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Transgenic carrier with function of automatic control and elimination of selection marker and application thereof in zea mays marker-free transgenic breeding

A technology of transgenic vectors and screening markers, which is applied in the fields of application, genetic engineering, and the use of vectors to introduce foreign genetic materials, etc. It can solve the problems of complex operations and inability to apply in large quantities, and achieve expansion of scope, avoiding toxic effects, and improving screening efficiency Effect

Inactive Publication Date: 2018-09-28
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the successful application of this technology in corn
[0010] In maize transgenic operations, mainly rely on the co-transformation method and transposon tagging method, both of which need to eliminate the screening marker genes through sexual crossing and backcrossing in the offspring of transgenic plants. The operation is complicated and cannot be applied in a large number easily

Method used

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  • Transgenic carrier with function of automatic control and elimination of selection marker and application thereof in zea mays marker-free transgenic breeding
  • Transgenic carrier with function of automatic control and elimination of selection marker and application thereof in zea mays marker-free transgenic breeding
  • Transgenic carrier with function of automatic control and elimination of selection marker and application thereof in zea mays marker-free transgenic breeding

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Transgenic vector pHZM1N-Rsc construction, the steps are as follows:

[0056] Design of the transgenic vector pHZM1N-Rsc:

[0057] With the development of transgenic technology, the optimization of selection markers has always been an important part of developing efficient transformation systems. The fusion gene of green fluorescent protein gene (EGFP) and isopentenyltransferase (IPT) gene was used to replace the bar gene on the pEGAD vector as a screening marker. The marker functions in two stages of the transgenic process: in the screening process, green fluorescence is used as a visual selection marker to accurately screen transformed cells, and the expression of IPT2 gene is used to promote the rapid growth of transformed cells, so as to quickly obtain transformed cells. damage tissue. After the screening marker was eliminated, the green fluorescent disappearance was used to select the callus that was eliminated by the screening marker, and in the regeneration sta...

Embodiment 2

[0062] Example 2 Application of Screening Marker Autonomous Control and Elimination of Transgenic Vectors in Maize Marker-Free Transgenic Breeding

[0063] (1) Preparation of carrier pHZM1N-Rsc Agrobacterium engineering bacteria:

[0064] 1.1) Competent preparation of Agrobacterium

[0065] Shake 50ml of EHA105 bacterial liquid until OD600=0.5 or so;

[0066] Centrifuge at 5000rpm for 5min, discard the supernatant;

[0067] Add 40ml of pre-cooled 10% glycerol, oscillate to resuspend the bacteria, and place on ice for 30min;

[0068] Centrifuge at 5000rpm for 5min at 4°C and discard the supernatant;

[0069] Add 30ml pre-cooled 10% glycerol, shake the resuspended bacteria;

[0070] Centrifuge at 5000rpm for 5min at 4°C and discard the supernatant;

[0071] Repeat steps 5-6;

[0072] Add 2ml of 10% glycerol to the EP tube, oscillate to resuspend the bacteria, and then distribute 200μl per tube into 1.5ml EP tubes.

[0073] 1.2) Agrobacterium electrotransformation and prep...

Embodiment example 1

[0108] Aspirate the infection liquid in the 50ml EP tube that the callus is housed, add the infection liquid carrying the Agrobacterium engineering bacterium in the implementation case 1;

[0109] Shake for 1 min, then stand at room temperature for 5 min;

[0110] Pour the infected callus into a plate with a filter paper at the bottom, dry it moderately, transfer it to a co-cultivation medium, and culture it in the dark at 19°C for 3 days;

[0111] Transfer the co-cultured callus into a 50ml EP tube, and wash it 3-5 times with sterile water containing 0.2g / L carbenicillin;

[0112] Pour the cleaned callus into a plate with a filter paper pad at the bottom, blow dry moderately, transfer to the recovery medium, and culture in the dark at 28°C for 7-10 days;

[0113] The calli after recovery culture were transferred to the subculture medium containing 0.2 g / L carbenicillin, and subcultured in the dark at 28°C for 21 days.

[0114] Fluorescence screening of transgene positive ca...

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Abstract

The invention provides a transgenic carrier with function of automatic control and elimination of selection marker and application thereof in zea mays marker-free transgenic breeding, and belongs to the technical field of breeding of zea mays molecules. The carrier uses a pEGAD carrier as a skeleton carrier, and a fusion gene of EGFP (enhanced green fluorescent protein) and IPT (isopentenyl transferase) genes is used for replacing a bar gene and is used as the selection marker; an expression framework of the selection marker is integrated into a Cre / lox site specific recombination system. Theapplication comprises the following steps of transferring the carrier into a zea mays embryonic callus, obtaining the zea mays embryonic callus with the green fluorescent expression effect; after heatshock, culturing the zea mays embryonic callus without the green fluorescent expression effect by differentiating and rooting, so as to obtain a transgenic plant. The carrier has the advantages thatthe selection efficiency is improved, the removal of the transgene selection marker is realized, and the theoretical and application basis is provided for the large-range development of marker-free zea mays transgene.

Description

technical field [0001] The invention belongs to the technical field of corn molecular breeding, and in particular relates to a screening marker self-controlling elimination transgene carrier and its application in marker-free transgenic breeding of corn. Background technique [0002] Maize (Zea mays L.) is one of the most important crops in the world. As a food crop in many regions of the world and a major feed crop in most regions, it is widely planted all over the world. At the same time, corn is also an important industrial raw material, which is widely used in fermentation industry, alcohol production and providing bioenergy. Maize breeding improvement and agricultural production are important factors affecting food security in my country. In recent decades, with the gradual improvement and popularization of new breeding technologies such as mutagenesis breeding technology, transgenic technology, double haploid technology and molecular marker-assisted selection technolo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/65A01H5/00A01H6/46
CPCC12N15/65C12N15/8261
Inventor 张方东杜邓襄
Owner HUAZHONG AGRI UNIV
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