Method and application of CRISPR-Cas9 specific knockout of soybean lipoxygenase gene

A lipoxygenase and specific technology, applied in the field of soybean molecular breeding and biology, can solve the problems of time-consuming and laborious, and achieve the effect of avoiding high cost and avoiding long cycle.

Active Publication Date: 2021-08-03
武汉艾迪晶生物科技有限公司
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AI Technical Summary

Problems solved by technology

In conventional breeding, it is often necessary to lox It is a time-consuming and labor-intensive process for mutants to be crossed with elite varieties, and then through multiple generations of backcrossing or selfing to breed non-beany flavor soybean varieties

Method used

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  • Method and application of CRISPR-Cas9 specific knockout of soybean lipoxygenase gene
  • Method and application of CRISPR-Cas9 specific knockout of soybean lipoxygenase gene
  • Method and application of CRISPR-Cas9 specific knockout of soybean lipoxygenase gene

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Experimental program
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Effect test

Embodiment 1

[0029] Example 1: Construction of CRISPR-Cas9 Gene Knockout Vector and Stable Transformation of Soybean

[0030] Main reagents and sources of materials:

[0031] The Escherichia coli DH5α competent cells, Agrobacterium rhizogenes K599 competent cells and Agrobacterium GV3101 competent cells used in this study were all preserved by our laboratory; the CRISPR-Cas9 gene editing vector pGES201 used was obtained from pCAMBIA1300 ( Cambia, Australia), the pGmU6 promoter promotes the expression of sgRNA, the soybean endogenous promoter pM4 promotes the expression of Cas9, and the empty vector carries its own gene "Escherichia coli toxin-antitoxin system toxin protein ccdB (control of cell division or death system)", and there are enzyme cutting sites BsaI ( figure 1 ).

[0032] PCR Mix enzyme PCR Master Mix was purchased from Qingke Biotechnology Co., Ltd.; BsaI restriction endonuclease was purchased from NEB (UK); T4 ligase was purchased from Beijing Quanshijin Biotechnology Co.,...

Embodiment 2

[0084] Example 2: Separation of T-DNA and Editing Sites GLox Full deletion mutant screening

[0085] We used methods such as leaf smearing Basta, Bar Primer PCR (BPP), sgRNA Specific PCR (SSP), and PCR product sequencing to screen positive plants, identify sgRNA distribution, and detect targeted gene editing in stably transformed plants. The results showed that: 76 strains of T 0 Among the stable transformed seedlings, 60 were positive plants, and 27 of them contained only sgRNA- GmLox1 / 2 , 22 strains contained only sgRNA- GmLox3 , 11 strains also contain sgRNA- GmLox1 / 2 and sgRNA- GmLox3 ; 22 positive plants occurred GLox Gene editing (gene editing efficiency is 36.7%), in which positive plants Gmlox -28 and Gmlox -60 knocked out 3 at the same time GLox Gene( image 3 ).

[0086] take T 0 Positive plants Gmlox -28 and Gmlox -60 harvested seeds, planted in pots in the grow room, gain T 1 Substitute plants. take T 1 Extract DNA from leaves of generati...

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Abstract

The invention discloses a method for specifically knocking out soybean lipoxygenase gene by CRISPR-Cas9 and its application, and belongs to the fields of soybean molecular breeding and biotechnology. The method comprises: designing and simultaneously targeting a highly homologous gene coding region of soybean lipoxygenase GmLox1 and GmLox2 sgRNA‑ GmLox1 / 2, and targeting GmLox3 sgRNA‑ GmLox3 ; Construct the CRISPR-Cas9 gene knockout vector separately for each sgRNA, and then mix and infect soybean cotyledon nodes; after soybean stable genetic transformation, the plants Gmlox‑28 and Gmlox-60 Simultaneous knockout of 3 GLox Gene; after two generations of selfing, at T 2 In the generation plants, 5 strains with T-DNA and editing site were screened GLox full deletion mutant. This method provides a new strategy for the direct and targeted rapid creation of new soybean germplasm without beany odor.

Description

technical field [0001] The invention belongs to the field of soybean molecular breeding and biotechnology, in particular to a method for specifically knocking out soybean lipoxygenase gene by CRISPR-Cas9 and its application. Background technique [0002] soybeans ( Glycine max (L.) Merr.) originated in China and is one of the main cultivated crops in my country and even in the world. It is also an important source of vegetable protein and fat intake for humans, poultry and livestock, and plays a pivotal role in my country's agricultural production (Han Lide et al., 2003 ; Bi Yingdong et al., 2014; Pan Wenhua and Xu Shiwei, 2014; Zhang Yanan, 2017). However, during the storage and processing of soybeans and their products, lipoxygenase (Lipoxygenase, LOX) in soybean cotyledons can specifically catalyze the oxygenation reaction of polyunsaturated fatty acids with cis-1.4-pentadiene structure, forming over Hydrogen oxide derivatives (Axelrod et al., 1981), finally decompose i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/53A01H5/00A01H5/10A01H6/54
CPCC12N9/0069C12N15/8213C12Y113/11012
Inventor 关跃峰王杰匡华琴
Owner 武汉艾迪晶生物科技有限公司
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