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Method for gaining woody datura stramonium hairy root generating hyoscyamine and hyoscine

A technology of scopolamine and datura, applied in the field of woody datura hairy root and its cultured progeny, can solve the problems of uncertain commercial prospects, excessive pesticide residues, and low active ingredients

Inactive Publication Date: 2008-07-30
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, the main techniques for obtaining woody mandala are wild materials and artificial cultivation, but there are disadvantages such as inconvenient natural reproduction, long production cycle, endangered wild resources, excessive pesticide residues and low active ingredients, making the commercial prospect of this method not yet clear. clear

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Obtaining of sterile explants of Datura japonica

[0022] Method: Using explants to establish axenic explants of Datura woody

[0023] Collect young young leaves of Datura arborica, soak them in 0.1% washing powder for 30 minutes, rinse them with tap water for several times; then soak them with 75% (V / V) ethanol for 30 seconds, rinse them with sterile water for 3 times; %(M / V) Mercury Lime (HgCl 2 ) solution for 9 minutes, rinsed with sterile water for 5 times; then blotted the surface moisture with sterile paper, cut it into 0.5 × 0.5 cm pieces, and inoculated them in sterile cluster bud induction medium (medium containing In a 150mL Erlenmeyer flask, sterilized at 121°C for 20 minutes), the medium formula is: MS+6-BA 3.0mg·L -1 +NAA0.2mg·L -1 , 30g·L -1 Sucrose, pH value is 5.8, then add 8.5g·L -1 of agar powder. Cultivate the leaves of Datura woody in the light incubator, the cultivation conditions are: 25°C, 12h·d -1 Lighting, the light intensity is 55μmol....

Embodiment 2

[0025] Genetic Transformation of Datura woody with Agrobacterium rhizogenes to Obtain Hairy Roots

[0026] 1. Agrobacterium rhizogenes C58C1. Take it out from the ultra-low temperature refrigerator before use, and inoculate it in 50ml YEB liquid medium (adding rifampicin to a final concentration of 40mg L -1 ), 28°C, 200rpm shaking and culturing twice to revive the cells.

[0027] 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 it reaches 0.3, add acetosyringone, continue 28°C, 200rpm shaking culture, the bacterial solution OD 600 When it reaches 0.5, it can be used for conversion.

[0028] 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 for 30 minutes, so that the concentration of the bacterial solution reac...

Embodiment 3

[0033] Molecular detection of the hairy roots of the woody Datura datura

[0034] 1. The extraction of Genomic DNA from the hairy root of Datura woody, the method is as follows:

[0035] (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.

[0036](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.

[0037] (3) Centrifuge at 4°C, 12,000 rpm for 10 minutes.

[0038] (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.

[0039] (5) Centrifuge at 12,000 rpm for 10 minutes at room temperature.

[0040] (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 un...

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Abstract

The invention provides a method of genetically transforming the datura arborea by genetic engineering and obtaining the hairy root for producing hyoscyamine and scopolamine. The invention relates to a method of genetically transforming the datura arborea by agrobacterium rhizogenes. The genetically transforming can obtain the hairy root of the datura arborea used for producing hyoscyamine and scopolamine. The method can provide a continuous novel medicine source used for producing hyoscyamine and scopolamine.

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 obtaining hyoscyamine and scopolamine-producing hairy roots of Datura woody by using transgenic technology, in particular to the genetic transformation of Agrobacterium rhizogenes Specific procedures for presenting Datura datura and obtaining hyoscypolamine and scopolamine hairy roots in woody Datura datura. The invention also provides the woody Datura datura hairy roots with high hyoscine and scopolamine yield obtained by the genetic engineering technology and the 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...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82A01H1/00A01H4/00
Inventor 孙敏雷桅杨雪清
Owner SOUTHWEST UNIVERSITY
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