阅读说明：本技术 利用CRISPR/Cas9修饰GmFAD2-1A基因获得高油酸含量大豆的方法 (Method for obtaining soybean with high oleic acid content by modifying GmFAD2-1A gene through CRISPR/Cas9 ) 是由 陈莉 侯文胜 付明雪 蔡宇鹏 孙�石 吴存祥 韩天富 于 2020-12-18 设计创作，主要内容包括：本发明公开了利用CRISPR/Cas9修饰GmFAD2-1A基因获得高油酸的大豆的方法。本发明提供的方法,包括如下步骤：1)用CRISPR/Cas9系统对出发植物中的GmFAD2-1A基因进行基因编辑,且使所述GmFAD2-1A基因发生突变导致翻译蛋白提前终止,得到转基因植物；2)所述转基因植物自交,得到纯合植物,即为目的植物；本发明的实验证明,利用CRISPR/Cas9介导的基因编辑技术对大豆脂肪酸去饱和酶编码基因GmFAD2-1A进行特定靶点的定点敲除,抑制油酸向亚油酸的转化,为高油酸大豆品种选育提供新材料,为加快高油酸大豆品种的选育具有积极的促进作用。(The invention discloses a method for obtaining high-oleic-acid soybean by modifying GmFAD2-1A gene with CRISPR/Cas 9. The method provided by the invention comprises the following steps: 1) carrying out gene editing on a GmFAD2-1A gene in a starting plant by using a CRISPR/Cas9 system, and enabling the GmFAD2-1A gene to be mutated to cause the termination of a translation protein in advance to obtain a transgenic plant; 2) selfing the transgenic plant to obtain a homozygous plant, namely a target plant; experiments prove that the specific target spot knockout is carried out on the soybean fatty acid desaturase coding gene GmFAD2-1A by using the CRISPR/Cas9 mediated gene editing technology, the conversion from oleic acid to linoleic acid is inhibited, a new material is provided for the breeding of high-oleic acid soybean varieties, and the positive promotion effect is achieved for accelerating the breeding of the high-oleic acid soybean varieties.)

1. A method for producing a target plant having a high oleic acid content, comprising the steps of:

1) carrying out gene editing on a GmFAD2-1A gene in a starting plant by using a CRISPR/Cas9 system, and enabling the GmFAD2-1A gene to be mutated to cause the termination of a translation protein in advance to obtain a transgenic plant;

2) selfing the transgenic plant to obtain a homozygous plant, namely a target plant;

the oleic acid content of the target plant is higher than that of the starting plant;

the GmFAD2-1A gene is a DNA molecule of (a1) or (a2) or (a3) as follows:

(a1) the coding region is a DNA molecule shown as a sequence 1 in a sequence table;

(a3) a DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in (b1) or (b2) and encodes the protein;

(a4) a DNA molecule derived from soybean and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to the DNA molecule defined in (b1) or (b2) and encoding said protein;

the target point of CRISPR/Cas9 system gene editing is the 649-672 th site of the sequence 1 or the 649-672 th site of the corresponding sequence 1.

2. The method of claim 1, wherein:

the CRISPR/Cas9 system includes the following 1) or 2):

1)sgRNA，

the target sequence of the sgRNA is the 649-672 th site of the sequence 1 or the 649-672 th site of the corresponding sequence 1;

2) a CRISPR/Cas9 vector expressing the sgRNA.

3. The method of claim 2, wherein:

the gene is edited by introducing the CRISPR/Cas9 vector into the starting plant.

4. A method according to any one of claims 1-3, characterized in that: the oleic acid content of the target plant seed is higher than that of the starting plant seed.

5. The method according to any one of claims 1-4, wherein:

the plant is a monocotyledon or a dicotyledon.

6. The method according to any one of claims 1-5, wherein:

the mutant form of mutating the GmFAD2-1A gene leading to premature termination of the translated protein is as follows:

1) adding a base T between 667 th and 668 th positions of the sequence 1, and keeping other nucleotide residues unchanged,

or, a base T is added corresponding to the middle of 667 th and 668 th positions of the sequence 1, and other nucleotide residues are kept unchanged;

2) deletion of 665-666 site of the sequence 1 and keeping other nucleotide residues unchanged,

or, corresponding to deletion at position 665-666 in the sequence 1, and keeping other nucleotide residues unchanged;

3) deletion of 667 th position of the sequence 1 and keeping the other nucleotide residues unchanged,

or, corresponding to deletion 667 of sequence 1, with the remaining nucleotide residues unchanged.

7. A method for making a plant having a gene editing function of GmFAD2-1A gene, comprising step 1) of any one of the methods of claims 1-6.

8. A substance for improving the oleic acid content of plants is a CRISPR/Cas9 system for gene editing GmFAD2-1A gene;

the target point of CRISPR/Cas9 system gene editing is the 649-672 th site of the sequence 1 or the 649-672 th site of the corresponding sequence 1.

9. The substance of claim 7, wherein: the CRISPR/Cas9 system includes the following 1) or 2):

1)sgRNA，

the target sequence of the sgRNA is the 649-672 th site of the sequence 1 or the 649-672 th site of the corresponding sequence 1;

2) a CRISPR/Cas9 vector expressing the sgRNA.

10. Use of the substance of claim 8 or 9 for the preparation of a target plant or cultivation of a target plant with a high oleic acid content;

or a GmFAD2-1A mutant gene is obtained by taking the 649-672 th site of the sequence 1 or the 649-672 th site corresponding to the sequence 1 as a target point to carry out CRISPR/Cas9 system gene editing;

or, a method for cultivating a plant having a high oleic acid content, which is the objective plant of the method of any one of claims 1 to 6.

Technical Field

The invention belongs to the technical field of biology, and relates to a method for obtaining high-oleic-acid soybean by modifying GmFAD2-1A gene with CRISPR/Cas 9.

Background

Soybean oil has great economic value and is widely applied to food, feed and industry. Soybean oil contains the largest proportion of polyvalent unsaturated fatty acids, while oleic acid is only about 20% and only one-fourth of the unsaturated fatty acids. Oleic acid is a monounsaturated fatty acid, has strong oxidation resistance and good stability, and is beneficial to health. The soybean variety with high oleic acid has the characteristics of higher oxidation stability, long storage life and the like. Therefore, the method reduces the content of polyvalent unsaturated fatty acid, improves the content of oleic acid, and the cultivation of high-oleic-acid soybean varieties is an important target of soybean quality breeding. Genetic variation related to the oleic acid content in germplasm resources and mutation materials is limited, and the soybean material with high oleic acid content is difficult to obtain by a conventional breeding method, so that great difficulty is brought to the cultivation of high oleic acid varieties, and the research progress is slow.

Disclosure of Invention

It is an object of the present invention to provide a method for producing a target plant having a high oleic acid content.

The method provided by the invention comprises the following steps:

1) carrying out gene editing on a GmFAD2-1A gene in a starting plant by using a CRISPR/Cas9 system, and enabling the GmFAD2-1A gene to be mutated to cause the termination of a translation protein in advance to obtain a transgenic plant;

2) selfing the transgenic plant to obtain a homozygous plant, namely a target plant;

the oleic acid content of the target plant is higher than that of the starting plant;

the GmFAD2-1A gene is a DNA molecule of (a1) or (a2) or (a3) as follows:

(a1) the coding region is a DNA molecule shown as a sequence 1 in a sequence table;

(a3) a DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in (b1) or (b2) and encodes the protein;

(a4) a DNA molecule derived from soybean and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to the DNA molecule defined in (b1) or (b2) and encoding said protein;

the target point of CRISPR/Cas9 system gene editing is the 649-672 th site of the sequence 1 or the 649-672 th site of the corresponding sequence 1 (correspondingly, the target point refers to the position of the sequence with high homology with the sequence 1 corresponding to the 649-672 th site of the sequence 1).

In the above method, the CRISPR/Cas9 system comprises the following 1) or 2):

1)sgRNA，

the target sequence of the sgRNA is the 649-672 th site of the sequence 1 or the 649-672 th site of the corresponding sequence 1;

2) a CRISPR/Cas9 vector expressing the sgRNA.

In the method, the gene editing is to introduce the CRISPR/Cas9 vector into the starting plant, in the embodiment of the invention, the CRISPR/Cas9 vector is a recombinant vector Cas9-sgRNA, the vector expresses the sgRNA, and the coding sequence of the target sequence binding region of the sgRNA is the 649-672 bit of the sequence 1, and the specific construction method is shown in the embodiment.

In the method, the oleic acid content of the target plant grain is higher than that of the starting plant grain.

In the above method, the plant is a monocotyledon or dicotyledon, and in the embodiment of the present invention, specifically soybean.

In embodiments of the invention, the mutation that mutates the GmFAD2-1A gene results in premature termination of the translational protein is any of the following:

1) adding a base T between 667 th and 668 th positions of the sequence 1, and keeping other nucleotide residues unchanged,

or, a base T is added corresponding to the middle of 667 th and 668 th positions of the sequence 1, and other nucleotide residues are kept unchanged;

2) deletion of 665-666 site of the sequence 1 and keeping other nucleotide residues unchanged,

or, corresponding to deletion at position 665-666 in the sequence 1, and keeping other nucleotide residues unchanged;

3) deletion of 667 th position of the sequence 1 and keeping the other nucleotide residues unchanged,

or, corresponding to deletion 667 of sequence 1, with the remaining nucleotide residues unchanged.

It is still another object of the present invention to provide a method for producing a plant having gene editing of the GmFAD2-1A gene.

The method provided by the invention comprises the step 1) in the first target method.

It is a further object of the present invention to provide a material for increasing the oleic acid content of plants.

The substance provided by the invention is a CRISPR/Cas9 system for gene editing GmFAD2-1A gene;

the target point of CRISPR/Cas9 system gene editing is the 649-672 th site of the sequence 1 or the 649-672 th site of the corresponding sequence 1.

In the above substance, the CRISPR/Cas9 system comprises the following 1) or 2):

1) sgRNA and cas9 protein,

the target sequence of the sgRNA is the 649-672 th site of the sequence 1 or the 649-672 th site of the corresponding sequence 1;

2) a CRISPR/Cas9 vector expressing the sgRNA and Cas9 proteins.

The application of the substances in preparing the target plants with high oleic acid content or cultivating the target plants with high oleic acid content is also within the protection scope of the invention.

The invention also provides a GmFAD2-1A mutant gene, which is obtained by using the 649-672 th site of the sequence 1 or the 649-672 th site corresponding to the sequence 1 as a target spot to carry out CRISPR/Cas9 system gene editing; specific examples in the embodiment of the present invention are any of the following:

1) adding a base T between 667 th and 668 th positions of the sequence 1, and keeping other nucleotide residues unchanged,

or, a base T is added corresponding to the middle of 667 th and 668 th positions of the sequence 1, and other nucleotide residues are kept unchanged;

2) deletion of 665-666 site of the sequence 1 and keeping other nucleotide residues unchanged,

or, corresponding to deletion at position 665-666 in the sequence 1, and keeping other nucleotide residues unchanged;

3) deletion of 667 th position of the sequence 1 and keeping the other nucleotide residues unchanged,

or, corresponding to deletion 667 of sequence 1, with the remaining nucleotide residues unchanged.

The invention also provides a method for cultivating the plant with high oleic acid content, which is the target plant obtained by the method for cultivating the first target.

Experiments prove that the specific target spot knockout is carried out on the soybean fatty acid desaturase coding gene GmFAD2-1A by using a CRISPR/Cas9 mediated gene editing technology, the conversion from oleic acid to linoleic acid is inhibited, a soybean mutant material with high oleic acid content and oleic acid content of about 40% is obtained, a new material is provided for breeding high oleic acid soybean varieties, and the active promotion effect is achieved for accelerating the breeding of the high oleic acid soybean varieties.

Drawings

FIG. 1 shows the mutation pattern of fad2-1A mutant at the GmFAD2-1A target.

FIG. 2 shows the relative fatty acid content in seeds of fad2-1a mutant.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.

The experimental procedures in the following examples are conventional unless otherwise specified.

The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Cultivated soybean Jack, described in the following documents: chen L, Cai Y, Liu X, Yao W, Guo C, Sun S, Wu C, Jiang B, Han T, Hou W (2018), Improvement of microbial Agrobacterium-mediated transformation efficiency by addition of glutamine and antisense in the culture medium of International Journal of Molecular sciences19,3039, publicly available from the institute of crop science, China academy of agricultural sciences.

Agrobacterium tumefaciens EHA105, described in: cai Y, Chen L, Liu X, Guo C, Sun S, Wu C, Jiang B, Han T and Hou W (2018a), CRISPR/Cas9-mediated targeted mutagenesis of GmFT2a delys watering time in soybean bean plant Biotechnol J16, 176-.

MS salt: phyto Tech, catalog No.: and M524.

MS organic: phyto Tech, catalog No.: and M533.

B5 organic: photostech corporation, catalog No.: G219.

b5 salt: photostech corporation, catalog No.: G768.

the YEP solid culture medium consists of a solvent and a solvent; the solutes and their concentrations in YEP solid medium were: NaCl 5g/L, yeast extract 5g/L, tryptone 10g/L, 15g/L agar; the solvent is water. YEP solid medium pH 7.0.

Germination medium (ph 5.8): 3.12g/L B5 salt, 1ml/L B5 organic, 20g/L sucrose, 7.5g/L agar, and the balance water.

Liquid medium (ph 5.4): 0.43g/L MS salt, 1ml/L B5 organic, 40mg/L acetosyringone, 150mg/L dithiothreitol, 100mg/L L-cysteine, 30g/L sucrose, 3.9 mg/L2-morpholine ethanesulfonic acid, and the balance of water.

Co-culture medium (ph 5.4): 0.43g/L MS salt, 1ml/L B5 organic, 40mg/L acetosyringone, 150mg/L dithiothreitol, 100mg/L L-cysteine, 30g/L sucrose, 7.5g/L agar, 3.9 mg/L2-morpholine ethanesulfonic acid, and the balance of water.

Recovery medium (ph 5.4): 3.1g/L B5 salt, 1ml/L B5 organic, 30g/L sucrose, 150mg/L cefuromycin, 150mg/L timentin, 1 mg/L6-BA, 0.98 g/L2-morpholine ethanesulfonic acid, 7.5g/L agar, 4ml/L Fe salt (200X), 50mg/L L-asparagine, 50mg/L L-glutamine, and the balance water.

Screening medium (ph 5.4): 3.1g/L B5 salt, 1ml/L B5 organic, 0.98 g/L2-morpholine ethanesulfonic acid, 30g/L sucrose, 150mg/L cefuroxime, 150mg/L timentin, 1 mg/L6-BA, 6mg/L glufosinate, 7.5g/L agar, 4ml/L Fe salt (200X), 50mg/L L-asparagine, 50mg/L L-glutamine, and the balance water.

Elongation medium (ph 5.6): 4.0g/L MS salt, 1ml/L B5 organic, 0.6 g/L2-morpholine ethanesulfonic acid, 30g/L sucrose, 150mg/L cefuromycin, 150mg/L timentin, 0.1mg/L IAA, 0.5mg/L GA, 1 mg/L6-BA, 6mg/L glufosinate-phosphine, 7.5g/L agar, 4ml/L Fe salt (200X), 50mg/L L-asparagine, 50mg/L L-glutamine, and the balance water.

Rooting medium (ph 5.7): 2.165g/L MS salt, 1ml/L B5 organic, 0.6 g/L2-morpholine ethanesulfonic acid, 20g/L sucrose, 7.5g/L agar, 50mg/L L-asparagine, 50mg/L L-glutamine, and the balance water.

Example 1 construction of GmFAD2-1A Gene editing CRISPR vector

First, obtaining gRNA

The soybean GmFAD2-1A (SEQ ID NO: 1) genomic sequence was obtained from the Phytozome database. GmFAD2-1A is located on chromosome 10. The selection of GmFAD2-1A sgRNA target site sequence was performed using CRISPR-P (http:// cbi. hzau. edu. cn/cgi-bin/CRISPR) online webpage tool. The target point is located in the second exon region of GmFAD2-1A, and the target sequence is GTGGCCAAAGTGGAAGTTCAAGG (649-672 th site of sequence 1).

After the target is designed, it needs to be integrated into the vector. Firstly, synthesizing a target primer of the sgRNA, wherein the primer sequence is as follows:

FAD2-1A-F：5′-TTGGTGGCCAAAGTGGAAGTTCA-3′

FAD2-1A-R：5′-AACTGAACTTCCACTTTGGCCAC-3′

(sgRNA with 20bp sequence underlined)

FAD2-1A-F and FAD2-1A-R primers are respectively added into a 25ul system for 5ul, water is added into the system for 15ul, the temperature is 95 ℃ for 3min, the annealing is carried out at 0.1 ℃/s until the temperature is 16 ℃, the annealing is kept for 10min at 16 ℃, and the annealing is finished to obtain a gRNA annealing product with a sticky end.

Preparation of sgRNA expression vector

1ul of the gRNA annealing product with sticky ends obtained in the previous step was taken and T4-linked to Cas9/gRNA vector (Beijing Vital bioscience, Inc., Cat: VK005-15, containing Cas9 protein expression unit) to obtain recombinant vector Cas9-sgRNA, which expresses sgRNA with the coding sequence of the target sequence binding region in the sgRNA being 649-672 th order of sequence 1.

Example 2 obtaining of fad2-1a mutant

Preparation of recombinant bacteria

The recombinant vector Cas9-sgRNA prepared in example 1 was transformed into E.coli DH5 α and plated on LB + Kan solid medium. And (4) selecting a monoclonal extracted plasmid, and sequencing.

Sequencing primer SQ: TGAAGTGGACGGAAGGAAGGAGG, the plasmid with the correct fragment was identified and named recombinant plasmid FAD 2-1A-sgRNA.

Transforming the recombinant plasmid FAD2-1A-sgRNA into agrobacterium EHA105 by an electrotransformation method, extracting the plasmid for sequencing verification, and naming the recombinant strain with correct sequencing verification as EHA-FAD 2-1A-sgRNA.

Second, Agrobacterium mediated transformation

The constructed EHA-FAD2-1A-sgRNA is transformed into a soybean variety Jack (hereinafter referred to as wild soybean) by utilizing an agrobacterium-mediated method, and the specific method comprises the following steps:

1. seed sterilization

1) Completely spreading the full, uniform and dry soybean variety Jack seeds without diseases and insect pests and spots in a culture dish, and then putting the culture dish into a dryer.

2) And after the step 1) is finished, putting a beaker with the volume of 100ml into the dryer, pouring 80ml of 12M sodium hypochlorite aqueous solution into the beaker, slowly adding 4ml of concentrated hydrochloric acid, quickly covering the dryer, sealing with vaseline, standing for 16h, and carrying out chlorine sterilization.

2. Preparation of infection bacterial liquid

1) Culturing the EHA-FAD2-1A-sgRNA bacterial solution obtained in the previous step at 28 ℃, and resuspending the bacterial solution in a liquid culture medium to obtain OD_600nm0.6 of the infected bacterial liquid.

2) And (3) putting the seeds treated in the step (1) into an ultra-clean bench, peeling off seed coats under a microscope, separating two cotyledons along a long axis, reserving the cotyledons with complete hypocotyls, scratching the joint of the hypocotyls and the cotyledons, generally scratching one cotyledon for 3-5 times, and then dip-dyeing for 2 hours in an incubator at 28 ℃.

3) And spreading the cotyledon with inner surface (smooth surface) facing upwards on the co-culture medium paved with sterile filter paper, and culturing at 22 deg.C in dark for 5 d.

4) After co-cultivation for 5 days, the explant was elongated to 2cm, and the hypocotyl was cut off to leave 0.5 cm. The treated explants were placed in recovery medium and cultured for 7d at 28 ℃ under 16h light/8 h dark conditions.

5) And taking out the explant from the recovery culture medium, removing a new bud, cutting off a part of embryonic axis, reserving 0.5cm of embryonic axis, transferring the trimmed explant into a screening culture medium, and culturing for 21 days under the conditions of 28 ℃, 16h of illumination and 8h of darkness.

6) And after screening induction for 21d, the explant generates a large amount of adventitious buds, the cotyledon and brown leaves are stripped, and the rest part is transferred into an elongation culture medium for culture at the temperature of 28 ℃ under the condition of 16h light/8 h dark.

7) In an elongation culture medium, when 5-8cm of caulicles are extracted from the cluster buds, cutting the base parts of the adventitious buds; dipping the stem base in 1mg/L IBA solution for 1min, transferring the stem base into a rooting medium for culture at 28 ℃, culturing for one week under 16h light/8 h dark conditions, transplanting the stem base into a pot after a large number of roots are generated, and obtaining a plant of T0 generation transformed soybean.

Thirdly, molecular detection of edited plants

DNA of T0 transformed soybean leaf is extracted as a template for PCR molecular detection, and wild soybean is used as a control.

PCR primers are designed near target sites of GmFAD2-1A genes, and PCR amplification and sequencing are carried out. Wherein, the primer FMA-F: 5'-TTGAGGGATTGTAGTTCTGTTGG-3' and FMA-R: 5'-ACACCAGTGAGAAGGCAACC-3' the GmFAD2-1A gene was amplified. And (3) PCR reaction system: 2X Phanta Max buffer12.5ul, dNTP Mix (10mM)0.5ul, DNA (200ng/ul)1ul, F (10pmol/ul)1ul, R (10pmol/ul)1ul, Super-Fidelity DNA Polymerase 0.5ul, ddH₂O8.5 ul, total volume 25 ul. An amplification reaction system: 3min at 95 ℃; 30sec at 95 ℃,30 sec at 58 ℃, 1min at 72 ℃ and 35 cycles; 5min at 72 ℃. The PCR product was sent to the company for sequencing verification.

The heterozygous editing plant is named as T0 generation GmFAD2-1A soybean when the mantle peak appears near the target position.

After the T0 generation GmFAD2-1A soybean is sowed, seeds of the T1 generation GmFAD2-1A soybean are harvested and cultivated, and the T1 generation GmFAD2-1A soybean is obtained.

The three PCR molecular detection methods are adopted to detect soybeans transformed from T1 to GmFAD2-1A, and sequencing results show that in the soybeans transformed from T1 to GmFAD2-1A, a mutant plant (FAD2-1A) generates the following mutations near a target site, so that protein translation is terminated early, and the GmFAD2-1A gene mutation types in the mutant plant comprise 3 types: -2bp, +1bp, -1bp (FIG. 1).

The GmFAD2-1A gene mutation type-2 bp in the T1 generation GmFAD2-1A soybean mutant plant is deletion of the 665-position 666 in the sequence 1 in the sequence table, and other nucleotides are kept unchanged.

The GmFAD2-1A gene mutation type +1bp in a T1 generation GmFAD2-1A soybean mutant plant is that a base T is added between 667 th and 668 th sites of a sequence 1 in a sequence table, and other nucleotides are kept unchanged.

The mutation type-1 bp of the GmFAD2-1A gene in a T1 generation GmFAD2-1A soybean mutant plant is deletion of 667 th site of a sequence 1 in a sequence table, and other nucleotides are kept unchanged.

And continuously culturing the T1 generation GmFAD2-1A soybean mutant plants with various GmFAD2-1A gene mutation types until T2 generation GmFAD2-1A soybeans are obtained, and harvesting seeds of T3 generation GmFAD2-1A soybeans (fad2-1A pure and mutant) after sowing.

Fourthly, determination of fatty acid content

1. Method for measuring fatty acid content

The material to be tested: soybean Jack (wild type), T3 generation GmFAD2-1A soybean homozygous lines SEA-73, SEA-81 and SEA-122.

Selecting seeds (fad2-1A pure and mutant) of T3 generation GmFAD2-1A soybean seeds respectively, and determining the mutation types of the seedsTable 1. Gas chromatography for T of fad2-1a pure and mutant₃The relative contents of various fatty acid components in the seeds were determined. The content of five fatty acids including palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2) and linolenic acid (C18:3) is mainly detected.

Table 1 shows the number of mutants used in the assay and their mutation types

The content of the soybean fatty acid is determined by a SHIMADZU gas chromatograph (GC-2010), and the sample processing and determining method is as follows:

extracting fatty acid from soybean powder: fatty acid extraction was performed by heating methyl esterification extraction (fangchin et al, 2015), and 20 full soybean seeds were selected for each soybean line, ground into fine powder with a sample grinder (Retsch ZM100, Φ 1.0mm, Rheinische, germany), and then sample treatment was performed:

weighing about 0.03g of soybean fine powder, and placing in a 2.0mL sterile centrifuge tube;

1.0mL of n-hexane was aspirated into the centrifuge tube, and the tube was placed in a water bath at 60 ℃ for 20 minutes with shaking every 5 minutes.

Sucking 1.0mL of sodium methoxide aqueous solution (0.5mol/L) into each centrifuge tube, leaching in a water bath at 60 ℃, and shaking for 10 minutes to ensure that the methanol is completely esterified; upon centrifugation at 13000rpm/min for 2 minutes, a clear stratification was observed. Pipette 200. mu.L of supernatant into a dedicated sample vial for chromatography and place in a refrigerator at 4 ℃.

Transgenic soybean lines were set up in 3 replicates and each replicate was tested 3 times. The mean of the 3 replicates was judged as the fatty acid component content and the fatty acid component content of each transgenic soybean line was normalized to the mean of the 3 replicates. Peaks of 5 fatty acid components appear in gas chromatographic analyzer detection, the peak emergence time of different gasified fatty acids is used for distinguishing five main fatty acid components, and the content (relative content) of different fatty acids is expressed by the percentage of the peak area of different fatty acids/the peak area of five main fatty acids.

Analyzing and detecting conditions by a gas chromatograph: column RTX-Wax (30 m.times.0.25. times.0.25); the temperature of an injection port is 250 ℃, nitrogen is 54ml/min, hydrogen is 40ml/min, air is 400ml/min, and a temperature programming mode is adopted. The temperature of the detector is 300 ℃; the contents of five main fatty acids (palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid) were calculated using an area normalization method.

Five fatty acid methyl ester standards, methyl palmitate (methyl palmitate), methyl stearate (methyl stearate), methyl oleate (methyl oleate), methyl linoleate (methyl linoleate), and methyl linolenate (methyl linolenate), were obtained from Sigma.

2. Fatty acid changes in mutant seed

The results of the gas chromatography are shown in FIG. 2: compared with a receptor variety Jack (wild type), the content of palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2) and linolenic acid (C18:3) in the mutant line is changed, and the peak-appearance retention time of each fatty acid is consistent with that of a standard product.

Compared with wild type control, the oleic acid and linoleic acid contents in the seeds of the mutant individual plants are remarkably different, and the oleic acid contents in the fad2-1a pure seeds and the mutant seeds are increased to 2 times of that of Jack and reach about 40 percent (Table 2).

Table 2 shows the relative content (%)% of major fatty acids in soybean seeds of wild type and fad2-1a mutant

The results show that the soybean GmFAD2-1A gene knocked out by using the CRISPR/Cas9 gene editing technology can obviously improve the content of oleic acid in soybean seeds. Meanwhile, the mutant with 1bp increased (SEA-81) is found to have much higher oleic acid content than other mutant types.

SEQUENCE LISTING

<110> institute of crop science of Chinese academy of agricultural sciences

<120> method for obtaining soybean with high oleic acid content by modifying GmFAD2-1A gene through CRISPR/Cas9

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1977

<212> DNA

<213> Artificial sequence

<400> 1

ttatgacatg taattgaatt ttttaattat aaaaaataat aaaacttaat tacgtactat 60

aaagagatgc tcttgactag aattgtgatc tcctagtttc ctaaccatat actaatattt 120

gcttgtattg atagcccctc cgttcccaag agtataaaac tgcatcgaat aatacaagcc 180

actaggcatg gtaaattaaa ttgtgcctgc acctcgggat atttcatgtg gggttcatca 240

tatttgttga ggaaaagaaa ctcccgaaat tgaattatgc atttatatat cctttttcat 300

ttctagattt cctgaaggct taggtgtagg cacctagcta gtagctacaa tatcagcact 360

tctctctatt gataaacaat tggctgtaat gccgcagtag aggacgatca caacatttcg 420

tgctggttac tttttgtttt atggtcatga tttcactctc tctaatctct ccattcattt 480

tgtagttgtc attatcttta gatttttcac tacctggttt aaaattgagg gattgtagtt 540

ctgttggtac atattacaca ttcagcaaaa caactgaaac tcaactgaac ttgtttatac 600

tttgacacag ggtctagcaa aggaaacaac aatgggaggt agaggtcgtg tggccaaagt 660

ggaagttcaa gggaagaagc ctctctcaag ggttccaaac acaaagccac cattcactgt 720

tggccaactc aagaaagcaa ttccaccaca ctgctttcag cgctccctcc tcacttcatt 780

ctcctatgtt gtttatgacc tttcatttgc cttcattttc tacattgcca ccacctactt 840

ccacctcctt cctcaaccct tttccctcat tgcatggcca atctattggg ttctccaagg 900

ttgccttctc actggtgtgt gggtgattgc tcacgagtgt ggtcaccatg ccttcagcaa 960

gtaccaatgg gttgatgatg ttgtgggttt gacccttcac tcaacacttt tagtccctta 1020

tttctcatgg aaaataagcc atcgccgcca tcactccaac acaggttccc ttgaccgtga 1080

tgaagtgttt gtcccaaaac caaaatccaa agttgcatgg ttttccaagt acttaaacaa 1140

ccctctagga agggctgttt ctcttctcgt cacactcaca atagggtggc ctatgtattt 1200

agccttcaat gtctctggta gaccctatga tagttttgca agccactacc acccttatgc 1260

tcccatatat tctaaccgtg agaggcttct gatctatgtc tctgatgttg ctttgttttc 1320

tgtgacttac tctctctacc gtgttgcaac cctgaaaggg ttggtttggc tgctatgtgt 1380

ttatggggtg cctttgctca ttgtgaacgg ttttcttgtg actatcacat atttgcagca 1440

cacacacttt gccttgcctc attacgattc atcagaatgg gactggctga agggagcttt 1500

ggcaactatg gacagagatt atgggattct gaacaaggtg tttcatcaca taactgatac 1560

tcatgtggct caccatctct tctctacaat gccacattac catgcaatgg aggcaaccaa 1620

tgcaatcaag ccaatattgg gtgagtacta ccaatttgat gacacaccat tttacaaggc 1680

actgtggaga gaagcgagag agtgcctcta tgtggagcca gatgaaggaa catccgagaa 1740

gggcgtgtat tggtacagga acaagtattg atggagcaac caatgggcca tagtgggagt 1800

tatggaagtt ttgtcatgta ttagtacata attagtagaa tgttataaat aagtggattt 1860

gccgcgtaat gactttgtgt gtattgtgaa acagcttgtt gcgatcatgg ttataatgta 1920

aaaataattc tggtattaat tacatgtgga aagtgttctg cttatagctt tctgcct 1977