Conversion of ion-beam mediated Chinese ephedra general DNA in microzyme and acquired tr-gene yeast engineering fungus

A yeast and transgenic technology, applied in the field of genetic transformation, can solve the problems such as the inability to construct ephedrine, and achieve the effects of easy cultivation, convenient genetic manipulation, and overcoming genetic transformation

Inactive Publication Date: 2007-09-05
XINJIANG UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, just can't construct the engineering bacterium that produces ephedrine and pseudoephedrine according to conventional genetic engineering method

Method used

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  • Conversion of ion-beam mediated Chinese ephedra general DNA in microzyme and acquired tr-gene yeast engineering fungus
  • Conversion of ion-beam mediated Chinese ephedra general DNA in microzyme and acquired tr-gene yeast engineering fungus
  • Conversion of ion-beam mediated Chinese ephedra general DNA in microzyme and acquired tr-gene yeast engineering fungus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Introduction and transformation of the total DNA of Ephedra to obtain Hansenula anomaly engineering strain 0451

[0048] (1), collection of medicinal plant ephedra, preparation of total DNA

[0049] Use 10 times the amount of color-changing silica gel (origin: Shanghai) of the blue ephedra (Ephedra glauca) sample and put the blue ephedra twigs with scaly triangular leaves (collected from the desert area of ​​Yamalik Mountain, Xinjiang) into a sealed bag at room temperature. Guaranteed to be dry within 12 hours. If the silica gel has changed from dark blue to pink, replace it in time. After returning to the laboratory, immediately seal the dried ephedra material and store it in a desiccator.

[0050] After the castor ephedra material was quick-frozen in liquid nitrogen, it was co-researched with quartz sand, and the total DNA was extracted by CTAB method (see reference 7). Detection of DNA purity by UV spectrophotometer A 260 / A 280 It was 1.8, and it was a...

Embodiment 2

[0065] Example 2: Introduction and transformation of total DNA of Ephedra to obtain Saccharomyces cerevisiae engineered strain 1179

[0066] (1), collection of medicinal plant ephedra, preparation of total DNA

[0067] Total DNA was prepared in the same manner as described in Part (1) of Example 1, except that shoots of Ephedra sinica (collected from the northern desert area of ​​the Tarim Basin, Xinjiang) with scaly triangular leaves were used. Detection of DNA purity by UV spectrophotometer A 260 / A 280 It was 1.9, and it was a single band in agarose electrophoresis (Fig. 5b).

[0068] (2), the treatment of yeast cells

[0069] Put the strains of Saccharomyces cerevisiae (purchased in Beijing: China General Microorganism Culture Collection and Management Center, No. 2.1882) into the wort culture solution (see reference 8) on the slant, and put it on a rotary shaker at 230r / min, and cultured at 30°C for 12 hours. With 0.1% soluble starch (domestic, analytically pure) an...

Embodiment 3

[0080] Example 3: The introduction and transformation of the total DNA of ephedra to obtain the fermenting Pichia pastoris engineered strain 3161

[0081] (1), collection of medicinal plant ephedra, preparation of total DNA

[0082] Part (1) of Example 1 was repeated to extract total DNA, except that shoots of Ephedra intermadia (collected from the western desert area of ​​Jungar Basin, Xinjiang) with scaly triangular leaves were used. Detection of DNA purity by UV spectrophotometer A 260 / A 280 It was 1.7, and it was a single band in agarose electrophoresis (Fig. 5c).

[0083] (2), the treatment of yeast cells

[0084] Put the strains of fermenting Pichia fermentans (purchased in Beijing: China General Microorganism Culture Collection and Management Center, No. 2.1706) into the wort culture solution (see reference 8), and put it on a rotary shaker 230r / min, 30°C for 12 hours. With 1.0% soluble starch (domestic, analytically pure) and 1.0% glucose (C 6 h 12 o 6 ·H 2 O...

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Abstract

A method for converting Chinese ephedra total DNA into yeasts, the transfer gene yeasts thereof, a culture medium for separating the transfer gene yeasts and liquid culture medium for transfer gene yeasts are disclosed.

Description

technical field [0001] The invention relates to the technical field of the genetic transformation of active naked DNA macromolecules mediated by ion beams. Specifically, the present invention relates to a method for genetic transformation of ephedra total DNA in yeast mediated by ion implantation, and a transgenic yeast engineered strain producing ephedrine and pseudoephedrine through genetic transformation. Background technique [0002] Most of the active ingredients contained in medicinal plants are their secondary metabolites, which are generally controlled by multiple genes. It is not yet possible to construct a plasmid vector for the target gene without fully understanding its genetic background. Therefore, it has important theoretical and practical significance to study the genetic transformation of the total DNA of medicinal plants. In recent years, the technology of total plant DNA transformation has made great progress, especially the genetic transformation of acti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/29C12N1/19C12N15/87
Inventor 毛培宏娄恺金湘王志方魏东张军
Owner XINJIANG UNIVERSITY
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