A method for rapid screening of non-transgenic site-directed mutation plants

A non-transgenic, site-directed mutation technology, applied in botany equipment and methods, biochemical equipment and methods, plant products, etc., can solve the problems of increasing manpower input, reduce time and labor, and avoid false positive effects

Active Publication Date: 2022-02-01
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method requires long-term planting and screening of offspring, which increases human input.

Method used

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  • A method for rapid screening of non-transgenic site-directed mutation plants
  • A method for rapid screening of non-transgenic site-directed mutation plants
  • A method for rapid screening of non-transgenic site-directed mutation plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] In this embodiment, a mutant of the non-transgenic tobacco Nbxylt gene was screened using positive and negative screening markers, and the process was as follows:

[0032] 1. Construction of positive and negative selection markers (coda::nptii or hpt::coda) suitable for tobacco knockout vectors

[0033](1) Construction of pCNS-Cas9 vector: it contains coda::nptii positive and negative selection markers, 35S promoter drives the expression of Cas9, and AtU6-26 promoter drives the expression of sgRNA, which is suitable for tobacco gene knockout. Design primers 411-F SEQ ID NO.3: 5'-AGTAGATGCCGACCGGATCTGTC-3' and RB-R SEQ ID NO.4: 5'-TGACAGGATATATTGGCGGGTAAAC-3', using pCambia1300 as a template (pCambia1300 can be purchased from Youbao Biological Company), and expand Fragment 1 containing the backbone of the Agrobacterium vector was amplified; primers RB-F SEQ ID NO.5: 5'-GTTTACCCCGCCAATATATCCTGTCA-3' and 411-R SEQ ID NO.6: 5'-ATCTCATTGCCCCCCGGATC-3' were designed, using pH...

Embodiment 2

[0059] In this example, the Arabidopsis thaliana AtBRI1 gene was site-directedly mutated using a positive and negative selection marker vector containing hpt::coda, and the process was as follows:

[0060] 1. Construction of the positive and negative selection marker hpt::coda knockout vector pHCE-Cas9 suitable for Arabidopsis

[0061] pHCE-Cas9 vector: contains hpt::coda positive and negative selection markers, EC1.1 promoter to drive the expression of Cas9, and AtU6-26 promoter to drive the expression of sgRNA, suitable for gene knockout in Arabidopsis. Design primers 411-F SEQ ID NO.30: 5'-AGTAGATGCCGACCGGATCTGTC-3' and RB-R SEQ ID NO.31: 5'-TGACAGGATATATTGGCGGGTAAAC-3', using pCambia1300 as a template to amplify the Agrobacterium vector backbone Fragment 1; design primers RB-F SEQ ID NO.32: 5'-GTTTACCCCGCCAATATATCCTGTCA-3' and hyg-R SEQ ID NO.33: 5'-CTTTGCCCTCGGACGAGTGCTGG-3', using pHEE401 as a template (pHEE401 plasmid: in the literature "Hui -LiXing, Li Dong, Zhi-Ping ...

Embodiment 3

[0081] In this example, a vector containing a positive and negative selection marker (coda::nptii or hpt::coda) is used to site-directed mutation of the rice Osglossy2 gene, and the process is as follows:

[0082] 1. The construction of a knockout vector containing positive and negative selection markers (coda::nptii or hpt::coda) suitable for rice

[0083](1) Construction of the pCNU-Cas9 vector: it contains coda::nptii positive and negative selection markers, the maize Ubi-1 promoter drives the expression of Cas9, and the OsU3 promoter drives the expression of sgRNA, which is suitable for gene knockout in rice. Design primers 411-F SEQ ID NO.41: 5'-AGTAGATGCCGACCGGATCTGTC-3' and RB-R SEQ ID NO.42: 5'-TGACAGGATATATTGGCGGGTAAAC-3', using pCambia1300 as a template to amplify a fragment containing the backbone of the Agrobacterium vector 1; Design primers RB-F SEQ ID NO.43: 5'-GTTTACCCCGCCAATATATCCTGTCA-3' and 411-R SEQ ID NO.44: 5'-ATCTCATTGCCCCCCGGATC-3', using pHUN411 as a te...

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Abstract

The invention discloses a method for rapidly screening non-transgenic site-directed mutation plants, which belongs to the field of biotechnology. The method comprises the following steps: (1) introducing sgRNA targeting a specific gene target site into a gene editing vector containing positive and negative selection markers (coda::nptii or hpt::coda); (2) using the gene editing vector as Basic construction of gene knockout vectors and transformation of plants to be treated with Agrobacterium, implementation of transgenes, and positive selection through nptii or hygromycin to obtain the first generation (Arabidopsis is called T1 generation, tobacco, rice, etc. are called T0 generation) Gene site-directed mutation materials containing transgenic elements; (3) planting the first-generation mutants obtained in step (2), and harvesting seeds. (4) The harvested seeds were screened using the negative selection marker codA, and finally the site-directed mutation materials without transgenic components were obtained.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for rapidly screening non-transgenic site-directed mutation plants. Background technique [0002] Genome editing technology can generate site-directed mutations in the genome. It is an emerging molecular biology technology in the past decade, which mainly relies on sequence specific nucleases (Sequence Specific Nucleases, SSNs) to generate DNA double-strand breaks (DNA double -strandbreaks, DSBs). DSBs are mainly repaired by non-homologous end joining (NHEJ) and homologous recombination (HR). The NHEJ repair pathway will generate a few base insertions or deletions, resulting in frameshift mutations, resulting in gene knockout; the HR repair pathway will cause complete genome repair or base substitution or site-specific insertion under the condition of providing a template. At present, commonly used SSNs mainly include zinc finger nucleases (Zinc-finger nucleases, ZFNs), tr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/29A01H5/10A01H6/46A01H6/82A01H6/54A01H6/20
CPCC12N15/8216C12N15/8209
Inventor 梁振
Owner SHANXI UNIV
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