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Agrobacterium rhizogenes mediated genetically modified Erigeron breviscapus hairy root transformation method

A technology of Agrobacterium rhizogenes and Dengzhanhua, which is applied in the field of plant genetic engineering, can solve the problems such as the establishment of the genetic transformation system of Dengzhanhua not yet.

Inactive Publication Date: 2016-01-20
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no report on the establishment of a genetic transformation system of Breviscapita

Method used

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  • Agrobacterium rhizogenes mediated genetically modified Erigeron breviscapus hairy root transformation method
  • Agrobacterium rhizogenes mediated genetically modified Erigeron breviscapus hairy root transformation method
  • Agrobacterium rhizogenes mediated genetically modified Erigeron breviscapus hairy root transformation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1: Extraction of the recombinant plasmid carrying the gene of interest

[0075] After activating the Trans1-T1 Escherichia coli carrying the PHB-Flag recombinant plasmid of the target gene CHI, a single colony was picked and cultured overnight in LB liquid medium containing 75 mg / L Kanamycin (Kan) at 37°C and 200 rpm. Using a plasmid extraction kit (alkaline lysis method) (the kit was purchased from Quanshijin Biological Co., Ltd.), the recombinant plasmid was extracted according to its instructions to obtain a purified recombinant plasmid.

Embodiment 2

[0076] Example 2: Genetic transformation mediated by Agrobacterium rhizogenes and induction of transgenic hairy roots

[0077] MS and B5 medium used in the examples were purchased from PhytoTechnology Laboratoties Company, YEB medium preparation reagents were purchased from Sangon Biotech Co., Ltd., sucrose was purchased from Sinopharm Chemical Reagent Co., Ltd., and agar powder was purchased from Shanghai Jiafeng Horticultural Supplies Co., Ltd.

[0078] The method of the present invention is as image 3 As shown, all operations of this embodiment are carried out under aseptic conditions, and described steps are the same as image 3 The order of the pictures corresponds.

[0079] 1. After sterilizing the breviscapus seeds with 0.1% mercury liter for 10 minutes, rinse them with sterile water for 3-5 times, absorb the residual water, inoculate them on MS medium, and inoculate them at a temperature of 25±1°C, a light intensity of 4000 lx, and a photoperiod of Cultivate for 45-...

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Abstract

The invention belongs to the technical field of plant genetic engineering in the plant biotechnology, and particularly relates to an agrobacterium rhizogenes mediated genetically modified Erigeron breviscapus hairy root transformation method. Pre-cultivated Erigeron breviscapus aseptic seedling blade explant is soaked in a C58C1 agrobacterium rhizogenes bacterial liquid carrying recombinant plasmid, hairy roots are cultivated on a selection medium containing hygromycin B (Hyb) for selection after infection, co-culture and bacteria removing, and a liquid medium is used for cultivating after a PCR amplification method is used for further identifying genetically modified roots carrying target genes. With the adoption of the technical scheme, the target genes can be guided into Erigeron breviscapus, and compared with genetic transformation of chemical methods of polyethylene glycol and the like, protoplast separation and cultivation segments are reduced; compared with genetic transformation of physical methods of a gene gun and the like, expensive instrument and equipment are not required, and the operation technique is simple.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering in plant biotechnology, and in particular relates to a method for genetic transformation of transgenic scutellaria breviscapus hairy roots mediated by Agrobacterium rhizogenes. Background technique [0002] Genetic transformation of plant cells is a key link in plant genetic engineering. Since Abel et al. (1986) introduced tobacco mosaic virus (TMV) capsid protein cDNA into tobacco cells and obtained transgenic tobacco plants resistant to TMV virus, many plant genetic transformation methods and technologies have been developed successively. [0003] Erigeron breviscapus (Vant.) Hand-Mazz. Also known as Erigeron breviscapus (Vant.) Hand-Mazz, is a perennial herb belonging to the family Asteraceae. Its main active ingredient is scutellarin, which is a mixture of scutellarin and a small amount of scutellarin. Breviscapine can relax blood vessels, improve microcirculation, inhibit ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/06
Inventor 张磊黄鑫陈瑞兵刘江华
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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