CN117210432A - Application of soybean phosphatidylcholine glycerol di-lipid choline phosphotransferase in regulation and control of seed oleic acid - Google Patents

Abstract

The application discloses an application of soybean phosphatidylcholine diglyceride choline phosphotransferase in regulating seed oleic acid, belonging to the technical field of genetic engineering breeding. The application discloses application of a GmLHB1 protein and a GmLHB2 protein in regulating and controlling oleic acid content of plant seeds, wherein the GmLHB1 protein is a protein with an amino acid sequence shown as a sequence 5 in a sequence table; the GmLHB2 protein is a protein with an amino acid sequence shown as a sequence 8 in a sequence table. The application also prepares the target soybean with improved oleic acid content of the seed by knocking out encoding genes of the GmLHB1 protein and the GmLHB2 protein in the soybean. The double knockout mutant of the GmLHB1 and GmLHB2 genes can remarkably improve the oleic acid content in soybean seeds, and the GmLHB1 and GmLHB2 genes as genes for regulating and controlling the oleic acid synthesis pathway can be applied to cultivation of high oleic acid soybean varieties.

Description

Application of soybean phosphatidylcholine diglyceride choline phosphotransferase in regulating seed oleic acid

Technical Field

The invention belongs to the technical field of genetic engineering breeding, and specifically relates to the application of soybean phosphatidylcholine diglyceride choline phosphotransferase in regulating seed oleic acid

Background Art

Phosphatidylcholine (PC) is a major type of phospholipid in eukaryotic organisms and is present in all biological membrane systems. As the main phospholipid that makes up biological membranes, PC participates in a wide range of physiological processes. In recent years, with the deepening of lipid metabolism research, the study of phosphatidylcholine and plant oil synthesis pathways has received increasing attention.

Phosphatidyl cholinediacylglycerolcholinephosphotransferase (LHB) catalyzes the transfer of phosphorylcholine groups from PC to DAG to form new PC and DAG. The action of LHB can make DAG containing 18:1 (18:1-DAG) generate 18:1-PC, and PC containing polyunsaturated fatty acids (18:2-PC 18:3-PC) generate 18:2-DAG or 18:3-DAG. Then DAG rich in polyunsaturated fatty acids participates in the biosynthesis of TAG, so that polyunsaturated fatty acids are accumulated in TAG. At present, the identification and preliminary functional studies of LHB genes are limited to a few model plants. The existence and function of the LHB family in other plants have not been analyzed, especially in soybean. Therefore, it is necessary to pay attention to the role of the soybean LHB gene family in regulating seed oleic acid synthesis.

Summary of the invention

The technical problem to be solved by the present invention is: how to increase the oleic acid content of plant seeds.

To solve the above technical problems, in the first aspect, the present invention provides the use of a protein or a substance regulating the expression of a gene or a substance regulating the activity or content of the protein in the following A1-A3), wherein the gene encodes the protein, and the protein is GmLHB1 protein and/or GmLHB2 protein;

A1) Application in regulating the content of fatty acids in plant seeds;

A2), application in the preparation of products for regulating the content of components of fatty acids in plant seeds;

A3), application in regulating the content of oleic acid in plant seeds;

A4), application in the preparation of products for regulating the oleic acid content of plant seeds;

A5) Application in regulating the linoleic acid content in plant seeds;

A6), application in the preparation of products for regulating the linoleic acid content in plant seeds;

A7) Application in regulating the content of linolenic acid in plant seeds;

A8), application in the preparation of products for regulating the content of linolenic acid in plant seeds;

A9) Application in regulating the content of stearic acid in plant seeds;

A10), application in the preparation of products for regulating the stearic acid content of plant seeds;

A11), plant breeding or assisted plant breeding;

The GmLHB1 is any one of the following proteins a1)-a3):

a1) The amino acid sequence is the protein shown in Sequence 5 in the sequence list;

a2), a protein having more than 80% identity with the amino acid sequence shown in a1) and related to the oleic acid content of plant seeds, obtained by substituting and/or deleting and/or adding amino acid residues of the amino acid sequence shown in a1);

a3), a fusion protein obtained by connecting a tag to the N-terminus or/and C-terminus of a1) or a2);

The GmLHB2 is any one of the following proteins b1)-b3):

b1) The amino acid sequence is the protein shown in Sequence 8 in the sequence list;

b2) a protein obtained by substituting and/or deleting and/or adding amino acid residues of the amino acid sequence shown in b1) and having more than 80% identity with the amino acid sequence shown in b1) and related to the oleic acid content of plant seeds;

b3) A fusion protein obtained by connecting a tag to the N-terminus and/or C-terminus of b1) or b2).

Furthermore, in the above application, the GmLHB1 protein and/or GmLHB2 protein is derived from soybeans.

Furthermore, in the above application, the substance that regulates the expression of the protein encoding gene or the substance that regulates the activity or content of the protein is a biological material, and the biological material is any one of the following:

B1), a nucleic acid molecule that inhibits or reduces the expression of the gene encoding the above protein or the activity of the above protein;

B2), an expression cassette containing the nucleic acid molecule described in B1);

B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

B4), a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

B5), a transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2), or a transgenic plant cell line containing the recombinant vector described in B3);

B6), transgenic plant tissue containing the nucleic acid molecule described in B1), or transgenic plant tissue containing the expression cassette described in B2), or transgenic plant tissue containing the recombinant vector described in B3);

B7), a transgenic plant organ containing the nucleic acid molecule described in B1), a transgenic plant organ containing the expression cassette described in B2), or a transgenic plant organ containing the recombinant vector described in B3);

B8), a nucleic acid molecule encoding the above protein;

B9), an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line containing the nucleic acid molecule described in B8).

Furthermore, in the above application, the nucleic acid molecule in B1) is a DNA molecule expressing a gRNA targeting the protein-coding gene in a1) or b1) or a gRNA targeting the protein-coding gene in a1) or b1);

B8) The nucleic acid molecule is a DNA molecule as shown in any one of g1) to g4) below:

g1), the coding sequence of the coding strand is a DNA molecule of sequence 4 in the sequence list;

g2), a DNA molecule that has more than 80% identity with the DNA molecule described in g1), and encodes a DNA molecule that regulates the quality of seed oleic acid-related protein w;

g3), the coding sequence of the coding strand is the DNA molecule shown in Sequence 7 in the sequence table;

g4), having more than 80% identity with the DNA molecule described in g3), and encoding a DNA molecule that regulates seed oleic acid-related proteins.

Furthermore, in the above application, the gRNA includes sgRNA1 or sgRNA2, the nucleotide sequence of the target sequence of the sgRNA1 is SEQ ID No.1, and the nucleotide sequence of the target sequence of the sgRNA2 is SEQ ID No.2.

Furthermore, the nucleotide sequence of the sgRNA1 is SEQ ID No.10, and the nucleotide sequence of the sgRNA2 is SEQ ID No.11.

In the above application, the plant is selected from any one of the following:

C1), dicotyledonous plants;

C2), Leguminosae;

C3) Soybean

C4) soybeans.

To solve the above technical problems, in a second aspect, the present invention provides a method for regulating the oleic acid content of plant seeds, the method comprising regulating the oleic acid content of plant seeds by regulating the expression of the gene encoding the GmLHB1 protein in the above application of the plant and/or the gene encoding the GmLHB2 protein in the above application or regulating the activity or content of the GmLHB1 protein and/or the GmLHB2 protein.

Furthermore, in the above method, the plant is soybean, and the method includes introducing the gene encoding the gRNA molecule and the Cas protein in the above application into the recipient soybean to inhibit or reduce the expression of the GmLHB1 protein and/or GmLHB2 protein encoding gene in the recipient soybean or inhibit or reduce the activity or content of the GmLHB1 protein and/or GmLHB2 protein in the recipient soybean, thereby obtaining the target soybean whose oleic acid content in the seeds is different from that of the recipient soybean or the target soybean whose oleic acid content in the seeds is higher than that of the recipient soybean.

To solve the above technical problems, in a third aspect, the present invention provides a method for increasing the oleic acid content of soybean seeds, the method comprising introducing the gene of the above gRNA molecule and the coding gene of the Cas protein into the recipient soybean to inhibit or reduce the expression of the GmLHB1 protein and/or GmLHB2 protein coding gene in the recipient soybean or inhibit or reduce the activity or content of the GmLHB1 protein and/or GmLHB2 protein in the recipient soybean, thereby obtaining the target soybean having a higher oleic acid content in the seeds than that of the recipient soybean.

Furthermore, in the above method, the expression of the gene encoding the GmLHB1 protein and/or the gene encoding the GmLHB2 protein in the recipient soybean or the activity or content of the GmLHB1 protein and/or the GmLHB2 protein in the recipient soybean is inhibited or reduced by performing at least one of the following mutations on the gene encoding the GmLHB1 protein whose nucleotide sequence of the coding chain is Sequence 3 and the gene encoding the GmLHB2 protein whose nucleotide sequence of the coding chain is Sequence 6 in the recipient soybean genome:

T1), deletion of nucleotide A at position 574 of SEQ ID No.3, deletion of nucleotides 438-444 of SEQ ID No.6;

T2), adding one nucleotide T between positions 445 and 446 and between positions 574 and 575 of SEQ ID No.3, respectively, and deleting nucleotides 316-443 of SEQ ID No.6;

T3), adding a nucleotide A between positions 445 and 446 of SEQ ID No.3, deleting nucleotides 570-575 of SEQ ID No.3, and deleting nucleotides 316-443 of SEQ ID No.6.

To solve the above technical problems, in a fourth aspect, the present invention provides the above protein and/or the above biomaterial.

In the present invention, the gene encoding the above-mentioned gRNA molecule and the Cas protein into the recipient plant is introduced by introducing the recombinant expression vector pCBSG015-GmLHB into the recipient plant.

The beneficial technical effects achieved by the present invention are as follows:

In this study, GmLHB1 (Glyma.08g213100) and GmLHB2 (Glyma.07g029800), members of the soybean LHB gene family, were used as research objects. The T2 generation double gene knockout mutants m3-1-1, m6-2-1, and m6-2-2 were obtained by CRISPR/Cas9 technology to measure the seed quality and observe the phenotype. Using the wild-type soybean Dongnong 50 (CK) as the control, the results showed that the oleic acid content in the fatty acid content of the GmLHB1 and GmLHB2 gene double knockout mutant soybean lines was significantly increased, and there were no significant differences in plant height, main stem node number, number of branches per plant, leaf shape, flower color, seed coat color, hilum color, growth period, etc. compared with the control varieties. There was no significant difference in total protein and fat content between the mutant soybeans and the control varieties, indicating that the knockout of GmLHB1 and GmLHB2 genes had no effect on the agronomic phenotype of transgenic soybeans, and had no effect on the accumulation of total protein and total fat in soybean seeds. These results indicate that the double knockout mutant of GmLHB1 and GmLHB2 can significantly increase the oleic acid content in soybean seeds. GmLHB1 and GmLHB2, as genes regulating the oleic acid biosynthesis pathway, can be used to breed high-oleic soybean varieties.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the soybean genetic transformation process.

Figure 2 shows the results of positive identification of genetic transformation.

FIG. 3 shows the genotype identification of the homozygous double gene knockout mutant strain.

FIG4 shows the relative expression levels of GmLHB1 and GmLHB2 genes.

FIG. 5 shows the genotype identification of homozygous single gene knockout mutant strains.

FIG6 shows the relative expression levels of GmLHB1 at different developmental stages.

FIG. 7 shows the relative expression levels of GmLHB2 at different developmental stages.

FIG8 shows the results of determination of protein and oil contents in wild-type Dongnong 50 and the double gene knockout mutant of GmLHB1 and GmLHB2.

FIG. 9 shows the results of determination of fatty acid component contents in wild-type Dongnong 50 and double gene knockout mutants of GmLHB1 and GmLHB2.

DETAILED DESCRIPTION

The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

Soybean seeds Dongnong 50 are preserved in this laboratory and disclosed in the document "Ying Zhao etc. Enhanced production of seed oil with improved fatty acid composition by overexpressing NAD ⁺ -dependent glycerol-3-phosphate dehydrogenase in soybean. Journal of Integrative Plant Biology (IF9.106) Pub Date: 2021-03-26, DOI: 10.1111/jipb.13094" (recorded as DN50 in the document). The public can obtain the above-mentioned biological material from the applicant. The obtained biological material can only be used for the verification of the embodiments of this application and cannot be used for other purposes.

SPSS 16.0 (SPSS Inc, Chicago, IL, USA) software was used for statistical analysis in the following examples, and One-way ANOVA test was used. * indicates a significant difference (P < 0.05), ** indicates a very significant difference (P < 0.01), and *** indicates a very significant difference (P < 0.001).

Example 1. Construction of soybean knockout vector

The preparation of soybean knockout strains was completed by Weimi Biotechnology (Jiangsu) Co., Ltd. The vector used was pCBSG015 (Basta) (provided by Weimi Biotechnology (Jiangsu) Co., Ltd.), which carries the marker gene aada and uses U6 promoter and enhanced 35S promoter, which can efficiently start sgRNA expression and efficiently express Cas9 protein, and is effectively used for dicotyledonous plant gene editing. First, the target gene Target sequences were designed and selected, and the CRISPR-P website was used to design targets in the homologous regions of the genomic sequences of GmLHB1 and GmLHB2 (http://crispr.hzau.edu.cn/CRISPR2/), and targets with high target scores, low off-target rates, and suitable positions were selected. The target sequences are shown in Sequence 1 and Sequence 2 in Table 1. The DNA fragment U6-26-sgRNA1-U6-26-sgRNA2 containing the sgRNA expression cassette was synthesized. U6-26-sgRNA1-U6-26-sgRNA2 was constructed into the expression vector of pCBSG015 (Basta) to obtain the recombinant expression plasmid pCBSG015 (Basta) -GmLHB. The nucleotide sequence of the expression plasmid pCBSG015 (Basta) -GmLHB is SEQ ID No.9. Among them, positions 274-1405 of SEQ ID No.9 are the expression cassettes of sgRNA, and positions 2412-6512 are the coding sequence of Cas9 protein. The expression plasmid pCBSG015 (Basta) -GmLHB expresses sgRNA1, sgRNA2 and Cas9 protein targeting GmLHB1 and GmLHB2 genes. The nucleotide sequence of sgRNA1 is SEQ ID No.10, and the nucleotide sequence of sgRNA2 is SEQ ID No.11.

The nucleotide sequence of the genome of the GmLHB1 gene is shown in Sequence 3 in the sequence list, its coding sequence is shown in Sequence 4 in the sequence list, and the amino acid sequence encoding the GmLHB1 protein is shown in Sequence 5 in the sequence list; the nucleotide sequence of the genome of the GmLHB2 gene is shown in Sequence 6 in the sequence list, its coding sequence is shown in Sequence 7 in the sequence list, and the amino acid sequence encoding the GmLHB2 protein is shown in Sequence 8 in the sequence list, and Sequence 9 is the full sequence of pCBSG015 (Basta) -GmLHB. See Table 1 for details.

Table 1 Sequence

Example 2: Genetic transformation of knockout vector

2.1 Genetic transformation

The vector was transferred into Agrobacterium EHA105 by electroporation and identified by PCR. Soybeans germinated for one day were used as materials and placed in a culture dish containing 50 ml of Agrobacterium suspension. About 150 explants were treated within 2 hours and placed at room temperature for 30 minutes for infection. After the infection, the Agrobacterium suspension was discarded and the explants were placed in a co-cultivation medium (1/10 concentration of B5 macro-salts and trace salts, 1/10 concentration of MS iron salts ^, B5 vitamins, 3% sucrose, 3.9 g·L ^-1 MES, 40 mg·L ^-1 AS, 0.25 μg·L ^-1 GA ₃ , 1 mM·L ^-1 DTT, 8.8 mM·L ^-1 L-cysteine, 1.70 mg·L ^-1 6-BA, 0.5% agar, pH = 5.4) and co-cultivated in the dark at 23°C for 3 days. After co-cultivation, the embryos were transferred to a resting medium (B5 macro- and trace salts, MS iron salts, B5 vitamins, 3% sucrose, 0.59 g·L ^-1 MES, 1.7 mg·L ^-1 6-BA, 100 mg·L ^-1 Cefotaxime, 0.8% agar, pH = 5.6), cultured in the light at 25°C for 7 days, and then placed on a screening medium containing glufosinate (B5 macro- and trace salts, MS iron salts, B5 vitamins, 3% sucrose, 0.59 g·L ^-1 MES, 1.7 mg·L ^-1 6-BA, 100 mg·L ^-1 Cefotaxime, 5 mg·L ^-1 PPT, 0.8% agar, pH = 5.6), and cultured for three weeks to induce resistant buds. Then, the seedlings were transferred to an elongation medium containing glufosinate (MS macro-salts and trace salts, MS iron salts, B5 vitamins, 3% sucrose, 0.59 g·L ^-1 MES, 0.5 mg·L ^-1 GA ₃ , 0.1 mg·L ^-1 IAA, 1 mg·L ^-1 ZR, 50 mg·L ^-1 L-Asparagine, 100 mg·L ^-1 L-Pyroglutamic, 100 mg·L ^-1 Cefotaxime, 0.8% agar, pH = 5.6.) and cultured in the light for 6-9 weeks. The regenerated elongated seedlings were rooted (1/2 concentration of B5 macro-salts and trace salts, MS iron salts, 2% sucrose, 0.59 g·L ^-1 MES, 1 mg·L ^-1 IBA, 0.8% agar, pH = 5.6). After rooting, the seedlings obtained by transplanting to nutrient soil according to the growth potential were T ₀ generation seedlings. The process of genetic transformation is shown in Figure 1. The seeds of the T ₀ generation mutant plants (T ₁ generation) were harvested for sowing. The GmLHB1 and GmLHB2 double gene knockout mutants finally obtained through gene editing and second-generation sequencing were used as T ₁ generation plants, and the T ₁ generation was self-pollinated to obtain the T ₂ generation.

2.2. Methods for identification of genetically transformed plants

(1) The aada gene was detected for each of the genetically transformed plants (T ₀ , T ₁ and T ₂ ). The plants that amplified the target fragment of 435 bp were positive plants that had been transformed with the gene editing vector. The primer information is as follows:

F: 5'-TCCGACATCGATCTCCTGGT-3';

R: 5'-CAGGGTGAGGACCACATTCC-3';

The reaction system (20ul) is as follows:

volume Components 10ul 2*PCR buffer 7ul H2O 1ul Upstream primer (10uM) 1ul Downstream primer (10uM) 1ul DNA

The reaction procedure is as follows:

(2) For genetically transformed plants that test positive for the aada gene, genome-specific primers are designed based on the target gene, and PCR amplification is performed (the amplification conditions refer to the aada gene PCR conditions) to obtain a DNA fragment containing the target site. The target fragment is subjected to Sanger sequencing to analyze the editing results of the target gene at the target site.

The specific primers are:

GmLHB1-F: 5’-AGGCGCAAACATCAAACAGC-3’;

GmLHB1-R: 5’-tgatgaatctcacCTGAGGCA-3’;

GmLHB2-F: 5’-ATCACAGGCGCAGACACCAA-3’;

GmLHB2-R: 5’-ttttgagaccaacccatttgc-3’.

2.3 Results of identification of genetically transformed plants

The knockout vector (pCBSG015-GmLHB) was transferred into the recipient variety Dongnong 50 by Agrobacterium-mediated soybean cotyledon node transformation. Ten _T0 mutant regenerated plants were obtained through explant infection, co-cultivation, recovery culture, cluster bud screening and elongation, rooting and seedling hardening. The seeds of the _T0 mutant plants ( _T1 generation) were harvested for sowing. After gene editing and sequencing, four GmLHB1 and GmLHB2 double gene knockout mutants (m3-1, m3-2, m6-1, and m6-2) were finally obtained. The homozygous _T2 generations obtained by selfing of the _T1 generation were named m3-1-1, m3-1-2, m3-2-1, m3-2-2, m6-1-1, m6-1-2, m6-2-1, and m6-2-2, respectively.

The results of preliminary screening by AADA gene detection according to the method shown in 2.2(1) are shown in Figure 2.

The mutant plants m3-1-1, m6-2-1, and m6-2-2 were randomly selected and the target mutant genotype was identified according to the method shown in 2.2(2). The results are shown in FIG3 , specifically:

Compared with the wild-type soybean Dongnong 50, the variant plant m3-1-1 has a GmLHB1 gene in two homologous chromosomes mutated into a GmLHB1/-1 gene, in which the 574th nucleotide A of sequence 3 in the sequence list is deleted, resulting in a frameshift mutation and premature termination of translation, thereby knocking out GmLHB1; for the GmLHB2 gene, the GmLHB2 gene in two homologous chromosomes mutated into a GmLHB2/-1 gene, in which the 438th to 444th nucleotides of sequence 6 in the sequence list are deleted, resulting in a frameshift mutation and premature termination of translation, thereby knocking out GmLHB2;

Compared with the wild-type soybean Dongnong 50, the variant plant m6-2-1 has a GmLHB1 gene in two homologous chromosomes mutated into a GmLHB1/-2 gene, in which a nucleotide T is added between positions 445 and 446 and between positions 574 and 575 of sequence 3 in the sequence list, respectively, resulting in a frameshift mutation and premature translation termination, thereby knocking out GmLHB1; for the GmLHB2 gene, the GmLHB2 gene in two homologous chromosomes mutated into a GmLHB2/-2 gene, in which nucleotides 316-443 of sequence 6 in the sequence list are deleted, resulting in a frameshift mutation and premature translation termination, thereby knocking out GmLHB2;

Compared with the wild-type soybean Dongnong 50, the variant plant m6-2-2 has a GmLHB1 gene in two homologous chromosomes mutated into a GmLHB1/-3 gene, in which a nucleotide A is added between positions 445 and 446 of sequence 3 in the sequence list, and nucleotides 570-575 are deleted, resulting in a frameshift mutation and premature translation termination, thereby knocking out GmLHB1; for the GmLHB2 gene, the GmLHB2 gene in two chromosomes mutated into a GmLHB2/-3 gene, in which nucleotides 316-443 of sequence 6 in the sequence list are deleted, resulting in a frameshift mutation and premature translation termination, thereby knocking out GmLHB2.

In the process of preparing double gene knockout mutant plants, single gene knockout GmLHB1 mutant plants m7-3-1, m8-1-1, m8-1-2 (numbered) and single gene knockout GmLHB2 mutant plants m10-1-1, m10-2-2 (numbered) were obtained. _T0 generation plants were self-pollinated to obtain _T1 generation, and _T1 generation self-pollination obtained 3 _T2 generation single gene knockout GmLHB1 homozygous mutant plants and 2 _T2 generation single gene knockout GmLHB2 homozygous mutant lines.

The gene mutation types of the single gene knockout mutant plants are shown in Figure 5, specifically:

Compared with the wild-type soybean Dongnong 50, the variant plant m7-3-1 has a GmLHB1 gene mutation in two homologous chromosomes into a GmLHB1/-4 gene. The GmLHB1/-4 gene is a deletion of nucleotides 446-573 of sequence 3 in the sequence list, resulting in a frameshift mutation and premature termination of translation, thereby knocking out GmLHB1.

Compared with the wild-type soybean Dongnong 50, the variant plant m8-1-1 has a GmLHB1 gene mutation in two homologous chromosomes into a GmLHB1/-5 gene. The GmLHB1/-5 gene is a deletion of the 574th nucleotide A of sequence 3 in the sequence list, resulting in a frameshift mutation and premature termination of translation, thereby knocking out GmLHB1.

Compared with the wild-type soybean Dongnong 50, the variant plant m8-1-2 has a GmLHB1 gene mutation in two homologous chromosomes into a GmLHB1/-6 gene. The GmLHB1/-6 gene is a deletion of the 574th nucleotide A of sequence 3 in the sequence list, resulting in a frameshift mutation and premature termination of translation, thereby knocking out GmLHB1.

Compared with the wild-type soybean Dongnong 50, the variant plant m10-1-1 mutated the GmLHB2 gene in both chromosomes into the GmLHB2/-4 gene. The GmLHB2/-4 gene added a nucleotide C between positions 441 and 442 of sequence 6 in the sequence list, resulting in a frameshift mutation and premature termination of translation, thereby knocking out GmLHB2.

Compared with the wild-type soybean Dongnong 50, the variant plant m10-2-2 mutated the GmLHB2 gene in both chromosomes into the GmLHB2/-5 gene. The GmLHB2/-5 gene added a nucleotide C between positions 441 and 442 of sequence 6 in the sequence list, resulting in a frameshift mutation and premature termination of translation, thereby knocking out GmLHB2.

Example 3. Relative expression levels of GmLHB1 and GmLHB2 genes in soybean tissues and seeds at different developmental stages

3.1 Test methods

To determine whether the relative expression of GmLHB1 and GmLHB2 genes in tissues and grains at different developmental stages is different, organs (roots, stems, leaves, flowers, pods, nodules) and grains at different developmental stages (EM (early development), MM (middle development), LM (late development), DS (dry seeds)) of Dongnong 50 grown to R4 were sampled, snap-frozen in liquid nitrogen, and stored at -80°C for RNA extraction. Total RNA was extracted using TRIzol reagent (Invitrogen). Reverse transcription was performed using ReverTra AceqPCR RT Master Mix with gDNARemover (TOYOBO). Real-time quantitative PCR was performed using the Bio-Rad Chromo4 real-time PCR system. The reaction mixture contained 12.5 μl of 2×SYBR Green Realtime PCR MasterMix (Toyobo), 0.5 μM of upstream and downstream primers, and 2 μl of cDNA template (equivalent to 100 ng of RNA), with a total volume of 25 μl. The reaction conditions were: 94°C, 30 s; 45 cycles: 94°C, 12 s; 58°C, 30 s; 72°C, 30 s. Finally, 80°C, 1 s. Soybean Actin4 was used as an internal reference to normalize the data. The relative expression level was calculated using the 2- ^△△CT method. The expression level in flowers was used as a reference, and the other treatments were calculated accordingly. Each sample was subjected to three independent biological replicates, and each was subjected to three technical replicates. All primers are shown in Table 1.

Table 2. Real-time quantitative PCR primer sequences

3.2 Analysis of candidate gene tissue specificity and expression levels in grains at different developmental stages

Real-time quantitative PCR was used to analyze the relative expression levels of GmLHB1 and GmLHB2 genes in organs and tissues of wild-type soybean Dongnong 50 (roots, stems, leaves, flowers, pods, and nodules at the R4 developmental stage) and grains at different developmental stages (EM (early development), MM (middle development), LM (late development), and DS (dry seeds)). The results are shown in Figure 4. GmLHB1 and GmLHB2 genes are expressed in various tissues and at different developmental stages. The expression levels in different organs and tissues are from high to low: root > stem > pod > nodule > leaf, with the highest expression level in the root; the expression levels in different growth stages are from high to low: MM (middle development) > LM (late development) > DS (dry seeds) > EM (early development), among which MM (middle development) has the highest expression level.

Example 4. qRT-PCR detection of _T2 generation gene-edited soybean plants

4.1 Test methods

In order to analyze the expression change characteristics of gene-edited plants and control plants, mutant plants and control plants were planted under the same conditions, and total RNA from leaves of gene-edited plants and control plants was extracted and reverse transcribed into cDNA, which was used as a template, GmActin4 as an internal reference gene, and the expression level of Null (a plant that has undergone genetic transformation but is still negative) plants as a reference for qRT-PCR amplification. The reaction procedure and primer sequences used were the same as those in 3.1 of Example 3.

4.2 qRT-PCR analysis of _T2 gene-edited soybean plants

Real-time quantitative PCR was used to analyze the relative expression levels of GmLHB1 and GmLHB2 double gene knockout mutants at different developmental stages (EM (early development), MM (middle development), LM (late development)). The results are shown in Figures 6 and 7. The expression levels of GmLHB1 and GmLHB2 genes were significantly decreased in the mutants, and the expression levels of GmLHB1 and GmLHB2 in the mutants at different developmental stages were from high to low: MM (middle development) > LM (late development) > EM (early development). It can be seen that the expression levels of GmLHB1 and GmLHB2 are higher in the middle and late stages of grain development.

Example 5. Determination of protein and oil content in T2 _- generation gene-edited soybean grains

The mutant plants and control plants (Null) were planted in a greenhouse, and the _T2 generation gene-edited mature seeds were collected from the lines, and the protein and oil contents were determined using a FOSS near-infrared grain analyzer.

The mature seeds of the T2 _- generation gene-edited plants (20 seeds from each of the three homozygous _T2 -generation plants m3-1-1, m6-2-1, and m6-2-2) and wild-type soybean seeds (20 seeds, marked with WT) were collected and the protein and oil contents were determined using a FOSS near-infrared grain analyzer. As can be seen from Figure 8, there was no significant difference in the total protein and oil contents of the mutant soybeans and the control varieties.

Example 6: Determination of fatty acid content in _T2 generation gene-edited soybean seeds

6.1 Sample processing

(1) Fix the sample at 105°C for 20-30 minutes, then dry at 65°C for 12-14 hours, and grind into fine powder after drying.

⑵ Weigh about 10 mg of sample, record the weighed weight, put the weighed sample into a storage tube, and set up three replicates.

⑶ Add 1 mL of 2.5% concentrated sulfuric acid (dissolved in methanol), 5 uL of BHT (2,6-di-tert-butyl-4-methylphenol, 50 mg/mL, dissolved in methanol), and 50 μL of internal standard (heptadecanoic acid, 10 mg/mL, dissolved in ethyl acetate) to the sample, and tighten the storage tube cap.

(4) Place the sample in a 85°C water bath for 1.5 hours, inverting it thoroughly every ten minutes.

⑸ Take out the sample, cool it to room temperature, and add 160uL 9% NaCl solution and 700μL n-hexane.

⑹ Vortex for 3 minutes and centrifuge at 4500 rpm for 10 minutes at room temperature.

⑺Pipette 400 μL of the supernatant from each sample into a new centrifuge tube and place it in a fume hood to dry overnight.

⑻ Before measuring on the machine, add 400 μL of ethyl acetate to each sample to fully dissolve it.

6.2 Determination of fatty acid content in _T2 generation gene-edited soybean seeds

The fatty acid content of the seeds was determined by gas chromatography. About 10 mg of the sample was quickly immersed in 1 mL of 2.5% concentrated sulfuric acid. Then 5 uL of 50 mg/mL BHT reducing agent and 50 uL of 10 mg/mL heptadecanoic acid (as an internal standard) were added. The methylated fatty acids were extracted with 700 uL of n-hexane and 160 uL of 9% sodium chloride. Five fatty acids were mainly determined: palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid. As can be seen from Figure 9, in the wild soybean Dongnong 50, the average contents of palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid in the seed dry weight were 2.73%, 1.24%, 3.51%, 12.25%, and 2.22%, respectively; in the mutant m3-1-1, the average contents of palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid in the seed dry weight were 2.63%, 0.93%, 9.86%, 8.17%, and 0.07%, respectively; in the mutant m6-2-1, the average contents of palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid in the seed dry weight were 2.73%, 1.24%, 3.51%, 12.25%, and 2.22%, respectively. The average values of palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid in the mutant m6-2-2 were 2.50%, 0.79%, 8.25%, 7.15% and 0.05% respectively. It can be seen that compared with the wild-type soybean Dongnong 50, the proportion of oleic acid in the three GmLHB1 and GmLHB2 double gene knockout mutants was significantly increased by 180.91%, 129.91% and 135.04% respectively. The proportion of linolenic acid was significantly decreased by 96.85%, 97.75% and 97.75% respectively, and the proportion of linoleic acid was also significantly decreased by 33.31%, 41.06% and 41.63% respectively. The oleic acid content in the single-gene knockout mutants of GmLHB1 and GmLHB2 was also increased, and the linoleic acid and linolenic acid contents were also decreased, but the changes in the contents did not show the significant difference in the double-gene knockout mutants of GmLHB1 and GmLHB2.

Example 7: Determination of fatty acid content in soybean grains of the _T2 generation single gene knockout strain

The fatty acid content of soybean grains of the _T2 generation single gene knockout strain was determined by referring to the method shown in Example 6. 15 grains of _T2 generation single gene knockout GmLHB1 were randomly selected, and 15 grains of _T2 generation single gene knockout GmLHB2 were randomly selected. The experiment was repeated three times and the results were averaged.

The results showed that the average contents of palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid in the seed dry weight of the mutant with single gene knockout GmLHB1 were 1.82%, 0.88%, 5.1%, 5.93% and 0.02% respectively; the average contents of palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid in the seed dry weight of the mutant with single gene knockout GmLHB2 were 2.13%, 1.11%, 6.45%, 8.42% and 0.07% respectively. It can be seen that compared with the wild-type soybean Dongnong 50, the proportion of oleic acid in the mutants with single gene knockout GmLHB1 and GmLHB2 increased by 45.30% and 83.76% respectively. The proportion of linoleic acid decreased by 51.59% and 31.27% respectively. It can be seen that the increase in oleic acid content in the mutants with single gene knockout of GmLHB1 and single gene knockout of GmLHB2 is not as significant as that in the mutants with double gene knockout of GmLHB1 and GmLHB2.

Oleic acid is a monounsaturated fatty acid with stable properties and strong antioxidant effect. Edible oils with high oleic acid content are not easy to oxidize and deteriorate under high temperature conditions and can be stored for a long time; in addition, the increase in oleic acid content can reduce harmful cholesterol in human lipid metabolism, thereby slowing down atherosclerosis and effectively preventing the occurrence of cardiovascular diseases. Linoleic acid and linolenic acid are polyunsaturated fatty acids with poor thermal stability and antioxidant properties. When exposed to air or processed at high temperatures, they are prone to oxidation, which reduces the nutritional value and affects the quality of the oil. Industrial hydrogenation reactions are also prone to produce trans fatty acids that are harmful to the human body. It can be seen that the increase in oleic acid content and the reduction in linoleic acid and linolenic acid content effectively improve the quality of soybeans.

Through statistical analysis and phenotypic observation of the protein, oil and fatty acid content of soybeans with GmLHB1 and GmLHB2 genes knocked out, it was found that knocking out GmLHB1 and GmLHB2 genes can significantly increase the oleic acid content in soybean seeds. This conclusion provides a theoretical basis and genetic preparation for further using gene editing to cultivate high-oleic acid soybean varieties.

The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims (10)

1. The application of proteins or substances that regulate the expression of genes or substances that regulate the activity or content of said proteins in the following A1-A11), said genes encoding said proteins, said proteins being GmLHB1 protein and/or GmLHB2 protein; A1), application in regulating the content of fatty acids in plant grains; A2), application in preparing products for regulating the component content of plant grain fatty acids; A3), application in regulating the oleic acid content of plant grains; A4), application in preparing products for regulating the oleic acid content of plant seeds; A5), application in regulating linoleic acid content in plant grains; A6), application in preparing products for regulating linoleic acid content in plant grains; A7), application in regulating linolenic acid content in plant grains; A8), application in preparing products for regulating linolenic acid content in plant grains; A9), application in regulating stearic acid content in plant grains; A10), application in preparing products for regulating the stearic acid content of plant grains; A11), plant breeding or assisted plant breeding; The GmLHB1 is any one of the following proteins a1)-a3): a1) The amino acid sequence is the protein shown in sequence 5 in the sequence listing; a2) The amino acid sequence shown in a1) has more than 80% identity with the amino acid sequence shown in a1) and is related to the oleic acid content of plant seeds obtained by substituting and/or deleting and/or adding amino acid residues. protein; a3) A fusion protein obtained by connecting a tag to the N-terminus or/and C-terminus of a1) or a2); The GmLHB2 is any one of the following proteins b1)-b3): b1) The amino acid sequence is the protein shown in sequence 8 in the sequence listing; b2) The amino acid sequence shown in b1) is obtained by substituting and/or deleting and/or adding amino acid residues and has more than 80% identity with the amino acid sequence shown in b1) and is related to the oleic acid content of plant seeds. protein; b3) A fusion protein obtained by connecting a tag to the N-terminus or/and C-terminus of b1) or b2). 2. The application according to claim 1, characterized in that: the GmLHB1 protein and/or GmLHB2 protein is derived from soybeans. 3. The application according to claim 1 or 2, characterized in that: the substance that regulates the expression of the protein-coding gene or the substance that regulates the activity or content of the protein is a biological material, and the biological material is any one of the following kind: B1), a nucleic acid molecule that inhibits or reduces the expression of the gene encoding the protein described in claim 1 or the activity of the protein described in claim 1; B2), an expression cassette containing the nucleic acid molecule described in B1); B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4), a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5), a transgenic plant cell line containing the nucleic acid molecule described in B1) or a transgenic plant cell line containing the expression cassette described in B2) or a transgenic plant cell line containing the recombinant vector described in B3); B6), transgenic plant tissue containing the nucleic acid molecule described in B1) or transgenic plant tissue containing the expression cassette described in B2) or transgenic plant tissue containing the recombinant vector described in B3); B7), a transgenic plant organ containing the nucleic acid molecule described in B1) or a transgenic plant organ containing the expression cassette described in B2) or a transgenic plant organ containing the recombinant vector described in B3); B8), a nucleic acid molecule encoding the protein described in claim 1; B9), expression cassette, recombinant vector, recombinant microorganism or transgenic plant cell line containing the nucleic acid molecule described in B8). 4. Application according to claim 3, characterized in that: B1) The nucleic acid molecule is a DNA molecule expressing a gRNA targeting the protein-coding gene a1) or b1) of claim 1 or a DNA molecule targeting the protein-coding gene a1) or b1) of claim 1 gRNA; B8) The nucleic acid molecule is a DNA molecule represented by any one of the following g1)-g4): g1), the coding sequence of the coding chain is the DNA molecule of sequence 4 in the sequence listing; g2), the coding sequence of the coding chain is the DNA molecule shown in sequence 7 in the sequence listing; g3), has more than 80% identity with the DNA molecule described in g1) or g2), and encodes a DNA molecule that regulates seed oleic acid-related proteins. 5. The application according to claim 4, characterized in that: the gRNA includes sgRNA1 and/or sgRNA2, the nucleotide sequence of the target sequence of the sgRNA1 is SEQ ID No. 1, and the target sequence of the sgRNA2 is The nucleotide sequence is SEQ ID No. 2. 6. A method for regulating the oleic acid content of plant seeds, characterized in that: the method includes regulating the gene encoding the GmLHB1 protein in the application of claim 1 of the plant and/or the application of claim 1 The expression of the gene encoding the GmLHB2 protein or regulating the activity or content of the GmLHB1 protein and/or the GmLHB2 protein can regulate the oleic acid content of plant seeds. 7. The method according to claim 6, characterized in that: the plant is soybean, and the method includes introducing into the recipient soybean the gene of the gRNA molecule and the encoding of the Cas protein in the application of claim 5. Genes are used to inhibit or reduce the expression of the GmLHB1 protein and/or GmLHB2 protein coding genes in the recipient soybean or to inhibit or reduce the activity or content of the GmLHB1 protein and/or GmLHB2 protein in the acceptor soybean, and obtain seeds. A target soybean having a different oleic acid content from the recipient soybean or a target soybean having a seed having a higher oleic acid content than said recipient soybean. 8. A method for increasing the oleic acid content of soybean seeds, the method comprising introducing the coding gene of the gRNA molecule and the coding gene of the Cas protein described in claim 5 into the recipient soybean to inhibit or reduce the content of the oleic acid in the recipient soybean. The expression of the gene encoding the GmLHB1 protein and/or GmLHB2 protein inhibits or reduces the activity or content of the GmLHB1 protein and/or GmLHB2 protein in the recipient soybean, thereby obtaining a seed with a higher oleic acid content than the acceptor soybean. Purpose soybeans. 9. The method according to claim 7 or 8, characterized in that: the inhibition or reduction of the expression of the gene encoding the GmLHB1 protein and/or the gene encoding the GmLHB2 protein in the recipient soybean is inhibited or reduced. The activity or content of the GmLHB1 protein and/or GmLHB2 protein in the recipient soybean is The coding gene of the GmLHB1 protein whose nucleotide sequence of the coding chain in the recipient soybean genome is sequence 3 and the coding gene of the GmLHB2 protein whose nucleotide sequence of the coding chain is sequence 6 are subjected to at least one of the following steps: Kind of mutation: T1), the 574th nucleotide A of SEQ ID No.3 is deleted, and the 438-444th nucleotide of SEQ ID No.6 is deleted; T2), add one nucleotide T between positions 445 and 446 and between positions 574 and 575 of SEQ ID No. 3, and delete nucleotides 316-443 of SEQ ID No. 6; T3), add a nucleotide A between positions 445 and 446 of SEQ ID No. 3, delete nucleotides 570-575 of SEQ ID No. 3, and delete 316- of SEQ ID No. 6 Nucleotide 443. 10. The protein according to claim 1 or 2 or/and the biological material according to claims 3-5.