Gene mutant for improving soybean oleic acid content and application thereof

A soybean oleic acid, single-gene mutation technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problems of reducing nutritional value, affecting oil quality, poor stability, etc.

Pending Publication Date: 2021-03-30
ZHEJIANG FORESTRY UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Linoleic acid and linolenic acid are polyunsaturated fatty acids with poor stability. They are easily oxidized during high-temperature processing, which r

Method used

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  • Gene mutant for improving soybean oleic acid content and application thereof
  • Gene mutant for improving soybean oleic acid content and application thereof
  • Gene mutant for improving soybean oleic acid content and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1: Obtaining of transgenic soybean plants

[0040] Electric shock transformation of Agrobacterium tumefaciens: Take 1 mL each of the constructed CRISPR / Cas9 gene editing vectors EP and KP and add them to Agrobacterium tumefaciens EHA105 competent cells, mix well, and transfer to the electric shock cup. Put the electric shock cup into the sample slot of the electric shock instrument, and select the appropriate voltage for electric shock (2.0mm, 2500V) according to the distance between the electrodes of the electric shock cup. Add 1 mL of YEP liquid medium without antibiotics to the electroshocked Agrobacterium tumefaciens, mix well, transfer to a sterilized 1.5 mL centrifuge tube, and incubate at 28°C for about 3 hours. The bacterial solution was centrifuged at 4000rpm for 3min, and the supernatant was discarded. Mix the remaining YEP liquid medium and precipitated bacteria, evenly spread it on the YEP solid medium plate containing 12mg / L rifampicin and 50mg / ...

Embodiment 2

[0044] Example 2: Utilizing fatty acid component analysis method to screen mutated soybean grains

[0045] With reference to the method of Zheng et al. (2003; Plant Cell 15:1872-1887), the obtained T2 generation soybean seeds were analyzed for fatty acid components, and the specific implementation steps were as follows:

[0046] (1) Fix the soybean grains, cut off the cotyledons (about 40mg) accounting for 1 / 4 of the total volume of soybeans at the end far away from the hilum with a clean blade along the direction parallel to the hilum, chop them and place them in a 2ml imported centrifuge tube , add glass beads, and then add internal standard C17:0 (dissolved in n-hexane, ready to use). The rest of the seeds were planted in the pre-prepared substrate (about 1 cm in depth), with the hilum facing down, for subsequent molecular identification;

[0047] (2) Close the lid tightly, carry out crushing treatment in the tissue crushing instrument, and centrifuge;

[0048] (3) Transf...

Embodiment 3

[0051] Example 3: Detection of gene editing sites in transgenic positive plants FAD2-1A, FAD2-1B and FAD3A, while detecting the presence or absence of the Cas9 protein gene

[0052] After fatty acid component analysis, seeds with an oleic acid content greater than 80% were selected for planting, and the oleic acid content among different seeds in the same selected strain was the same or similar. The KP transgenic T2 generation plants were obtained, and the genomic DNA of the leaves of the KP transgenic T2 generation plants was extracted.

[0053] Then, use genomic DNA as a template, design primers so that the amplified product is a sequence of 150-250 bp near the target sequence, and carry out a conventional PCR reaction. The designed primers are shown in Table 2 below.

[0054] Table 2: List of primer sequences.

[0055]

[0056] Finally, the PCR product was detected by polyacrylamide gel electrophoresis for mutation of the target sequence. The specific electrophoresis me...

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Abstract

The invention relates to a gene mutant for improving soybean oleic acid content and application thereof, and belongs to the technical field of plant gene engineering. The gene mutant is a single-genemutant or a polygene mutant; the single-gene mutant is subjected to FAD2-1A gene mutation; the polygene mutant is subjected to joint mutation of an FAD2-1A gene and other genes, and the other genes are one or two of an FAD2-1B gene and an FAD3A gene; FAD2-1A gene mutation is that two bases are deleted at the 529th site and the 530th site of the gene; the FAD2-1B gene mutation is that 10 basic groups are deleted at the 265-274 site of the gene; the FAD3A gene mutation is that seven basic groups are deleted at the 55-61th site of the gene. The gene mutation site information provides a brand-new way for quality breeding work of soybeans or other oil crops with high oleic acid content, and has a good application prospect.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a gene mutant for increasing soybean oleic acid content and application thereof. Background technique [0002] Soybean (Glycine mas L.Merrill) originated in China, is one of the world's important oil crops and economic crops, and is also the main source of human vegetable oil and vegetable protein. With the improvement of living standard and improvement of diet structure, people's demand for high-quality soybean oil is increasing day by day, and cultivating high-quality soybean has become one of the important goals of soybean breeding. Soybean oil accounts for about 40% of the vegetable edible oil consumption in my country. The components and proportions of soybean fatty acids determine the quality of soybean oil. It is mainly composed of palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2) and linolenic acid (18:3), whi...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N15/10C12N15/82C12N15/84C12N15/55A01H5/10A01H6/54
CPCC12N9/0071C12N15/102C12N15/8218C12N15/8247C12N9/22C12Y114/19006C12Q2521/327C12Q2525/161
Inventor 郑月萍夏婷丁硕郑挺唐梦珍童红英沈志成林蓉李丹丹郑志富
Owner ZHEJIANG FORESTRY UNIVERSITY
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