Application of CSA1 gene in regulation and control of branching angle and petiole angle of leguminous plant and creation of close-planting-resistant high-yield plant type leguminous plant

Abstract

The invention discloses application of a CSA1 gene in regulating and controlling branching angles and petiole angles of leguminous plants and creating close-planting-resistant high-yield plant type leguminous plants. According to the invention, a CSA1 gene is cloned from soybean, and a CRISPR-Cas9 system is further utilized to edit the CSA1 gene, so that two different mutants are obtained. A result shows that the branching angle and the petiole angle of a mutant plant are remarkably reduced, and the yield of the mutant is remarkably higher than that of a wild type under a high-density planting condition, so that the CSA1 gene can regulate and control the branching angle and the petiole angle of the leguminous plant and create a close-planting-resistant leguminous plant, an important gene resource is provided for molecular breeding of the leguminous plant, and the application prospect is wide. And an effective means is provided for creating close-planting-resistant high-yield leguminous plants.

Description

Technical Field

[0001] The present invention relates to the technical fields of plant genetic engineering and genetic breeding technology. More specifically, it relates to the application of the CSA1 gene in regulating the branch angle and petiole angle of leguminous plants and creating high-yield leguminous plants with high density tolerance. Background Art

[0002] One of the main ways to increase soybean yield is to create soybean materials with high density tolerance and increase the soybean planting density. Previously, QTL loci and genes related to soybean plant type have been reported. Chen Xiaorui et al. (2024) successfully isolated GmBAS1 through the analysis of the soybean cytochrome P450 gene family. Further, transgenic materials GmBAS1a and GmBAS1b were created by overexpression, and the transgenic materials showed reduced plant height and smaller petiole angles. Su Bohong (2024) used the mutant material it1 obtained previously and Jidou 12 to create a genetic population, and cloned a pleiotropic gene GmIT1 that regulates the morphological development of soybean plant type, which has an impact on multiple traits of soybean plants. Clark et al. (2022) located a major convergent soybean locus qGmBa1 on chromosome 19 of soybean using the genetic population constructed by LD00-3309 and RIL1890, but the major gene was not cloned. Gao et al. (2017) analyzed the large petiole angle mutant Gmilpa1 obtained by mutagenesis and cloned the branch angle control gene GmILPA1 encoding APC8-like. Although some genes related to soybean plant type have been cloned in soybean, the specific mechanism is still unclear. Therefore, it is necessary to explore new genes related to soybean plant type and analyze the molecular mechanism to cultivate new soybean varieties with high density tolerance and high yield. Summary of the Invention

[0003] The purpose of the present invention is to overcome the above-mentioned defects and deficiencies existing in the prior art, and provide the application of the CSA1 gene in regulating the branch angle and / or petiole angle of leguminous plants.

[0004] The second purpose of the present invention is to provide the application of the CSA1 gene in creating leguminous plants with high density tolerance and high-yield plant type.

[0005] The third purpose of the present invention is to provide a method for creating high-yield leguminous plants with high density tolerance.

[0006] The above purposes of the present invention are achieved by the following technical solutions:

[0007] The present invention first cloned the CSA1 gene from soybeans. The nucleotide sequence thereof is shown in SEQ ID No.1, and the amino acid sequence encoded thereby is shown in SEQ ID No.2. Further, the CRISPR-Cas9 system was used to edit the CSA1 gene, and two different homozygous mutants were obtained. The results showed that the branching angle and petiole angle of the mutant plants were significantly reduced. Under high-density planting conditions, the yield of the mutants was significantly higher than that of the wild type, indicating that CSA1 can regulate the branching angle and petiole angle of leguminous plants and create dense-tolerant leguminous plants, which not only provides important gene resources for the molecular breeding of leguminous plants, but also provides effective germplasm resources and technical means for creating high-yield leguminous plants with dense planting tolerance.

[0008] Therefore, the present invention first provides the use of the CSA1 gene in regulating the branching angle and / or petiole angle of leguminous plants. The nucleotide sequence of the CSA1 gene is selected from one of the sequences in the following groups:

[0009] (a) The nucleotide sequence shown in SEQ ID No.1;

[0010] (b) The nucleotide sequence encoding the protein with the amino acid sequence shown in SEQ ID No.2;

[0011] (c) The nucleotide sequence encoding the protein with the amino acid sequence shown in SEQ ID No.2, which is substituted, deleted or added with one or several amino acids and has the same function;

[0012] (d) The nucleotide sequence having a homology of ≥90% with the sequence shown in SEQ ID No.1 and encoding a protein having the same function as the protein shown in SEQ ID No.2;

[0013] (e) The nucleotide sequence truncated or added with 1-60 nucleotides at the 5' end and / or 3' end of the polynucleotide shown in SEQ ID No.1 and encoding a protein having the same function as the protein shown in SEQ ID No.2;

[0014] (f) The nucleotide sequence complementary to any one of the nucleotide sequences in (a)-(e).

[0015] The present invention also provides the use of the CSA1 gene in creating high-yield leguminous plants with dense planting tolerance. The nucleotide sequence of the CSA1 gene is selected from one of the sequences in the above groups (a)-(f).

[0016] The present invention also provides a method for creating a leguminous plant with high yield and tolerance to high planting density. By means of genetic engineering, the CSA1 gene or its promoter of the leguminous plant is modified to cause the loss or weakening of the gene function of CSA1 or a decrease in the expression level, thereby reducing the branching angle and / or petiole angle, making the plant type of the leguminous plant convergent and tolerant to high planting density, increasing the number of leguminous plant individuals per unit planting area, and enhancing the yield per unit planting area, so as to create a leguminous plant material with a plant type of high yield and tolerance to high planting density; the nucleotide sequence of the CSA1 gene is selected from one of the sequences in groups (a) to (f) above.

[0017] Further, the method includes: using the CSA1 gene as a target, designing a CRISPR-Cas-based sgRNA sequence, ligating a DNA fragment encoding the sgRNA sequence into a CRISPR / Cas vector, transforming the leguminous plant, and further obtaining a transgenic soybean with the loss or weakening of the gene function of CSA1 or a decrease in the expression level.

[0018] Preferably, the action site of the sgRNA is located in the coding region of the CSA1 gene.

[0019] Preferably, the nucleotide sequences of the action targets of the sgRNA are shown as SEQ ID No.5 and SEQ ID No.6 respectively.

[0020] More preferably, the amplification primers of the sgRNA are shown as SEQ ID No.7-8 and SEQ ID No.9-10 respectively.

[0021] Preferably, the CRISPR / Cas vector is pGES201.

[0022] Preferably, the transformation is carried out by the Agrobacterium-mediated method.

[0023] Preferably, the leguminous plant is soybean.

[0024] The present invention also provides the application of the transgenic leguminous plant obtained by any of the above methods in plant breeding.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention provides the application of the CSA1 gene in regulating the branching angle and petiole angle of leguminous plants and creating high-yield leguminous plants with a plant type suitable for high-density planting. The present invention uses the CRISPR-Cas9-mediated gene editing technology to edit the CSA1 gene and obtains two different homozygous mutants. The results show that the branching angle and petiole angle of the mutant plants are significantly reduced, and under the condition of high-density planting, the yield of the mutants is significantly higher than that of the wild type, indicating that the CSA1 gene can regulate the branching angle and petiole angle of leguminous plants and create high-density-tolerant leguminous plants, which not only provides important gene resources for the molecular breeding of leguminous plants, but also provides an effective means for creating high-yield leguminous plants suitable for high-density planting. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is the mapping and cloning of the CSA1 gene in Example 1 of the present invention. Among them, A and B are Manhattan plots of the branching angle trait; C is the 5 haplotypes of the CSA1 gene; D is the statistical chart of the branching angle phenotypes of the 5 haplotypes of the CSA1 gene.

[0028] Figure 2 It is the phenotypic statistical chart of the gene editing mutant and the gene editing vector and the wild type in Example 2 of the present invention; among them, A is the gene editing target; B is the mutation situation of the mutant; C is the branching angle diagram of the wild type and mutant plants in the field; D and E are the statistical charts of the branching angle and petiole angle of the wild type and mutant, respectively.

[0029] Figure 3 It is the yield statistics of the wild type and mutants under different density tests in Example 3 of the present invention. Among them, A is the harvested plant diagram of the wild type Young and mutants Gmcsa1-1 and Gmcsa1-2; B is the statistical chart of the branching angle of the wild type and mutants under different density conditions; C and D are the yield statistical charts of the wild type and mutants at different densities in the Guangzhou pilot site and Yingde pilot site, respectively. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0030] The present invention provides a gene CSA1 related to the high-yield plant type suitable for high-density planting of soybeans.

[0031] The present invention adopts the following technical solutions:

[0032] GWAS is carried out by combining genotype and branching angle phenotype data, and by combining haplotypes and homologous gene function annotations, a major gene CSA1 that controls the branching angle of soybeans is cloned by means of molecular biology and comparative genomics.

[0033] The CSA1 gene was edited using the CRISPR-Cas9 system, and two different mutants were obtained. It was found that the branching angle and petiole angle of the mutant plants were significantly reduced. Thus, it can be reasonably inferred that knocking out the CSA1 gene, or silencing the coding gene, or reducing the expression of the coding gene can significantly reduce the branching angle and petiole angle of soybeans, thereby making soybeans more suitable for close planting.

[0034] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following embodiments can be used as a guide for those of ordinary skill in the art to make further improvements and do not constitute any limitation to the present invention in any way.

[0035] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0036] The soybean material Young was disclosed in the literature Abdel-Haleem H, Jr T, Rufty T W, et al. Quantitative trait loci controlling aluminum tolerance in soybean: Candidate gene and single nucleotide polymorphism marker discovery[J]. Molecular Breeding, 2013, 33(4): 851-862.) and is a soybean variety with a relatively large branching angle. The public can obtain the soybean material from the College of Agriculture, South China Agricultural University.

[0037] Example 1 Cloning of the Soybean High-Yield Gene CSA1 for Close Planting Tolerance

[0038] The inventors of the present invention isolated and cloned a gene CSA1 related to the high-yield plant type of soybeans suitable for close planting. The specific cloning method is as follows:

[0039] (1) Total RNA of the soybean material Young was extracted using a plant total RNA extraction kit (TR02, GeneMark), and its integrity was detected by 1% agarose gel electrophoresis; cDNA synthesis was carried out with reference to the instructions of the PrimeScript TM RT reagent Kit with gDNA Eraser kit.

[0040] (2) Using the above cDNA as a template, with the upstream primer F: 5’-ATGAAGATCTTCAATTGGGTTC-3’ (SEQ ID No.3); downstream primer R: 5’-TTAAATTGGAAGGAAATAAC-3’ (SEQ ID No.4) as primers, perform PCR amplification. Configure the PCR reaction solution (50 μL system) in the following component order: 2×Phanta Max Buffer (25 μL), ddH2O (19 μL), dNTP Mix (1 μL), primer F (2 μL), primer R (2 μL), cDNA (1 μL), Phanta Max Super-Fidelity DNA Polymerase (1 μL). The amplification program is: pre-denaturation at 95°C for 3 minutes, denaturation at 95°C for 15 seconds, annealing at 56°C for 15 seconds, extension at 72°C for 1 minute, for a total of 35 cycles; then final extension at 72°C for 5 minutes; store at 4°C.

[0041] (3) After the amplification is completed, purify and recover the PCR product with reference to the ordinary DNA product purification kit (DP204, Tiangen). After purification and recovery, ligate the pLB vector (pLB zero-background rapid ligation kit, Tiangen), transform Escherichia coli TOP10, and pick single colonies for shaking culture and sequencing.

[0042] The sequencing results show that the nucleotide sequence of the PCR amplification product is as shown in SEQ ID No.1, and its encoded amino acid sequence is the protein CSA1 as shown in SEQ ID No.2; the DNA shown in SEQ ID No.1 is named the CSA1 gene. The cloning results of the CSA1 gene are as Figure 1 shown, Figure 1 A and 1B show that the CSA1 gene is located at the front end of chromosome 16; Figure 1 C and 1D respectively show the haplotype of CSA1 and the branching angles of different haplotypes. These results comprehensively indicate that there are variations in CSA1 among soybean populations, and the branching angles of different variations are different.

[0043] Example 2 Obtaining of Homozygous Gene-Edited Mutants of the Gene CSA1 Related to the Dense-Planting Tolerant Plant Type of Soybean

[0044] 1. Construction of the gene editing vector

[0045] Design gene targets according to the CSA1 gene of the present invention to obtain target 1 and target 2:

[0046] Target 1 (SEQ ID No.5): 5’-TTTGAGCACAACTTTGAGGTGG-3’

[0047] Target 2 (SEQ ID No.6): 5’-CTTCATAAGAGATCACAGGTGG-3’

[0048] Design primers containing information on two targets.

[0049] The primer sequences designed according to Target 1 are as follows:

[0050] sgRNA1 Forward primer (SEQ ID No.7):

[0051] 5’-GGATTGTTTGAGCACAACTTTGAGG-3’;

[0052] sgRNA1 Reverse primer (SEQ ID No.8):

[0053] 5’-AAACCCTCAAAGTTGTGCTCAAACA-3’.

[0054] The primer sequences designed according to Target 2 are as follows:

[0055] sgRNA2 Forward primer (SEQ ID No.9):

[0056] 5’-GGATTGCTTCATAAGAGATCACAGG-3’;

[0057] sgRNA2 Reverse primer (SEQ ID No.10):

[0058] 5’-AAACCCTGTGATCTCTTATGAAGCA-3’.

[0059] Synthesize the primers designed above, dilute them to 10 μM with water and then anneal them to obtain double-stranded DNA fragments sgRNA with sticky ends, specifically:

[0060] sgRNA1. Anneal primer sgRNA1 Forward primer and sgRNA1 Reverse primer to obtain double-stranded DNA fragment sgRNA1 with sticky ends.

[0061] The reaction system is a 25 μl system: 5 μl each of sgRNA1 Forward primer and sgRNA1 Reverse primer, 15 μl of water.

[0062] Reaction conditions: 96 °C for 5 min, anneal to 12 °C at 0.1 °C / s and hold for 5 min to complete annealing, obtaining sgRNA1 with sticky ends.

[0063] sgRNA2. Anneal the primers sgRNA2 Forward primer and sgRNA2 Reverse primer to obtain the double-stranded DNA fragment sgRNA2 with sticky ends.

[0064] The reaction system is all 25 μl: 5 μl each of sgRNA2 Forward primer and sgRNA2 Reverse primer, and 15 μl of water.

[0065] Reaction conditions: 96 °C for 5 min, anneal at 0.1 °C / s to 12 °C and hold for 5 min to complete annealing, obtaining sgRNA2 with sticky ends.

[0066] Digest 1 μg of the soybean CRISPR / Cas9 vector pGES201 vector (Bai M, Yuan J, Kuang H, et al. Generation of a multiplex mutagenesis population via pooled CRISPR-Cas9 in soya bean. Plant Biotechnol J. 2020;18(3):721 - 731. doi:10.1111 / pbi.13239) with BsaI, and recover the linearized pGES201 vector for standby.

[0067] Ligate the obtained sgRNA1 with sticky ends and sgRNA2 with sticky ends, and the above-mentioned linearized pGES201 vector by molecular cloning to complete the construction of the final vector CRISPR / Cas9-sgRNA1-sgRNA2. CRISPR / Cas9-sgRNA1-sgRNA2 expresses sgRNA1 for target 1 and sgRNA2 for target 2.

[0068] 2. Obtaining of homozygous gene-edited mutants

[0069] Transfer the CRISPR / Cas9-sgRNA1-sgRNA2 vector obtained in step 1 into Escherichia coli competent cell DH5α, coat it on the solid medium of LB + Kan, pick monoclonal colonies to extract plasmids and perform sequencing verification, and obtain the correct CRISPR / Cas9-sgRNA1-sgRNA2 vector plasmid by sequencing alignment.

[0070] The CRISPR / Cas9-sgRNA1-sgRNA2 plasmid with correct sequencing verification was transferred into Agrobacterium tumefaciens EHA105 by electroporation transformation method. The plasmid was extracted and sequenced for verification. The strain with correct sequencing verification was named EHA105-CRISPR / Cas9-sgRNA1-sgRNA2.

[0071] Then, soybean was transformed by the Agrobacterium EHA105-mediated cotyledon node transformation method (Li et al. Optimization of Agrobacterium-Mediated Transformation in Soybean. Front Plant Sci. 2017;8:246. Published 2017 Feb 24. doi:10.3389 / fpls.2017.00246). The medium formulations used were the same as those in the reference, as follows: Seed sterilization: Uniform soybean seeds were selected, sterilized with chlorine gas for 12 hours, placed in a laminar flow hood to blow off the chlorine gas, and then stored in a 4°C refrigerator for later use. The receptor used was wild-type soybean Young. Cotyledon node infection: The EHA105 strain containing the recombinant plasmid was streaked on YEP solid medium + spectinomycin (50 mg / L). After culturing at 28°C for 36 hours, single colonies were picked and cultured in YEP liquid medium + spectinomycin (50 mg / L) overnight with shaking at 220 r / min and 28°C to prepare the bacterial liquid infection solution. The sterilized seeds were soaked in sterilized water for 12 hours and then the cotyledon nodes were scratched with a scalpel in a laminar flow hood. After scratching, the scratched cotyledon nodes were placed in the bacterial liquid infection solution for 1 hour. Co-culture: The infected cotyledon nodes were placed on the co-culture medium CCM for 5 days until the cotyledons turned green. Bud induction: The co-cultured cotyledons were transferred to the bud induction medium SI for 15 days and then transferred to the bud induction medium SI again for 15 days, and then clustered buds could be seen. Bud elongation: The cotyledon explants with clustered buds after two co-cultures were placed in the bud elongation medium SE for culture, with a 15-day culture cycle. Rooting: When the buds grew to about 5 cm, they were transferred to the rooting medium for culture until the roots grew larger and then transferred to the soil for culture until soybean seeds were harvested. Transgenic line identification: The harvested transgenic seeds were sown in the soil. When the first trifoliate leaves grew, glufosinate was applied for positive identification of transgenic lines.

[0072] A total of 2 gene-edited mutants were obtained, and the two gene-edited mutant lines were named Gmcsa1-1 and Gmcsa1-2 respectively. The mutation situations of the mutants are as Figure 2As shown in B, where Gmcsa1-1 has a deletion of 3 bp at the first target site and 2 bp at the second target site; Gmcsa1-2 has a deletion of 4 bp at the first target site and 4 bp at the second target site. The phenotypic analysis of Gmcsa1-1 and Gmcsa1-2 is as Figure 2 shown in C-E. The branch angle and petiole angle of Gmcsa1-1 and Gmcsa1-2 plants are significantly smaller than those of the wild type. Figure 2 C is a photo of the wild type (WT), Gmcsa1-1, and Gmcsa1-2 planted in the field, showing that the branch angle and petiole angle of Gmcsa1-1 and Gmcsa1-2 are smaller than those of the wild type. Figure 2 C and Figure 2 D are the statistical data of the branch angle and petiole angle respectively, showing that the branch angle and petiole angle of Gmcsa1-1 and Gmcsa1-2 are significantly smaller than those of the wild type. Combining these results, it shows that after the loss of function of the CSA1 gene, the branch angle and petiole angle of the plant are significantly reduced.

[0073] Example 3 Functional verification of the gene CSA1 related to the plant type of soybean with high density tolerance

[0074] The two gene-edited mutants Gmcsa1-1 and Gmcsa1-2 prepared in Example 2 and the wild-type soybean Young were planted at different densities, and the branch angle and plot yield were measured after maturity.

[0075] The experiments used in this example were repeated 3 times, which is statistically significant.

[0076] The results are as Figure 3 shown. The branch angle of the gene-edited mutants Gmcsa1-1 and Gmcsa1-2 is significantly smaller than that of the wild type Young ( Figure 3 A, 3B). At low densities of 150,000 plants per hectare and 212,500 plants per hectare, the yield of the mutants is lower than that of the wild type, but at high densities of 275,000 plants per hectare and 412,500 plants per hectare, the yield of the mutants is significantly higher than that of the wild type ( Figure 3 C, 3D), indicating that after the loss of function of CSA1, it has a significant effect of increasing soybean yield under high-density planting conditions.

[0077] The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application but are made using conventional techniques known in the art. The application of some basic features can be made within the scope of the appended claims below.

Claims

1. Application of CSA1 gene in regulating branch angle and / or petiole angle of leguminous plants, characterized in that: The nucleotide sequence of the CSA1 gene is selected from one of the following groups of sequences: (a) the nucleotide sequence shown in SEQ ID No. 1; (b) a nucleotide sequence encoding a protein having an amino acid sequence as shown in SEQ ID No. 2; (c) a nucleotide sequence encoding a protein having the same function as the one shown in SEQ ID No. 2, wherein one or more amino acids are replaced, deleted or added; (d) a nucleotide sequence having a homology of ≥90% with the sequence shown in SEQ ID No.1 and encoding a protein having the same function as the protein shown in SEQ ID No.2; (e) a nucleotide sequence in which 1 to 60 nucleotides are truncated or added to the 5' end and / or 3' end of the polynucleotide shown in SEQ ID No. 1 and the protein encoded by the nucleotide sequence has the same function as the protein shown in SEQ ID No. 2; (f) A nucleotide sequence complementary to the nucleotide sequence described in any one of (a) to (e).

2. Application of CSA1 gene in creating leguminous plants with high yield and high density tolerance, characterized in that: The nucleotide sequence of the CSA1 gene is selected from one of the following groups of sequences: (a) the nucleotide sequence shown in SEQ ID No. 1; (b) a nucleotide sequence encoding a protein having an amino acid sequence as shown in SEQ ID No. 2; (c) a nucleotide sequence encoding a protein having the same function as the one shown in SEQ ID No. 2, wherein one or more amino acids are replaced, deleted or added; (d) a nucleotide sequence having a homology of ≥90% with the sequence shown in SEQ ID No.1 and encoding a protein having the same function as the protein shown in SEQ ID No.2; (e) a nucleotide sequence in which 1 to 60 nucleotides are truncated or added to the 5' end and / or 3' end of the polynucleotide shown in SEQ ID No. 1 and the protein encoded by the nucleotide sequence has the same function as the protein shown in SEQ ID No. 2; (f) A nucleotide sequence complementary to the nucleotide sequence described in any one of (a) to (e).

3. A method for producing high-yield leguminous plants that are resistant to dense planting, characterized in that: By genetic engineering means, the CSA1 gene or its promoter of the legume plant is modified so that the CSA1 gene function is lost or weakened, or the expression level is reduced, thereby reducing the branch angle and / or petiole angle, and obtaining a legume plant with a convergent plant shape and tolerance to dense planting; the nucleotide sequence of the CSA1 gene is selected from one of the following groups of sequences: (a) the nucleotide sequence shown in SEQ ID No. 1; (b) a nucleotide sequence encoding a protein having an amino acid sequence as shown in SEQ ID No. 2; (c) a nucleotide sequence encoding a protein having the same function as the one shown in SEQ ID No. 2, wherein one or more amino acids are replaced, deleted or added; (d) a nucleotide sequence having a homology of ≥90% with the sequence shown in SEQ ID No.1 and encoding a protein having the same function as the protein shown in SEQ ID No.2; (e) a nucleotide sequence in which 1 to 60 nucleotides are truncated or added to the 5' end and / or 3' end of the polynucleotide shown in SEQ ID No. 1 and the protein encoded by the nucleotide sequence has the same function as the protein shown in SEQ ID No. 2; (f) A nucleotide sequence complementary to the nucleotide sequence described in any one of (a) to (e).

4. The method according to claim 3, characterized in that: The method comprises: taking the CSA1 gene as a target, designing an sgRNA sequence based on the CRISPR-Cas system, connecting a DNA fragment containing the encoding sgRNA sequence to a vector carrying CRISPR / Cas, transforming legumes, and thereby obtaining legumes with a missing or weakened CSA1 gene function or a reduced expression level.

5. The method according to claim 4, characterized in that: The sgRNA action site is located in the coding region of the CSA1 gene.

6. The method according to claim 5, characterized in that: The nucleotide sequences of the sgRNA target sites are shown in SEQ ID No. 5 and SEQ ID No.

6.

7. The method according to claim 4, characterized in that: The CRISPR / Cas vector is pGES201.

8. The method according to claim 4, characterized in that: The transformation was carried out by Agrobacterium-mediated method.

9. The use according to claim 1 or 2 or the method according to any one of claims 3 to 8, characterized in that: The leguminous plant is soybean.

10. Use of the transgenic leguminous plant obtained by the method according to any one of claims 3 to 8 in plant breeding.

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