4.6 mm × 250 mm, 5 μm); mobile phase: methanol-0.05% trifluoroacetic acid (v / v 10:90); flow rate: 0.8 mL / min; column temperature: 25 °C; detection wavelength: 280 nm; injection volume: 4 μL.

[0026] Example 3 The stable callus tissue from Example 1 was transferred to a new subculture medium (MS + 0.5 mg / L NAA + 0.5 mg / L 6-BA). The subculture medium was first sterilized and then cooled to approximately 50°C in a clean bench. Sterilized methyl jasmonate and tryptophan were added to the medium, bringing the final concentrations of methyl jasmonate and tryptophan to 200 μM and 5 µg / mL, respectively. The culture was carried out in the dark at 25 ± 2°C for 10–20 days. The cultured callus tissue was harvested at 10, 15, and 20 days.

[0027] Take 0.05-0.1g of induced and cultured callus tissue, grind it with liquid nitrogen, add 2 mL of methanol, sonicate for 5 min, then transfer it to a centrifuge tube, centrifuge at 5000 rpm for 10 min, take the supernatant, add 2 mL and 1 mL of methanol to the precipitate and repeat the above steps twice, centrifuge, combine the supernatants, filter through a 0.22μm filter membrane to obtain crude serotonin extract.

[0028] The crude serotonin extract was purified by high performance liquid chromatography (HPLC). The chromatographic conditions were as follows: C18 column (4.6 mm × 250 mm, 5 μm); mobile phase: methanol-0.05% trifluoroacetic acid (v / v 10:90); flow rate: 0.8 mL / min; column temperature: 25 °C; detection wavelength: 280 nm; injection volume: 4 μL.

[0029] Example 4 The stable callus tissue from Example 1 was transferred to a new subculture medium (MS + 0.5 mg / L NAA + 0.5 mg / L 6-BA). The subculture medium was first sterilized and then cooled to approximately 50°C in a clean bench. Sterilized methyl jasmonate and tryptophan were added to the medium, bringing the final concentrations of methyl jasmonate and tryptophan to 200 μM and 5 µg / mL, respectively. The medium was then incubated in the dark at 25 ± 2°C for 10–20 days. The cultured callus tissue was harvested at 10, 15, and 20 days.

[0030] Take 0.05-0.1g of induced and cultured callus tissue, grind it with liquid nitrogen, add 2 mL of methanol, sonicate for 5 min, then transfer it to a centrifuge tube, centrifuge at 5000 rpm for 10 min, take the supernatant, add 2 mL and 1 mL of methanol to the precipitate and repeat the above steps twice, centrifuge, combine the supernatants, filter through a 0.22μm filter membrane to obtain crude serotonin extract.

[0031] The crude serotonin extract was detected by high performance liquid chromatography (HPLC). The chromatographic conditions were as follows: C18 column (4.6 mm × 250 mm, 5 μm); mobile phase: methanol-0.05% trifluoroacetic acid (v / v 10:90); flow rate: 0.8 mL / min; column temperature: 25 °C; detection wavelength: 280 nm; injection volume: 4 μL.

[0032] Example 5: Blank Control The stable callus tissue from Example 1 was transferred to a fresh subculture medium (MS + 0.5 mg / L NAA + 0.5 mg / L 6-BA) and cultured in the dark at 25 ± 2 °C for 10–20 days. The cultured callus tissue was harvested at 10, 15, and 20 days of culture.

[0033] Take 0.05-0.1g of induced and cultured callus tissue, grind it with liquid nitrogen, add 2 mL of methanol, sonicate for 5 min, then transfer it to a centrifuge tube, centrifuge at 5000 rpm for 10 min, take the supernatant, add 2 mL and 1 mL of methanol to the precipitate and repeat the above steps twice, centrifuge, combine the supernatants, filter through a 0.22μm filter membrane to obtain crude serotonin extract.

[0034] The crude serotonin extract was detected by high performance liquid chromatography (HPLC). The chromatographic conditions were as follows: C18 column (4.6 mm × 250 mm, 5 μm); mobile phase: methanol-0.05% trifluoroacetic acid (v / v 10:90); flow rate: 0.8 mL / min; column temperature: 25 °C; detection wavelength: 280 nm; injection volume: 4 μL.

[0035] The expression levels of serotonin synthase genes (JsTDC1 and JsT5H) in Examples 2-5 were detected using qRT-PCR. -ΔΔCT The relative expression levels of the gene were calculated using the method described above, with the GADPH gene as an internal reference. Gene-specific primers are shown in Table 1.

[0036] Table 1 Primer Correspondence Table The results showed that when the deep-striped walnut callus was treated with MeJA and its precursor simultaneously, the serotonin content in Example 4 was significantly higher than that in Examples 2, 3, and 5 at 10, 15, and 20 days of induction. In particular, the serotonin content was the highest at 10-15 days, demonstrating that the combination of 200 μM methyl jasmonate and 5 μg / mL tryptophan has a synergistic effect and is the optimal solution for promoting serotonin synthesis in deep-striped walnut callus.

[0037] Expression levels of serotonin synthase genes (JsTDC1 and JsT5H) were detected. Results showed that treatment with 200 μM methyl jasmonic acid and 5 μg / mL tryptophan (MeJA + Trp) did not significantly differ from treatment with 200 μM methyl jasmonic acid alone. However, treatment with 200 μM methyl jasmonic acid and 5 μg / mL tryptophan (MeJA + TAM) resulted in higher JsTDC1 expression levels compared to treatments with 200 μM methyl jasmonic acid alone and 200 μM methyl jasmonic acid and 5 μg / mL tryptophan. In conclusion, treatment with 200 μM methyl jasmonic acid and 5 μg / mL tryptophan in *Hedysarum sylvestris* callus can promote the production of large amounts of serotonin.

[0038] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A method for promoting serotonin synthesis from walnut callus using methyl jasmonate and tryptophan, characterized in that: The method specifically includes the following steps: (1) Select the embryos of mature fresh walnut seeds, disinfect them and place them in an induction culture medium. After 14 days of induction culture, white callus tissue will form. (2) After the subculture medium is sterilized and cooled to 50°C, sterilized methyl jasmonate and tryptophan are added. After cooling, the callus tissue described in step (1) is transferred to the subculture medium for culture. (3) After culturing for 10-20 days, serotonin is extracted from the callus tissue.

2. The method for promoting serotonin synthesis from walnut callus using methyl jasmonate and tryptophan according to claim 1, characterized in that: The subculture medium described in step (2) is MS solid medium supplemented with 0.5 mg / L NAA and 0.5 mg / L 6-BA.

3. The method for promoting serotonin synthesis from walnut callus using methyl jasmonate and tryptophan according to claim 1, characterized in that: The induction culture conditions described in step (1) are: culture at 25±2℃ in the dark.

4. The method for promoting serotonin synthesis from walnut callus using methyl jasmonate and tryptophan according to claim 1, characterized in that: In step (2), the final concentration of methyl jasmonate is 200 μM and the final concentration of tryptophan is 5 µg / mL.

5. The method for promoting serotonin synthesis from walnut callus using methyl jasmonate and tryptophan according to claim 1, characterized in that: The culture conditions described in step (3) are: culture at 25±2℃ in the dark for 10 days.

6. The method for promoting serotonin synthesis from walnut callus using methyl jasmonate and tryptophan according to claim 1, characterized in that: The extraction method of serotonin in step (3) is as follows: freeze-dry the treated callus tissue and then crush it. Add 2 mL of methanol, shake for 5 min, centrifuge at 5000 rpm for 10 min, remove the supernatant, add 2 mL and 1 mL of methanol to the precipitate and repeat the extraction twice. Centrifuge and combine the supernatants, filter through a 0.22 μm filter membrane to obtain serotonin extract.

7. The method for promoting serotonin synthesis from walnut callus using methyl jasmonate and tryptophan according to claim 1, characterized in that: The walnuts mentioned are deep-striped walnuts.

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