Agrobacterium tumefaciens mediated efficient glycyrrhiza glabra genetic transformation method

A genetic transformation method, Agrobacterium-mediated technology, applied in the field of Agrobacterium-mediated high-efficiency Glycyrrhizae glabra genetic transformation, to meet the needs of large-scale factory production in the pharmaceutical industry, simple and easy to operate, and solve the effects of lack of drug sources

Inactive Publication Date: 2018-11-02
SHIHEZI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the research on Glycyrrhiza glabra mainly focuses on the medicinal components, physiological mechanism, extraction process of metabolites and pharmacological analysis, and there is no report on the establishment of genetic transformation system of Glycyrrhiza glabra

Method used

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  • Agrobacterium tumefaciens mediated efficient glycyrrhiza glabra genetic transformation method
  • Agrobacterium tumefaciens mediated efficient glycyrrhiza glabra genetic transformation method
  • Agrobacterium tumefaciens mediated efficient glycyrrhiza glabra genetic transformation method

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Experimental program
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Embodiment 1

[0035] Example 1. Establishment of Glycyrrhiza glabra genetic transformation system

[0036] 1. Construction of eukaryotic expression vector

[0037] 1. Obtaining the target gene ipt

[0038] (1) Plasmid extraction

[0039] The Escherichia coli strain containing the vector pCAMBIA1301 was preserved and provided by the Key Laboratory of Agricultural Technology of Xinjiang Shihezi University. The wild-type Agrobacterium tumefaciens was inoculated into LB liquid medium, shaken at 28°C and cultured at 200rpm / min overnight, and the plasmid of Escherichia coli strain containing the vector pCAMBIA1301 was extracted using the TIANprep Mini Plasmid Kit. The method is as follows:

[0040] . Add 500 μL of equilibrium solution BL to the adsorption column CP3 (the adsorption column is placed in the collection tube), centrifuge at 12,000 rpm for 1 min, pour off the waste liquid in the collection tube, put the adsorption column back into the collection tube, and balance the adsorption co...

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Abstract

The invention belongs to the technical field of radix glycyrrhizae genetic engineering and particularly relates to an agrobacterium tumefaciens mediated efficient glycyrrhiza glabra genetic transformation method. The method includes the steps of firstly, introducing plasmids containing target DNA into agrobacterium tumefaciens to obtain recombinant agrobacterium tumefaciens; secondly, culturing the recombinant agrobacterium tumefaciens to serve as transformation infection liquid; thirdly, soaking glycyrrhiza glabra explants with the transformation infection liquid; fourthly, co-culturing the explants in a differential medium; fifthly, culturing the co-cultured explants in a screening and differential medium, and removing the agrobacterium tumefaciens; sixthly, using kanamycin as the screening pressure to obtain multiple shoots; seventhly, inoculating the multiple shoots to a root induction culture medium to obtain resistant regenerated glycyrrhiza glabra seedlings, wherein PCR identification shows that the resistant seedlings are positive transgenic seedlings. The method has the advantages that the method is simple and practicable to operate, high in transformation efficiency and widely applicable to fields such as glycyrrhiza glabra genetic breeding, cell engineering and metabolic engineering, and the transformation, differentiation and seedling formation and rooting can be completed within 55 days.

Description

technical field [0001] The invention relates to a genetic transformation method in the biological field, in particular to an Agrobacterium-mediated high-efficiency Glycyrrhiza glabra genetic transformation method and application thereof. Background technique [0002] Glycyrrhiza glabra (Lyciumruthenicum Murr.) is a perennial herb of the genus Glycyrrhiza in the genus Leguminosae. It is widely distributed in Europe, the Mediterranean region, Kazakhstan, Uzbekistan, Kyrgyzstan, Tajikistan, Siberia, Russia and Mongolia, and only in Xinjiang in China. Glycyrrhiza glabra is one of the original plants of traditional Chinese medicine licorice. It has many pharmacological effects such as clearing heat and detoxifying, moistening the lungs and relieving cough, reducing inflammation and expectorating phlegm, relieving asthma and spasm, anti-tumor, anti-virus and improving immunity. It is widely used in medicine and health care. production of goods. At present, the research on Glycyr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/00A01H6/54
CPCC12N15/8205
Inventor 沈海涛刘海亮祝建波毕权何大俊
Owner SHIHEZI UNIVERSITY
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