Method for efficiently inducing hairy roots of euphorbia helioscopia for producing ingenol

Abstract

The invention discloses a method for efficiently inducing hairy roots of begonia for producing ingenol. The obtained hairy roots can be used for extracting an active substance ingenol with the content higher than that in an original plant. The method comprises the following steps: 1) preparing a sterile explant; (2) activating agrobacterium and preparing a bacterium solution; (3) inducing hairy roots; (4) carrying out degerming induction culture; (5) subculture; and 6) amplification culture of hairy roots. By applying the induction culture method disclosed by the invention, efficient induction and large-scale culture of the euphorbia hairy roots can be realized, and the hairy roots of the euphorbia which grow quickly, are stable and good in state and contain a large amount of ingenol are obtained; the technical support is provided for the production of the active euphorbia pekinensis diterpenoid compound of the medicinal plant, namely the lepidogramme, and the development and utilization of medicinal resources.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a method for inducing and culturing hairy roots of malus ferox by inducing agrobacterium rhizogenes. The content of active substance ingenol in the hairy roots of malus ferox is higher than that in the original plant. Background Art

[0002] Begonia Euphorbia milii Ch. des Moulins] is a genus of the Euphorbiaceae family (Euphorbiaceae). Euphorbia ) is a perennial creeping shrub, also known as tiger thorn plum, unicorn thorn, etc. It is native to Africa and widely cultivated in tropical and temperate regions. It is mostly cultivated as an ornamental plant in the north and south of my country, and is commonly seen in parks, botanical gardens and courtyards. The iron begonia is also a medicinal plant. Its stems, leaves, roots and latex are used as medicine. It is bitter and cool in nature, slightly toxic, and enters the heart meridian. It has the effects of detoxification, blood circulation, pus discharge and water expulsion. Studies have shown that the chemical components of the iron begonia are mainly flavonoids, triterpenes, diterpenes and phenols, among which are rich in ingenol ester diterpenoid compounds unique to Euphorbiaceae plants. The parent nucleus ingenol of this type of compound is of great value and is the raw material for the industrial semi-synthetic production of ingenol methyl butylate, a drug for the treatment of actinic keratosis. However, ingenol is only distributed in a few Euphorbia plants, and the yield of ingenol isolated from plants is very low, so the cost of semi-synthetic ingenol methyl butylate has remained high. In 2013, LEO Pharmaceuticals and the Scripps Research Institute in the United States developed a 14-step total synthesis method for ingenol, but due to the difficulty of the process and the extremely low yield (only about 1%), it is difficult to commercialize.

[0003] Hairy roots are generated by inoculating Agrobacterium rhizogenes ( Agrobacterium rhizogenes ) The T-DNA fragment on the Ri plasmid is inserted, integrated into the genome of the plant cell and expressed, inducing adventitious roots to grow from the plant cells. The hairy roots basically maintain the characteristics of the original roots of the plant in morphology, and have the complete metabolic pathways in the original roots in physiology. They can synthesize the secondary metabolites in the original plant, and the yield of secondary metabolites synthesized by the hairy roots may be higher than that of the original plant. In addition, hairy root culture also has the advantages of rapid growth, physiological and biochemical and genetic stability, absence of chimeras, and hormone autotrophy. The production of active secondary metabolites using hairy root culture technology has been successfully applied in many medicinal plants, such as Mongolian Astragalus, Licorice, Uncaria, and Vaccaria segetalis.

[0004] At present, there is no report on the induction and cultivation technology of hairy roots of thorn begonia. The present invention provides a method for efficiently inducing and cultivating hairy roots of thorn begonia, and the obtained hairy roots can be used to extract ingenol with a higher content than that in the original plant. The present invention provides a new way to efficiently obtain active euphorbia diterpenoid compounds including ingenol, and also lays a foundation for the further development and utilization of medicinal resources of thorn begonia. Summary of the invention

[0005] The invention aims to provide a method for efficiently inducing hairy roots of Malus ferox to produce ingenol, and a large amount of ingenol can be extracted from the hairy roots obtained by the method.

[0006] In order to achieve the above object, the present invention provides the following technical solutions: A method for efficiently inducing and culturing hairy roots of Malus ferox comprises the following steps: 1) Preparation of sterile explants: Use the pedicels of Malus ferox as explants, sterilize them in 75% ethanol for 45 seconds, rinse them with sterile water twice, sterilize them with 0.1% mercuric chloride for 5 minutes, rinse them with sterile water 4-5 times, cut them into 1-1.5 cm lengths, and inoculate them into 1 / 2MS solid culture medium supplemented with 30 g / L sucrose and 2.5-3.0 g / L phytagel, with a pH of 5.8. After inoculation, pre-culture them at 25°C in the dark for 5-7 days. 2) Activation of Agrobacterium and preparation of bacterial solution: Take 50 μL of Agrobacterium rhizogenes strain Ar A4 stored at -80℃ and add it to 5mL YEB liquid medium containing 50 mg / L kanamycin. After shaking culture at 28℃ and 180-200 r / min for 24 h, take 1mL of bacterial solution and inoculate it into 50mL YEB liquid medium containing 50 mg / L kanamycin. Shake and culture under the same conditions until the bacterial solution OD reaches 0. 600 = 0.6-0.8, centrifuge at 5000 r / min for 8-10 min, discard the supernatant, and resuspend the bacteria in 50 mL of MS liquid medium containing 80-100 μmol / L acetosyringone to obtain activated Agrobacterium rhizogenes culture solution; 3) Induction of hairy roots: Place the pre-cultured explants into the prepared infection solution for 4-6 min, remove the explants, dry the surface solution with sterile filter paper, place on 1 / 2MS solid medium in the same step 1), and culture at 25°C in the dark for 3 days; 4) Sterilization induction culture: the explants after the co-culture are placed on a sterilization medium for sterilization induction culture, the sterilization medium is: 1 / 2MS medium + ceftriaxone sodium, the initial concentration of ceftriaxone sodium is 400 mg / L, the medium is changed every 5 to 7 days and the concentration of ceftriaxone sodium is gradually reduced until the Agrobacterium rhizogenes is completely removed and no more ceftriaxone sodium is added; 5) Subculture: taking the hairy roots after thorough sterilization and inoculating them on a subculture medium for subculture, wherein the subculture medium is 1 / 2MS solid culture medium; 6) Hairy root expansion culture: The hairy roots from step 5) were transferred into 1 / 2MS liquid culture medium and expanded at 25°C and 100-120 rpm / min. The culture was subcultured every 10 days to obtain a large number of growing hairy roots of Malus ferox.

[0007] The Agrobacterium rhizogenes strain used for infection in step 2) is Ar A4, and the bacterial solution is shaken and cultured until OD 600 =0.6-0.8, centrifuge, discard the supernatant and resuspend the bacteria, add MS liquid culture medium containing acetosyringone at a concentration of 80-100 μmol / L.

[0008] The above step 4) the sterilization induction culture process specifically includes: the explants after co-culture are first placed on a sterilization culture medium of 1 / 2MS solid basic culture medium + 400 mg / L ceftriaxone sodium for sterilization culture, and after hairy roots are visibly grown and there are no visible bacterial plaques on the surface of the culture medium and the explants, the concentration of ceftriaxone sodium can be gradually reduced during subculture, each time by 50 to 100 mg / L, until no ceftriaxone sodium is added and hairy roots that are completely sterilized are obtained.

[0009] The beneficial effects of the invention are as follows: the invention provides a method for efficiently inducing and culturing hairy roots of Malus ferox, wherein the pedicels of Malus ferox are used as explants, and the hairy roots of Malus ferox with rapid growth and good stability are obtained by induction with rhizogenes Ar A4; after the hairy roots are collected and freeze-dried, ingenol and a variety of euphorbia diterpenoid compounds can be extracted, thereby providing technical support for the production of active secondary metabolites of the medicinal plant Malus ferox and the further development and utilization of medicinal resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The hairy roots grow from the pedicels of Malus truncatum infected by Agrobacterium rhizogenes Ar A4; Figure 2 The hairy roots of Malus ferox grown on 1 / 2MS solid medium; Figure 3 The hairy roots of Malus ferox grown on 1 / 2MS liquid medium; Figure 4 Hairy roots rolB and rolC Gene PCR identification results, in the figure: M is DL2000 marker; 1 is the original root of non-transformed A. ferox (negative control); 2 and 3 are different root systems of A. ferox hairy roots; 4 is the plasmid of Agrobacterium rhizogenes (positive control);

[0011] Figure 5 This is a comparative bar graph of the determination of ingenol content in the hairy roots of Malus thomsoniae and various parts of the original plant. Implementation

[0012] The present invention is described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention. Example

[0013] 1. Preparation of sterile explants: The pedicels of the Chinese thorn begonia were used as explants, sterilized in 75% ethanol for 45 seconds, rinsed twice with sterile water, sterilized with 0.1% mercuric chloride for 5 minutes, rinsed five times with sterile water, cut into 1 cm lengths, inoculated into 1 / 2MS medium, supplemented with 30 g / L sucrose and 2.8 g / L phytagel, pH 5.8, and pre-cultured at 25°C in the dark for 5 days; 2. Preparation of infection solution for inducing hairy roots: Take 50 μL of Agrobacterium rhizogenes strain Ar A4 stored at -80℃, add it to 5mL YEB liquid medium containing 50 mg / L kanamycin, and culture it at 28℃, 180 r / min for 24 h. Take 1mL of bacterial solution and inoculate it into 50mL YEB liquid medium containing 50 mg / L kanamycin, and culture it under the same shaking conditions. 600 = 0.6, centrifuge at 5000 r / min for 8 min, discard the supernatant, and resuspend the bacteria in 50 mL of MS liquid medium containing 80 μmol / L acetosyringone to obtain activated Agrobacterium rhizogenes culture medium; 3. Induction of hairy roots: Place the pedicels of Malus truncatum after pre-culture into the infection solution. Take out the explants after 6 minutes of infection, dry the surface of the bacterial solution with sterile filter paper, place them on 1 / 2MS solid culture medium, and culture them in the dark at 25℃ for 3 days. 4. Sterilization induction culture of hairy roots: After co-culture, the explants were transferred to sterilization medium for dark sterilization culture at 25°C. The sterilization medium was 1 / 2MS medium + cefotaxime sodium, with an initial concentration of 400 mg / L. The medium was changed every 7 days, and the concentration of cefotaxime sodium was reduced by 100 mg / L each time. White hairy roots were observed after 19 days of sterilization culture (see attached). Figure 1 After 4 subcultures, cefotaxime sodium was no longer added to the culture medium, and completely sterilized hairy roots were obtained (see Appendix Figure 2 ); 5. Subculture: Take the thoroughly sterilized hairy roots and inoculate them into 1 / 2MS solid culture medium for subculture, once every 10 days; 6. Expansion of hairy roots: Transfer the hairy roots of the thorn begonia to 1 / 2MS liquid culture medium, culture at 25℃ in the dark, 130 rpm / min shaking, subculture once every 10 days, and obtain a large number of fast-growing hairy roots of the thorn begonia (see attached Figure 3 ); 7. PCR detection of hairy roots of A. ferrum: TIANGEN kit (DP305) was used to extract genomic DNA of hairy roots and untransformed primary roots of A. ferrum. PCR amplification of the T-DNA on the Ri plasmid of Agrobacterium rhizogenes rol B and rol C gene sequence, used rol The upstream primer sequence for gene B was 5′-CGAGGGGATCCGATTTGCTTT-3′, and the downstream primer sequence was 5′-GACGCCCTCCTCGCCTTCCT-3′; rol The upstream primer sequence of the C gene was 5'-CTCCTGACATCAAACTCGTC-3', and the downstream primer sequence was 5'-TGCTTCGAGTTATGGGTACA-3'. The PCR reaction conditions were 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles; 72℃ final extension for 10 min. The amplified products were analyzed by 1.0% agarose gel electrophoresis. The PCR amplification results showed that the DNA of the hairy roots of Malus truncatum was amplified. rol B and rol C gene fragment, and amplified from Agrobacterium rhizogenes Ar A4 plasmid DNA rol B and rol C gene fragments are of the same size ( rol B: 622 bp; rol C: 586 bp), the original root DNA of normal plants was not amplified rol B and rol C gene fragment, proving that the T-DNA of the Ri plasmid in Agrobacterium rhizogenes Ar A4 has been successfully integrated into the hairy root gene of Malus ferrugide (see Appendix Figure 4 );

[0014] 8. Determination of the content of ingenol in the hairy roots of Euphorbia cerasifera: The hairy roots of Euphorbia cerasifera were quickly frozen in liquid nitrogen, and then placed in a freeze dryer for freeze drying. After freeze drying, they were ground into uniform powder using a powder grinder. Weigh 50 mg of freeze-dried powder of the hairy roots, add 10 mL of methanol and ultrasonically extract for 30 min, take 5 mL of the supernatant, add 5% KOH solution, adjust the pH to 13, place in a 45°C water bath for hydrolysis for 30 min, and adjust the pH to neutral with 0.2 mol / L acetic acid solution to obtain an alkaline hydrolyzate. After adding water to 25 mL, pass through the SPE column, elute with 10 mL of methanol, and concentrate to dryness under reduced pressure. Dissolve in 5 mL of chromatographic methanol to prepare a sample with a concentration of 50 mg / mL for liquid quality detection. At the same time, different concentrations of ingenol standards were prepared to draw standard curves, and the ingenol content in the hairy roots of Euphorbia cerasifera was calculated. The results showed that the ingenol content in the hairy roots of Euphorbia cerasifera was significantly higher than the ingenol content in various tissues of the original plant (see Appendix Figure 5 ), reaching 593.14 mg / kg (dry weight). Example

[0015] 1. Preparation of sterile explants: The pedicels of the Chinese thorn begonia were used as explants, sterilized in 75% ethanol for 45 seconds, rinsed twice with sterile water, sterilized with 0.1% mercuric chloride for 5 minutes, rinsed six times with sterile water, cut into 1.5 cm lengths, inoculated into 1 / 2MS medium, supplemented with 30 g / L sucrose and 2.6 g / L phytagel, pH 5.8, and pre-cultured at 25°C in the dark for 7 days; 2. Preparation of infection solution for inducing hairy roots: Take 50 μL of Agrobacterium rhizogenes strain Ar A4 stored at -80℃, add it to 5mL YEB liquid medium containing 50 mg / L kanamycin, and culture it at 28℃ and 200 r / min for 24 h. Take 1mL of the bacterial solution and inoculate it into 50mL YEB liquid medium containing 50 mg / L kanamycin, and culture it under the same shaking conditions. When the bacterial solution OD600 = 0.8, centrifuge it at 5000 r / min for 8 min, discard the supernatant, and resuspend the bacteria in 50mL MS liquid medium containing 100 μmol / L acetosyringone to obtain the activated Agrobacterium rhizogenes bacterial solution; 3. Induction of hairy roots: Place the pre-cultured pedicels of Malus ferox in the infection solution, take them out after 8 minutes of infection, dry the surface solution with sterile filter paper, place them on 1 / 2MS solid culture medium, and culture them in the dark at 25℃ for 3 days; 4. Sterilization induction culture of hairy roots: After co-culture, the explants were transferred to sterilization medium for dark sterilization culture at 25°C. The sterilization medium was 1 / 2MS medium + cefotaxime sodium, with an initial addition concentration of 400 mg / L. The medium was changed every 5 days, and the concentration of cefotaxime sodium was reduced by 50 mg / L each time. After 20 days of sterilization culture, white hairy roots were observed to grow. After 7 subcultures, cefotaxime sodium was no longer added to the culture medium, and completely sterilized hairy roots were obtained. 5. Subculture: Inoculate the thoroughly sterilized hairy roots onto the subculture medium for subculture at 25°C in the dark. The subculture medium is 1 / 2MS solid medium. 6. Expansion culture of hairy roots: transfer the hairy roots of the thorn begonia to 1 / 2MS liquid culture medium, culture at 25℃ in the dark, 110 rpm / min shaking, subculture once every 14 days, and obtain a large number of fast-growing hairy roots of the thorn begonia; 7. PCR detection of hairy roots of A. ferrum: TIANGEN kit (DP305) was used to extract genomic DNA of hairy roots and untransformed primary roots of A. ferrum. PCR amplification of the T-DNA on the Ri plasmid of Agrobacterium rhizogenes rol B and rol C gene sequence, used rol The upstream primer sequence for gene B was 5′-CGAGGGGATCCGATTTGCTTT-3′, and the downstream primer sequence was 5′-GACGCCCTCCTCGCCTTCCT-3′; rol The upstream primer sequence of the C gene was 5'-CTCCTGACATCAAACTCGTC-3', and the downstream primer sequence was 5'-TGCTTCGAGTTATGGGTACA-3'. The PCR reaction conditions were 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles; 72℃ final extension for 10 min. The amplified products were analyzed by 1.0% agarose gel electrophoresis. The PCR amplification results showed that the DNA of the hairy roots of Malus truncatum was amplified. rol B and rol C gene fragment, and amplified from Agrobacterium rhizogenes Ar A4 plasmid DNA rol B and rol C gene fragments are of the same size ( rol B: 622 bp; rol C: 586 bp), the original root DNA of normal plants was not amplified rol B and rol C gene fragment, proving that the T-DNA of the Ri plasmid in Agrobacterium rhizogenes Ar A4 has been successfully integrated into the hairy root genes of Malus ferrugide;

[0016] 8. Determination of the content of ingenol in the hairy roots of Euphorbia cerasifera: The determination method is the same as in Example 1. First, the hairy roots of Euphorbia cerasifera are put into liquid nitrogen for quick freezing, and then placed in a freeze dryer for freeze drying. After freeze drying, they are beaten into uniform powder with a powdering machine. Weigh 50 mg of the hairy root freeze-dried powder, add 10 mL of methanol for ultrasonic extraction for 30 min, take 5 mL of the supernatant, add 5% KOH solution, adjust the pH to 13, place in a 45°C water bath for hydrolysis for 30 min, and adjust the pH to neutral with 0.2 mol / L acetic acid solution to obtain an alkaline hydrolyzate. After adding water to 25 mL, pass through the SPE column, elute with 10 mL of methanol, and concentrate to dryness under reduced pressure. After dissolving with 5 mL of chromatographic methanol, a sample with a concentration of 50 mg / mL is prepared for liquid quality detection. At the same time, different concentrations of ingenol standard solutions are prepared to draw a standard curve to calculate the ingenol content in the hairy roots of Euphorbia cerasifera. The results showed that the ingenol content in the hairy roots of Malus thunbergii was significantly higher than that in various tissues of the original plant, reaching 536.82 mg / kg (dry weight).

[0017] The above embodiments are only preferred implementations of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for efficiently inducing hairy roots of Malus ferox to produce ingenol, characterized in that The following steps are involved: 1) Preparation of sterile explants: Use the pedicels of Malus ferox as explants, sterilize them in 75% ethanol for 45 seconds, rinse them twice with sterile water, sterilize them with 0.1% mercuric chloride for 5 minutes, rinse them 5-6 times with sterile water, cut them into 1-1.5 cm lengths, and inoculate them into 1 / 2MS solid culture medium supplemented with 30 g / L sucrose and 2.5-3.0 g / L phytagel, with a pH of 5.

8. After inoculation, pre-culture them at 25°C in the dark for 5-7 days. 2) Activation of Agrobacterium and preparation of bacterial solution: Take 50 μL of Agrobacterium rhizogenes strain Ar A4 stored at -80℃ and add it to 5 mL of YEB liquid medium containing 50 mg / L kanamycin. After shaking culture at 28℃ and 180-200 r / min for 24 h, take 1 mL of bacterial solution and inoculate it into 50 mL of YEB liquid medium containing 50 mg / L kanamycin. Shake and culture under the same conditions until the bacterial solution OD reaches 0. 600 = 0.6-0.8, centrifuge at 5000 r / min for 8 min, discard the supernatant, and resuspend the bacteria in 50 mL of MS liquid medium containing 80-100 μmol / L acetosyringone to obtain activated Agrobacterium rhizogenes culture liquid; 3) Induction of hairy roots: Place the pre-cultured explants into the prepared infection solution for 4-6 minutes, remove the explants, dry the surface solution with sterile filter paper, place on 1 / 2MS solid medium in the same step 1), and culture at 25°C in the dark for 3 days; 4) Sterilization induction culture: the explants after the co-culture are placed on a sterilization medium for sterilization induction culture at 25°C in the dark. The sterilization medium is: 1 / 2MS medium + cefotaxime sodium. The initial concentration of cefotaxime sodium is 400 mg / L. The medium is changed every 5 to 7 days and the concentration of cefotaxime sodium is gradually reduced until the rhizogenes Agrobacterium is completely removed and no cefotaxime sodium is added. 5) Subculture: Take the thoroughly sterilized hairy roots and inoculate them on the subculture medium, which is 1 / 2MS solid medium, subculture once every 7 to 10 days, and culture at 25°C in the dark; 6) Expansion culture of hairy roots: Transfer the hairy roots from step 5) into 1 / 2MS liquid culture medium and culture them at 25°C in the dark with shaking at 110-130 rpm / min. Subculture every 10-14 days to obtain a large number of fast-growing hairy roots of Malus ferox.

2. The method according to claim 1, characterized in that The Agrobacterium rhizogenes strain used for infection in step 2) is Ar A4.

3. The method according to claim 1, characterized in that Step 2) Incubate the Agrobacterium rhizogenes solution with shaking until the OD 600 =0.6~0.8, and the concentration of acetosyringone added when resuspending the bacteria is 80~100 μmol / L.

4. The method according to claim 1, characterized in that Step 4) The sterilization induction culture process specifically includes: the explants after co-culture are first placed on a sterilization culture medium of 1 / 2MS solid basic culture medium + 400 mg / L ceftriaxone sodium for sterilization culture, and after hairy roots are visibly grown and there are no visible bacterial plaques on the surface of the culture medium and the explants, the concentration of ceftriaxone sodium can be gradually reduced during subculture, each time by 50 to 100 mg / L, until no ceftriaxone sodium is added and hairy roots that are completely sterilized are obtained.

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