A method for promoting biosynthesis of JuA in the root system of elaeagnus pungens seedlings

Abstract

The application belongs to the technical field of plant culture and relates to a method for promoting JuA biosynthesis of sour jujube seedling roots. By exploring exogenous substances for regulating JuA biosynthesis, it is found that adenosine triphosphate (ATP) and abscisic acid (ABA) can be used as exogenous substances for regulating JuA biosynthesis of sour jujube seedling roots, and then the optimal application concentration and method of the exogenous substances ATP or ABA in JuA biosynthesis are obtained, the production efficiency of JuA is improved, and a foundation is laid for the industrial production of JuA.

Description

Technical Field

[0001] This invention belongs to the field of plant culture technology, and more specifically relates to a method for promoting the biosynthesis of JuA in the roots of jujube seedlings. Background Technology

[0002] Sour jujube seed (Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex HFChow) is a traditional Chinese medicine with calming and sleep-aiding effects. Its main pharmacologically active component is jujuboside A (JuA), which exhibits significant pharmacological activities in sedation, sleep improvement, neuroprotection, antioxidation, and anti-inflammation. With the accelerating pace of life and the rapid aging of the population, sleep disorders are becoming increasingly prominent, leading to a growing market demand for sour jujube seed with good calming and sleep-aiding effects, resulting in a severe supply shortage. Besides sour jujube seed, JuA is also found in leaves, bark, root bark, and other tissues and organs, indicating that leaves, bark, and root bark can be potential sources for JuA production, but these sources suffer from low and unstable content. Exploring a technology for efficient JuA production is a core technology in this field. Previous studies have shown that the JuA synthesis activity in the roots of jujube seedlings is significantly affected by exogenous substances. Therefore, exploring the components of exogenous substances that regulate JuA biosynthesis and their optimal application concentration in JuA biosynthesis, so as to improve JuA production efficiency and lay an experimental foundation for industrialized JuA production, is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention provides a method for promoting JuA biosynthesis in the roots of jujube seedlings. By exploring exogenous substances that regulate JuA biosynthesis, and thereby obtaining the optimal application concentration and method of exogenous substances ATP or ABA in JuA biosynthesis, the efficiency of JuA production is improved, laying the foundation for industrialized JuA production.

[0004] To achieve the above objectives, the present invention provides a method for promoting JuA biosynthesis in the roots of jujube seedlings, comprising:

[0005] After applying exogenous substances ATP or ABA to the jujube seedlings, they were cultured again.

[0006] Furthermore, the concentration of the exogenous substance ATP or ABA is 0.01–0.50 mM.

[0007] Preferably, the concentration of the exogenous substance ATP or ABA is 0.05 to 0.10 mM.

[0008] Furthermore, the jujube seedlings are jujube seedlings with 2 to 4 true leaves.

[0009] Furthermore, the method for cultivating the jujube seedlings includes the following steps:

[0010] Sow jujube seeds in a culture medium, cover with a film after sowing, and incubate in an incubator for 28–30 days.

[0011] Preferably, the jujube seeds are jujube seeds with smooth seed coats, no cracks, plump, free from mold, free from insect infestation, and uniform in size.

[0012] Preferably, the culture medium comprises nutrient soil and vermiculite in a mass ratio of 1:1 and a moisture content of 25-35%.

[0013] Preferably, the sowing depth is 3 cm.

[0014] Preferably, the incubation temperature in the incubator is 25°C, and the light intensity is 150 μmol·m⁻¹. -2 ·s -1 The photoperiod is 12 hours of light and 12 hours of darkness, and the soil moisture is 30%.

[0015] Furthermore, the duration of the continued culture is 28–30 days.

[0016] On the other hand, the present invention also provides the application of exogenous substance ATP in promoting the biosynthesis of JuA in jujube roots.

[0017] On the other hand, the present invention also provides the application of exogenous substance ABA in promoting the biosynthesis of JuA in jujube roots.

[0018] It is worth noting that studies have found that ATP and ABA have no significant effect on the JuA content in jujube leaves, but both have a significant effect on the JuA content in the roots of jujube seedlings. This suggests that the receptors for ATP and ABA may only exist in the roots, and not in the above-ground tissues and organs, ultimately leading to an increase in JuA content in jujube roots without a significant impact on the JuA content in the leaves and jujube kernels.

[0019] As can be seen from the above technical solution, compared with the existing technology, it has the following beneficial effects:

[0020] This invention points out that ATP and ABA can be used as exogenous substances to regulate JuA biosynthesis in the roots of jujube seedlings. At the same time, by regulating the concentration of ATP and ABA, the JuA content in the roots of jujube seedlings is significantly increased, the JuA production efficiency is improved, and an experimental foundation is laid for the industrial production of JuA. Attached Figure Description

[0021] The accompanying drawings, which form part of this application, are used to provide a further understanding of this application. The illustrative embodiments and descriptions of this application are used to explain this application and do not constitute an undue limitation of this application. In the drawings:

[0022] Figure 1 The chromatogram is for a 20 μL JuA standard.

[0023] Figure 2 The image shows a chromatogram of JuA in the roots of jujube seedlings that were not treated with ATP or ABA in Comparative Example 1.

[0024] Figure 3 The image shows a chromatogram of JuA in the roots of jujube seedlings treated with 0.01 mM ATP in Example 1.

[0025] Figure 4 The image shows a chromatogram of JuA in the roots of jujube seedlings treated with 0.05 mM ATP in Example 2.

[0026] Figure 5 The image shows a chromatogram of JuA in the roots of jujube seedlings treated with 0.10 mM ATP in Example 3.

[0027] Figure 6 The image shows a chromatogram of JuA in the roots of jujube seedlings treated with 0.50 mM ATP in Example 4.

[0028] Figure 7 The image shows the JuA chromatogram of the roots of jujube seedlings treated with 0.01 mM ABA in Example 5.

[0029] Figure 8 The image shows the JuA chromatogram of the roots of jujube seedlings treated with 0.05 mM ABA in Example 6.

[0030] Figure 9 The image shows the JuA chromatogram of the roots of jujube seedlings treated with 0.10 mM ABA in Example 7.

[0031] Figure 10 The image shows the JuA chromatogram of the roots of jujube seedlings treated with 0.50 mM ABA in Example 8. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] In the examples and comparative examples, the culture medium consisted of nutrient soil and vermiculite in a mass ratio of 1:1.

[0034] Example 1

[0035] Methods to promote JuA biosynthesis in the roots of jujube seedlings:

[0036] Select jujube seeds that are smooth, free of cracks, plump, free of mold, free of insect damage, and uniform in size. Sow them in a seedling tray filled with culture medium at a depth of 3 cm. Maintain the humidity of the culture medium at (30±5)% and cover with a transparent lid. Place the seedling tray at 25℃ with a light intensity of 150 μmol·m². -2 ·s -1 The seedlings were cultured in an incubator with a photoperiod of 12h / 12h (light / dark) and a soil moisture of 30%. When the seedlings had 2-4 true leaves after 28-30 days of cultivation, ATP was added to the culture medium at a concentration of 0.01mM. Other conditions remained unchanged, and the seedlings were cultured for another 28-30 days.

[0037] Example 2

[0038] Methods to promote JuA biosynthesis in the roots of jujube seedlings:

[0039] Select jujube seeds that are smooth, free of cracks, plump, free of mold, free of insect damage, and uniform in size. Sow them in a seedling tray filled with culture medium at a depth of 3 cm. Maintain the humidity of the culture medium at (30±5)% and cover with a transparent lid. Place the seedling tray at 25℃ with a light intensity of 150 μmol·m². -2 ·s -1 The seedlings were cultured in an incubator with a photoperiod of 12h / 12h (light / dark) and a soil moisture of 30%. When the seedlings had 2-4 true leaves after 28-30 days of cultivation, ATP was added to the culture medium at a concentration of 0.05mM. Other conditions remained unchanged, and the seedlings were cultured for another 28-30 days.

[0040] Example 3

[0041] Methods to promote JuA biosynthesis in the roots of jujube seedlings:

[0042] Select jujube seeds that are smooth, free of cracks, plump, free of mold, free of insect damage, and uniform in size. Sow them in a seedling tray filled with culture medium at a depth of 3 cm. Maintain the humidity of the culture medium at (30±5)% and cover with a transparent lid. Place the seedling tray at 25℃ with a light intensity of 150 μmol·m². -2 ·s -1The seedlings were cultured in an incubator with a photoperiod of 12h / 12h (light / dark) and a soil moisture of 30%. When the seedlings had 2-4 true leaves after 28-30 days of cultivation, ATP was added to the culture medium at a concentration of 0.10mM. Other conditions remained unchanged, and the seedlings were cultured for another 28-30 days.

[0043] Example 4

[0044] Methods to promote JuA biosynthesis in the roots of jujube seedlings:

[0045] Select jujube seeds that are smooth, free of cracks, plump, free of mold, free of insect damage, and uniform in size. Sow them in a seedling tray filled with culture medium at a depth of 3 cm. Maintain the humidity of the culture medium at (30±5)% and cover with a transparent lid. Place the seedling tray at 25℃ with a light intensity of 150 μmol·m². -2 ·s -1 The seedlings were cultured in an incubator with a photoperiod of 12h / 12h (light / dark) and a soil moisture of 30%. When the seedlings had 2-4 true leaves after 28-30 days of cultivation, ATP was added to the culture medium at a concentration of 0.50mM. Other conditions remained unchanged, and the seedlings were cultured for another 28-30 days.

[0046] Example 5

[0047] Methods to promote JuA biosynthesis in the roots of jujube seedlings:

[0048] Select jujube seeds that are smooth, free of cracks, plump, free of mold, free of insect damage, and uniform in size. Sow them in a seedling tray filled with culture medium at a depth of 3 cm. Maintain the humidity of the culture medium at (30±5)% and cover with a transparent lid. Place the seedling tray at 25℃ with a light intensity of 150 μmol·m². -2 ·s -1 The seedlings were cultured in an incubator with a photoperiod of 12h / 12h (light / dark) and a soil moisture of 30%. When the seedlings had 2-4 true leaves after 28-30 days of cultivation, ABA was added to the culture medium at a concentration of 0.01mM. Other conditions remained unchanged, and the seedlings were cultured for another 28-30 days.

[0049] Example 6

[0050] Methods to promote JuA biosynthesis in the roots of jujube seedlings:

[0051] Select jujube seeds that are smooth, free of cracks, plump, free of mold, free of insect damage, and uniform in size. Sow them in a seedling tray filled with culture medium at a depth of 3 cm. Maintain the humidity of the culture medium at (30±5)% and cover with a transparent lid. Place the seedling tray at 25℃ with a light intensity of 150 μmol·m². -2 ·s -1The seedlings were cultured in an incubator with a photoperiod of 12h / 12h (light / dark) and a soil moisture of 30%. When the seedlings had 2-4 true leaves after 28-30 days of cultivation, ABA was added to the culture medium at a concentration of 0.05mM. Other conditions remained unchanged, and the seedlings were cultured for another 28-30 days.

[0052] Example 7

[0053] Methods to promote JuA biosynthesis in the roots of jujube seedlings:

[0054] Select jujube seeds that are smooth, free of cracks, plump, free of mold, free of insect damage, and uniform in size. Sow them in a seedling tray filled with culture medium at a depth of 3 cm. Maintain the humidity of the culture medium at (30±5)% and cover with a transparent lid. Place the seedling tray at 25℃ with a light intensity of 150 μmol·m². -2 ·s -1 The seedlings were cultured in an incubator with a photoperiod of 12h / 12h (light / dark) and a soil moisture of 30%. When the seedlings had 2-4 true leaves after 28-30 days of cultivation, ABA was added to the culture medium at a concentration of 0.10mM. Other conditions remained unchanged, and the seedlings were cultured for another 28-30 days.

[0055] Example 8

[0056] Methods to promote JuA biosynthesis in the roots of jujube seedlings:

[0057] Select jujube seeds that are smooth, free of cracks, plump, free of mold, free of insect damage, and uniform in size. Sow them in a seedling tray filled with culture medium at a depth of 3 cm. Maintain the humidity of the culture medium at (30±5)% and cover with a transparent lid. Place the seedling tray at 25℃ with a light intensity of 150 μmol·m². -2 ·s -1 The seedlings were cultured in an incubator with a photoperiod of 12h / 12h (light / dark) and a soil moisture of 30%. When the seedlings had 2-4 true leaves after 28-30 days of cultivation, ABA was added to the culture medium at a concentration of 0.50mM. Other conditions remained unchanged, and the seedlings were cultured for another 28-30 days.

[0058] Comparative Example 1

[0059] Cultivation of jujube seedlings:

[0060] Select jujube seeds that are smooth, free of cracks, plump, free of mold, free of insect damage, and uniform in size. Sow them in a seedling tray filled with culture medium at a depth of 3 cm. Maintain the humidity of the culture medium at (30±5)% and cover with a transparent lid. Place the seedling tray at 25℃ with a light intensity of 150 μmol·m². -2 ·s -1Cultured in an incubator with a photoperiod of 12h / 12h (light / dark) and soil moisture of 30% for 56-60 days.

[0061] Test case

[0062] 1. Preparation of reagents

[0063] Acetonitrile and methanol were chromatographic grade (Simark, Germany); adenosine triphosphate (ATP), abscisic acid (ABA), petroleum ether (60–90 °C), and anhydrous ethanol were analytical grade; JuA standard was purchased from Nanjing Yuanzhi Biotechnology Co., Ltd., batch number yz081921; ultrapure water.

[0064] 2. Determination of JuA

[0065] The preparation of standard solutions and the determination of samples are carried out according to the following method, the specific steps of which include:

[0066] (1) Take the roots of the cultured jujube seedlings as samples, wash them, dry them in an 80℃ drying oven to constant weight, and grind them into powder using a mortar and pestle.

[0067] (2) Accurately weigh 1.000g of powdered sample, place it in a wire-tethered filter paper bag, and put it in a Soxhlet extractor. Add 125mL of petroleum ether to a flat-bottomed flask and reflux to remove impurities. Discard the petroleum ether after 4 hours.

[0068] (3) After the petroleum ether in the sample powder has completely evaporated, transfer it to a 100mL round-bottom flask, add 20mL of anhydrous ethanol and 3-4 boiling stones, and heat under reflux for 2h.

[0069] (4) Place two layers of filter paper in the Buchner funnel and filter the refluxed solution.

[0070] (5) Pour the filtrate into a flask and rinse the flask with a small amount of 70% ethanol solution several times. Combine the washing solution with the filtrate.

[0071] (6) Evaporate the solution under vacuum of 1 MPa and 35-45°C until the solution is dry;

[0072] (7) Dissolve the solute that is coated on the flask wall after rotary evaporation in 3 mL of methanol, shake 3 to 5 times, and sonicate at 40 kHz for 10 min.

[0073] (8) Pipette the sample solution into a centrifuge tube, centrifuge for 1 min, and bring the supernatant to a final volume of 5 mL with methanol.

[0074] (9) The content of JuA was determined by high performance liquid chromatography (LC-16P, HPLC). The chromatograph was an Agilent 1220 Infinity LC with octadecylsilane-bonded silica gel as the packing material. The chromatographic column was an EclipsePlus 95AC18 (4.6 mm × 250 mm, 5 μm). The mobile phase was acetonitrile (A)-water (B); the flow rate was 1.0 mL·min. -1 The column temperature was 25°C, the evaporative light scattering detector was a Sanotac ELSD6000, the drift tube temperature was 65°C, and the gas flow rate was 1.00 L / min. -1 Inject 10 μL of sample and perform gradient elution according to the procedure in Table 1;

[0075] Table 1

[0076]

[0077] (10) Accurately weigh 0.1 mg of JuA standard and dissolve it in 1 mL of methanol. Precisely inject 5 μL and 20 μL of the standard solution into the HPLC system. The chromatogram of the 20 μL JuA standard is shown below. Figure 1 As shown, the logarithmic equation (Equation 1) is obtained using the external standard two-point method.

[0078] lnA = 1.334lnC + 4.238 (Equation 1)

[0079] In Equation 1, A is the peak area; C is the injection volume (mg).

[0080] 3. Experimental Results

[0081] 3.1 JuA content in the roots of jujube seedlings that have not been treated with ATP or ABA

[0082] The JuA content in the roots of jujube seedlings cultured in Comparative Example 1 was determined by HPLC. The result showed that the JuA content was (0.00431±0.00124) mg·g⁻¹. -1 .

[0083] The JuA chromatogram of the roots of jujube seedlings that were not treated with ATP or ABA in Comparative Example 1 is shown below. Figure 2 As shown.

[0084] 3.2 Effects of exogenous ATP on JuA content in the roots of Ziziphus jujuba seedlings

[0085] The JuA content in the roots of the jujube seedlings cultured in Examples 1-4 was determined by HPLC, and the results are shown in Table 2.

[0086] Table 2

[0087]

[0088] The chromatogram of JuA in the roots of jujube seedlings treated with 0.01 mM ATP in Example 1 is shown below. Figure 3 As shown;

[0089] The chromatogram of JuA in the roots of jujube seedlings treated with 0.05 mM ATP in Example 2 is shown below. Figure 4 As shown;

[0090] The chromatogram of JuA in the roots of jujube seedlings treated with 0.10 mM ATP in Example 3 is shown below. Figure 5 As shown;

[0091] The chromatogram of JuA in the roots of jujube seedlings treated with 0.50 mM ATP in Example 4 is shown below. Figure 6 As shown.

[0092] Using the JuA content of the jujube seedling roots in Comparative Example 1 as a control group, the data in Table 2 show that the JuA content in Example 1 increased by 20.77 times, the JuA content in Example 2 increased by 327.34 times, the JuA content in Example 3 increased by 326.52 times, and the JuA content in Example 4 increased by 3.35 times. This indicates that treatment with 0.05 mM and 0.10 mM ATP can significantly promote the JuA content of jujube seedling roots. When the concentration is too low (0.01 mM), the promoting effect is not obvious. However, when treated with 0.50 mM ATP, the jujube leaves showed obvious curling and wilting, resulting in a significant reduction in root quality and affecting the JuA content of the roots.

[0093] 3.3 Effects of exogenous ABA on JuA content in the roots of jujube seedlings

[0094] The JuA content in the roots of the jujube seedlings cultured in Examples 5-8 was determined by HPLC, and the results are shown in Table 3.

[0095] Table 3

[0096]

[0097] The chromatogram of JuA in the roots of jujube seedlings treated with 0.01 mM ABA in Example 5 is shown below. Figure 7 As shown;

[0098] The chromatogram of JuA in the roots of jujube seedlings treated with 0.05 mM ABA in Example 6 is shown below. Figure 8 As shown;

[0099] The chromatogram of JuA in the roots of jujube seedlings treated with 0.10 mM ABA in Example 7 is shown below. Figure 9 As shown;

[0100] The chromatogram of JuA in the roots of jujube seedlings treated with 0.50 mM ABA in Example 8 is shown below. Figure 10 As shown.

[0101] Using the JuA content of the jujube seedling roots in Comparative Example 1 as a control group, the data in Table 3 show that the JuA content in Example 5 increased by 7.84 times, the JuA content in Example 6 increased by 382.50 times, the JuA content in Example 7 increased by 37.68 times, and the JuA content in Example 8 increased by 2.96 times. This indicates that 0.05 mM and 0.10 mM ABA treatments can significantly promote the JuA content of jujube seedling roots. The promoting effect is most obvious after 0.05 mM ABA treatment. When the concentration is too low (0.01 mM), the promoting effect is not obvious. When treated with 0.50 mM ABA, the jujube leaves showed obvious curling and wilting, resulting in a significant reduction in root quality and affecting the JuA content of the roots.

[0102] As can be seen from the above experiments, ATP and ABA can be used as exogenous substances to regulate JuA biosynthesis in the roots of jujube seedlings. When the exogenous substance in the roots of jujube seedlings is ATP, the applicable concentration includes 0.01mM to 0.50mM, with 0.05mM being the preferred and 0.10mM being the next best. When the exogenous substance in the roots of jujube seedlings is ABA, the applicable concentration includes 0.01mM to 0.50mM, with 0.05mM being the preferred and 0.10mM being the next best.

[0103] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0104] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for promoting JuA biosynthesis in the roots of jujube seedlings, characterized in that, include: The seedling culture medium for jujube seedlings was supplemented with exogenous ATP and cultured continuously. The concentration of the exogenous substance ATP applied is 0.05 mM to 0.10 mM; The application of exogenous ATP is performed when the jujube seedlings have grown to have 2-4 true leaves. The duration of continued culture is 28-30 days.

2. The method according to claim 1, characterized in that, The method for culturing jujube seedlings before applying exogenous ATP includes the following steps: Sow jujube seeds in a culture medium, cover with a film after sowing, and incubate in an incubator for 28-30 days.

3. The method according to claim 2, characterized in that, The culture medium consists of nutrient soil and vermiculite in a 1:1 mass ratio, with a humidity of 25%–35%; the sowing depth is 3 cm; the incubation temperature is 25°C, and the light intensity is 150 μmol·m⁻². -2 ·s -1 The photoperiod is 12 hours of light and 12 hours of darkness, and the soil moisture is 30%.

4. The application of the method as described in any one of claims 1-3 in promoting JuA biosynthesis in jujube roots.

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