A method for site-directed mutation of alfalfa gene using CRISPR/Cas9 system

A gene site-directed mutation, alfalfa technology, applied in the field of gene editing, can solve the problems of incomplete RNAi silencing, limited application, unstable characteristics, etc., and achieve the effect of thorough technology, high mutation efficiency, and accelerated breeding.

Active Publication Date: 2021-07-09
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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  • A method for site-directed mutation of alfalfa gene using CRISPR/Cas9 system
  • A method for site-directed mutation of alfalfa gene using CRISPR/Cas9 system
  • A method for site-directed mutation of alfalfa gene using 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-directed mutation of alfalfa gene by using CRISPR / Cas9 system. The present invention uses CRISPR / Cas9 technology in the cross-pollination autotetraploid plant alfalfa for the first time, and obtains mutant plants; for the first time, the MtU6 promoter is used to initiate sgRNA transcription in alfalfa to improve transcription efficiency; and natural mutation and traditional Compared with artificially induced mutations, the present invention accurately introduces mutations with higher mutation efficiency, and can achieve a mutation rate of 52%, of which the homozygous mutation rate reaches 12%. The invention directly mutates the gene coding sequence, which opens up a new field for gene editing in alfalfa, and lays the foundation for subsequent simultaneous silencing of multiple genes, deletion of large chromosome fragments, precise insertion of foreign genes into target sites, gene regulation, etc. technical basis.

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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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/84C12N15/113C12N9/22A01H5/00A01H6/54
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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