Gmtps1 protein and its encoding gene in improving soybean plant type traits and yield

Abstract

This invention discloses the application of the GmTPS1 protein and its encoding gene in improving soybean plant architecture and yield. This invention belongs to the field of biotechnology and relates to the application of the GmTPS1 protein and its encoding gene in improving soybean plant architecture and yield. This invention provides the application of the GmTPS1 protein, or substances regulating its gene expression, or substances regulating its activity or content, in regulating plant architecture. The GmTPS1 protein has the amino acid sequence SEQ ID No. 3. This invention utilizes CRISPR / Cas9-mediated gene editing technology to perform site-directed mutations or knockouts at specific targets in the soybean plant architecture encoding gene GmTPS1, providing new materials for the breeding of soybean varieties with improved plant architecture and playing a positive role in accelerating the breeding of soybean varieties with improved plant architecture.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the application of GmTPS1 protein and its encoding gene in improving soybean plant type traits and yield. Background Technology

[0002] Soybeans, as an important crop used for both grain and oilseed production, play a crucial role in ensuring my country's food security and agricultural trade. With the continuous improvement of people's living standards, the demand for vegetable oil and feed protein has increased dramatically, making my country's insufficient soybean production capacity increasingly prominent. Compared with major soybean-producing countries such as the United States, Brazil, and Argentina, my country's soybean production is low and cannot meet daily production and living needs, leading to a year-on-year increase in soybean imports. Given the limited soybean planting area in my country, how to rapidly and effectively improve soybean varieties and increase soybean yield through modern bio-breeding technologies is a critical production problem that urgently needs to be solved and a breeding technology bottleneck that urgently needs to be overcome.

[0003] Crop plant architecture plays a decisive role in the morphogenesis of individual plants and crop populations, and is a crucial factor influencing plant yield, crop production level, and economic benefits. Crop plant architecture includes plant height, branching (tillering), leaf shape, and spike type (pod-setting habit). Crop plant architecture domestication or improvement plays a vital role in achieving significant breakthroughs in crop yield. However, current research on the regulatory mechanisms of soybean plant architecture and key genes involved in this regulation is still in the exploratory stage, with few related research reports. The molecular mechanisms, key genes, and functional networks affecting soybean plant architecture regulation remain unclear. In particular, due to limitations in research materials, there is a lack of research on which plant architecture is more conducive to increasing soybean yield under field production conditions. Therefore, further exploration of more plant architecture regulatory genes is not only of significant theoretical value for discovering superior soybean plant architecture regulatory genes and cultivating high-yielding ideal plant architectures; but also, through the creation of specific materials, allows for the systematic evaluation and selection of ideal soybean plant architectures under production conditions, which has significant practical application value for ultimately realizing breeding applications and improving soybean yield. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to improve the soybean plant type to obtain a target plant that is conducive to increasing soybean yield.

[0005] To address the aforementioned technical problems, this invention first provides the application of GmTPS1 protein or a substance regulating the expression of a gene, or a substance regulating the activity or content of said GmTPS1 protein, in regulating plant architecture. The gene encodes the GmTPS1 protein, and the GmTPS1 protein is any one of the following proteins:

[0006] (a1) Proteins with amino acid sequences as shown in SEQ ID No. 3;

[0007] (a2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No. 3, which has more than 80% identity with the protein shown in a1) and has the function of regulating plant architecture.

[0008] (a3) A fusion protein obtained by attaching a tag to the end of the protein in (a1) or (a2).

[0009] In the above applications, the GmTPS1 protein is derived from soybeans.

[0010] In the above applications, the substance regulating gene expression or the substance regulating the activity or content of the GmTPS1 protein is a biological material related to the protein, and the biological material is any one of the following:

[0011] B1) The nucleic acid molecule encoding the GmTPS1 protein;

[0012] B2) Nucleic acid molecules that inhibit, reduce, or silence the expression of the gene encoding the GmTPS1 protein;

[0013] B3) An expression cassette containing the nucleic acid molecules described in B1) and / or B2);

[0014] B4) A recombinant vector containing the nucleic acid molecules described in B1) and / or B2), or a recombinant vector containing the expression cassette described in B3);

[0015] B5) Recombinant microorganisms containing the nucleic acid molecules described in B1) and / or B2), or recombinant microorganisms containing the expression cassette described in B3), or recombinant microorganisms containing the recombinant vector described in B4);

[0016] B6) A transgenic plant cell line containing the nucleic acid molecules described in B1) and / or B2), or a transgenic plant cell line containing the expression cassette described in B3);

[0017] B7) Transgenic plant tissue containing the nucleic acid molecules described in B1) and / or B2), or transgenic plant tissue containing the expression cassette described in B3);

[0018] B8) A transgenic plant organ containing the nucleic acid molecules described in B1) and / or B2), or a transgenic plant organ containing the expression cassette described in B3).

[0019] In the above applications, the nucleic acid molecule described in B1) is any of the following:

[0020] B11) The coding sequence of the coding strand is the cDNA molecule or DNA molecule of SEQ ID No. 2.

[0021] B12) The nucleotides encoding the chain are the cDNA molecule or DNA molecule of SEQ ID No. 1;

[0022] In the above applications, the nucleic acid molecule described in B2) is any of the following:

[0023] B21) A gRNA targeting the gene encoding the GmTPS1 protein, wherein the target sequence of the gRNA is a DNA molecule whose nucleotide sequence is from position 282 to 301 of SEQ ID No. 1;

[0024] B22) DNA molecules that express the gRNA described in B21).

[0025] In this invention, the regulation can be increased, enhanced, or raised. The regulation can also be decreased, weakened, or reduced.

[0026] The present invention also provides a method for altering soybean plant architecture.

[0027] The present invention provides a method for altering soybean plant architecture, comprising downregulating or weakening or reducing the activity and / or content of the GmTPS1 protein in the target soybean, and / or the expression level of the gene encoding the GmTPS1 protein, to alter soybean plant architecture.

[0028] In the above method, the downregulation or reduction of the activity and / or content of the GmTPS1 protein in the target soybean, and / or the expression level of the gene encoding the GmTPS1 protein, includes knocking out the gene encoding the GmTPS1 protein in the genome of the target soybean using a CRISPR / Cas9 system.

[0029] In the above method, the CRISPR / Cas9 system includes a vector expressing sgRNA that targets the gene encoding the GmTPS1 protein.

[0030] In the above method, the target sequence of the sgRNA is the DNA molecule whose nucleotide sequence is from position 282 to 301 of SEQ ID No. 1.

[0031] In the above method, the soybean plant architecture is altered by mutating the gene encoding the GmTPS1 protein, and the mutation can be any of the following:

[0032] 1) Delete nucleotides 296-299 of SEQ ID No. 1, while keeping the other nucleotides of SEQ ID No. 1 unchanged;

[0033] 2) Add a nucleotide T between nucleotides 298 and 299 of SEQ ID No.1, while keeping the other nucleotide residues of SEQ ID No.1 unchanged.

[0034] The application of the aforementioned GmTPS1 protein or regulatory gene expression substances, or substances that regulate the activity or content of the GmTPS1 protein, in soybean breeding also falls within the scope of protection of this invention.

[0035] The purpose of soybean breeding may include altering soybean plant architecture.

[0036] The changes may include increasing soybean plant height and / or decreasing the number of soybean plant branches and / or increasing the number of soybean plant nodes, or increasing the number of pods per plant, or increasing the number of grains per plant.

[0037] This invention demonstrates that modifying the soybean GmTPS1 gene using CRISP / Cas9 gene editing technology can increase soybean plant height and number of nodes, reduce the number of branches, and increase the number of pods and seeds per soybean plant. Attached Figure Description

[0038] Figure 1 This refers to the GmTPS1 gene editing target and homozygous mutation type.

[0039] Figure 2 These are wild-type and homozygous mutant potted plant types. Detailed Implementation

[0040] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. All quantitative experiments in the following examples were performed in triplicate.

[0042] The cultivated soybean Jack in the following examples is described in the following literature: Chen L, Cai Y, Liu X, Yao W, Guo C, Sun S, Wu C, Jiang B, Han T, Hou W (2018), Improvement of soybean Agrobacterium-mediated transformation efficiency by adding glutamine and asparagine into the culture media. International Journal of Molecular Sciences 19, 3039. The biological material is available to the public from the applicant and is used only for repeating the experiments of the present invention and shall not be used for other purposes.

[0043] The Agrobacterium tumefaciens EHA105 used in the following examples is described in the following literature: Cai Y, Chen L, Liu X, Guo C, Sun S, Wu C, Jiang B, Han T and Hou W (2018a), CRISPR / Cas9-mediated targeted mutationnesis of GmFT2a delays flowering time in soya bean. Plant Biotechnol J16, 176-185. This biological material is available to the public from the applicant and is intended solely for the purpose of replicating the experiments of this invention and shall not be used for any other purpose.

[0044] MS salts (basal salts for MS medium): PhytoTech, catalog number: M524.

[0045] MS Organic: PhytoTech, Catalog No.: M533.

[0046] B5 Organic: Phytotech, catalog number: G219.

[0047] B5 Salt: Phytotech, Catalog No.: G768.

[0048] YEP solid medium consists of a solvent and a solute; the solutes and their concentrations in YEP solid medium are as follows: NaCl 5 g / L, yeast extract 5 g / L, tryptone 10 g / L, and agar 15 g / L; the solvent is water. The pH of YEP solid medium is 7.0.

[0049] Germination medium (pH 5.8): 3.12 g / L B5 salt, 1 mL / L B5 organic, 20 g / L sucrose, 7.5 g / L agar, with the remainder being water.

[0050] Liquid culture medium (pH 5.4): 0.43 g / L MS salt, 1 mL / L B5 organic, 40 mg / L acetylsuccinone, 150 mg / L dithiothreitol, 100 mg / L L-cysteine, 30 g / L sucrose, 3.9 mg / L 2-morpholinoethanesulfonic acid, balance water.

[0051] Co-culture medium (pH 5.4): 0.43 g / L MS salt, 1 mL / L B5 organic, 40 mg / L acetylsuccinone, 150 mg / L dithiothreitol, 100 mg / L L-cysteine, 30 g / L sucrose, 7.5 g / L agar, 3.9 mg / L 2-morpholinoethanesulfonic acid, balance water.

[0052] Recovery medium (pH 5.4): 3.1 g / L B5 salt, 1 mL / L B5 organic, 30 g / L sucrose, 150 mg / L cephalosporin, 150 mg / L termethin, 1 mg / L 6-BA, 0.98 g / L 2-morpholinoethanesulfonic acid, 7.5 g / L agar, 4 mL / L Fe salt (200×), 50 mg / L L-asparagine, 50 mg / L L-glutamine, balance water.

[0053] Screening medium (pH 5.4): 3.1 g / L B5 salt, 1 mL / L B5 organic, 0.98 g / L 2-morpholinoethanesulfonic acid, 30 g / L sucrose, 150 mg / L cephalosporin, 150 mg / L termethin, 1 mg / L 6-BA, 6 mg / L glufosinate, 7.5 g / L agar, 4 mL / L Fe salt (200×), 50 mg / L L-asparagine, 50 mg / L L-glutamine, balance water.

[0054] Elongation medium (pH 5.6): 4.0 g / L MS salt, 1 mL / L B5 organic, 0.6 g / L 2-morpholinoethanesulfonic acid, 30 g / L sucrose, 150 mg / L cephalosporin, 150 mg / L termethin, 0.1 mg / L IAA, 0.5 mg / L GA, 1 mg / L 6-BA, 6 mg / L glufosinate, 7.5 g / L agar, 4 mL / L Fe salt (200×), 50 mg / L L-asparagine, 50 mg / L L-glutamine, balance water.

[0055] Rooting medium (pH 5.7): 2.165 g / L MS salt, 1 mL / L B5 organic, 0.6 g / L 2-morpholinoethanesulfonic acid, 20 g / L sucrose, 7.5 g / L agar, 50 mg / L L-asparagine, 50 mg / L L-glutamine, with the remainder being water.

[0056] The data in the following examples were processed using SPSS 11.5 statistical software. Experimental results are expressed as mean ± standard deviation. One-way ANOVA was used, with P < 0.05 (*) indicating a significant difference compared to the control, and P < 0.01 (**) indicating a highly significant difference compared to the control.

[0057] Example 1: Construction of GmTPS1 gene editing CRISPR vector

[0058] I. Obtaining sgRNA

[0059] (1) The soybean GmTPS1 (SEQ ID No. 1) genome sequence was obtained from the Phytozome database. GmTPS1 is located on chromosome 2. The GmTPS1 sgRNA target sequence was selected using the online CRISPR-P tool (http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR). The target site is located in the first exon region of GmTPS1, and the target sequence is 5'-CTGGAGAGGCTCCGTCATTT-3' (positions 282 to 301 of SEQ ID No. 1, corresponding to positions 41-60 of the coding sequence of SEQ ID No. 2).

[0060] (2) The vector pUC57-sgRNA was double-digested with restriction endonucleases NHeI and BbsI in a 50uL system at 37℃ for 3 hours. The target band (approximately 3201bp in size) was detected by 1% agarose gel electrophoresis.

[0061] (3) Synthesize primers according to the target sequence of sgRNA. The sequences are as follows (lowercase letters are the target sites): GmTPS1-Cas9-F:5'-TCGAAGTAGTGATTGctggagaggctccgtcatttGTTTTAGAGCTAGAA-3'; GmTPS1-Cas9-R:5'-TTCTAGCTCTAAAACaaatgacggagcctctccagAATCACTACTTCGA-3'.

[0062] (4) Add 5 μL of the corresponding F / R primers (10 μM each) to a centrifuge tube, and add 15 μL of ddH2O to bring the reaction volume to 25 μL. Anneal at 95℃ for 3 min, then at 0.1℃ / s to 16℃, and hold at 16℃ for 10 min to complete the annealing process and form oligo dimers. The oligo dimer formed by GmTPS1-Cas9-F and GmTPS1-Cas9-R encodes an sgRNA targeting GmTPS1 (named sgRNA-GmTPS1).

[0063] (5) Take 3.5 μL of the annealing product and 1.5 μL of the enzyme-digested pUC57-sgRNA vector (nucleotide sequence SEQ ID No. 4) and gel recover the product using... The Ultra One Step Cloning Kit (Nanjing Novizan Biotechnology Co., Ltd., catalog number C115-02) was used for ligation. *E. coli* DH5α was transformed using the freeze-thaw method, plated on LB+Amp solid medium, and incubated overnight at 37°C. Single colonies were picked, shaken, and sequenced. The sequencing primers were pSgRNA-CX: 5'-CGCCAGGGTTTTCCCAGTCACGAC-3'. Bacterial cultures with correct sequencing results were used for plasmid extraction. The extracted plasmid was named pUC57-sgRNA-GmTPS1.

[0064] (6) Double digestion with restriction endonucleases PacI and PmeI was performed in a 50 μL system at 37°C for 3 hours. The pUC57-sgRNA-GmTPS1 plasmid was digested with PacI and PmeI, and the empty vector PTF101-SpCas9 plasmid was digested with PmeI and PmeI, and the empty vector PTF101-SpCas9 plasmid was digested with PmeI and PmeI, and the empty vector PTF101-SpCas9 plasmid was digested with PmeI and PmeI, and the empty vector PTF101-SpCas9 plasmid was digested with PmeI and PmeI, and the empty vector PTF101-SpCas9 plasmid was digested with PmeI and PmeI, and the empty vector PTF101-SpCas9 plasmid was digested with PmeI and PmeI, and the empty vector PTF101-SpCas9 plasmid was digested with PmeI and PmeI, and the digested fragments were then transformed into *E. coli* DH5α using the freeze-thaw method. The ligation was plated on LB+Spe solid medium and incubated overnight at 37°C. Single clones were picked, cultured, and sequenced to verify successful ligation. The sequencing primers were sgRNA-TYJC: 5'-TGGGAATCTGAAAGAAGAGAAGCA-3'. The plasmid that was verified to be correct was named pGmTPS1-sgRNA.

[0065] The structure of the recombinant plasmid pGmTPS1-sgRNA is described as follows: It is a recombinant expression vector in which the expression cassette element (nucleotide sequence SEQ ID No. 5) containing the target sequence of GmTPS1 replaces the nucleotide sequence between the PacI and Pme restriction sites on the PTF101-SpCas9 plasmid (nucleotide sequence SEQ ID No. 6), while keeping the other nucleotide sequences of the PTF101-SpCas9 plasmid unchanged.

[0066] Example 2: Obtaining and Phenotypic Identification of the gmtps1 Mutant

[0067] I. Preparation of Recombinant Bacteria

[0068] The recombinant vector pGmTPS1-sgRNA prepared in Example 1 was transformed into Agrobacterium EHA105 by electroporation. The plasmid was extracted and sequenced for verification. The recombinant strain that was correctly sequenced was named EHA-GmTPS1-sgRNA.

[0069] II. Agrobacterium-mediated transformation

[0070] The constructed EHA-GmTPS1-sgRNA was transformed into the soybean variety Jack (hereinafter referred to as wild-type soybean) using Agrobacterium-mediated transformation. The specific method is as follows:

[0071] 1. Seed sterilization

[0072] 1) Take healthy, plump, uniform, and dry Jack soybean seeds that are free from pests, diseases, and spots, spread them evenly in a petri dish, and then place the petri dish in a desiccator.

[0073] 2) After completing step 1), place a 100mL beaker in the desiccator, pour 80mL of 12M sodium hypochlorite aqueous solution into the beaker, then slowly add 4mL of concentrated hydrochloric acid, and then quickly cover the desiccator, seal it with petroleum jelly, and place it for 16 hours for chlorine sterilization.

[0074] 2. Preparation of infecting bacterial solution

[0075] 1) Incubate the EHA-GmTPS1-sgRNA bacterial culture obtained above at 28℃, resuspend it in liquid culture medium, and obtain OD. 600nm =0.6% of the infecting bacterial solution.

[0076] 2) Place the seeds treated in 1) into a clean bench, peel off the seed coat under a microscope, separate the two cotyledons along the long axis, and keep the cotyledon with the complete hypocotyl. Make scratches at the junction of the hypocotyl and cotyledon, usually 3-5 scratches per cotyledon. Then soak in a 28℃ incubator for 2 hours.

[0077] 3) Place the cotyledons with the inner (smooth) side up on a co-culture medium lined with sterile filter paper, and incubate in the dark at 22°C for 5 days.

[0078] 4) After 5 days of co-culture, the hypocotyl of the explants elongated to 2 cm. Part of the hypocotyl was cut off, leaving 0.5 cm. The treated explants were then placed in recovery medium and cultured at 28°C under 16 h light / 8 h dark conditions for 7 days.

[0079] 5) Remove the explants from the recovery medium, remove the new shoots, cut off part of the hypocotyl, leaving 0.5 cm of the hypocotyl, and then transfer the trimmed explants into the selection medium and culture them at 28℃ for 21 days under 16h light / 8h dark conditions.

[0080] 6) After 21 days of selection and induction, the explants produced a large number of adventitious buds. The cotyledons and brown leaves were removed, and the remaining parts were transferred to elongation medium for culture at 28°C under 16h light / 8h dark conditions.

[0081] 7) In the elongation medium, when the clustered buds produce 5-8cm young stems, cut them off from the base of the adventitious buds; dip the stem base in 1mg / L IBA solution for 1min, and then transfer it to the rooting medium for culture. Culture at 28℃ under 16h light / 8h dark conditions for one week. After a large number of roots are produced at the base of the stem, transplant them into pots. The resulting plants are T0 generation transformed soybeans.

[0082] III. Molecular Detection of Edited Plants

[0083] DNA was extracted from leaves of T0 generation transformed soybeans as a template for PCR molecular detection, with wild-type soybeans as a control.

[0084] PCR primers were designed near the target site of the GmTPS1 gene. Soybean genomic DNA was used as a template for PCR amplification and sequencing. Primers FMA-F (5'-TTGGTTTACCCTTCGTGGTGT-3') and FMA-R (5'-TGGTCTCCTCTGAGCCCTTAT-3') amplified the GmTPS1 gene. The PCR reaction mixture consisted of 12.5 μL of 2×PhantaMax Buffer, 0.5 μL of dNTP Mix (10 mM), 1 μL of DNA (200 ng / μL), 1 μL of FMA-F (10 pmol / μL), 1 μL of FMA-R (10 pmol / μL), 0.5 μL of Super-Fidelity DNA Polymerase, and 8.5 μL of ddH2O, for a total volume of 25 μL. The amplification reaction was performed at 95℃ for 3 min; followed by 35 cycles of 95℃ for 30 sec, 58℃ for 30 sec, and 72℃ for 1 min; and then 72℃ for 5 min. The PCR products were sent to the company for sequencing verification.

[0085] The plants exhibiting overlapping peaks near the target location were identified as heterozygous edited plants and named T0 generation GmTPS1 soybeans.

[0086] After sowing T0 generation GmTPS1 soybeans, the seeds of T1 generation GmTPS1 soybeans were harvested and cultivated to obtain T1 generation GmTPS1 soybeans.

[0087] Using the above-mentioned PCR molecular detection method, T1 generation GmTPS1 transgenic soybeans were tested. Sequencing results showed that in T1 generation GmTPS1 transgenic soybeans, the mutant plants named gmtps1 homozygous mutant 1 and soybean gmtps1 homozygous mutant 2 (gmtps1) had the following mutations near the target site, causing premature termination of protein translation. The GmTPS1 gene mutation types in the mutant plants included two types: -4bp and +1bp. Figure 1 ).

[0088] Compared to wild-type soybean, the T1 generation soybean gmtps1 homozygous mutant plant showed the following mutation in both homologous chromosomes of the GmTPS1 protein-coding gene (GmTPS1 gene): deletion of nucleotides 55-58 of SEQ ID No. 2, while keeping the other nucleotides of SEQ ID No. 2 unchanged. This GmTPS1 gene mutation type is -4bp. This results in the production of the TGA stop codon, prematurely terminating translation.

[0089] The +1bp mutation in the GmTPS1 gene of T1 generation soybean homozygous mutant 2 involves adding a T base between positions 57 and 58 of SEQ ID No. 2 in the sequence listing, while keeping other nucleotides unchanged. This +1bp nucleotide insertion results in the formation of the TGA stop codon, prematurely terminating translation.

[0090] The T1 generation soybean gmtps1 homozygous mutant 1 and the T2 generation soybean gmtps1 homozygous mutant 2 were further cultivated. By using PAT test strips, T2 generation transgenic gmtps1 homozygous mutant 1 and homozygous mutant 2 without transgenic elements were screened and propagated to the T4 generation, and phenotypic identification was performed.

[0091] IV. Mutant strain type identification

[0092] Plant type traits (plant height, number of nodes, number of branches, number of pods per plant, and number of grains per plant) of wild-type plants (control), T4 generation soybean gmtps1 homozygous mutant 1 (mutant 1) and T4 generation soybean gmtps1 homozygous mutant 2 (mutant 2) were statistically analyzed.

[0093] In Beijing, summer field planting is carried out with a plant spacing of 10cm and a row spacing of 0.5m. Each mutant strain has 10-15 plants.

[0094] The results showed that, in terms of plant type, compared with the control plant height of 147.8 cm, mutant 1 plants had an average height of 150.2 cm, and mutant 2 plants had an average height of 153.4 cm (the height of mutant plants was increased compared with the control, but not significantly); in terms of branching phenotype, the control plant had 2.6 branches, mutant 1 plants had an average of 1.3 branches, and mutant 2 plants had an average of 1.8 branches, with the number of branches in mutant plants being significantly reduced; in terms of the number of nodes, the control... The average number of nodes per plant was 25.9 for mutant 1, 28.2 for mutant 1, and 28.1 for mutant 2, showing a significant increase in the number of nodes per plant. Regarding yield per plant, the control plant averaged 89.4 pods and 210.7 seeds per plant, mutant 1 averaged 93.0 pods and 235.2 seeds per plant, and mutant 2 averaged 102.0 pods and 254.5 seeds per plant, showing a significant increase in the number of seeds per plant in the mutants.

[0095] Table 1. Statistics on soybean plant type data

[0096]

[0097] The above results indicate that modifying the soybean GmTPS1 gene using CRISP / Cas9 gene editing technology can significantly increase the number of nodes in soybean plants and improve the number of grains per plant.

[0098] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Application of a substance that knocks out the GmTPS1 protein encoding gene in regulating plant architecture, wherein the gene encodes the GmTPS1 protein, and the GmTPS1 protein is a protein with an amino acid sequence as shown in SEQ ID No. 3; The method of regulating plant plant type is to increase the number of plant nodes, increase the number of grains per plant, and reduce the number of branches; The plant in question is soybean.

2. Application of substances that reduce GmTPS1 protein content in regulating plant architecture, wherein the GmTPS1 protein is a protein with an amino acid sequence as shown in SEQ ID No. 3; The method of regulating plant plant type is to increase the number of plant nodes, increase the number of grains per plant, and reduce the number of branches; The plant in question is soybean.

3. The application according to claim 1 or 2, characterized in that: The GmTPS1 protein is derived from soybean.

4. The application according to claim 1 or 2, characterized in that: The substance is a biomaterial related to the protein, and the biomaterial is any one of the following: B1) A nucleic acid molecule that inhibits, reduces, or silences the expression of the gene encoding the GmTPS1 protein as described in claim 1 or 2; 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) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms 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); B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2); 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).

5. The application according to claim 4, characterized in that: The gene encoding the GmTPS1 protein is any of the following nucleic acid molecules: B11) The coding sequence is the cDNA molecule of SEQ ID No.

2. B12) The nucleotide is the DNA molecule of SEQ ID No. 1; B1) The nucleic acid molecule is any one of the following: B21) Targeting the gRNA of the gene encoding the GmTPS1 protein as described in claim 1 or 2; B22) DNA molecules that express the gRNA described in B21).

6. A method for altering soybean plant architecture, characterized in that: This includes lowering or reducing the content of the GmTPS1 protein as described in claim 1 or 2 in the target soybean, and / or the expression level of the gene encoding the GmTPS1 protein as described in claim 1 or 2, to change the soybean plant type; the change in soybean plant type is to increase the number of nodes per soybean plant, increase the number of grains per plant, and reduce the number of branches.

7. The method according to claim 6, characterized in that: The reduction or decrease of the content of the GmTPS1 protein in the target soybean as described in claim 1 or 2, and / or the expression level of the gene encoding the GmTPS1 protein as described in claim 1 or 2, includes knocking out the gene encoding the GmTPS1 protein in the genome of the target soybean using a CRISPR / Cas9 system.

8. The method according to claim 7, characterized in that: The CRISPR / Cas9 system includes a vector expressing sgRNA that targets the gene encoding the GmTPS1 protein.

9. The method according to claim 7 or 8, characterized in that: The method alters soybean plant architecture by mutating the gene encoding the GmTPS1 protein, wherein the mutation is any one of the following: 1) Delete nucleotides 296-299 of SEQ ID No. 1, while keeping the other nucleotides of SEQ ID No. 1 unchanged; 2) Add a nucleotide T between nucleotides 298 and 299 of SEQ ID No.1, while keeping the other nucleotide residues of SEQ ID No.1 unchanged.

10. The use of any one of the substances described in claims 1-4 in soybean breeding; The purpose of the breeding is to select soybean varieties with increased plant node number, higher number of grains per plant, and reduced number of branches.

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