Method for producing medicinal ingredient aucubin by induction and culture of eucommia ulmoides hairly root

A technology of aucubin and medicinal ingredients, which is applied in the field of eucommia eucommia hairy root induction and cultivation to produce medicinal ingredient aucubin, which can solve the problem of non-temporary effects of restoration of production, long growth cycle of eucommia ulmoides, endangered eucommia, etc. problem, to achieve the effect of high-quality sustainable use

Inactive Publication Date: 2009-02-04
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to excessive digging by people, Eucommia ulmoides is on the verge of extinction in most areas. In addition, Eucommia ulmoides has a long growth cycle, and the restoration of production will not be effective immediately.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Obtaining sterile explants of Eucommia ulmoides

[0033] Method: Using explants to establish sterile explants of Eucommia ulmoides

[0034] Fresh Eucommia ulmoides seeds (discarded seed coat) of the same year, 0.4mg·L -1 Soak in gibberellin (GA) for 24 hours; then use 0.1% (M / V) mercury liter (HgCl 2 ) solution for 12 minutes, rinsed with sterile water for 5 times; then blot the surface moisture with sterile paper, and inoculate it in the basic medium (the medium is contained in a 150mL Erlenmeyer flask, sterilized at 121°C for 20 minutes) , the formula of the medium is: MS+0.4mg·L -1 GA, 30g·L -1 Sucrose, pH value is 5.8, then add 7.5g·L -1 of agar powder. Cultivate leaves of Eucommia ulmoides in a light incubator under the following conditions: 25°C, 12h·d -1 Lighting, the light intensity is 55μmol·m -2 ·s -1 . After 40 days, the Eucommia ulmoides seeds go through three stages of imbibition, dew whitening and seedling growth to induce sterile Eucommia ulmo...

Embodiment 2

[0036] Obtaining Hairy Roots from Eucommia Eucommia by Genetic Transformation with Agrobacterium rhizogenes

[0037] 1. Agrobacterium rhizogenes C58C1 (cultivated by the Department of Bioengineering and Biotechnology, Southwest University). Take out 200 μl from the ultra-low temperature refrigerator before use, and inoculate it in 25ml YEB liquid medium (add rifampicin to a final concentration of 40mg L -1 ), 28°C, 200rpm shaking and culturing twice to revive the cells.

[0038] 2. Add acetosyringone two hours before the end of the second activation culture to make the final concentration reach 100 μmol L -1 OD 600 When = 0.3, add acetosyringone, continue 28°C, 200rpm shaking culture, bacterial solution OD 600 =0.5, it can be used for conversion.

[0039] 3. Centrifuge at 4000 rpm for 10 minutes at room temperature, discard the supernatant, and use an equal volume of MS liquid medium (containing 100 μmol L -1 Acetosyringone) suspension, at 28 ° C, 200rp shaking culture ...

Embodiment 3

[0044] Molecular detection of hairy roots of Eucommia ulmoides

[0045] 1. Extraction of Eucommia hairy root genomic DNA, the method is as follows:

[0046] (1) Take 200 mg of hairy roots cultured in liquid for two weeks, filter, wash with 10 mL of distilled water, blot fully, freeze in liquid nitrogen, and grind into powder.

[0047] (2) Add 500 μL of extraction buffer (3% mercaptoethanol) to a 1.5 mL Eppendorf tube, fully shake in a 65°C water bath for 50 minutes, and mix by inverting every 5 minutes.

[0048] (3) Centrifuge at 4°C, 12,000 rpm for 10 minutes.

[0049] (4) Aspirate the supernatant, add 500 μL of phenol: chloroform: isoamyl alcohol (25:24:1), mix gently, and let stand for 5 minutes to separate layers.

[0050] (5) Centrifuge at 12,000 rpm for 10 minutes at room temperature.

[0051] (6) Aspirate about 350 μL of the supernatant, add an equal volume of chloroform:isoamyl alcohol (24:1), mix gently, and let stand for 5 minutes until the layers are separated....

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Abstract

The present invention provides a method for producing aucubin used as medicinal ingredient by the induction of Eucommia fairy root, which obtains the Eucommia fairy root by genetic transformation for the production of the aucubin. The method includes the following processes: Agrobacterium rhizogenes are used to genetically transform Eucommia ulmoides Oliv. in order to obtain the Eucommia fairy root, which is determined as the genetically transformed fairy root by molecular detection, and HPLC detection indicates that the Eucommia fairy root obtained by genetic transformation can produce the aucubin. The method can provide novel sustainable medicine source for the production of the aucubin used as medicinal ingredient.

Description

technical field [0001] The invention belongs to the fields of molecular biology, physiology, breeding, genetic engineering and the like, and relates to a method for inducing and cultivating hairy roots of Eucommia ulmoides to produce the medicinal ingredient aucubin, in particular to Agrobacterium rhizogenes genetically transforming eucommia and obtaining the produced Specific procedure of aucubin in the hairy roots of Eucommia ulmoides. The invention also provides hairy root of Eucommia ulmoides with high yield of aucubin obtained by genetic engineering technology and its cultured progeny. Background technique [0002] Plant secondary metabolites have very important economic functions, and have broad application prospects in industrial and agricultural production and people's daily life. Among them, their status in the development of natural medicines is particularly eye-catching. However, no effective or effective ones have been found so far. Sophisticated synthesis metho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N5/10C12P17/06A01H1/00A01H5/06
Inventor 孙敏雷桅杨蕊孙一铭
Owner SOUTHWEST UNIVERSITY
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