Genetic transformation, gene editing and analysis methods of main soybean varieties

A method of genetic transformation and gene editing technology, which is applied in the field of genetic transformation, gene editing and analysis of main soybean varieties, and can solve the problems that no successful reports of soybean varieties have been seen, and the difficulty of transforming good soybean varieties.

Pending Publication Date: 2020-04-28
北大荒垦丰种业股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the difficulty in transforming good soybean varieties, there have been

Method used

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  • Genetic transformation, gene editing and analysis methods of main soybean varieties
  • Genetic transformation, gene editing and analysis methods of main soybean varieties
  • Genetic transformation, gene editing and analysis methods of main soybean varieties

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] This example discloses the construction of the gene editing transformation vector KF57, which specifically includes the following steps:

[0091] (1) Cloning of soybean translation elongation factor gene GmEF2 promoter: Agrobacterium transformation vector for gene editing is based on pCambia3301 backbone, using eukaryotic cells encoding optimized Streptococcus pyogenes (Streptococcus pyogenes) Cas9 endonuclease and single The guide RNA (sgRNA) was expressed with the same carrier to realize the targeted cut-off of the target gene locus, and the transformed plants were screened using the Barnase gene expression of glufosinate-resistant herbicide (Glufosinate, phosphinothricin). The soybean Williams82 genome Glycine max Wm82.a2.v1 (phytozome.jgi.doe.gov) collected by the US Department of Energy contains multiple high-level constitutively expressed translation elongation factor genes including Glyma.05G089000.1, and its promoter is suitable for High-level expression of the ...

Embodiment 2

[0095] Example 2 Genetic Transformation of Kenbao Xiaoliudou No. 1

[0096] Gene editing can only be achieved by transforming the gene editing vector into cells for expression. In order to edit the Fad2 gene of Kenfeng’s self-owned excellent variety Kenbao Xiaoliudou No. 1, the Agrobacterium-mediated method was used to expand and germinate Kenbao Xiaoliudou No. 1. The seeds are explants, and the gene editing vector KF57 is transformed into soybean cells for knockout editing of the GmFad2-1A and GmFad2-1B genes. The specific operation process is as follows:

[0097] (1) Preparation of explants: Select Kenbao Xiaolidou No. 1 plump seeds with a smooth surface and no scars, put them in a petri dish and put them in a desiccator, add 96 ml of 10% sodium hypochlorite and 4 ml of concentrated hydrochloric acid to generate chlorine gas, and airtightly sterilize Seeds about 16 hours. Open the cover of the sterilized seeds in the ultra-clean bench and blow off the chlorine gas, add ster...

Embodiment 3

[0104] Embodiment 3 Genetic Transformation of Longken 314

[0105] Longken 314 is another Kenfeng self-owned fine variety that can be transformed by Agrobacterium-mediated transformation. Similarly, the transformation of KF57 can realize the knockout and editing of GmFad2-1A and GmFad2-1B genes, and obtain high oleic acid traits. The transformation process mediated by Agrobacterium is similar to that of Kenbao Xiaoliudou No. 1 mentioned above. Glufosinate at different concentrations of 4, 8, 12, 16, 20, and 30 mg / L was tested for screening, and it was found that Longken 314 Agrobacterium infection was suitable Concentration and the appropriate concentration of transforming screening agent are all slightly lower, and the two concentrations are too high to cause browning and death of explants and cluster buds respectively. 20mg / L glufosinate was used to screen the induction and elongation of clustered shoots, and 2 healthy plants were obtained from the transformation of 100 expl...

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Abstract

The invention belongs to the field of crops, and particularly relates to genetic transformation, gene editing and analysis methods of main soybean varieties. The invention discloses a gene editing andtransforming vector which is based on a CRISPR/Cas9 system and takes an agrobacterium transforming vector pCambia3301 as a skeleton, and a promoter is designed for improving expression of Cas9 and gRNA. According to the invention, rapid and effective genetic transformation of the main soybean varieties is realized for the first time, and a simple and rapid PCR amplification sequencing analysis method for identifying the gene editing effect is developed. Through genetic separation and screening by molecular biology analysis technology, gene editing offspring plants which do not carry exogenousgenes of the soybean varieties for production are successfully obtained, and editing sites are stably inherited.

Description

technical field [0001] The invention belongs to the field of crops, and in particular relates to genetic transformation, gene editing and analysis methods of main soybean varieties. Background technique [0002] Soybean originated in China and is one of the main crops that humans rely on for survival. It is an important source of vegetable protein and edible oil, and occupies an important position in the agricultural production of the world and China. According to the statistics of World Agricultural Production Analysis (World Agricultural Production, USDA WAP 5-19) released by the United States Department of Agriculture in May 2019, in 2018, the global soybean planting area was 124.6 million hectares, with a total output of 362.08 million tons, and the average yield per unit area was 2.88 tons per hectare. The planting area in the United States is 35.66 million hectares, about 95% of which are genetically modified varieties such as herbicide resistance. The total output is...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/00A01H6/54
CPCC07K14/415C12N15/8218
Inventor 李忠森赵超越刘丹谷月胡喜平
Owner 北大荒垦丰种业股份有限公司
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