阅读说明：本技术 创制高产大豆的方法 (Method for creating high-yield soybean ) 是由 年海 葛良法 蔡占东 冼沛琪 马启彬 程艳波 周强华 于 2021-07-20 设计创作，主要内容包括：本发明公开了对大豆GmJAGGED1-1基因和GmJAGGED1-2基因同时进行编辑修饰,获得高产大豆的方法。具体利用CRISPR/Cas9系统对出发大豆中GmJAGGED1-1和GmJAGGED1-2基因进行同时编辑,且使所述GmJAGGED1-1和GmJAGGED1-2基因同时发生突变导致翻译蛋白提前终止,得到转基因大豆；实现出发大豆中GmJAGGED1-1和GmJAGGED1-2基因同时基因编辑。本发明利用CRISPR/Cas9介导的基因编辑技术对大豆GmJAGGED1-1和GmJAGGED1-2基因进行特定靶点的定点敲除,获得产量较对照提高8％以上的高产大豆突变体材料,为高产大豆品种选育提供新材料。(The invention discloses a method for simultaneously editing and modifying soybean GmJAGGED1-1 gene and GmJAGGED1-2 gene to obtain high-yield soybean. Specifically, a CRISPR/Cas9 system is used for simultaneously editing GmJAGGED1-1 and GmJAGGED1-2 genes in starting soybeans, and the GmJAGGED1-1 and GmJAGGED1-2 genes are simultaneously mutated to cause premature termination of translation proteins, so that transgenic soybeans are obtained; realizes the simultaneous gene editing of GmJAGGED1-1 and GmJAGGED1-2 genes in the starting soybean. The invention utilizes CRISPR/Cas9 mediated gene editing technology to carry out fixed point knockout of specific targets on soybean GmJAGGED1-1 and GmJAGGED1-2 genes, obtains high-yield soybean mutant materials with yield improved by more than 8% compared with a control, and provides new materials for breeding high-yield soybean varieties.)

1. Use of a substance which regulates the activity or content of protein a and protein B or a substance which regulates the expression of a gene encoding said protein a and a gene encoding said protein B, characterized in that: the application is any one of the following:

p1, use in regulating or increasing soybean yield;

p2, in regulating and controlling the number of soybean seeds or increasing the number of soybean seeds;

p3, in regulating and controlling the weight of soybean seeds or increasing the weight of the soybean seeds;

p4, application in soybean breeding;

the protein A is the protein A1), A2) or A3) as follows:

A1) the amino acid sequence is protein of a sequence 3 in a sequence table;

A2) a protein which is derived from A1) and has the same function, or has 80% or more identity with the protein shown in A1) and has the same function, and is obtained by substituting and/or deleting and/or adding more than one amino acid residue in an amino acid sequence shown in A1);

A3) a fusion protein obtained by attaching a protein tag to the N-terminus or/and the C-terminus of A1), A2) or A3);

the protein B is the following protein B1), B2) or B3):

B1) the amino acid sequence is protein of a sequence 4 in a sequence table;

B2) a protein which is derived from B1) and has the same function or more than 80% of identity with the protein shown in B1) and has the same function, wherein the protein is obtained by substituting and/or deleting and/or adding more than one amino acid residue in the amino acid sequence shown in B1);

B3) a fusion protein obtained by connecting protein tags at the N-terminal or/and the C-terminal of B1), B2) or B3).

2. Use according to claim 1, characterized in that: the protein A and the protein B are both derived from soybean.

3. Use according to claim 1 or 2, characterized in that: the coding gene of the protein A is a DNA molecule shown as a1) or a2) or a 3):

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

a2) a DNA molecule which has 90% or more than 90% of identity with the nucleotide sequence defined by a1) and codes the protein A;

a3) a DNA molecule which hybridizes with the nucleotide sequence defined by a1) or a3) under strict conditions and codes for the protein A;

the coding gene of the protein B is a DNA molecule shown as B1) or B2) or B3):

b1) the coding sequence is a DNA molecule shown in a sequence 2 in a sequence table;

b2) a DNA molecule which has 90% or more than 90% of identity with the nucleotide sequence defined by B1) and codes the protein B;

b3) a DNA molecule which hybridizes with the nucleotide sequence defined by B1) or B3) under strict conditions and codes for the protein B.

4. Use according to any one of claims 1-3, characterized in that: the substance for regulating the activity or the content of the protein A and the protein B or the substance for regulating the expression of the coding gene of the protein A and the coding gene of the protein B is any one of the following substances c1) -c 4):

c1) a nucleic acid molecule that inhibits or reduces the expression of the protein a-encoding gene and the protein B-encoding gene;

c2) an expression cassette comprising the nucleic acid molecule of c 1);

c3) a recombinant vector comprising the nucleic acid molecule of c1) or a recombinant vector comprising the expression cassette of c 2);

c4) a recombinant microorganism containing c1) the nucleic acid molecule, or a recombinant microorganism containing c2) the expression cassette, or a recombinant microorganism containing c3) the recombinant vector.

5. Use according to any one of claims 1-4, characterized in that: c1) the nucleic acid molecule is sgRNA targeting the gene encoding protein A according to any one of claims 1 to 3 and the gene encoding protein B according to any one of claims 1 to 3, or a DNA molecule expressing the sgRNA.

6. Use according to claim 5, characterized in that: the sgRNA is named sgRNA1 and the sgRNA is named sgRNA2, the target sequence of the sgRNA1 is shown as the 405-th and 426-th position of the sequence 1 in the sequence table, and the target sequence of the sgRNA2 is the 592-th and 613-th position of the sequence 1 in the sequence table.

7. A method for increasing soybean yield and/or soybean seed number and/or soybean seed weight, comprising increasing soybean yield and/or soybean seed number and/or soybean seed weight by suppressing or reducing the expression level of the gene encoding protein a according to any one of claims 1 to 3 and the gene encoding protein B according to any one of claims 1 to 3 in the genome of soybean.

8. The method of claim 7, wherein: the method comprises introducing into the soybean a substance which reduces the activity or content of the protein A according to claim 1 and the protein B according to claim 1 or a substance which reduces the expression of the gene encoding the protein A according to any one of claims 1 to 3 and the gene encoding the protein B according to any one of claims 1 to 3; the substance for reducing the activity or content of the protein A according to claim 1 and the protein B according to claim 1 or the substance for reducing the expression of the gene encoding the protein A according to any one of claims 1 to 3 and the gene encoding the protein B according to any one of claims 1 to 3 is any one of the following substances c1) to c 4):

c1) a nucleic acid molecule that inhibits or reduces the expression of the protein a-encoding gene and the protein B-encoding gene;

c2) an expression cassette comprising the nucleic acid molecule of c 1);

c3) a recombinant vector comprising the nucleic acid molecule of c1) or a recombinant vector comprising the expression cassette of c 2);

c4) a recombinant microorganism containing c1) the nucleic acid molecule, or a recombinant microorganism containing c2) the expression cassette, or a recombinant microorganism containing c3) the recombinant vector.

9. The method of claim 8, wherein: c1) the nucleic acid molecule is sgRNA targeting the gene encoding protein A according to any one of claims 1 to 3 and the gene encoding protein B according to any one of claims 1 to 3, or a DNA molecule expressing the sgRNA.

10. The method of claim 7, wherein: the method for inhibiting or reducing the expression quantity of the protein A coding gene of any one of claims 1 to 3 and the protein B coding gene of any one of claims 1 to 3 in the soybean genome comprises the steps of replacing the protein A coding gene shown in a sequence 1 in the soybean genome with a Gmjagged1-1 gene, replacing the protein B coding gene shown in a sequence 2 with a Gmjagged1-2 gene, wherein the Gmjagged1-1 gene is a DNA molecule obtained by deleting the 419-420 th nucleotide CA and the 597-560-th nucleotide GAGT in the sequence 1 in the sequence table and keeping other nucleotides in the sequence 1 in the sequence table unchanged; the Gmjagged1-2 gene is a DNA molecule obtained by deleting the 235 th and 238 th nucleotides CATG and the 393 th nucleotide A of the sequence 2 in the sequence table and keeping other nucleotides of the sequence 2 in the sequence table unchanged.

Technical Field

The invention relates to the technical field of plant biology, in particular to a method for obtaining high-yield soybeans by modifying GmJAGGED1-1 and GmJAGGED1-2 through gene editing simultaneously.

Background

Soybeans, commonly known as soybeans, are one of important food crops and oil crops. Yield-related traits are important factors in determining high yield of soybeans, for example, increasing the number of single pods can lead to an increase in the number of seeds per plant, which in turn leads to an increase in the weight of seeds per plant, ultimately resulting in an increase in soybean yield. Therefore, the gene which can improve the traits related to the yield and increase the soybean yield is significant.

The ln locus of soybean controls the shape of soybean leaves and the number of single pod. The soybean material carrying the ln locus often appeared as narrow leaves and was dominated by three pods with four pods. Map-based cloning showed that the single base mutation of GmJAGGED1-2 located on chromosome 20 of soybean is the major gene controlling this site (Fang, et al., 2013; Jeong et al., 2012). Breeding practices have shown that new soybean varieties can be created by introducing ln loci into soybean material that does not contain the loci, and that yield can be increased by 8% to 10% (Liu et al, 2020). GmJAGGED1-1 is located on soybean chromosome 10 and is highly homologous with GmJAGGED 1-2. Until now, soybean materials and related phenotypes simultaneously mutated with GmJAGGED1-1 and GmJAGGED1-2 are not reported, so that the improvement of the yield of soybeans by creating soybean materials simultaneously mutated with GmJAGGED1-1 and GmJAGGED1-2 has a profound practical significance.

Reference to the literature

Fang,C.,Li,W.,Li,G.,Wang,Z.,Zhou,Z.,Ma,Y.,Shen,Y.,Li,C.,Wu,Y.,Zhu,B.,Yang,W.and Tian,Z.(2013)Cloning of Ln gene through combined approach of map-based cloning and association study in soybean.J Genet Genomics 40,93-96.

Jeong,N.,Suh,S.J.,Kim,M.-H.,Lee,S.,Moon,J.-K.,Kim,H.S.andJeong,S.-C.(2012)Ln is a key regulator of leaflet shape and number of seeds per pod in soybean.The Plant cell 24,4807-4818.

Liu,S.,Zhang,M.,Feng,F.and Tian,Z.(2020)Toward a"Green Revolution"for Soybean.Molecular plant 13,688-697.

Disclosure of Invention

The technical problem to be solved by the invention is how to regulate the yield of the soybean or how to regulate the seed number and/or seed weight of the soybean.

In order to solve the above technical problems, the present invention provides, in a first aspect, an application of a substance that regulates activities or contents of protein a and protein B or an expression substance that regulates a gene encoding the protein a and a gene encoding the protein B, the application being any one of:

p1, use in regulating or increasing soybean yield;

p2, in regulating and controlling the number of soybean seeds or increasing the number of soybean seeds;

p3, in regulating and controlling the weight of soybean seeds or increasing the weight of the soybean seeds;

p4, application in soybean breeding;

the protein A can be the following protein A1), A2) or A3):

A1) the amino acid sequence is protein of a sequence 3 in a sequence table;

A2) a protein which is derived from A1) and has the same function, or has 80% or more identity with the protein shown in A1) and has the same function, and is obtained by substituting and/or deleting and/or adding more than one amino acid residue in an amino acid sequence shown in A1);

A3) a fusion protein obtained by attaching a protein tag to the N-terminus or/and the C-terminus of A1), A2) or A3);

the protein B can be the following proteins B1), B2) or B3):

B1) the amino acid sequence is protein of a sequence 4 in a sequence table;

B2) a protein which is derived from B1) and has the same function or more than 80% of identity with the protein shown in B1) and has the same function, wherein the protein is obtained by substituting and/or deleting and/or adding more than one amino acid residue in the amino acid sequence shown in B1);

B3) a fusion protein obtained by connecting protein tags at the N-terminal or/and the C-terminal of B1), B2) or B3).

In the above application, the protein A and the protein B can be both derived from soybean.

In the application, the sequence 3 in the sequence table is composed of 257 amino acid residues, and the sequence 4 in the sequence table is composed of 256 amino acid residues.

The one or more amino acid residues may specifically be within ten amino acid residues.

In the above applications, the 80% or greater identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, or 99% identity.

In the above application, the gene encoding protein A may be a DNA molecule represented by a1) or a2) or a3) as follows:

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

a2) a DNA molecule which has 90% or more than 90% of identity with the nucleotide sequence defined by a1) and encodes the protein A described above;

a3) a DNA molecule which hybridizes with the nucleotide sequence defined by a1) or a3) under strict conditions and codes for the protein A.

In the above application, the encoding gene of the protein B can be a DNA molecule shown as B1) or B2) or B3) as follows:

b1) the coding sequence is a DNA molecule shown in a sequence 2 in a sequence table;

b2) a DNA molecule having 90% or more 90% identity to the nucleotide sequence defined in B1) and encoding the protein B as described above;

b3) a DNA molecule which hybridizes with the nucleotide sequence defined by B1) or B3) under stringent conditions and encodes the protein B described above.

In the above application, the substance for regulating the expression of the gene encoding protein a and the gene encoding protein B may be a substance for regulating at least one of the following 6 types of regulation: 1) regulation at the level of transcription of said gene; 2) regulation after transcription of the gene (i.e., regulation of splicing or processing of a primary transcript of the gene); 3) regulation of RNA transport of the gene (i.e., regulation of nuclear to cytoplasmic transport of mRNA of the gene); 4) regulation of translation of the gene; 5) regulation of mRNA degradation of the gene; 6) post-translational regulation of the gene (i.e., regulation of the activity of a protein translated from the gene).

In the above application, the substance for regulating the activity or content of protein a and protein B may be a substance for knocking out the coding gene of protein a and the coding gene of protein B and/or a substance for regulating the expression of the coding gene of protein a and the coding gene of protein B.

In the above application, the substance for regulating the expression of the gene encoding protein a and the gene encoding protein B may be the substance for inhibiting or reducing the expression of the genes, and the inhibition or reduction of the expression of the genes may be achieved by gene knockout or by gene silencing.

The gene knockout (geneknockout) refers to a phenomenon in which a specific target gene is inactivated by homologous recombination. Gene knockout is the inactivation of a specific target gene by a change in the DNA sequence.

The gene silencing refers to the phenomenon that a gene is not expressed or is under expression under the condition of not damaging the original DNA. Gene silencing is premised on no change in DNA sequence, resulting in no or low expression of the gene. Gene silencing can occur at two levels, one at the transcriptional level due to DNA methylation, differential staining, and positional effects, and the other post-transcriptional gene silencing, i.e., inactivation of a gene at the post-transcriptional level by specific inhibition of a target RNA, including antisense RNA, co-suppression (co-suppression), gene suppression (quelling), RNA interference (RNAi), and micro-RNA (mirna) -mediated translational suppression, among others.

In the above application, the substance for regulating the expression of the gene encoding protein a and the gene encoding protein B may be an agent for inhibiting or reducing the expression of the genes. The agent that inhibits or reduces the expression of the gene can be an agent that knocks out the gene, such as an agent that knocks out the gene by homologous recombination or an agent that knocks out the gene by CRISPR-Cas 9. The agent that inhibits or reduces expression of the gene may comprise a polynucleotide that targets the gene, such as an siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

In the above application, the substance for regulating the activity or content of protein a and protein B or the substance for regulating the expression of the gene encoding protein a and the gene encoding protein B may be any of the following substances c1) to c 4):

c1) a nucleic acid molecule that inhibits or reduces the expression of the protein a-encoding gene and the protein B-encoding gene described above;

c2) an expression cassette comprising the nucleic acid molecule of c 1);

c3) a recombinant vector comprising the nucleic acid molecule of c1) or a recombinant vector comprising the expression cassette of c 2);

c4) a recombinant microorganism containing c1) the nucleic acid molecule, or a recombinant microorganism containing c2) the expression cassette, or a recombinant microorganism containing c3) the recombinant vector.

c1) The nucleic acid molecule may be a sgRNA expressed while targeting the expression of the protein a-encoding gene and the protein B-encoding gene described above or a DNA molecule expressing the sgRNA.

The sgRNA is named sgRNA1 and the sgRNA is named sgRNA2, the target sequence of the sgRNA1 is the 405-th and 426-th position (the 221-242-th position of the sequence 2 in the same sequence table) of the sequence 1, and the target sequence of the sgRNA2 is the 592-613-th position (the 387-408-th position of the sequence 2 in the sequence table) of the sequence 1.

The term "identity" refers to sequence similarity to a native nucleic acid sequence. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences. The identity of 90% or greater than 90% can be at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity.

In order to solve the above-mentioned technical problems, the present invention also provides a method for increasing soybean yield and/or increasing soybean seed number and/or increasing soybean seed weight, comprising increasing soybean yield and/or increasing soybean seed number and/or increasing soybean seed weight by inhibiting or reducing the expression amount of the above-mentioned protein a-encoding gene and protein B-encoding gene in soybean genome.

The reduction or inhibition of the expression level of the protein a-encoding gene and the protein B-encoding gene in soybean can be achieved by any means known in the art, such that the genes are subjected to deletion mutation, insertion mutation or base transition mutation, thereby achieving the reduction or loss of gene function, specifically, chemical mutagenesis, physical mutagenesis, RNAi, genome site-directed editing or homologous recombination, and the like.

In the above-mentioned genome site-directed editing method, Zinc Finger Nuclease (ZFN) technology, Transcription activator-like effector nuclease (TALEN) technology, Clustered regularly spaced short palindromic repeats and their related systems (Clustered regularly interspaced short palindromic repeats/CRISPR associated, CRISPR/Cas9 system) technology, and other technologies capable of realizing genome site-directed editing can be used. In any case, the entire gene encoding the protein described above may be targeted, and each element regulating the expression of the gene encoding the protein described above may be targeted, as long as the loss or reduction of the function of the gene can be achieved. For example, the exon or 5' UTR of the gene encoding the protein described above may be targeted.

The method described above may comprise introducing into the soybean a substance that reduces or inhibits the activity of protein a and protein B or the expression of the gene encoding protein a and the gene encoding protein B described above. The substance which reduces or inhibits the activities of protein A and protein B or the expression of the gene encoding protein A and the gene encoding protein B as described above may be any of the following substances c1) to c 4):

c1) a nucleic acid molecule that inhibits or reduces the expression of the protein a-encoding gene and the protein B-encoding gene described above;

c2) an expression cassette comprising the nucleic acid molecule of c 1);

c3) a recombinant vector comprising the nucleic acid molecule of c1) or a recombinant vector comprising the expression cassette of c 2);

c4) a recombinant microorganism containing c1) the nucleic acid molecule, or a recombinant microorganism containing c2) the expression cassette, or a recombinant microorganism containing c3) the recombinant vector.

c1) The nucleic acid molecule is sgRNA targeting the protein A coding gene and the protein B coding gene or DNA molecule expressing the sgRNA.

The sgRNA is named sgRNA1 and the sgRNA is named sgRNA2, the target sequence of the sgRNA1 is the 405-th and 426-th position (the 221-242-th position of the sequence 2 in the same sequence table) of the sequence 1, and the target sequence of the sgRNA2 is the 592-613-th position (the 387-408-th position of the sequence 2 in the sequence table) of the sequence 1.

The step of inhibiting or reducing the expression quantity of the protein A coding gene and the protein B coding gene in the soybean genome is to replace the protein A coding gene shown in a sequence 1 in the soybean genome with a Gmjagged1-1 gene and replace the protein B coding gene shown in a sequence 2 with a Gmjagged1-2 gene, wherein the Gmjagged1-1 gene is a DNA molecule obtained by deleting the 419-420 nucleotide CA and the 597-560-nucleotide GAGT in the sequence 1 in a sequence table and keeping other nucleotides in the sequence 1 in the sequence table unchanged; the Gmjagged1-2 gene is a DNA molecule obtained by deleting the 235 th and 238 th nucleotides CATG and the 393 th nucleotide A of the sequence 2 in the sequence table and keeping other nucleotides of the sequence 2 in the sequence table unchanged.

The invention discloses a method for simultaneously editing and modifying soybean GmJAGGED1-1 gene and GmJAGGED1-2 gene to obtain high-yield soybean. Specifically, a CRISPR/Cas9 system is used for simultaneously editing GmJAGGED1-1 and GmJAGGED1-2 genes in starting soybeans, and the GmJAGGED1-1 and GmJAGGED1-2 genes are simultaneously mutated to cause premature termination of translation proteins, so that transgenic soybeans are obtained; realizes the simultaneous gene editing of GmJAGGED1-1 and GmJAGGED1-2 genes in the starting soybean. The invention utilizes CRISPR/Cas9 mediated gene editing technology to carry out fixed point knockout of specific targets on soybean GmJAGGED1-1 and GmJAGGED1-2 genes, obtains high-yield soybean mutant materials with yield improved by more than 8% compared with a control, and provides new materials for breeding high-yield soybean varieties.

Drawings

FIG. 1 shows the mutation types of the Gmjagged1-1 gene and the Gmjagged1-2 gene of the mutant jag2-7 at two target points.

FIG. 2 shows statistics of the number of grains per plant and the weight of grains per plant of the mutant (jag2-1) and the wild type (Huachun No. 6) in summer and spring. Wherein, A in figure 2 is the statistics of the single plant grain number of the mutant (jag2-1) and the wild type (Huachun No. 6) in summer; b in FIG. 2 is the statistics of individual grain weight of mutant (jag2-1) and wild type (Huachun No. 6) in summer; FIG. 2C shows statistics of the number of single plants in spring for mutant (jag2-1) and wild type (Huachun No. 6); FIG. 2D shows the statistics of individual grain weight of mutant (jag2-1) and wild type (Huachun No. 6) in spring. Significance was calculated using t-ttest, representative of significance at the 0.01 level.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The terms used in the following methods of practice and examples generally have the meanings commonly understood by those of ordinary skill in the art, unless otherwise specified.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

In the following examples, soybean variety huachun 6 was a soybean variety obtained by breeding at the college of agriculture of southern China university, and was approved by the second national crop variety approval committee at 28 days 7 and 7 of 2009, at the third meeting, and the approval number was national approved bean 2009012.

In the following examples, the soybean CRISPR/Cas9 vector pGES201 is described in non-patent documents "Mengyan Bai1, Juehui Yuan1, Huaqin Kuang, Pingging Gong, Suning Li, Zhuhui Zhuang, Bo Liu, Jianing Sun, Maoxiang Yang, Lan Yang, Dong Wang, Shikui Song, Yuefeng Guan (2020) Generation of a multiple mutagenesis amplification video needle assembled CRISPR-Cas9 in plant biotechnology J18, 721-.

Example 1

Construction of CRISPR/Cas9 vector for simultaneously knocking out soybean GmJAGGED1-1 and GmJAGGED1-2 genes

1. Obtaining of sgRNA

Genomic sequences of the soybean GmJAGGED1-1 and GmJAGGED1-2 genes were obtained from the Phytozome database. The GmJAGGED1-1 gene is positioned on the No. 10 chromosome of soybean (49647129-49649853), the genome has the full length of 2725bp and is shown as a sequence 1 in a sequence table, and an encoded amino acid sequence is a protein shown as a sequence 3 in the sequence table; the GmJAGGED1-2 gene is located on the No. 20 chromosome of soybean (35827672-35830107), the genome has the full length of 2436bp and is shown as a sequence 2 in a sequence table, and an encoding amino acid sequence is a protein shown as a sequence 4 in the sequence table. The CRISPR-P (http:// CRISPR. hzau. edu. cn/CRISPR2/) tool is used for target design and selection of common targets that can edit GmJAGGED1-1 and GmJAGGED1-2 genes simultaneously. And finally obtaining a target point 1 and a target point 2:

target 1: 5'-CTCTCTGTCGGTACCATGTAGG-3' (located at the position 405-426 of the sequence 1 in the sequence table and located at the position 221-242 of the sequence 2 in the sequence table);

target 2: 5'-CCGATGAGTACTCTAGGGATGG-3' (located at position 592-613 of sequence 1 in the sequence table and at position 387-408 of sequence 2 in the sequence table).

Primers were designed containing information on two targets:

the primer sequences designed according to the target 1 are as follows:

sgRNA1 Forwardprimer：5’-GGATTGCTCTCTGTCGGTACCATGT-3’；

sgRNA1 Reverseprimer：5’-AAACACATGGTACCGACAGAGAGCA-3’。

the primer sequences designed according to target 2 are as follows:

sgRNA2 Forwardprimer：5’-GGATTGCCATCTCTAGAGTACTCAT-3’；

sgRNA2 Reverseprimer：5’-AAACATGAGTACTCTAGAGATGGCA-3’。

synthesizing the designed primer, diluting the primer to 10 mu M with water, and annealing to obtain a double-stranded DNA fragment sgRNA with a sticky end, wherein the method specifically comprises the following steps:

(1)sgRNA1

annealing the primers sgRNA1 Forward primer and sgRNA1 Reverse primer to obtain a double-stranded DNA fragment sgRNA1 with sticky ends.

The reaction system is a 25. mu.l system: sgRNA1 Forward primer and sgRNA1 Reverse primer were each 5. mu.l, water 15. mu.l.

Reaction conditions are as follows: annealing is completed after annealing at 96 ℃ for 5min and 0.1 ℃/s to 12 ℃ for 5min, and sgRNA1 with a sticky end is obtained.

(2)sgRNA2

Annealing the primers sgRNA2 Forward primer and sgRNA2 Reverse primer to obtain a double-stranded DNA fragment sgRNA2 with sticky ends.

The reaction systems were all 25. mu.l systems: sgRNA2 Forward primer and sgRNA2 Reverse primer were each 5. mu.l, water 15. mu.l.

Reaction conditions are as follows: annealing is completed after annealing at 96 ℃ for 5min and 0.1 ℃/s to 12 ℃ for 5min, and sgRNA2 with a sticky end is obtained.

2. Preparation of vector for expression of sgRNA

Digesting 1 mu g of the soybean CRISPR/Cas9 vector pGES201 vector by BsaI enzyme, and recovering to obtain a pGES201 linearized vector for later use.

The sgRNA1 with sticky ends and the sgRNA2 with sticky ends obtained in step 1 and the pGES201 linearized vector are connected through the system in table 1 to complete the construction of the final vector CRISPR/Cas9-sgRNA1-sgRNA 2. The CRISPR/Cas9-sgRNA1-sgRNA2 expresses sgRNA1 targeting target 1 and sgRNA2 targeting target 2.

TABLE 1

sgRNA1	1μl
		sgRNA2	1μl
pGES201 linearized vector	2μl
		10 XT 4 DNA ligase Buffer	2μl
T4 DNA ligase	1μl
		Sterile water	13μl

II, obtaining double mutants of soybean GmJAGGED1-1 and GmJAGGED1-2

1. Transferring the CRISPR/Cas9-sgRNA1-sgRNA2 vector obtained in the step one into escherichia coli competent DH5 alpha, coating the escherichia coli competent DH5 alpha on a LB + Kan solid culture medium, selecting a monoclonal to extract a plasmid, and sequencing, wherein sequencing primers are sgRNA1 Forward primer and sgRNA2 Reverse primer.

sgRNA1 Forward primer：5’-GGATTGCTCTCTGTCGGTACCATGT-3’；

sgRNA2 Reverse primer：5’-AAACATGAGTACTCTAGAGATGGCA-3’。

The sequencing alignment was completed to obtain the correct CRISPR/Cas9-sgRNA1-sgRNA2 vector plasmid.

2. The recombinant plasmid CRISPR/Cas9-sgRNA1-sgRNA2 is transferred into Agrobacterium tumefaciens EHA105 by an electric shock transformation method, the plasmid is extracted for sequencing verification, and recombinant strains with correct sequencing verification (containing a sgRNA1 coding sequence and a sgRNA2 coding sequence) are respectively named as EHA105-CRISPR/Cas9-sgRNA1-sgRNA 2.

3. Genetic transformation of Huachun No. 6 soybean:

with soybean cultivar Huachun No. 6 (publicly available from Guangdong center for Soybean improvement, national institute of agriculture, southern China), as a recipient material, stable Transformation was performed by the cotyledonary node method, with references "Li S, Cong Y, Liu Y, Wang T, Shuai Q, Chen N, Gai J, Li Y.optimization of Agrobacterium-media Transformation in Soybean. front Plant Sci.2017 Feb 24; 8:246, doi:10.3389/fpls.2017.00246, PMID: 28286512; PMCID pmcs5323423 ", briefly modified according to the references of the soybean conversion process as follows: placing the soybeans infected by the agrobacterium into a dedifferentiation culture medium for culturing for 5 days, then transferring the soybeans to a redifferentiation culture medium for culturing for 14 days, then transferring the soybeans to a resistant redifferentiation culture medium containing Basta for culturing for 14 days, then transferring the soybeans to a resistant redifferentiation culture medium containing phytohormone and Basta for culturing for 28 days, and finally transferring the soybeans to a root induction culture medium for culturing. The plants obtained were transferred to soil for growth and grown in a 1: and spraying and screening the positive seedlings by using 1000-diluted herbicide Basta to obtain T0 positive soybean plants.

4. GmJAGGED1-1 and GmJAGGED1-2 double-mutant screening and identification

DNA of leaves of T0 positive soybean plants is extracted to be used as a template for PCR molecular detection, and wild soybean Huachun No. 6 is used as a control. Designing PCR primers containing target spots sgRNA1 and sgRNA2, carrying out PCR amplification, and sequencing the amplified product. Wherein the primers for amplifying the GmJAGGED1-1 are as follows: GmJAGGED1-1F (as primer 1) and GmJAGGED1-1R (as primer 2); the primers for amplifying the GmJAGGED1-2 are as follows: GmJAGGED1-2F (as primer 1) and GmJAGGED1-2R (as primer 2).

GmJAGGED1-1F：5’-TCAACCAAACCAGACGAAGC-3’；

GmJAGGED1-1R：5’-AACCACGGCAGTGTTCAACT-3’。

GmJAGGED1-2F：5’-AAAGTCACGAGACCCACTGTC-3’；

GmJAGGED1-2R：5’-TGAAAGGCTTCCCATGGTGG-3’。

The PCR reagent system is shown in table 2 below:

TABLE 2

2×Phanta Max Buffer	25μl
		dNTP Mix(10mM)	1μl
Super-Fidelity DNA Polymerase	1μl
		Primer 1	1μl
Primer 2	1μl
		Sterile water	21μl

The PCR reaction system is as follows: 3min at 95 ℃; 30sec at 95 ℃, 30sec at 58 ℃, 2min at 72 ℃ and 35 cycles; 5min at 72 ℃.

The PCR product was sent to the company for sequencing verification. Sequencing results show that plants with mantle peaks near the target position are heterozygous editing plants and are named as T0 GmJAGGED1-1 and GmJAGGED1-2 double editing soybeans.

Planting seeds of T1 generations harvested after selfing of T0 GmJAGGED1-1 and GmJAGGED1-2 double-editing soybeans to obtain T1-generation plants, and amplifying and sequencing by adopting the PCR system in the step 4 to obtain a soybean plant jag2-7(T1 generation) edited at the sgRNA1 and sgRNA2 positions of GmJAGGED1-1 and GmJAGGED1-2 genes, wherein the soybean plant jag2-7 is shown in a figure 1: the GmJAGGED1-1 gene of the material deletes the 419 nd 420 nd nucleotide CA in the sequence 1 in the sequence table at the position of a target spot 1, deletes 2bp altogether, deletes the 597 nd 560 nd nucleotide GAGT in the sequence 1 in the sequence table at the position of the target spot 2, deletes 4bp altogether, causes the deletion of CDS starting codon and the shift mutation of a second exon, finally causes the loss of the function of GmJAGGED1-1, generates Gmjagged1-1 gene, and then knocks out the GmJAGGED1-1 gene.

The GmJAGGED1-2 gene of the material deletes 235 th and 238 th nucleotides CATG in a sequence 2 in a sequence table at a target point 1 position, deletes 4bp altogether, deletes 393 th nucleotides A in the sequence table at the target point 2 position, deletes 1bp altogether, causes CDS initiation codon deletion and second exon frameshift mutation, finally causes GmJAGGED1-2 function loss, and generates Gmjagged1-2 gene.

Third, GmJAGGED1-1 and GmJAGGED1-2 double-mutant jag2-7 yield-related trait determination

Selfing T1 generation jag2-7, and harvesting T2 generation jag2-7 seeds. The soybean seeds of T2 generation jag2-7 and wild type soybean Huachun No. 6 (WT) were planted in a soybean isolation area of a southern China agricultural university test base in Guangzhou, Guangdong at the beginning of 3 months and at the beginning of 6 months, the plant spacing was 12cm, the row spacing was 50cm, the row length was 1.0m, three biological replicates were set, and 3 rows were each biological replicate. And respectively counting the grain number and the grain weight of each plant in the harvest season.

The results are shown in FIG. 2, and the number of grains per plant of jag2-7 was significantly increased, 17.63% and 10.59% respectively, compared to the recipient material, Huachun No. 6, in both spring and summer. Compared with the acceptor material Huachun No. 6, the single-plant grain weight of jag2-7 is remarkably increased by 8.81% and 8.67%, respectively.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> southern China university of agriculture

<120> method for producing soybean with high yield

<130> GNCSY211772

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2725

<212> DNA

<213> Soybean (Glycine max)

<400> 1

tttgcacttt ataattctgt ctgtcatttc ccaagtctca accaaaccag acgaagctag 60

ctagcaattt gcaaaaaaac tgattaagtt aagcccccca cttccactac cacactcacg 120

ctctcttttt aaagtcacga gacccactgt cccattgttc attcaatttt tggtgtgggg 180

ggagataaga gagagagaga gagagtggca gaggaaccga tagggaactt tcaaagctct 240

ttatcttcta ccatcactca actagcctct atattgcagt ctcaaactga aagtctcaca 300

aattttgtag aaaactgtag ttttatcccc acccccctcc cccatctgaa agaaagagta 360

tttgcctcgc atcatttttc ttttctttct ctctttctgt ctctctctct gtcggtacca 420

tgtaggtctt cccccactac tacaccttca cacccttctt ctcatcctcc tctttctcct 480

tctctaactc taagcccttt taactttctc tctatctttt tctctctctt atgactttgt 540

cgttccttta caggagacca gaacgaaacc cattagatct taacaatttg cccgatgagt 600

actctaggga tggcaaacaa gtcctcgaag accatacctc ttcacccggt aaacttcatg 660

atcatatcta tctatctatc tatatatata tatagaagca cgctagaata tcaaaggaat 720

actttttttc tttatatatt ccgaattcta tttcacaaac acacaccatg ggaagccttt 780

cacttttcac aggaaacaaa gttgaacact gccgtggttt gttacttacc tctgtggtat 840

attccttgac catgaaacag tgttcaccac acttcttttt ttattctttt ggttttgtcc 900

ttgtatacgt ttccttctct cctttttctt tttttcctct tcttctctta aaagataaaa 960

gaaaaggaaa aaaaagaaga agcataaaag tagttaggtg atagaactta aaataaagaa 1020

aagtaattgt tggggcgagc gattagttaa ttaattaaaa tgaattgaat tgatttgagg 1080

gatttgggta atcgagaatg aggcgtgaaa gggaaagaag gtggtgtcat ggaagttgct 1140

gtaattggat ggttatggtt gcaggttgca ggaaaaagaa aagcggcggg aaggatggaa 1200

aagacgagtg tgggaaggtc tacgagtgta gattttgttc cctcaagttc tgcaagtctc 1260

aggctcttgg gggacacatg aaccgccacc gccaaggcta gtatatacca cctattatat 1320

cttttcgttt ttcttcctct ttactctcac aagaaccaaa tcttcactgc ccgttaatga 1380

tttggccatg aagggttgtc atttcaatca aaaaaagttg agttagctga aattcgatcc 1440

agagggtccc ttcagctgtt tttctttttt tcttttttta attctttctt tctttttgct 1500

ttgcttttat tattttgttt tgagcagaga gggaaacgga gacgctgaac caggctcgtc 1560

aactggtatt tcgtagcgat catatcattg ctccacaagg tgcccctcac ttagggtatg 1620

cacccattac ttccatgcag ctattctctt ccaatttgtt gttgttgttc caccctaata 1680

ttacactatc attcccagtt ctttcttaaa tacttgtttt tctctaggtt ctttagcatt 1740

tgttaacgtt gttttttcaa gttaacgcat aatattaggg tgcgcagcat gacactgttt 1800

gatacaattg actgtgataa tattcagatg ctgccagcca ataggaacgg ggggttatca 1860

cccatcagga gacccaacag tgcctctaag attcccgaga tacttctcag gttcatcctc 1920

aactcacatg ccaccaccgc cgccgccgcc gccgccaccg caacaatcac acctatacgc 1980

atcaccttcg aggccagtgt catttgggtc atcacacttc ccccaccagc atgctgtgaa 2040

cgattactat gtgggccacg tgatgagtgg tgggagccac ggacactatg ttggaggaga 2100

gagcacgagt agttacacgt acattggtgc cccggtgggg caagctggtg gattcgctgg 2160

tggtggtaag gaggggtcag cagtgcagga ggaagggttg agttggggaa ggagctattc 2220

aggaggagca cagcatcgtt tggatcctcc ctcagcgatc aatcggtttc atgatggttt 2280

ctaatgagat gagatgagat gagagattct tttgtttgag tgtttttggt ttgcgttttg 2340

tgttatgtta ttttatgtgg tatcatcata aagacacgca atccagagag agagacataa 2400

caagctgaca catggggtca tggagaggag ggtatagcta aggctaggat gaagagagag 2460

agtgtgcgtg agaggtggtt tttggtttat ctacagtaac caggaacagc ttttgagctc 2520

atggacacgc tcagctactt tggcttggag ttatgggtgg gatttcttct caacatcgcc 2580

ttttgtattg gtagctacct atcttggaca accagttaga tatgaactga aactttccaa 2640

actccttttg ctttgcatgc caaattacaa ccattttcct tctaatttgc ttctttcaac 2700

atactactat tactatttta tttat 2725

<210> 2

<211> 2436

<212> DNA

<213> Soybean (Glycine max)

<400> 2

gggggcgggg ggagataaga gagagagaga gagtggcaga ggaactgata gagaactttc 60

aaagctcttt atcttctacc atcactcaac cagcctctat attgcagtct caaactgaaa 120

gtctaaattt ttttgtaaaa agctttagtt ttatccctac ccccacccca tctgaaagaa 180

agagtgtttg cctcacatca tttttcccct ttctgtctct ctctctgtcg gtaccatgta 240

ggtcttcccc cactactaca ccttcacacc cttcttttct tcctcctctt tctccttctc 300

taaacccttt aactttctct ctcttatgac tttgttgttc ctttacagga gaccagaacg 360

aaacccctta gatcttaaca atttgcccga tgagtactct agagatggca aacaagtcct 420

cgaagaccat acctcttcat ccggtaaact tcatgatcat accaatatat atatatatgc 480

acgctgaaat atcaaaggaa cacttttttt ttctttctat ttaccgaatt ctatttcaca 540

atcacacacc accatgggaa gcctttcact tttcacagga aacaaagttg aacactgccc 600

gtggtttgtt acccatccct gtggtatatt ccttgaccat gaaacagtgt tcaccacact 660

tttttttcct cattttttta ttctttttgt tttgtccttg tatacgtttc ttttcttttt 720

ttgtttcttc tccttttcct cttctacttc gttatagtat ctcttaaaag ttatggaaaa 780

aataaaataa gaaaaacata aaagtagtta ggtgatagaa tagaacttaa aaaaaagaaa 840

agtagttgtt tgggggcgag cggtaagtta attaattgaa ttgatttgat ggaggatttg 900

ggtaatcgag aatggggagt taaaggcaaa gaagggtgtc atggaagttg ctttaattgg 960

atggttatag ttgcaggttg caggaaaaag aaaagcggcg ggaaggatgg aaaagacgag 1020

tgtgggaagg tctacgagtg tagattttgt tccctcaagt tctgcaagtc tcaggctctt 1080

gggggacaca tgaaccgcca ccgccaaggc tagtaccacc tatattatta tactttatat 1140

tttcttcctc ttttctttta ccactccttc tcacaagaac caaatctctt ctggccgtta 1200

atgatttggc catgaaagct tgtcatttca atcacaaaat gttgtgttgt tggattgctg 1260

aaattcgatc atcgagaggg ccccttcagc tgtttttttt ttttcttttt gctttgcttt 1320

attatttgtt tttgtgattg ttttgagcag agagggaaac ggagacgctg aaccaggctc 1380

gtcaactggt ctttcgttgt gatcataaca ttgctgcaca aggtgcccct cacttagggt 1440

atgcacccat tacttccatg cagcgattct cttcctattt cttcttcttg ttcaacattt 1500

atatatttca tttctcaaat acttgttttt ctgtgggttc tttagcgttt gttaacgttg 1560

ttttttcaag gtataacaca taatatttgg gtactcagca tgacactgtt tgatactgcg 1620

atttatttgt gataatattc aattcagatg ctgccaaaca ataggaacgg ggggttatca 1680

tccctcagga gacccaacag tgcctctaag attcccaaga tacttctcag gttcatcctc 1740

aactcacatg ccaccatccc cgccaccgcc gccgccaccg caacgaccat acctataccc 1800

ttcacctacg aggccagtgt catttgggtc atcacacttc cctctccagc atgcagtgaa 1860

cgattactat gtgggccacg tgatgagtgg tggcagccac ggacactatg ttggaggaga 1920

gagcacaagg agttacacgt gcattggtgc accggtgggg caaggtggcg gattcgctgg 1980

tggtaaggag gggtctgcag tgcaggagga agggttgagt acttggggaa ggggctattc 2040

aggtgcacag gatcgtttgg atcctccctc agcgatcaat cggtttcaag atggtttcta 2100

aagagatgag agattctttg tttgagtggt ttttggtttg tgttatgttt ctgttatgtt 2160

atgtggtatc atcataaaga cacgcaatcc agagagagag agaggtggtt tttggtttat 2220

ctacagtaac cagggacagc ttttgagctc atggacacgc tcagctactt tggcttggag 2280

ttgtgggtgg gatttcttct caacatcgcc ttttgtattg gtagctagct agctagcttg 2340

gacaaccagt tagatttgaa ctgaaacttt ccaaactcct tttcctttgc atgccaaatt 2400

acaaccattt tccttctcag taatttgttc tttcaa 2436

<210> 3

<211> 257

<212> PRT

<213> Soybean (Glycine max)

<400> 3

Met Arg Pro Glu Arg Asn Pro Leu Asp Leu Asn Asn Leu Pro Asp Glu

1 5 10 15

Tyr Ser Arg Asp Gly Lys Gln Val Leu Glu Asp His Thr Ser Ser Pro

20 25 30

Gly Cys Arg Lys Lys Lys Ser Gly Gly Lys Asp Gly Lys Asp Glu Cys

35 40 45

Gly Lys Val Tyr Glu Cys Arg Phe Cys Ser Leu Lys Phe Cys Lys Ser

50 55 60

Gln Ala Leu Gly Gly His Met Asn Arg His Arg Gln Glu Arg Glu Thr

65 70 75 80

Glu Thr Leu Asn Gln Ala Arg Gln Leu Val Phe Arg Ser Asp His Ile

85 90 95

Ile Ala Pro Gln Gly Ala Pro His Leu Gly Cys Cys Gln Pro Ile Gly

100 105 110

Thr Gly Gly Tyr His Pro Ser Gly Asp Pro Thr Val Pro Leu Arg Phe

115 120 125

Pro Arg Tyr Phe Ser Gly Ser Ser Ser Thr His Met Pro Pro Pro Pro

130 135 140

Pro Pro Pro Pro Pro Pro Gln Gln Ser His Leu Tyr Ala Ser Pro Ser

145 150 155 160

Arg Pro Val Ser Phe Gly Ser Ser His Phe Pro His Gln His Ala Val

165 170 175

Asn Asp Tyr Tyr Val Gly His Val Met Ser Gly Gly Ser His Gly His

180 185 190

Tyr Val Gly Gly Glu Ser Thr Ser Ser Tyr Thr Tyr Ile Gly Ala Pro

195 200 205

Val Gly Gln Ala Gly Gly Phe Ala Gly Gly Gly Lys Glu Gly Ser Ala

210 215 220

Val Gln Glu Glu Gly Leu Ser Trp Gly Arg Ser Tyr Ser Gly Gly Ala

225 230 235 240

Gln His Arg Leu Asp Pro Pro Ser Ala Ile Asn Arg Phe His Asp Gly

245 250 255

Phe

<210> 4

<211> 256

<212> PRT

<213> Soybean (Glycine max)

<400> 4

Met Arg Pro Glu Arg Asn Pro Leu Asp Leu Asn Asn Leu Pro Asp Glu

1 5 10 15

Tyr Ser Arg Asp Gly Lys Gln Val Leu Glu Asp His Thr Ser Ser Ser

20 25 30

Gly Cys Arg Lys Lys Lys Ser Gly Gly Lys Asp Gly Lys Asp Glu Cys

35 40 45

Gly Lys Val Tyr Glu Cys Arg Phe Cys Ser Leu Lys Phe Cys Lys Ser

50 55 60

Gln Ala Leu Gly Gly His Met Asn Arg His Arg Gln Glu Arg Glu Thr

65 70 75 80

Glu Thr Leu Asn Gln Ala Arg Gln Leu Val Phe Arg Cys Asp His Asn

85 90 95

Ile Ala Ala Gln Gly Ala Pro His Leu Gly Cys Cys Gln Thr Ile Gly

100 105 110

Thr Gly Gly Tyr His Pro Ser Gly Asp Pro Thr Val Pro Leu Arg Phe

115 120 125

Pro Arg Tyr Phe Ser Gly Ser Ser Ser Thr His Met Pro Pro Ser Pro

130 135 140

Pro Pro Pro Pro Pro Pro Gln Arg Pro Tyr Leu Tyr Pro Ser Pro Thr

145 150 155 160

Arg Pro Val Ser Phe Gly Ser Ser His Phe Pro Leu Gln His Ala Val

165 170 175

Asn Asp Tyr Tyr Val Gly His Val Met Ser Gly Gly Ser His Gly His

180 185 190

Tyr Val Gly Gly Glu Ser Thr Arg Ser Tyr Thr Cys Ile Gly Ala Pro

195 200 205

Val Gly Gln Gly Gly Gly Phe Ala Gly Gly Lys Glu Gly Ser Ala Val

210 215 220

Gln Glu Glu Gly Leu Ser Thr Trp Gly Arg Gly Tyr Ser Gly Ala Gln

225 230 235 240

Asp Arg Leu Asp Pro Pro Ser Ala Ile Asn Arg Phe Gln Asp Gly Phe

245 250 255