Method for generating alfalfa genome edited homozygous plant

A genome editing and alfalfa technology, applied in the field of plant genetic engineering, can solve the problems of prolonging the test cycle and long plant growth cycle, and achieve the effects of simplifying the workload, growing rapidly, and shortening the screening cycle

Inactive Publication Date: 2017-05-17
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
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Problems solved by technology

However, most of the materials generated during the CRISPR / Cas9 genome editing process are heterozygous edits. How to use a simple and fast method to obtain homozygous edits can be used to solve the problem of prolonging the test period due to the long growth cycle of plants and the need for large-scale editing after genome editing. The problem of screening and identification has become a very important issue in genome editing research

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  • Method for generating alfalfa genome edited homozygous plant
  • Method for generating alfalfa genome edited homozygous plant
  • Method for generating alfalfa genome edited homozygous plant

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Experimental program
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Embodiment 1

[0049] Embodiment 1: CRISPR / Cas9-PDS expression vector construction and transformation obtain Medicago truncatula (R108) hairy root root system, which comprises the following steps:

[0050] 1) Cloning and gRNA design of Medicago truncatula (R108) PDS gene:

[0051] Cloning of PDS gene: Searched by NCBI and Phytozome, there is only one complete PDS gene sequence (gene number Medtr3g084830) in Medicago truncatula genome. The sequence of this gene was used as a standard to design primers, and the DNA of Medicago truncatula (R108) was used as a template for PCR amplification and sequencing.

[0052] gRNA design: use CRICPR-P website, use Medicago truncatula (Mt4.0v2) as TargetGenome, use Medtr3g084830 gene as Locus Tag, Submit. Note that gRNA with suitable restriction sites adjacent to the PAM region is selected as the target site for site-directed editing.

[0053] 2) Construction of genome editing expression vector pYLCRISPR / Cas9P35S-B-tRNA-PDS:

[0054] PUC57 vector plasmid...

Embodiment 2

[0073] Example 2: Rapid screening of homozygous hairy root systems where the PDS gene of Medicago truncatula (R108) was site-specifically edited. The main steps are as follows:

[0074] 1) Propagation of hairy roots: The resistant hairy roots induced in Example 1 were randomly numbered, and the root systems were randomly numbered 1S0, 2S0, 3S0... in order to facilitate tracking and purification. The resistant root system grows rapidly and can be subcultured every 10-14 days. In the S1 and S2 generations, 5-10 single hairy roots were randomly selected for continuous subculture, and the remaining root segments after subculture were collected for DNA extraction and reserved for identification. The temperature of the incubator was 25±2°C, and the culture was carried out in the dark.

[0075] 2) Enzyme digestion identification of hairy root: Extract DNA from the resistant hairy root sample obtained in step (1), PCR amplify the target gene including gRNA, and perform single enzyme ...

Embodiment 3

[0082]Example 3: Establishment of the hairy root regeneration system and the homozygous editing of the PDS gene to obtain the albino seedlings of Medicago truncatula (R108), the main features of which include the following steps:

[0083] 1) Hairy root induction of callus: Transfer the homozygous root system of genome editing identified by screening to callus induction medium (SHC), induce it for 3-5 weeks, and the callus produced can proceed to the next step of differentiation and regeneration. The temperature of the incubator was 25±2°C, and the culture was carried out in the dark.

[0084] 2) Hairy root differentiation and regeneration: the callus obtained in step (1) is transferred to the differentiation medium (MSR), and bud points can be differentiated within one week, and regenerated shoots can be obtained after 3-5 weeks of differentiation. The temperature of the incubator is 25±2°C, and the light time is 16h light / 8h dark every day (see Image 6 ).

[0085] 3) Rooti...

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Abstract

The invention discloses a method for generating an alfalfa genome edited homozygous plant, and belongs to the technical field of plant genetic engineering. The method mainly comprises: establishing an alfalfa hairy root system; converting alfalfa on a CRISPR / Cas9 over-expression vector, and obtaining resistant hairy roots; quickly filtering a homozygous hairy root system having an alfalfa genome edited by a fixed point; establishing a hairy root regeneration system, and obtaining a genome edited homozygous alfalfa plant. The method is mainly used for solving the problems of large workload and long period for obtaining a CRISPR / Cas9 genome edited homozygous plant. By means of the method of the invention, an alfalfa genome edited homozygous hairy root system can be obtained within 1-2 months; all alfalfa plants obtained by regenerating the homozygous hairy root system are homozygous edited plants.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a method for producing alfalfa CRISPR / Cas9 genome editing homozygous plants. Background technique [0002] As a high-quality legume forage, alfalfa plays an important role in livestock and poultry production. It has strong adaptability, long cultivation history, wide distribution range, stable productivity, long service life, high nutritional value, good palatability, good feeding effect for livestock and poultry, water storage and soil conservation, wind and sand fixation, soil improvement and fertilization, post-crop It has application value such as increasing yield, restoring vegetation, and protecting ecology. It is known as the "king of forage grass" and plays an irreplaceable role in agricultural and animal husbandry production. [0003] Bioactive ingredients in alfalfa, such as alfalfa polysaccharides, alfalfa saponins and alfalfa flavonoids,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/00
CPCC12N15/8205
Inventor 付春祥张海玲曹英萍尚晨李佶恺王建丽孙德全申忠宝
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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