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Preparation method of peanut mutant, peanut mutant gene, protein encoded by peanut mutant gene and application

A technology for mutant genes and mutants is applied in the field of primers for detecting peanut mutant genes and the preparation of peanut mutants, which can solve problems such as large workload, and achieve the effects of convenient operation, fast mutation speed and high efficiency.

Active Publication Date: 2020-11-17
深圳市深花生物科技有限公司
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Problems solved by technology

[0007] The purpose of the present invention is to provide a preparation method of peanut mutants, a peanut mutant gene and its encoded protein and application, and a primer for detecting peanut mutant genes, aiming to solve the existing problem of high oleic acid peanut There are technical problems with heavy workload in breeding

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  • Preparation method of peanut mutant, peanut mutant gene, protein encoded by peanut mutant gene and application
  • Preparation method of peanut mutant, peanut mutant gene, protein encoded by peanut mutant gene and application
  • Preparation method of peanut mutant, peanut mutant gene, protein encoded by peanut mutant gene and application

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preparation example Construction

[0035] The embodiment of the present invention provides a kind of preparation method of peanut mutant, it comprises the following steps:

[0036] S1. Provide the coding region sequence of peanut desaturated fatty acidase gene AhFAD2A, CRISPR / Cas9 gene editing vector and peanut embryogenic callus;

[0037] S2. According to the sequence of the coding region of the peanut desaturated fatty acidase gene AhFAD2A, the sgRNA target sequence T1-T4 is obtained;

[0038] S3. According to the sgRNA target sequence T1-T4, synthesize the sgRNA target sequence nucleotide fragments containing cohesive ends and their complementary strands respectively, and mix and anneal to obtain double-stranded DNA T1-T4 containing cohesive ends at both ends;

[0039] S4. Ligate the double-stranded DNA T1-T4 with the CRISPR / Cas9 gene editing vector to obtain the gene editing vector T1-T4;

[0040] S5. Transforming peanut embryogenic callus with at least one of the gene editing vectors T1-T4, and culturing ...

Embodiment

[0075] 1. Cloning and sequencing of peanut desaturated fatty acidase gene AhFAD2A

[0076] According to the cultivated peanut (Arachis hypogaea) omega-6desaturase (FadS) mRNA sequence (GenBank: AF030319.1) design PCR primer FAD2A-f and FAD2A-r, its nucleotide sequence is respectively as SEQ ID NO: 6 and SEQ ID NO: 7.

[0077] The peanut leaves were taken, the kit extracted the genomic DNA, and the AhFAD2A gene was amplified by PCR. The PCR reaction system is as follows:

[0078] The 50μl reaction system includes: ddH 2 O: 13 μl, Primer: 5 μl, DNA: 5 μl, 2×Taq mix: 25 μl, Mg 2+ : 1 μl, glycerol: 1 μl.

[0079] The reaction conditions are as follows: pre-denaturation at 95°C for 5 min, followed by a cycle of denaturation at 95°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 1 min. After 40 cycles, complete extension at 72°C for 10 min, and storage at 4°C.

[0080] DNA sequencing was performed on the PCR product to obtain the sequence of the coding region o...

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Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a preparation method of a peanut mutant, a peanut mutant gene, protein encoded by the peanut mutant gene and an application. By designing SgRNA T1-T4, a CRISPR / Cas9 genome editing system is constructed, a Cas9 enzyme guided by sgRNA cuts a peanut desaturated fatty acid enzyme gene AhFAD2A at a gene target, and gene sequence insertion or base mutation is generated after DNA repair, so that targeted mutation of the peanut desaturated fatty acid enzyme gene AhFAD2A is completed, the gene cannot synthesize a functional desaturated fatty acid enzyme and cannot catalyze oleic acid dehydrogenation to generate linoleic acid, peanuts with high oleic acid content can be obtained, and the quality of peanut varieties can be improved.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a preparation method of a peanut mutant, a peanut mutant gene, its encoded protein and its application, and a primer for detecting the peanut mutant gene. Background technique [0002] As the raw material of high-quality edible oil, peanut is an important oil crop and economic crop in my country. Its total output and consumption account for more than 40% of the world, and its export volume accounts for more than 55% of the world (United Nations Data Center, 2012). Has a strong competitiveness. Peanut seeds are very rich in oil, with an average oil content of about 51%, and are widely welcomed by the market. The main components of peanut oil are monounsaturated fatty acid oleic acid (C18:1, Δ9), polyunsaturated fatty acid linoleic acid (C18:2, Δ9, Δ12) and saturated fatty acids palmitic acid (C16:0) and stearic acid ( C18:0), in which unsaturated fatty aci...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82C12N15/113C12N15/11C07K14/415C12Q1/6895A01H4/00A01H5/00A01H6/54
CPCC07K14/415C12N15/8247C12N15/8218C12N15/113C12Q1/6895A01H4/00
Inventor 于为常张旺
Owner 深圳市深花生物科技有限公司
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