Application of soybean GmSMS6 protein and coding gene thereof in regulation and control of soybean grain weight and grain shape

Abstract

The invention provides an application of soybean GmSMS6 protein and a coding gene thereof in regulation and control of soybean grain weight and grain shape. According to the invention, the function of soybean GmSMS6 in regulation and control of soybean grain weight is proved through a CRSIPR-Cas9 system for the first time. GmSMS6 is a negative regulation gene of the soybean grain weight, and the soybean grain weight can be remarkably increased by knocking out the gene; the hundred-grain weight of an overexpressed plant is remarkably reduced compared with that of a wild plant. The phenotype of a transgenic plant shows that the GmSMS6 can negatively regulate and control the soybean grain weight and can be used for high-yield breeding of soybeans. The invention provides important gene resources and theoretical guidance for soybean high-yield molecular breeding and germplasm innovation.

Description

Technical Field

[0001] This invention belongs to the field of mutation or genetic engineering, specifically relating to the application of soybean GmSMS6 protein and its encoding gene in regulating soybean grain weight and grain shape. Background Technology

[0002] Soybeans Soybean Soybean is an important dual-purpose crop, providing humans and animals with abundant oils, proteins, and other nutrients. Therefore, there is an urgent need to cultivate high-yielding soybean varieties to increase soybean production and meet the demands of soybean consumption and industrial development.

[0003] Seed weight is a key trait determining soybean yield, but the regulatory mechanisms by which soybean plants control final seed weight remain poorly understood. Seed size and weight are precisely regulated by internal genetic information and environmental signals. The ubiquitin-proteasome pathway, G protein signaling, mitogen-activated protein kinase (MAPK) signal transduction, and transcriptional regulators control seed development through a variety of complex physiological processes. In recent years, several factors regulating soybean seed size / weight have been identified. Comparative transcriptomic analysis of cultivated and wild soybean seeds identified GmGA20OX as a positive regulator of soybean seed weight. PP2C-1 interacts with GmBZR1 and mediates its dephosphorylation, thereby activating brassinolide signaling and ultimately regulating seed size / weight. (SPINDLY homolog gene) GmSSS1 Seed enlargement is promoted by positively regulating cell expansion and proliferation. ST1 regulates pectin biosynthesis by converting UGluA to UGaLA, ultimately promoting an increase in 100-seed weight. POWR1 regulates seed size by modulating nutrient transport. GmSWEET10a and GmSWEET10b promote seed size increase by increasing seed sugar content, and the expression of GmSWEET10a is influenced by... GmST05 In general, research on the soybean grain weight regulatory network is still in the early stages of gene mining. There is a need to discover new genes that regulate soybean grain weight and elucidate their molecular mechanisms, providing genetic resources and a theoretical basis for soybean molecular breeding.

[0004] 14-3-3 proteins are a family of proteins widely distributed and conserved in eukaryotes. They play crucial roles in plant growth and development, participating in stress responses, hormone regulation, signal transduction, primary metabolic regulation, and gene expression regulation. Specifically, 14-3-3 proteins regulate plant growth and development, including flowering, root growth, phototropism, and seed development. They also participate in plant responses to stresses such as drought, cold, salt, and biotic stress. Exploring the effects and regulatory mechanisms of soybean 14-3-3 proteins on seed development is of great significance and valuable for improving soybean yield and quality. Summary of the Invention

[0005] The technical problem this application aims to solve is how to regulate the traits of plant seeds.

[0006] Therefore, this invention provides applications of biomaterials, which may be the application of the biomaterials in regulating plant grain traits and / or preparing products that regulate plant grain traits, or the application of the biomaterials in plant breeding and / or preparing plant breeding products; the indicators of the plant breeding include the grain traits. The purpose of the plant breeding includes regulating the grain traits of plants.

[0007] The biological material is a protein, a substance that regulates the expression of the gene encoding the protein, or a substance that regulates the content of the protein. The protein is GmSMS6, and is any one of the following: A1) The amino acid sequence is that of the protein shown in sequence 2. A2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein described in A1), which has more than 80% identity with the protein shown in A1) and can regulate plant grain traits. A3) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1) or A2).

[0008] The proteins mentioned above can be derived from soybeans.

[0009] In the aforementioned proteins, the protein tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0010] In the above-mentioned proteins, identity refers to the identity of the amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences to calculate the identity value (%), then the identity value can be obtained.

[0011] In the aforementioned proteins, the 80% or more identity can be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 95%, 96%, 98%, 99%, or 100% identity.

[0012] The amino acid sequence of the above protein is sequence 2 in the sequence listing, consisting of 262 amino acid residues, and it is named GmSMS6 protein or protein GmSMS6. Its encoding gene is... GmSMS6 Gene. Sequence 2 (SEQ ID No. 2) is as follows: MAASAPTPREEFVYMAKLAEQAERYEEMVEFMEKVSASAESEELTVEERNLLSVAYKNVIGARRASWRIISSIEQKEESRGNEDHVAVIRDYRSKIEAELSNICDGILKLLDTRLVPSAASGDSKVFYLKM KGDYHRYLAEFKTGADRKEAAESTLSAYKAAQDIANTELPPTHPIRLGLALNFSVFYYEILNSPDRACSLAKQAFDEAIAELDTLGEESYKDSTLIMQLLRDNLTLWTSDMQDDGADEIKEAAPKGDGEQN.

[0013] The substance mentioned above may be any of the following: B1) RNA molecules that inhibit, reduce, or downregulate the expression of genes encoding the above-mentioned proteins, or RNA molecules that inhibit, reduce, or downregulate the activity or content of the proteins. B2) Expresses the gene encoding the RNA molecule described in B1); B3) An expression cassette containing the gene described in B2); B4) A recombinant vector containing the gene described in B2) or a recombinant vector containing the expression cassette described in B3); B5) Recombinant microorganisms containing the gene described in B2), or recombinant microorganisms containing the expression cassette described in B3), or recombinant microorganisms containing the recombinant vector described in B4); B6) A transgenic plant cell line containing the gene described in B2), or a transgenic plant cell line containing the expression cassette described in B3), or a transgenic plant cell line containing the recombinant vector described in B4); B7) Transgenic plant tissue containing the gene described in B2), or transgenic plant tissue containing the expression cassette described in B3), or transgenic plant tissue containing the recombinant vector described in B4); B8) A transgenic plant organ containing the gene described in B2), or a transgenic plant organ containing the expression cassette described in B3), or a transgenic plant organ containing the recombinant vector described in B4); B9) Nucleic acid molecules that encode the above proteins; B10) contains an expression cassette containing the nucleic acid molecule described in B9); B11) A recombinant vector containing the nucleic acid molecule described in B9) or a recombinant vector containing the expression cassette described in B10); B12) Recombinant microorganisms containing the nucleic acid molecules described in B9), or recombinant microorganisms containing the expression cassette described in B10), or recombinant microorganisms containing the recombinant vector described in B11); B13) A transgenic plant cell line containing the nucleic acid molecule described in B9) or a transgenic plant cell line containing the expression cassette described in B10) or a transgenic plant cell line containing the recombinant vector described in B11); B14) Transgenic plant tissue containing the nucleic acid molecule described in B9) or transgenic plant tissue containing the expression cassette described in B10) or transgenic plant tissue containing the recombinant vector described in B11); B15) A transgenic plant organ containing the nucleic acid molecule described in B9) or a transgenic plant organ containing the expression cassette described in B10) or a transgenic plant organ containing the recombinant vector described in B11).

[0014] The transgenic plant is a plant obtained through biological methods such as recombinant DNA technology in genetic engineering.

[0015] B1) The RNA molecule targets the gRNA of the protein-coding gene, wherein the coding gene is located at positions 325-344 and 489-508 in sequence 1.

[0016] Furthermore, in the biological material, the recombinant microorganisms in B5) and / or B12) can specifically be yeast, bacteria, algae, and fungi.

[0017] Furthermore, the recombinant microorganism may be Agrobacterium. Specifically, Agrobacterium is EHA105.

[0018] Furthermore, in the biological material, the plant tissues described in B7) and / or B14) may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos, and / or anthers.

[0019] Furthermore, in the biological material, the transgenic plant organs described in B8) and / or B15) may be the roots, stems, leaves, flowers, fruits, and seeds of the transgenic plant.

[0020] The present invention also provides a method for improving the grain traits of seed plants, the method comprising improving the grain traits of seed plants by inhibiting, reducing or downregulating the expression of the coding gene containing the above-mentioned protein in a target seed plant; the grain traits being grain weight and / or grain length and / or grain width and / or grain thickness.

[0021] The improvement of seed plant grain traits may be to increase or enhance the grain weight and / or grain length and / or grain width and / or grain thickness of the target seed plant.

[0022] The present invention also provides a method for producing seed plants with altered grain traits, the method comprising the step of regulating the expression of the coding gene in a target seed plant containing the coding gene of the above-mentioned protein to obtain seed plants with altered grain traits; wherein the grain traits are grain weight and / or grain length and / or grain width and / or grain thickness.

[0023] In this application, the regulation can be up-regulation, enhancement, or increase, or it can be down-regulation, suppression, or reduction.

[0024] The regulation is at least one of the following six types of regulation: C1) Regulation at the transcriptional level of the encoded gene. C2) Regulation that occurs post-transcriptionally in the encoded gene C3) Regulation of RNA transport in the encoded gene. C4) regulates the translation of the encoded gene. C5) regulates the degradation of the mRNA encoding the gene. C6) Post-translational regulation of the gene.

[0025] In the above text, the seed characteristics are selected from at least one of the following: D1) plant seed weight, D2) plant seed length, D3) plant seed width, D4) plant seed thickness.

[0026] The weight of the grain or the weight of the plant seed can be the weight of 100 grains.

[0027] The plant mentioned above may be any of the following: E1) a dicotyledonous plant, E2) a Rosales plant, E3) a legume, E4) a soybean, E5) soybean.

[0028] This invention provides soybeans GmSMS6 The gene has applications in one or more of the following areas: 1. regulating soybean grain weight; 2. regulating soybean seed length; 3. regulating soybean seed width; 4. regulating soybean seed thickness. This invention has discovered and cloned a novel gene that regulates soybean grain weight and grain shape. GmSMS6 Proving genes using CRISPR-Cas9 technology GmSMS6 This gene negatively regulates soybean grain weight and grain shape; knocking out this gene increases soybean grain weight and yield. Overexpression of this gene significantly reduces grain weight in transgenic plants. The results indicate that this gene negatively regulates soybean 100-grain weight and grain shape, and knocking out this gene increases soybean grain length, width, thickness, and weight, providing genetic resources and theoretical guidance for high-yield soybean breeding. Attached Figure Description

[0029] Figure 1 Showing Glyma.02g208700 (also referred to in this article) GmSMS6 Location and cloning of ). Where a represents the BSA (bulked segregant analysis) location analysis result for the 100-grain weight trait. B represents... GmSMS6 The gene structure diagram.

[0030] Figure 2 Showing GmSMS6 PCR detection results of transgenic materials (using the Bar gene as the detection marker). Among them, M is 2000 plus DNA marker; 1-3 are WT; 4-6 are GmSMS6 mutant plants; 7-9 are GmSMS6 overexpressing plants.

[0031] Figure 3 Showing GmSMS6 A schematic diagram of the acquisition of gene-edited materials and sequencing results.

[0032] Figure 4 Showing GmSMS6 Detection of gene expression levels in transgenic soybean lines.

[0033] Figure 5 Showing GmSMS6 Seed phenotype, 100-seed weight, seed length, seed width, and seed thickness of transgenic soybean materials were measured. Among them, ad represents the seed weight and seed shape-related phenotype of gene-edited plants; eh represents the seed weight and seed shape-related phenotype of overexpression plants. Detailed Implementation

[0034] 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.

[0035] 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.

[0036] The following examples used SPSS 11.5 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA was used. P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a highly significant difference, and P < 0.001 (***) indicates a highly significant difference.

[0037] This invention uses Jack as the receptor material.

[0038] In the following examples, the soybean “Jack” was kindly provided by Researcher Wensheng Hou of the Institute of Crop Science, Chinese Academy of Agricultural Sciences, and is referred to as “the soya bean cultivar Jack” in the following literature: Cai Y, Chen L, Liu X, Guo C, Sun S, Wu C, Jiang B, Han T, Hou W. CRISPR / Cas9-mediated targeted mutagenesisof GmFT2a delays flowering time in soya bean. Plant Biotechnol J. 2018 Jan;16(1):176-185. doi: 10.1111 / pbi.12758. Epub 2017 Jun 20. PMID: 28509421; PMCID:PMC5785355.

[0039] The vector pGES201 is the last three to four segments in the right column on page 728 of the following literature. Its full sequence can be found in the Supporting information section of that literature. Supplemental Figure 1: The sequence of pGES201 vector: Mengyan Bai1, Juehui Yuan1, Huaqin Kuang, Pingping Gong, Suning Li, Zhihui Zhang, Bo Liu, Jiafeng Sun, Maoxiang Yang, Lan Yang, Dong Wang, Shikui Song and Yuefeng Guan. Generation of a multiplex mutagenesis population via pooled CRISPR-Cas9 in soya bean. Plant Biotechnol J. doi: 10.1111 / pbi.13239.

[0040] Vector pTF101-eGFP Reference: Cai, Z., Xian, P., Cheng, Y., Zhong, Y., Yang, Y., Zhou, Q., Lian, T., Ma, Q., Nian, H., and Ge, L. (2023). MOTHER-OF-FT-AND-TFL1 regulates the seed oil and protein content in soybean. NewPhytol. 239: 905-919. The aforementioned biological materials are available to the public from the Institute of Botany, Chinese Academy of Sciences. These biological materials are intended solely for repeating the relevant experiments of this invention and may not be used for any other purpose.

[0041] This invention provides a gene related to plant grain weight. GmSMS6.

[0042] The present invention adopts the following technical solution: Using genotype and 100-grain weight phenotype data, QTL mapping and map-based cloning were performed. Combined with sequence analysis and homologous gene functional annotation, a gene affecting soybean grain weight was cloned and named... GmSMS6 Its amino acid sequence is shown in SEQ ID NO.2, and its cDNA sequence is shown in SEQ ID NO.1.

[0043] Using the CRISPR-Cas9 system to analyze the...GmSMS6 Gene editing was performed on the gene, and the results showed that the soybean seeds of the mutant plants had significantly increased seed length, seed width, seed thickness, and 100-seed weight. This indicates that this gene negatively regulates soybean 100-seed weight and seed shape, suggesting that silencing or reducing the expression of this gene can increase soybean seed size and weight. Conversely, overexpression of this gene can decrease soybean seed size and weight.

[0044] The regulating plant preferably includes legumes; the legume preferably includes soybean.

[0045] This invention uses Jack as the receptor material.

[0046] Example 1: Soybeans GmSMS6 Gene knockout vector construction soybeans GmSMS6 The gene sequence was obtained from phytozome (https: / / phytozome-next.jgi.doe.gov / ). TargetDesign (http: / / skl.scau.edu.cn / targetdesign / ) was used for sequencing. GmSMS6 The design of sgRNA target sequences yielded two sgRNAs: sgRNA1: 5'-GTCGCCTACAAGAACGTAAT-3'; sgRNA2: 5'-CAAGCTCCTCGACACGCGCC-3', both located in... GmSMS6 On the first exon. Synthesize the corresponding forward and reverse primers for the sgRNA generated by the CRISPR-P 2.0 website.

[0047] soybeans GmSMS6 The genome sequence (SEQ ID NO.1, 2441bp) is as follows:

[0048] In SEQ ID NO.1, positions 1-165 are 5'UTR, positions 2003-2441 are 3'UTR; positions 166-654, 1336-1458, 1722-1838, and 1943-2002 are exons, and the rest are introns.

[0049] soybeans GmSMS6 The amino acid sequence of the protein GmSMS6 encoded by the gene is sequence 2 (SEQ ID NO. 2, 262aa) in the sequence listing, as follows: MAASAPTPREEFVYMAKLAEQAERYEEMVEFMEKVSASAESEELTVEERNLLSVAYKNVIGARRASWRIISSIEQKEESRGNEDHVAVIRDYRSKIEAELSNICDGILKLLDTRLVPSAASGDSKVFYLKM KGDYHRYLAEFKTGADRKEAAESTLSAYKAAQDIANTELPPTHPIRLGLALNFSVFYYEILNSPDRACSLAKQAFDEAIAELDTLGEESYKDSTLIMQLLRDNLTLWTSDMQDDGADEIKEAAPKGDGEQN.

[0050] soybeans GmSMS6 The nucleic acid sequence (SEQ ID NO.3, 789bp) of the gene's coding sequence (CDS) is sequence 3 in the sequence listing, as follows: 5'--3'.

[0051] Build as follows GmSMS6 Gene knockout vector: The upstream primer for target 1 is B1-F:5'- GGAT TGTCGCCTACAAGAACGTAAT-3' (underscore base for forming sticky ends), downstream primer is B1-R: 5'- AAACATTACGTTCTTGTAGGCGACA-3' (underlined base for forming sticky ends), upstream primer B2-F for target 2: 5'- GGAT TGGCGCGTGTCGAGGAGCTTG-3', downstream primer is B2-R: 5'- AAAC CAAGCTCCTCGACACGCGCCA-3'.

[0052] B1-F and B1-R were annealed to form the annealed product B1-FR (a double-stranded DNA fragment with sticky ends). pGES201 was digested with BsaI to obtain a linearized vector of pGES201. B1-FR was then ligated into the linearized vector of pGES201 to obtain a vector expressing Cas9 and sgRNA targeting sgRNA1. GmSMS6 Gene knockout vector pCRISPR-Cas9-sgRNA1- GmSMS6 .

[0053] B2-F and B2-R were annealed to form the annealed product B2-FR (a double-stranded DNA fragment with sticky ends). pGES201 was digested with BsaI to obtain a linearized vector of pGES201. B2-FR was then ligated into the linearized vector of pGES201 to obtain a vector expressing Cas9 and sgRNA targeting sgRNA2. GmSMS6 Gene knockout vector pCRISPR-Cas9-sgRNA2- GmSMS6 .

[0054] pCRISPR-Cas9-sgRNA1- GmSMS6 Recombinant Agrobacterium tumefaciens EHA105 was obtained by introducing it into competent cells. GmSMS6 pCRISPR-Cas9-sgRNA2- GmSMS6 Recombinant Agrobacterium tumefaciens EHA105 was obtained by introducing it into competent cells. GmSMS6 .

[0055] Example 2 GmSMS6 Gene cloning and overexpression vector construction GmSMS6 The specific gene cloning method is as follows: Total RNA was extracted from soybean cultivar Jack, and cDNA was obtained by reverse transcription. Using this cDNA as a template, upstream cloning primer F: 5'-AACCCATTTGTTGCTTGCTGCGAG-3' and downstream cloning primer R: 5'-GCAATACCAAGTTTGCTAGAGA-3' were used for cloning.GmSMS6 Genes and connected to pEASY On the ®-Blunt Cloning Vector (Beijing TransGen Biotech Co., Ltd., catalog number CB101-01), we obtained a sample containing... GmSMS6 The gene plasmid was then used as a template to amplify a recombinant fragment with XbaI and SacI restriction endonucleases at both ends using the upstream primer 5'-gagaacacgggggactctagaATGGCAGCGAGCGCTCCC-3' and the downstream primer 5'-cgatcggggaaattcgagctcTTAATTTTGTTCCCCGTCACCT-3'. XbaI and SacI The linearized vector pTF101-eGFP was ligated with the recombinant fragment to obtain pTF101- GmSMS6 Recombinant plasmid. pTF101- GmSMS6 The recombinant plasmid is a copy of the pTF101-eGFP plasmid. XbaI and SacI Fragments between enzyme recognition sites are replaced with GmSMS6 The recombinant vector was obtained by extracting the coding sequence (CDS) of the gene while keeping other nucleotide sequences of pTF101-eGFP unchanged. GmSMS6 Driving in recombinant plasmids GmSMS6 The promoter for gene transcription is the CaMV35S promoter.

[0056] pTF101- GmSMS6 The recombinant plasmid was introduced into competent cells of Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium tumefaciens EHA105 / pTF101- GmSMS6 .

[0057] Example 3 GmSMS6 Obtaining and identifying gene knockout and overexpression plants 3.1 Obtaining transgenic plants After transforming the constructed gene editing and gene overexpression vector into Agrobacterium EHA105, soybean transformation was prepared.

[0058] (1) Soybean seed cleaning and disinfection: Using the soybean variety Jack as the recipient material, select seeds that are plump, uniform in size and free of disease spots on the surface, and use sodium hypochlorite and concentrated hydrochloric acid to react and release chlorine gas for overnight disinfection.

[0059] (2) Explant preparation and bacterial infection: The treated soybean seeds were placed in a clean conical flask and soaked overnight in sterile water. In a clean bench, the seed coat was cut along the hilum with a scalpel to separate the embryo into two halves.

[0060] Cotyledonary explants of soybean variety Jack were placed in a resuspending solution at OD. 600nm The recombinant Agrobacterium EHA105 / pCRISPR-Cas9-sgRNA1- of Example 2, with a value of 0.6-0.8, GmSMS6 and EHA105 / pCRISPR-Cas9-sgRNA2- GmSMS6 The explants were mixed with a 1:1 (volume ratio) bacterial solution, then sonicated for 3 minutes. The conical flask containing the explants was then placed under a vacuum pump and treated for 10 minutes (0.6 Pa). Finally, it was placed on a shaker and incubated at 23°C and 110 rpm for 40 minutes. After discarding the bacterial solution, the explants for Jack gene editing were obtained.

[0061] Cotyledonary explants of soybean variety Jack were placed in a resuspending solution at OD. 600nm Recombinant Agrobacterium EHA105 / pTF101 with a value of 0.6-0.8 GmSMS6 The explants were placed in the bacterial culture and then sonicated for 3 minutes. The conical flask containing the explants was then placed under a vacuum pump and treated for 10 minutes (0.6 Pa). Finally, it was placed on a shaker and incubated at 23°C and 110 rpm for 40 minutes. After discarding the bacterial culture, the explants for overexpression in the soybean variety Jack were obtained.

[0062] (3) Co-culture: The explants used for Jack gene editing and the explants used for overexpression of the soybean variety Jack were treated as follows: They were spread on clean filter paper, dried, and spread on co-culture medium (B5 medium + 3% sucrose + 0.8% Agar + 3mM MES + 0.25mg / L gibberellin (GA3) + 1.67mg / L benzylaminopurine (6-BA) + 580mg / L cysteine ​​+ 154.2mg / L dithiothreitol + 200µM acetoyl eugenol (AS), pH 5.4), and placed in an incubator. After 3 days of dark culture at 23℃, they were transferred to a tissue culture room at 23-25℃ with a photoperiod of 16 h light / 8 h dark for 1 day.

[0063] (4) Recovery culture: The hypocotyl of the explant was partially removed, leaving 3-4 mm, and inserted obliquely into the recovery culture medium (MS medium + 3% sucrose + 3mM MES + 0.8% Agar + 1.67 mg / L benzylaminopurine (6-BA) + 100 mg / L cephalosporin (Cef), pH 5.7), and sealed. It was placed in the tissue culture room with a photoperiod of 16 h light / 8 h darkness for 7 days.

[0064] (5) Bud induction and selection culture: Excessively long hypocotyls were removed, and explants were obliquely inserted into the selection medium (MS medium + 3% sucrose + 3mM MES + 0.8% Agar + 1.67mg / L benzylaminopurine (6-BA) + 5mg / L glutfosinate + 100mg / L cephalosporin (Cef), pH 5.7). The explants were placed in the tissue culture room with a photoperiod of 16 h light / 8 h dark for 21 days.

[0065] (6) Bud elongation culture: Remove the cotyledons of the explant, retaining the clustered buds and base, and make a new wound at the base. Place the explant on bud induction medium (MS medium + 3% sucrose + 3mM MES + 0.8% Agar + 50mg / L asparagine + 50mg / L glutamine + 0.1mg / L indoleacetic acid (IAA) + 0.5mg / L gibberellin (GA3) + 1mg / L zeatin + 5mg / L glutfosinate + 500mg / L termethin (Tim) + 100mg / L cephalosporin (Cef), pH 5.7), and place it in a tissue culture room at 23-25℃ with a photoperiod of 16 h light / 8 h darkness for 2-8 weeks. During this period, change the medium or induce root growth according to the growth status of the explant.

[0066] (7) Root induction culture: When the shoots elongate to 3-5 cm, cut the elongated shoots from the base of the tissue, soak the cut end in auxin solution for 2 minutes, and then transfer them into rooting medium (MS medium + 3% sucrose + 3mM MES + 0.8% Agar + 1mg / LIBA, pH 5.7). Place them in the tissue culture room for root induction, which generally takes about 2 weeks.

[0067] (8) Seedling transplanting: transplant the seedlings into the soil, cover them with a film and place them in the light. After the plants have grown normally for 3-5 days, remove the film to obtain the T0 generation soybean variety Jack gene-edited plants (plants obtained from explants used for Jack gene editing) and the overexpression plants of soybean variety Jack (plants obtained from explants used for Jack overexpression).

[0068] Harvest T0 generation seeds, self-pollinate T0 generation seeds to obtain T1 generation seeds, plant T1 generation seeds to obtain T1 generation seedlings and perform the following tests.

[0069] 3.2 Identification of soybean variety Jack after gene editing and overexpression The bar gene was examined in soybean variety Jack, its T1 generation Jack gene-edited plants obtained in 3.1, and T1 generation Jack overexpression plants. The results are as follows: Figure 2As shown, 1-3 are WT (Jack) lines, denoted as WT_1, WT_2, and WT_3; 4-6 are gene-edited lines, denoted as KO_1, KO_2, and KO_3; and 7-9 are overexpression lines, denoted as OE_1, OE_2, and OE_3.

[0070] Using the genomic DNA of the T1 soybean variety Jack gene-edited plant obtained in 3.1 as a template, PCR amplification of the plant was performed using upstream primer F: 5'-CAATCCCAGCCTTTCGGTT-3' and downstream primer R: 5'-TCAATTTTCAACATCCATCGCTT-3'. Sanger sequencing was then performed on the PCR products to identify the mutation status of the target site in the T1 generation plants. PCR and Sanger sequencing techniques revealed three editing modes at the target site in the T1 generation soybean variety Jack gene-edited plant (…). Figure 3 ).

[0071] Specifically, in cr1 (KO_1), compared to the soybean variety Jack, the genome has... GmSMS6 The region corresponding to the gene underwent the following changes: the thymine deoxyribonucleotide at position 341 of the sequence shown in SEQ ID NO.1 was knocked out, resulting in a frameshift mutation and premature termination of amino acid translation.

[0072] In cr2(KO_2), compared to the soybean variety Jack, the genome has GmSMS6 The region corresponding to the gene underwent the following change: a thymine deoxyribonucleotide was inserted between positions 341 and 342 of the sequence shown in SEQ ID NO.1, resulting in a frameshift mutation and premature termination of amino acid translation.

[0073] In cr3 (KO_3), compared to the soybean variety Jack, the genome has GmSMS6 The region corresponding to the gene underwent the following changes: positions 341 to 492 of the sequence shown in SEQ ID NO.1 were knocked out, resulting in a frameshift mutation and premature termination of amino acid translation.

[0074] Gene expression in overexpressing plants was identified by qRT-PCR. Total RNA was extracted from T1 generation soybean cultivar Jack overexpressing plants and seedlings of the recipient soybean cultivar Jack. After reverse transcription, real-time quantitative PCR was performed on the transgenic plants using quantitative primers F: 5'-GGACATTGCTAACACCGAGC-3' and R: 5'-GGGTAAGGTTATCACGAAGA-3'. Soybean... Actin ( Glyma18g52780) as an internal reference gene (upstream internal reference primer ACT3-F: 5'-GCACCACCGGAGAGAAAATA-3'; downstream internal reference primer ACT3-R: 5'-GTGCACAATTGATGGACCAG-3'), using 2 -△△CT Data processing and detection GmSMS6 Gene expression in various transgenic lines. Results are as follows: Figure 4 As shown, compared with the recipient material soybean variety Jack, in transgenic lines OE_1, OE_2, and OE_3... GmSMS6 Gene expression was upregulated by tens of times ( Figure 4 The expression of the target gene was significantly increased in overexpressing plants.

[0075] Example 4 GmSMS6 Phenotypic identification of gene knockout and overexpression plants 4.1 Phenotypic identification of soybean variety Jack after gene editing and overexpression After harvesting mature soybean plants (KO_1, KO_2, KO_3) and overexpressing plants (OE_1, OE_2, OE_3) of the Jack gene-edited strain, they were grown alongside the wild-type soybean variety Jack in a natural experimental field at the Xiangshan Molecular Base, Institute of Botany, Chinese Academy of Sciences (Beijing). Soybean seed phenotypes were analyzed upon maturity, with at least six biological replicates for each line. Results are as follows: Figure 5 As shown.

[0076] 100-seed weight statistics: After the above-mentioned potted soybean seeds matured, the seeds harvested from each plant were dried at 37℃ for one week until the seeds reached constant weight. The 100-seed weight (i.e., the weight of 100 seeds after thorough drying) of the recipient control soybean variety Jack (WT) was measured. The 100-seed weights of the gene knockout plants KO_1, KO_2, and KO_3 and the overexpression plants OE_1, OE_2, and OE_3 of the soybean variety Jack were compared. At least three individual plants were measured for each line, and each individual plant was randomly measured three times. The comparison results of the soybean variety Jack with the gene knockout plants KO_1, KO_2, and KO_3 are as follows: Figure 5 The results of comparing soybean variety Jack with overexpressing plants of soybean variety Jack (OE_1, OE_2, OE_3) are as follows: Figure 5 e. Seed length, width, and thickness statistics: After the above-mentioned potted soybean seeds matured, the seeds harvested from a single plant were dried at 37℃ for one week. After the seeds reached constant weight, the length, width, and thickness of the seeds of the recipient control soybean variety Jack were measured. The length, width, and thickness of the seeds of the gene knockout plants KO_1, KO_2, and KO_3 and the overexpression plants of the soybean variety Jack OE_1, OE_2, and OE_3 were compared (i.e., the length, width, and thickness of the thoroughly dried seeds). At least 30 seeds were measured for each line.

[0077] The grain length measurement results of the recipient control soybean variety Jack (WT) and gene knockout plants KO_1, KO_2, and KO_3 are as follows: Figure 5 The particle width measurement results for b are as follows: Figure 5 The particle thickness measurement results are as follows: Figure 5 d.

[0078] The grain length measurement results of the recipient control soybean variety Jack (WT) and overexpressing plants OE_1, OE_2, and OE_3 are as follows: Figure 5 The particle width measurement results are as follows: Figure 5 The particle thickness measurement results are as follows: Figure 5 h.

[0079] As can be seen, compared with the control WT, the seeds of the three gene knockout lines showed significant increases in seed weight (14.9%-20.4%), seed length (6.8%-9.4%), seed width (5.7%-7.6%), and seed thickness (5.4%-8.7%). Conversely, the seeds of the overexpression lines showed significant decreases in seed weight (7.8%-9.1%), seed length (5.8%-6.9%), seed width (3.4%-5.5%), and seed thickness (5.1%-6.6%) compared with the control WT. These results indicate... GmSMS6 Genes negatively regulate soybean grain weight-related phenotypes, including grain length, grain width, and grain thickness.

[0080] 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. The application of biomaterials, characterized in that, The application is any one of the following: A1) The application of the biomaterials described herein in regulating plant grain traits and / or in preparing products that regulate plant grain traits. A2) The application of the biomaterials described herein in plant breeding and / or the preparation of plant breeding products; The biological material is a protein, a substance that regulates the expression of the gene encoding the protein, or a substance that regulates the content of the protein. The protein is GmSMS6, and is any one of the following: B1) The amino acid sequence of the protein is shown in sequence 2. B2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein described in B1), which has more than 80% identity with the protein shown in B1) and can regulate plant grain traits. B3) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of B1) or B2).

2. The application according to claim 1, characterized in that, The protein is derived from soybeans.

3. The application according to claim 1 or 2, characterized in that, The substance is any one of the following: C1) RNA molecules that inhibit, reduce, or downregulate the expression of the gene encoding the protein of claim 1 or 2, or RNA molecules that inhibit, reduce, or downregulate the activity or content of the protein. C2) expresses the gene encoding the RNA molecule described in C1); C3) contains an expression cassette containing the gene described in C2); C4) A recombinant vector containing the gene described in C2) or a recombinant vector containing the expression cassette described in C3); C5) Recombinant microorganisms containing the gene described in C2), or recombinant microorganisms containing the expression cassette described in C3), or recombinant microorganisms containing the recombinant vector described in C4); C6) A transgenic plant cell line containing the gene described in C2), or a transgenic plant cell line containing the expression cassette described in C3), or a transgenic plant cell line containing the recombinant vector described in C4); C7) Transgenic plant tissue containing the gene described in C2), or transgenic plant tissue containing the expression cassette described in C3), or transgenic plant tissue containing the recombinant vector described in C4); C8) A transgenic plant organ containing the gene described in C2), or a transgenic plant organ containing the expression cassette described in C3), or a transgenic plant organ containing the recombinant vector described in C4); C9) is a nucleic acid molecule that encodes the protein described in claim 1 or 2; C10) contains an expression cassette containing the nucleic acid molecule described in C9); C11) A recombinant vector containing the nucleic acid molecule described in C9) or a recombinant vector containing the expression cassette described in C10); C12) Recombinant microorganisms containing the nucleic acid molecules described in C9), or recombinant microorganisms containing the expression cassette described in C10), or recombinant microorganisms containing the recombinant vector described in C11); C13) A transgenic plant cell line containing the nucleic acid molecule described in C9) or a transgenic plant cell line containing the expression cassette described in C10) or a transgenic plant cell line containing the recombinant vector described in C11); C14) Transgenic plant tissue containing the nucleic acid molecule described in C9) or transgenic plant tissue containing the expression cassette described in C10) or transgenic plant tissue containing the recombinant vector described in C11); C15) A transgenic plant organ containing the nucleic acid molecule described in C9) or a transgenic plant organ containing the expression cassette described in C10) or a transgenic plant organ containing the recombinant vector described in C11).

4. The application as described in any one of claims 1-3, characterized in that, The grain trait is selected from at least one of the following: D1) Weight of plant seeds, D2) Plant seed length, D3) Plant seed width, D4) Plant seed thickness.

5. A method for improving the grain traits of seed plants, characterized in that, The method includes improving seed plant grain traits by inhibiting, reducing, or downregulating the expression of the coding gene in a target seed plant containing the coding gene of the protein described in claim 1 or 2; the grain traits being grain weight and / or grain length and / or grain width and / or grain thickness.

6. A method for producing seed plants with altered grain traits, characterized in that, The method includes the step of regulating the expression of the coding gene in a target seed plant containing the coding gene of the protein described in claim 1 or 2 to obtain a seed plant with altered grain traits; the grain traits are grain weight and / or grain length and / or grain width and / or grain thickness.

7. The method according to claim 6, characterized in that, The regulation refers to increasing, strengthening, or raising.

8. The method according to claim 6, characterized in that, The regulation refers to downregulation, suppression, or reduction.

9. The method according to any one of claims 5-8, characterized in that, The grain trait is selected from at least one of the following: E1) Weight of plant seeds, E2) Plant seed length, E3) Plant seed width, E4) Plant seed thickness.

10. The application as described in any one of claims 1-4 or the method as described in any one of claims 5-8, characterized in that, The plant is any one of the following: F1) Dicotyledons, F2) Plants of the Rosales order, F3) Leguminosae (family legumes) F4) Soybean species, F5) Soybeans.

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