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Novel efficient selectable marker-free corn transgenic vector system construction method and application thereof

A non-selectable marker, transgenic vector technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve the problem of low activity, and achieve the effect of improving transformation efficiency, important theoretical significance and application value, and high transposition activity.

Inactive Publication Date: 2015-08-12
ANHUI NORMAL UNIV
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Problems solved by technology

[0005] Aiming at the deficiencies of the above-mentioned prior art, the present invention provides a method for constructing a new high-efficiency non-selectable marker transgenic vector system for maize and its application, which overcomes the problem of low activity of the existing maize transposon system and can obtain more non-selectable transposon systems at the same time. Mark transgenic plants, improve the transformation efficiency of transgenes, and provide technical support for the large-scale transformation of important food crops such as corn and rice in the later stage, and obtain safe and non-selectable transgenic plants

Method used

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  • Novel efficient selectable marker-free corn transgenic vector system construction method and application thereof
  • Novel efficient selectable marker-free corn transgenic vector system construction method and application thereof
  • Novel efficient selectable marker-free corn transgenic vector system construction method and application thereof

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Embodiment 1

[0033] Cloning of Mu Transposon-Related Fragments in Highly Active Mu Lines in Maize

[0034] The full-length sequences and flanking sequences of the maize MuDR and Mu1 transposons were searched from the Genbank database of the NCBI website, primers were designed according to the conserved regions of the sequences, and PCR cloning techniques were used to amplify from the genomic DNA of the leaves of the high-activity single-copy Mu strains of maize. The complete non-autonomous Mu transposable element sequence (1754bp) and the cDNA complete fragment (2783bp) of the gene mudrA encoding the transposase (MUDR) were amplified in the phMR53 plasmid, and the cloned fragment was connected to the pUC-T vector by Sequencing verifies the accuracy of its sequence.

[0035] Construction of Novel Binary Plasmid Vectors pMuDR and pMuE

[0036] Using the pCAMBIO1300 plasmid vector as the backbone, refer to figure 1 As shown in the vector linear diagram, the new binary plasmid vectors pMuDR ...

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Abstract

The invention provides a novel efficient selectable marker-free corn transgenic vector system construction method and an application thereof. By a PCR cloning technology, related fragments of Mu transposons are obtained from highly active Mu lines of corn; a binary plasmid vector pCAMBIA1300 is used as a skeleton to respectively construct novel efficient binary plasmid vectors pMuE and pMuDR; and through an agrobacterium-mediated genetic transformation method, Luyuan 92 selfing line of corn is transformed, and a selectable marker-free transgenic plant is screened out from progeny. According to the invention, transformation efficiency of transgene can be enhanced. By the vector system, a marker-free bio-safety transformant can be directly obtained in future. The vector system especially can provide a reliable and effective tool for marker-free gene transformation of important grain crops such as corn, rice and the like. The method provided by the invention has important theoretical significance and application value.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, in particular to a construction method and application of a novel high-efficiency non-selectable marker transgenic vector system for maize. Background technique [0002] With the continuous emergence of commercial plant transgenic varieties, people pay more and more attention to the environmental safety and food safety of genetically engineered organisms. These problems are mainly due to the persistence of selectable marker genes for antibiotic or herbicide resistance in transgenic plants. With the development of plant genetic engineering, a large number of crops with desired traits have been produced. In order to speed up the progress of crop improvement, an effective transformation system is extremely important to introduce exogenous target genes into plants and select a relatively small number of transformed cells. So far, marker genes that have been widely used for selection are mai...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84A01H5/00
Inventor 钱叶雄席贻龙
Owner ANHUI NORMAL UNIV
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