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Method for fixed-point transformation of plants by virtue of gene transient expression

A technology of transient expression and fixed-point transformation, applied in the field of plant genetic engineering, can solve the problems of difficult genome transformation and inability to play its role, and achieve the effect of improving regeneration ability and high biological safety

Active Publication Date: 2016-07-27
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Because this method involves exogenous genes to be integrated into the plant genome first, and the process of transformation needs to add selection markers (selection pressure), for some plants that are difficult to transform genetically, such as wheat, corn, soybean, potato, etc. Genome transformation is relatively difficult, which makes gene editing technology unable to play its role in plant genome transformation

Method used

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  • Method for fixed-point transformation of plants by virtue of gene transient expression
  • Method for fixed-point transformation of plants by virtue of gene transient expression
  • Method for fixed-point transformation of plants by virtue of gene transient expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1. Obtaining non-transgenic tagasr mutants by transiently expressing CRISPR / Cas9 nuclease using the particle gun method

[0073] 1. Design of target site target-C5

[0074] Target-C5: 5'- CCG CCGGGCACCTACGGCAAC-3'; (No. 248-268 in the TaGASR7 gene shown in GenbankNo.EU095332)

[0075] 2. Preparation of pTaU6-gRNA plasmid containing C5 nucleotide fragment

[0076] C5 is the coding DNA of RNA that can complementarily bind to target-C5.

[0077] The following single-stranded oligonucleotides were synthesized with cohesive ends (underlined):

[0078] C5F: 5'- CTTG TTGCCGTAGGTGCCCGG-3';

[0079] C5R: 5'- AAAC CCGGGCACCTACGGCAA-3'.

[0080] After the oligonucleotide annealing procedure, a double-stranded DNA with cohesive ends was formed and inserted between the two BbsI restriction sites of the pTaU6-gRNA plasmid to obtain a pTaU6-gRNA plasmid containing C5. The positive plasmid was verified by sequencing. That is, the recombinant plasmid obtained after th...

Embodiment 2

[0126] Example 2, the use of particle gun method to transiently express TELEN nuclease to obtain heritable and non-transgenic Tamlo mutants

[0127] 1. Site-directed editing of wheat MLO gene by transient expression of T-MLO by gene gun method

[0128] The TELEN plasmid is a T-MLO vector, which can express a pair of TALEN proteins, and the TALEN proteins are composed of a DNA binding domain and a FokI domain that can recognize and bind to a target site. The target sites are:

[0129] TaMLO-A gene: TCGCTGCTGCTCGCCGTcacgca ggacc caatctcCGGGATATGCATCTCCCA;

[0130] TaMLO-B gene: TCGCTGCTGCTCGCCGTgacgca ggacc ccatctcCGGGATATGCATCTCCGA;

[0131] TaMLO-D gene: TCGCTGCTGCTCGCCGTgacgca ggacc caatctcCGGGATATGCATCTCCGA.

[0132] Wherein, the underlined part is the recognition sequence of the restriction endonuclease AvaII.

[0133] (1) Get the immature embryos of wheat variety Bobwhite and carry out hyperosmotic treatment with hyperosmotic medium for 4 hours;

[0134] (2) Use a ...

Embodiment 3

[0156] Example 3. Further verification of the gene editing method based on transient expression

[0157] The gene editing method of the present invention was further tested using 5 different wheat genes as targets.

[0158] First, edited the 1 Three homologous genes of TaGASR7 (TaGASR7-A1, TaGASR7-B1 and TaGASR7-D1). Each of the three homologous genes has three exons and two introns ( Figure 9 b). Design sgRNA targeting exon 3 because this exon is highly conserved. After initial testing of nuclease activity in protoplasts 2 , the most efficient sgRNA expression cassette (Table 5) combined with Cas9 in a single construct (pGE-TaGASR7, Figure 9 d). It was introduced into the immature embryos of two common wheat varieties (Bobwhite and Kenong199) by gene gun. Embryogenic callus appeared at 2 weeks, and a large number of seedlings (2-3 cm in height) regenerated from the callus at 4-6 weeks. Contrary to most genome editing experiments in plants, transgenic plants were sel...

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Abstract

The invention discloses a method for fixed-point transformation of plants by virtue of gene transient expression. The invention provides a method for fixed-point transformation of target sites in target genes of a plant. The method comprises the following steps: performing transient expression on nuclease with sequence specificity in cells or tissues of a target plant by taking the cells or tissues of the target plant as a transient expression object, wherein the nuclease with sequence specificity is specific to the target sites and is used for cutting the target sites, so that the fixed-point transformation of the target sites is completed by virtue of DNA repairing of the plant. According to the method disclosed by the invention, the transformation of the target genes is realized by virtue of transient expression of the nuclease with sequence specificity, the regenerative power of plants is improved, the generated mutation can be inherited stably to future generations, and more importantly, the integration of exogenous genes does not exist in a generated mutant plant, and the generated mutant plant is relatively high in biological safety.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and relates to a method for point-specific transformation of plants through gene transient expression. The invention specifically relates to a method for realizing plant genome fixed-point modification with high biological safety through a transient expression system. Background technique [0002] Modification of plant genome is the main method to study the function of plant genome or genetic improvement of crops. The current methods of transforming plant genomes mainly focus on traditional hybrid breeding methods and mutation breeding methods. Traditional hybrid breeding methods require multiple generations, are time-consuming and laborious, and are affected by reproductive isolation restrictions between species and adverse gene linkage. Physical or chemical mutagenesis methods, such as radiation mutagenesis, EMS mutagenesis, etc., can randomly generate a large number of mutation sites ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N5/04C12N5/10A01H5/00
CPCC12N5/04C12N15/8216C12N2800/80C12N15/8213C12N9/22C12N15/102C12N15/113A01H4/008C12N5/14
Inventor 高彩霞王延鹏张毅刘金星张康
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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