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Plant gene editing method independent on genotype

A technology for gene editing and transgenic plants, applied in the field of plant gene editing that does not depend on genotype, can solve problems such as the elimination of unfavorable offspring transgenic components, limited genetic variation, etc., to overcome the limitations of the recipient genotype and product safety Good results

Inactive Publication Date: 2019-01-11
JILIN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the problems that existing gene editing is limited by genotype and requires tissue culture to produce a large number of genetic variations, which is not conducive to the elimination of transgenic components in offspring, the present invention provides a plant gene editing method that does not depend on genotype
This method does not depend on genotype and does not require complicated tissue culture, and can quickly realize gene editing of plant genotypes, especially for difficult-to-transform genotype plant gene editing

Method used

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  • Plant gene editing method independent on genotype
  • Plant gene editing method independent on genotype
  • Plant gene editing method independent on genotype

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Experimental program
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Effect test

preparation example Construction

[0064] The preparation method of the gene editing intermediate vector tool mainly includes the following steps:

[0065] Step 1. Construct a plant expression vector containing gene editing functional elements, and the plant expression vector can realize effective editing of the target gene after entering the plant body.

[0066] Step 2, using the plant material capable of genetic transformation as the acceptor material, using the constructed plant expression vector to carry out plant genetic transformation, and cultivating transgenic plant plants.

[0067] Step 3: Select a single copy of the obtained transgenic plant plant A with stable expression of Cas9 as the male parent, select the strain B with haploid induction efficiency as the female parent for hybridization, and generate the F1 generation or further in the offspring. Screening positive plants with plant haploid induction ability and containing transgenic components is the intermediate vector tool for gene editing.

Embodiment 1

[0073] Example 1 Construction of gene editing test plant expression vector

[0074] A plant expression vector containing gene editing functional elements is constructed, and the plant expression vector can realize effective editing of the target gene after entering the plant. The gene editing functional element includes a gRNA sequence that guides the gene editing enzyme to the target site.

[0075] 1. Design of target fragment pds1-T1

[0076] For the convenience of observing the phenotype, the test trait was selected as the albinism trait. Maize albino seedling trait control gene pds1 (phytoene desaturase1) gene, Maizegdb database accession number: GRMZM2G410515, its sequence is shown in SEQ ID NO: 1 in the sequence list. Nucleotide sequencing results confirmed that the easy-to-transform recipient material HiII used in the study was consistent with the pds1 genome sequence of the maize inbred line B73, and gene editing targets could be designed based on the genome sequence...

Embodiment 2

[0084] Example 2 Gene Editing Test Plant Expression Vector Maize Genetic Transformation and Transformant Creation

[0085] 1. Transformation of recombinant plasmid pCAMBIA1300-bar-pYAO-Cas9-pds1-sgRNA into Agrobacterium and identification of positive clones

[0086] 1.1 Take 200 μL of Agrobacterium competent, add 1-2 μL of plasmid DNA, ice bath for 30 minutes, quick freeze in liquid nitrogen for 1 minute, 37 ° C water; bath for 5 minutes, add 800 μL of YEP medium;

[0087] 1.2 Under the condition of 28°C, resume culture at 100rpm for 3h, centrifuge at 5000g for 1min, and discard the supernatant;

[0088] 1.3 Add 100 μL of YEP medium; resuspend the bacteria, spread on the YEP plate containing the corresponding antibiotics, culture at 28°C for 36-48 hours;

[0089] 1.4 Pick a single colony in 5mLYEP medium (containing corresponding antibiotics), culture at 28°C, 200rpm for 48h, extract plasmid DNA, and perform PCR and enzyme digestion verification. Verify that the correct sing...

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Abstract

The invention discloses a plant gene editing method independent on genotype, relates to the field of plant genetic engineering and solves the problems that descendant transgenic ingredients are removed unfavorably as a result of great heritable variation because existing gene edition is limited by genotype and tissue culture is needed. The method comprises the following steps: preparing an intermediate carrier with a tool attribute for gene edition by taking a transgenic plant gene editing target point gRNA and a gene editing system element and a plant strain with haploid inducting efficiencyas materials; inducting a plant to generate haploids by taking the intermediate carrier as a male parent and finishing gene edition at the same time; and carrying out natural or manual doubling to finally obtain a plant new variety / strain homozygote of the targeted gene edition. The method is free of complex genetic transformation and tissue culture processes after preparing the gene editing intermediate carrier tool, and achieves gene edition of different genotypes of a same plant by means of manual cross pollination.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to a method for editing plant genes independent of genotype. Background technique [0002] The use of gene editing technology to knock out unfavorable genes in a targeted manner can achieve precise improvement of target traits, which is of great help to improve the efficiency of targeted genetic improvement of crops. In 2016, precise plant gene editing technology was listed as one of the top ten scientific and technological breakthroughs. Targeted genetic engineering based on CRISPR-Cas9 can precisely improve the characteristics of crops, and has shown great technical advantages in ensuring sustainable food production. and market potential. In terms of corn research, DuPont Pioneer has successfully used this technology to obtain gene-edited corn plants such as waxy corn, sulfonylurea herbicide resistance, male sterility and ligule deletion. In terms of other crop...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/00A01H6/46A01H6/54A01H1/02A01H1/04
CPCA01H1/02A01H1/04C12N15/8213C12N2810/10
Inventor 刘相国
Owner JILIN ACAD OF AGRI SCI
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