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Corn genetic transferring method conducted by agrobacterium rhizogenes

An Agrobacterium rhizogenes, genetic transformation technology, applied to other methods of inserting foreign genetic material, using a vector to introduce foreign genetic material, recombinant DNA technology, etc. Problems such as low gene conversion rate

Inactive Publication Date: 2011-09-14
JILIN NORMAL UNIV
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Problems solved by technology

Solve the key technical problems in the existing technology of corn transgenic engineering breeding, such as low conversion rate of exogenous genes, difficulty in screening transformants, poor genetic stability of offspring, easy inactivation, and low survival rate of transplanting

Method used

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  • Corn genetic transferring method conducted by agrobacterium rhizogenes
  • Corn genetic transferring method conducted by agrobacterium rhizogenes
  • Corn genetic transferring method conducted by agrobacterium rhizogenes

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Embodiment

[0045] 1. Transformation and clone screening: In the present invention, green fluorescent protein is used as an exogenous gene, and pBINm-gfp5-ER is purchased from Arabidopsis Biological Resource Center (ABRC) No. CS9342. Take 100 μl of competent cells with a sterile tip and place them in a 1.5ml pre-cooled sterile Eppendorf tube, add 10-1000ng pBINm-gfp5-ER, mix gently, and immediately place on ice for 30min. Place the tube in a constant temperature water bath at 42°C for heat shock for 90 seconds, immediately put it back on ice for 15 minutes, add 800 μl LB liquid medium without additional antibiotics, mix well, and culture for 45-60 minutes at 37°C for pre-expression. Use a sterile pipette to transfer 200 μl of the bacterial solution to an LB plate supplemented with corresponding selected antibiotics, spread evenly, incubate at 37°C for 12-16 hours, and select a single colony for amplification.

[0046] 2. Three-parent hybridization method: transfer pBINm-gfp5-ER into Agrob...

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Abstract

The invention discloses a novel method for Agrobacterium rhizogenes (Ar) mediated corn gene transformation. Ar is adopted to infect corn callus to induce the generation of hairy roots; through differentiation cultivation, the hairy roots regenerate transgene plants. Ri plasmid is taken as a vector to transfer one or a plurality of exogenous genes into the genome of the acceptor. The hairy-root transformation rate of the invention is as high as 78 percent; the hairy-roots regenerate plants (with high frequency) by 55 percent. The survival rate of the transplanted regenerated-plants is as high as 92 percent; objective clone screening is easy, suitable for genetic operation, and practical. During genetic transformation of the exogenous genes, the roots of the plants and drought resistance are very strong due to rol gene action of Ri plasmid. The method can be applied to the production of corn drought-resistant transgene plants.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering. It specifically relates to a new method for genetic transformation of maize mediated by Agrobacterium rhizogenes. Background technique [0002] At present, the methods of maize genetic transformation technology at home and abroad mainly include: gene gun-mediated method, Agrobacterium tumefaciens-mediated method, PEG-mediated method, pollen tube passage method, ovary injection method, ultrasonic method, cationic transformation method Wait. Gene gun-mediated method and Agrobacterium tumefaciens-mediated method are the most conventional methods widely used. Its disadvantages are: the gene gun-mediated method is not only relatively expensive, but also often rearranges and high-copy insertions occur in the process of gene integration. The genetic stability of foreign genes in offspring is poor, and it is difficult to screen out resistant genes. Strong strain, and it is easy to cause in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/87
Inventor 徐洪伟周晓馥
Owner JILIN NORMAL UNIV
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