Wheat genome site-specific modification method

A technology of genetic modification and wheat, applied in the field of plant genetic engineering, can solve the problems of time-consuming, labor-intensive, blindness, and high cost

Active Publication Date: 2013-10-09
SUZHOU QI BIODESIGN BIOTECHNOLOGY CO LTD
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AI Technical Summary

Problems solved by technology

At present, the creation and chromosome modification of wheat mutants are mainly obtained through EMS mutagenesis, physical mutagenesis and chromosome engineering, but these methods are time-consuming, laborious and blind
In the current plant genome engineering (Genome engineering) technology, zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are mainly used for genetic modification, but no gene-specific modification has been reported in wheat, and these The technical operation process is complicated, the cost is high, and the technology is difficult. Therefore, it is urgent to develop a more efficient, cheap and simple new technology for genetic modification of wheat

Method used

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preparation example Construction

[0037] 1. Preparation of Cas9 nuclease recombinant expression vector

[0038] Streptococcus pyogenes Cas9 (SpCas9, S.pyogenes Cas9) gene was codon optimized, and nuclear localization signal (NLS) and BamH Ⅰ / Mfe Ⅰ restriction enzyme cutting sites were added at both ends of the gene coding sequence to optimize The latter Cas9 can be better expressed and localized in wheat. The nucleotide coding sequence of Cas9 after adding NLS and restriction sites at both ends and codon optimization is shown in SEQ ID No. 1 in the sequence listing. In SEQ ID No.: 1, positions 1-6 are BamH Ⅰ restriction sites, positions 4213-4218 are Mfe Ⅰ restriction sites, positions 10-36 are N-terminal NLS sequences, positions 4162-4209 are The C-terminal NLS sequence, the 37th-4161st position is the coding gene of Cas9 protein. The coding frame in SEQ ID №: 1 is nucleotide 7-4212 of SEQ ID №: 1, which encodes the protein shown in SEQ ID №: 2, which is the Cas9 nuclease with a nuclear localization signal p...

Embodiment 1

[0044] Example 1. Site-directed mutagenesis of the wheat endogenous gene TaMLO by gRNA: Cas9 system

[0045] (1) Design of target fragment target-SP6

[0046] target-SP6: G GAGATTGGGTCCTGCGTGA CGG ; (Genbank No is the 429th-451st nucleotide in the negative strand of the TaMLO gene of AF384144)

[0047] (2) Preparation of pU6-gRNA plasmid containing SP6 nucleotide fragment

[0048] SP6 is the coding DNA of RNA that can complementarily bind to the target target-SP6.

[0049] Synthesize the following single-stranded primers with sticky ends (underlined):

[0050] SP6-F: CTTG GAGATTGGGTCCTGCGTGA;

[0051] SP6-R: AAAC TCACGCAGGACCCAATCTC.

[0052] After the primer annealing procedure, double-stranded DNA with cohesive ends was formed and inserted between the two BbsI restriction sites of the pU6-gRNA plasmid to obtain the pU6-gRNA plasmid containing SP6. The positive plasmid was verified by sequencing.

[0053] (3) Transformation of gRNA: Cas9 system into wheat protopla...

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Abstract

The invention discloses a wheat genome site-specific modification method which comprises the following steps: enabling wheat tissues to contain guide RNA (ribonucleic acid) and Cas9 nuclease; under the coaction of the guide RNA and Cas9 nuclease, cutting a double-chain target segment on the target gene; and by utilizing the DNA (deoxyribonucleic acid) restoration function of the wheat cells, finally implementing random insertion and / or random deletion on the target segment in the wheat target gene. The experiment proves that the method disclosed by the invention can successfully perform gene mutation on wheat.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering and relates to a wheat genome modification method. Background technique [0002] Wheat is one of the most important and widely planted crops in the world. The modification of wheat genome is beneficial to the research of wheat genome, which is of great significance to increase yield, enhance its resistance to diseases and insect pests and agricultural development. At present, the creation and chromosome modification of wheat mutants are mainly obtained through EMS mutagenesis, physical mutagenesis and chromosome engineering, but these methods are time-consuming, laborious and blind. In the current plant genome engineering (Genome engineering) technology, zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are mainly used for genetic modification, but no gene-specific modification has been reported in wheat, and these The technical operation process is...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12N15/82
Inventor 高彩霞王延鹏单奇伟陈坤玲
Owner SUZHOU QI BIODESIGN BIOTECHNOLOGY CO LTD
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