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Method for site-specific mutagenesis of medicago sativa gene by employing CRISPR/Cas9 system

A gene site-directed mutagenesis, alfalfa technology, applied in the field of gene editing, can solve problems such as limited application, unstable characteristics, and incomplete RNAi silencing

Active Publication Date: 2018-11-23
GUANGDONG SANJIE HERBAGE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As mentioned above, compared with ZFN, TALEN, CRISPR / Cas9 and other gene editing technologies, RNAi has limited the application of this technology due to its incomplete silencing and unstable characteristics
CRISPR / Cas9 technology has successfully obtained stable genetic homozygous mutants in rice, Arabidopsis, tomato, poplar, poplar, apple, wheat, corn, soybean and other species, but CRISPR has not been used in alfalfa / Reports on gene editing by targeted gene editing technologies such as Cas9

Method used

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  • Method for site-specific mutagenesis of medicago sativa gene by employing CRISPR/Cas9 system
  • Method for site-specific mutagenesis of medicago sativa gene by employing CRISPR/Cas9 system
  • Method for site-specific mutagenesis of medicago sativa gene by employing CRISPR/Cas9 system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Using the CRISPR / Cas9 system to mutate the PDS gene encoding phytoene dehydrogenase in alfalfa

[0059] Phytoene desaturase (PDS) is the main rate-limiting enzyme in the synthetic pathway of carotenoid pigments, which can catalyze colorless C40 phytoene to produce ζ-carotene, streptosporin, tomato Red pigment, 3,4-dehydrolycopene, 3,4,3'4'-dehydrolycopene or 3,4-dehydrostreptoerythrin, etc. The mutant plants of this gene show albino phenotype, which is convenient for observation. Using this gene as a target gene in plants is a convenient way to test whether the gene knockout system works and evaluate the working efficiency.

[0060] According to the PDS sequence in Medicago truncatula, a close relative of Medicago truncatula, primers were designed to amplify the PDS gene sequence of Medicago truncatula and sequenced. Based on the obtained PDS gene sequence and referring to the case in Medicago truncatula, the target site was designed; the target site was conn...

Embodiment 2

[0089] Example 2 PALM1 gene mutation in alfalfa

[0090] The difference from Example 1 is:

[0091] According to the PALM1 sequence in Medicago truncatula, a close relative of alfalfa, primers were designed to amplify the MsPALM1 gene sequence of alfalfa and sequenced, and the target site was designed according to the obtained MsPALM1 gene sequence; the target site was connected to the site of the vector MsCRISPR / Cas9 to obtain For the knockout vector MsCRISPR / Cas9::PALM1 of the MsPALM1 gene, the vector was transformed into alfalfa explants by the above-mentioned transformation method of Agrobacterium tumefaciens, and regenerated plants were obtained; the detection method was PCR-RE, and the obtained regenerated Genomic DNA samples were extracted from the plants, amplified with specific amplification primers containing the target sequence for the MsPALM1 gene, and screened for mutants by cutting PCR products with BstU I restriction endonuclease ( Figure 4 ); and by phenotypi...

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Abstract

The invention discloses a method for site-specific mutagenesis of medicago sativa genes by employing a CRISPR / Cas9 system. First of all, a binary expression vector MsCRISPR / Cas9 is constructed; the vector is designed and constructed for a target gene; then, agrobacterium tumefaciens-mediated transformation is used to achieve site-specific mutagenesis of a specific gene, and a mutant transformed plant with the target gene knocked out is obtained by screening; the method uses the CRISPR / Cas9 technology for cross-pollination autotetraploid plant medicago sativa for the first time, and obtains a mutant plant; a MtU6 promoter is used for the first time to initiate sgRNA transcription in the medicago sativa, thereby improving the transcription efficiency; the mutation rate can reach 52%; the method realizes direct mutation on the coding sequence of the gene, and opens up a new field for gene editing in the medicago sativa, laying a technical foundation for simultaneous silencing of a plurality of genes, deletion of large chromosome fragments, precise insertion of foreign genes into targets, gene regulation and the like.

Description

technical field [0001] The invention belongs to the technical field of gene editing, and in particular relates to a method for site-directed mutation of alfalfa gene by using a CRISPR / Cas9 system. Background technique [0002] Alfalfa (Medicago sativa.) is a widely planted legume forage, rich in high-quality dietary fiber, edible protein, multivitamins (including vitamin B, vitamin C, vitamin E, etc.), a variety of beneficial minerals, saponins, flavonoids Bioactive components such as carotenoids, carotenoids, phenolic acids, etc., have high protein content and good palatability, which are of great significance for improving the feed level of livestock. In addition to being used as a forage crop, the well-developed root system of alfalfa plays an important role in the prevention and control of soil erosion and environmental governance. In addition, alfalfa also plays an important role in improving soil fertility because of the symbiosis of a large number of nitrogen-fixing ...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N9/22C12N15/70A01H5/00A01H6/54
CPCC12N9/22C12N15/70C12N15/8213C12N2310/20C12N15/743A01H5/10C12N15/8209A01H6/544C12N15/11C12N15/8205C12N2800/80
Inventor 陈海涛王文谢雄平邱强尚占环苏克先何辉
Owner GUANGDONG SANJIE HERBAGE BIOTECH CO LTD
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