Method for obtaining high oleic acid rapeseed by means of gene editing technology

A technology of gene editing and high oleic acid, applied in the field of plant genetic engineering, can solve the problem of undiscovered oleic acid in Brassica napus, and achieve the effects of good application prospects, improved oil content and high editing efficiency

Inactive Publication Date: 2019-01-11
WUHAN ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] After searching, no reports were found on the regulation of oleic acid content in Brassica napus using CRISPR / Cas9 technology

Method used

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  • Method for obtaining high oleic acid rapeseed by means of gene editing technology
  • Method for obtaining high oleic acid rapeseed by means of gene editing technology
  • Method for obtaining high oleic acid rapeseed by means of gene editing technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: the acquisition of transgenic rape plant

[0029] Preparation of aseptic receptor materials: select plump and clean Brassica napus ZY50, 627R and DT, a total of 15 rapeseeds, soak in 76% ethanol for 3 minutes, then sterilize in 0.1% mercury liter for 15 minutes, rinse with sterile water 4 times , blot the excess water with sterile filter paper, inoculate on the germination medium, and culture in a cardboard box at 25-28°C for one week in the dark. Wherein the seed germination medium is: MS medium + agar powder 6.0-8.0 g / L, and the pH value is 5.8-6.0.

[0030] Donor strain culture: take out the strain tube stored at -80°C for a long time, put it on ice, scrape the surface of the frozen culture with an inoculation needle under aseptic conditions, and quickly streak the Agrobacterium adhered to the inoculation needle On the surface of LB agar plate containing 50mg / L kanamycin (Kan)+50mg / L gentamicin sulfate (Gen)+50mg / L rifampicin (Rfi). The inoculated aga...

Embodiment 2

[0036] Embodiment 2: the DNA extraction procedure of transformed plant

[0037] The DNA in the leaves of the T0 generation rape obtained in Example 1 was extracted by the CTAB sample method. Concretely include the following steps:

[0038] (1) Use a numbered 2mL centrifuge tube to take a small amount of young leaves of rapeseed;

[0039] (2) Place the sampled centrifuge tube on ice, open the lid and add clean steel balls and 100 μL 2% CTAB solution, put the lid on and grind it with a sample grinder adapter (TissulyserⅡ, QIAGEN) (28times / s, 30s );

[0040] (3) After the sample is ground, take out the centrifuge tube, open the lid, pour out the steel ball, and then add 300 μL CTAB solution;

[0041] (4) Place the centrifuge tube on the centrifuge box plate, place it in a constant temperature water bath at 55°C-60°C for 50min-60min, shake gently once every 10min, and place it on the workbench to cool to room temperature after the water bath;

[0042] (5) Add an equal volume (...

Embodiment 3

[0055] Example 3. Detection of the FAD2 gene editing site of a transgenic positive individual plant

[0056] Editing detection was performed on the genomic DNA of transgene-positive individual plants with target bands in both pairs of positive detection primers. Firstly, the target segment was amplified on the copy of the FAD2 gene in the transgene-positive individual plants using the primers (Table 3) whose PCR products partially overlapped each other. High-fidelity PMSF DNA polymerase (Phanta Max Super-Fidelity DNA Polymerase, Vazyme) was used to amplify the target segment, Two monoclonal antibodies that can inhibit its 5'→3' polymerase activity and 3'→5' exonuclease activity at room temperature are added to Max, which can perform highly specific hot-start PCR. The enzyme has 5'→3' polymerase activity and 3'→5' exonuclease activity, and the amplified product is blunt-ended. According to the PCR reaction preparation method in Table 4, the amplification of the target fragm...

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Abstract

The invention discloses a method for cultivating high oleic acid rapeseed by means of a gene editing technology, being characterized in that an FAD2 gene is site-directed edit by CRISPR/Cas9 system toobtain transgenic rapeseed plant, and comprising the follow steps: (1) selecting two gRNA target sites of FAD2 gene as S1 and S2 respectively, and synthesizing and construct vector-related primers; (2) constructing a double-target CRISPR/Cas9 gene editing vector; (3). extracting plasmid and transforming Agrobacterium tumefaciens; and (4) using Agrobacterium tumefaciens-mediated transformation totransfer the expression vector into rapeseed, and obtaining the transgenic plants with mutation of FAD2 gene. The oil content of the transgenic rapeseed plant is significantly higher than that of thewild-type rapeseed plant. Moreover, the method provided by the invention has high editing efficiency and good application prospect.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, in particular to editing the FAD2 gene of Brassica napus by using the CRISPR / Cas9 system, so as to obtain rape varieties with high oleic acid content. Background technique [0002] Brassica napus L., 2n=38, AACC is a Brassicaceae plant and is the oil crop with the largest planting area in China. After long-term natural selection and artificial selection, the agronomic traits of Brassica napus are constantly being improved in a direction that is more suitable for the daily needs of human beings. Good agronomic traits of Brassica napus mainly include high oil content, high unsaturated fatty acid content, low glucosinolates, low erucic acid, stress resistance and good plant type, etc. Among them, the breeding of "double-low" rapeseed with low erucic acid and low glucosinolate has become a research hotspot in Brassica napus breeding in recent years. In addition, the rapeseed oil of "double-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N9/22A01H5/00A01H6/20
CPCC12N9/22C12N15/8213C12N15/8247
Inventor 万丽丽王转茸辛强洪登峰杨光圣
Owner WUHAN ACADEMY OF AGRI SCI
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