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application of NTS/dNTS combination in preparation of plant mutants

A kind of technology of plants and variants, applied in the field of application in the preparation of plant mutants

Pending Publication Date: 2020-04-17
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in plants, there are very few reports on the use of P2A to couple target proteins with other marker proteins

Method used

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  • application of NTS/dNTS combination in preparation of plant mutants
  • application of NTS/dNTS combination in preparation of plant mutants
  • application of NTS/dNTS combination in preparation of plant mutants

Examples

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Embodiment 1

[0097] Example 1, Construction of TS / dTS, TS / dNTS and NTS / dNTS Corresponding Vectors and Their Application in Rice Gene Replacement

[0098] 1. Construction of recombinant expression vector and description of replacement principle

[0099] 1. Construction of recombinant expression vector

[0100] Artificially synthesize the following recombinant expression vectors, each of which is a circular plasmid:

[0101] For the W548 site, there are three vectors, namely W548-TS / dTS, W548-TS / dNTS, and W548-NTS / dNTS.

[0102] For the P565 site, there are three vectors, namely P565-TS / dTS, P565-TS / dNTS, and P565-NTS / dNTS.

[0103] Schematic diagram of the backbone structure of the recombinant expression vector figure 1 shown. The specific structure is described as follows:

[0104] The sequence of the W548-TS / dTS recombinant expression vector is sequence 1 in the sequence list. The 131-467th position of sequence 1 is the OsU3 promoter sequence, the 474-550th position is the tRNA sequ...

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Abstract

The invention discloses application of NTS / dNTS combination in preparation of plant mutants. The method comprises the following steps: esgRNA, Cas9 nicking enzyme, screening agent resistant protein and donor DNA are introduced into a plant, wherein esgRNA is used for targeting a target sequence of a DNA fragment A, the DNA fragment A target sequence is positioned on a transcription chain in a plant genome, a transcription chain of the donor DNA sequentially consists of a DNA fragment A target sequence, a DNA fragment B and a DNA fragment A target sequence, a DNA fragment B is a DNA molecule obtained by mutating the DNA fragment A; under the guidance of esgRNA, the Cas9 incision enzyme is used for generating a single-stranded DNA incision on a non-transcriptional strand of a plant genome, generating two single-stranded DNA incisions on a non-transcriptional strand of a donor DNA, and the DNA fragment A in the plant genome is replaced with the DNA fragment B through a repair mechanism ina plant body to realize plant gene replacement.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the application of NTS / dNTS combination in preparing plant mutants. Background technique [0002] The probability of precise gene replacement mediated by long-strand DNA templates in cells is very low, but introducing a DNA double-strand break (dsDNA break, DSB) near the site to be replaced can significantly increase the probability of replacement. CRISPR-Cas9 technology has become a powerful genome editing method and has been widely used in many tissues and cells. The CRISPR / Cas9protein-RNA complex is positioned on the target by the guide RNA (guide RNA), and the DNA is cleaved to generate DSBs, thereby increasing the efficiency of precise gene replacement mediated by long-strand DNA templates. After the generation of DSB, the organism will instinctively start the DNA repair mechanism. There are generally two repair mechanisms, one is non-homologous end joining (NHEJ),...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/54A01H5/00A01H6/46
CPCC12N15/8209C12N15/8213C12N15/8274C12N9/1022C12Y202/01006Y02A40/146
Inventor 徐雯杨进孝武莹张成伟康桂婷刘亚
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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