Soybean hundred kernel weight regulation gene and its encoded protein and application

Abstract

This invention relates to the fields of plant molecular genetics and genetic engineering, specifically disclosing a soybean 100-seed weight regulatory gene, its encoded protein, and its applications. The key technical points are: the soybean 100-seed weight regulatory gene GmSMS1 cloned in this invention has the nucleotide sequence shown in SEQ ID NO: 1, and the encoded protein amino acid sequence shown in SEQ ID NO: 2; this invention obtains the GmSMS1 gene through map-based cloning, and knockout of the gene yields a soybean mutant with a significantly reduced 100-seed weight, demonstrating that the GmSMS1 gene can effectively regulate soybean seed 100-seed weight. This invention also provides a recombinant vector containing this gene, an expression cassette, a transgenic cell line or recombinant bacteria, primers for amplifying the gene, and the application of this gene in cultivating transgenic plants with high 100-seed weight. Targeted improvement of plant 100-seed weight using the GmSMS1 gene is feasible and can provide genetic resources and theoretical support for the rapid and precise breeding of high-yield crop varieties.

Description

Technical Field

[0001] This invention relates to the fields of plant molecular genetics and genetic engineering, and more specifically, to genes regulating 100-seed weight in soybeans, their encoded proteins, and their applications. Background Technology

[0002] Soybean is an important crop in my country, used for both grain and oilseed production, and is a major source of high-quality plant protein, edible vegetable oil, and feed protein. However, my country relies on imports for over 80% of its soybeans, seriously threatening national food security. my country's soybean production capacity is constrained by both planting area and yield per unit area. Given the limited arable land in my country, increasing yield per unit area is the main way to increase the total soybean output. Soybean yield is mainly determined by three factors: 100-seed weight, number of seeds per plant, and planting density. Among these, 100-seed weight is a crucial target trait for high-yield soybean breeding. Soybean 100-seed weight is a quantitative trait controlled by multiple genes, with complex regulatory mechanisms. Currently, only about 10 genes regulating 100-seed weight have been cloned in soybeans, which is insufficient to support the needs of high-yield molecular breeding. Therefore, in-depth exploration of key genes regulating soybean 100-seed weight, analysis of their molecular mechanisms of action, and construction of a fine-grained regulatory network for soybean 100-seed weight will provide genetic resources and a theoretical foundation for breeding new high-yield soybean varieties.

[0003] 100-seed weight is one of the important yield traits of soybean. Since the release of the reference genome of soybean variety Williams 82 in 2010, research on the regulatory mechanism of soybean 100-seed weight has made some progress, with more than 300 related QTLs reported and more than 10 functional genes cloned. Cytochrome P450 family genes GmCYP78A72, GmCYP78A5, and GmCYP82C4 can all regulate soybean 100-seed weight. In wild soybean, the PP2C-1 allele promotes seed enlargement by regulating cell size. GmSWEET10a and GmSWEET10b are involved in glucose and hexose transport, thus affecting 100-seed weight. GmPDAT simultaneously regulates soybean 100-seed weight and oil content; overexpression lines show significantly increased 100-seed weight and oil content, while RNA interference lines show decreased 100-seed weight. GmKIX8-1 and GmSSS1 are both negative regulators of soybean 100-seed weight: GmKIX8-1 mutants have increased seed cell number and larger seeds; GmSSS1 mutants have both increased seed cell number and cell area, resulting in increased 100-seed weight. GmST05 is a positive regulator of 100-seed weight cloned from over 1800 soybean materials through GWAS analysis. GmGA3ox1 promotes gibberellin synthesis and increases soybean seed weight. GmPLATZ encodes a zinc finger transcription factor that directly binds to the promoter of GmGA20OX and regulates soybean 100-seed weight by activating its expression. HSW and SW16.1 are both QTLs regulating soybean seed weight; HSW loss-of-function mutants have increased seed weight; SW16.1 negatively regulates seed weight in wild soybean alleles, while positively regulating it in cultivated soybean alleles. Environmental factors also affect seed development; GmCOL2b and Dt1 both control soybean 100-seed weight in a light-dependent manner. A recent study, using combined GWAS and TWAS analysis, cloned the gene GmRWOS1, which simultaneously regulates 100-seed weight and oil content. The knockout mutant showed increased 100-seed weight and oil content. Currently, the number of cloned genes regulating 100-seed weight in soybeans remains relatively small, and a complete molecular regulatory network is far from being established.

[0004] A search revealed a method for obtaining high 100-seed weight transgenic soybeans by overexpressing the Gmcdf1 gene. This Gmcdf1 gene encodes a key transcription factor that regulates stomatal conductance and affects plant photosynthetic capacity. By constructing an overexpression vector and transforming the recipient soybean line "Bert-1," transgenic soybean plants with increased 100-seed weight can be obtained. This research provides a new approach to further enriching the gene resources regulating soybean 100-seed weight, but its regulatory mechanism involves the photosynthetic pathway and not the histone methylation modification pathway. In contrast, the regulatory role of histone methylation modification in crop seed development, which is the focus of this invention, lacks in-depth research in soybeans, and there are currently no reports of regulating soybean 100-seed weight using genes encoding components of the histone methylation complex. Therefore, identifying key genes in soybeans that regulate 100-seed weight through the histone methylation pathway and elucidating their molecular mechanisms remains of significant research value and application demand for constructing a complete fine-tuned regulatory network for soybean 100-seed weight and supplementing the existing gene library regulating 100-seed weight.

[0005] Methylation plays a crucial regulatory role in crop seed development. In Arabidopsis, ULT1 encodes a chromatin-modifying protein called TrxG; loss-of-function mutants exhibit enlarged leaves, flowers, siliques, and seeds. ULT1 can be recruited by transcription factors TCP14 / 15, promoting H3K4me3 modification of target genes and activating their expression to regulate organ development. In rice, the PcG family protein OsFIE2 possesses specific histone H3 methyltransferase activity; decreased expression of OsFIE2 affects the synthesis of plant hormones, signal transduction, and the expression of genes related to cell proliferation, leading to smaller grains, reduced fertility, and delayed germination. In wheat, the histone methyltransferase complex PRC2 is recruited by the transcription factor TaMADS-GS, affecting cytokinin synthesis through methylation modification of TaCKX, thereby regulating grain weight. In soybean, researchers examined the dynamic changes in DNA methylation levels during seed development: CHH methylation increased during seed development, while CG and CHG methylation levels remained unchanged. Some genes playing important roles in seed development were not modified by DNA methylation. Therefore, it is hypothesized that another mode of methylation modification during soybean seed development—histone methylation—may play a more important role. In-depth research into the mechanism by which histone methylation regulates 100-seed weight, and targeted regulation of key genes through metabolic engineering, has significant guiding implications for breeding high-yield soybeans and protecting national food security.

[0006] Therefore, this application proposes a gene regulating 100-grain weight of soybean, its encoded protein, and its application to solve the above problems. Summary of the Invention

[0007] The purpose of this invention is to solve the technical problems mentioned in the background section, and to provide a soybean 100-seed weight regulatory gene and its encoded protein and their applications. The soybean 100-seed weight regulatory gene GmSMS1 cloned in this invention has the nucleotide sequence shown in SEQ ID NO: 1, and the encoded protein amino acid sequence shown in SEQ ID NO: 2. This invention obtains the GmSMS1 gene through map-based cloning, and knockout of the gene yields a soybean mutant with a significantly reduced 100-seed weight, demonstrating that the GmSMS1 gene can effectively regulate the 100-seed weight of soybean seeds. This invention also provides a recombinant vector containing this gene, an expression cassette, a transgenic cell line or recombinant bacteria, primers for amplifying the gene, and a method for cultivating transgenic plants with altered 100-seed weight. Targeted improvement of plant 100-seed weight using the GmSMS1 gene is feasible and can provide gene resources and theoretical support for the rapid and precise breeding of high-yield crop varieties.

[0008] The above-mentioned objective of the present invention is achieved as follows:

[0009] The present invention provides a soybean 100-grain weight regulatory gene, namely GmSMS1, derived from soybean (Glycine max(L.) Merr), and the gene is a DNA molecule as follows (1) or (2) or (3):

[0010] (1) A DNA molecule with the sequence shown in SEQ ID NO.1;

[0011] (2) DNA molecules that have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with the DNA sequence in (1) and encode a protein associated with the 100-grain weight of a plant.

[0012] The present invention also provides a protein encoded by the soybean 100-seed weight regulatory gene GmSMS1, which is as follows:

[0013] (a) A protein consisting of an amino acid sequence as shown in SEQ ID NO.2;

[0014] (b) A protein derived from the sequence described in SEQ ID NO.2 by substitution and / or deletion and / or addition of one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.2, and which is related to the 100-grain weight of a plant.

[0015] Furthermore, the substitution and / or deletion and / or addition of one or more amino acid residues shall be the substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0016] The present invention also provides recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the above-mentioned genes.

[0017] Furthermore, the recombinant vector is a recombinant vector obtained by inserting the above-mentioned gene editing site into the KpnⅠ and XbaⅠ recognition sites of the V135 vector.

[0018] The present invention also provides primers for amplifying the full length of the above-mentioned gene or any fragment thereof.

[0019] The present invention also provides the application of the soybean 100-seed weight regulating gene GmSMS1 in the cultivation of transgenic plants with high 100-seed weight (a method for cultivating transgenic plants with altered 100-seed weight), wherein the gene is introduced into the target plant to obtain the transgenic plant; the transgenic plant has an increased 100-seed weight compared with the target plant.

[0020] Furthermore, the gene is introduced into the target plant using the aforementioned recombinant vector.

[0021] Furthermore, the target plant is a dicotyledonous or monocotyledonous plant, specifically soybean.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. This invention provides a novel soybean 100-grain weight regulatory gene resource, and for the first time clones the soybean 100-grain weight regulatory gene GmSMS1, which encodes a component of the histone methylation complex: polycomb protein EED. Existing cloned 100-grain weight regulatory genes such as GmCYP78A72, GmCYP78A5, GmCYP82C4, GmPDAT, GmSWEET10a, and GmSWEET10b mainly involve pathways such as cytochrome P450, lipid metabolism, and glucose transport. However, the mechanism of action of the histone methylation modification pathway involved in the regulation of soybean 100-grain weight has not been reported. The cloning of the GmSMS1 gene in this invention fills this technological gap and provides a novel molecular element for the soybean 100-grain weight regulatory network.

[0024] 2. This invention obtains the naturally occurring loss-of-function mutant sms1 through EMS mutagenesis. Using map-based cloning technology, the GmSMS1 gene (containing a SNP in its coding region causing a change from glycine to aspartic acid) is precisely identified through sequence alignment between the wild type and the mutant. Furthermore, using CRISPR / Cas9 gene editing technology, this gene is knocked out in the soybean variety Zhongji 602, resulting in two homozygous knockout mutant families, CR1 and CR2, with different editing types. Phenotypic results show that compared to the recipient control Zhongji 602 (average 100-seed weight 16.95 g), CR1's average 100-seed weight is 12.35 g, a decrease of 27%; CR2's average 100-seed weight is 13.58 g, a decrease of 20%, both differences being statistically significant. The above positive (mutant) and reverse (gene editing) genetic evidence corroborate each other, conclusively demonstrating the positive regulatory function of the GmSMS1 gene on soybean 100-seed weight.

[0025] 3. This invention demonstrates that the GmSMS1 gene is a positive regulator of soybean 100-grain weight. The knockout mutant of this gene has a significantly reduced 100-grain weight and can be used for the breeding of soybean varieties with different grain weight types. The gene resources, recombinant vectors, primers and transgenic methods provided by this invention can be directly applied to soybean molecular breeding practices, providing strong technical support and material basis for accelerating the breeding of new soybean varieties.

[0026] 4. This invention reveals the regulatory role of the gene GmSMS1, which encodes a component of the histone methylation complex in soybean, on seed size. This provides new experimental evidence for understanding the function of histone methylation modification in leguminous seed development and has important academic and theoretical value. Attached Figure Description

[0027] Figure 1 This invention provides a phenotypic analysis of the sms1 mutant in the following embodiments: (A) Phenotypic of wild-type (Zhongpin 661) and mutant (sms1) plants at the pod-setting stage, scale bar 10 cm; (B) Seed length phenotype of wild-type and mutant, scale bar 5 mm; (C) Seed width phenotype of wild-type and mutant, scale bar 5 mm; (DG) Statistical analysis of 100-seed weight (D), seed length (E), seed width (F), and seed thickness (G) of wild-type and mutant. The data shown are mean ± standard deviation (n≥20), and the P-value was obtained by t-test.

[0028] Figure 2 This invention relates to the cloning of the GmSMS1 gene in the embodiments of the present invention ((A) BSA localization analysis; (B) fine localization of the GmSMS1 gene; (C) differences in gene sequence and encoded protein sequence between wild-type and mutant GmSMS1 gene).

[0029] Figure 3This is a phenotypic analysis of GmSMS1 knockout transgenic soybeans in this embodiment of the invention ((A) Phenotyps of mature plants of Zhongji 602 and two knockout mutants, scale bar 10 cm; (B) Phenotyps of seed length of Zhongji 602 and two knockout mutants, scale bar 1 cm; (C) Phenotyps of seed width of Zhongji 602 and two knockout mutants, scale bar 1 cm; (D) Statistics of 100-seed weight of wild type and mutant (n=3); (E) Statistics of seed length of wild type and mutant (n=30); (F) Statistics of seed width of wild type and mutant (n=30); The data shown are mean ± standard deviation, and the p-values ​​are from one-way ANOVA corrected by Tukey). Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the following description is provided in conjunction with the appendix. Figure 1-3 The present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0031] This invention provides a novel soybean 100-grain weight regulatory gene, GmSMS1, its encoded protein, and its applications, aiming to solve the problem of insufficient existing 100-grain weight regulatory gene resources.

[0032] Unless otherwise specified, the experimental methods used in the following embodiments of the present invention are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. Agrobacterium tumefaciens EHA105 is described in *New Agrobacterium helper plasmids for gene transfer to plants. Hood, Elizabeth E; Gelvin, Stanton B; Melchers, Leo S; Hoekema, Andre. Transgenic Research, 2(4): p. 208-218-218 (1993). It is publicly available from the Institute of Crop Science, Chinese Academy of Agricultural Sciences.

[0033] The implementation of the present invention will be described in detail below with reference to specific embodiments.

[0034] Example 1: Localization and Cloning of Genes Regulating 100-Seed Weight in Soybeans

[0035] 1. Screening and phenotypic analysis of mutants

[0036] In this embodiment, a mutant with smaller seeds and a reduced 100-seed weight was screened from the EMS mutagenesis mutant library of soybean variety Zhongpin 661 and named sms1 (small seed 1). Compared with the wild type (WT), the sms1 mutant has smaller seeds, a lower 100-seed weight, and significantly reduced seed length, width, and thickness (e.g., ...). Figure 1 (As shown).

[0037] 2. Genetic analysis and gene mapping

[0038] In this embodiment, a genetic population was constructed using the sms1 mutant as the maternal parent and the wild type as the paternal parent. Statistical analysis of the seed phenotypes in the F2 segregating population revealed a segregation ratio of 3:1 between high and low 100-seed weight plants, indicating that the 100-seed weight phenotype is controlled by a pair of recessive nuclear genes, which were named GmSMS1. Then, 50 high-100-seed-weight plants and 50 low-100-seed-weight plants were selected from the F2 population to construct two DNA pools for BSA analysis. Based on the BSA results, the GmSMS1 gene was initially located within a 3.44 Mb region (e.g., ...). Figure 2 (As shown in A in the diagram).

[0039] To further refine the mapping, a mapping population was constructed using the sms1 mutant as the male parent and Jidou 12 as the female parent. The 100-seed weight of individual plants in the F2 population was measured, resulting in 150 plants with low 100-seed weight. Simultaneously, polymorphic markers between sms1 and Jidou 12 were screened within the initial mapping region, yielding six pairs of polymorphic markers. First, using polymorphic markers S5 and S188 located on either side of the region, the genotypes of the 150 F2 plants with low 100-seed weight were identified by polyacrylamide gel electrophoresis, detecting 37 exchange plants. Subsequently, using four polymorphic markers S98, S121, S125, and S139, the genotypes of the 37 exchange plants were further identified, finely mapping the GmSMS1 gene to a 99.6 kb region containing 12 open reading frames (ORFs) (e.g., ...). Figure 2 (As shown in B). The above six pairs of gene-localizing primers are: S5, S98, S121, S125, S139, and S188, and their sequences are as follows:

[0040] S5-F: GCAAATTACATGAAAGTGGCAT;

[0041] S5-R: GCATGTTGCTCACATGAGTGT;

[0042] S98-F: TGCATTGGTTACAATTGATTTGA;

[0043] S98-R: TTGATGCCAAAAATAGATTGGAG;

[0044] S121-F: ACCGAAATCGCCATGATTAG;

[0045] S121-R:AAACATTCAATTTGGGTTGTAAA;

[0046] S125-F: TTTGAGAATGTGTGCATTTAACA;

[0047] S125-R: AAATCCTCTCATTTCCCCGT;

[0048] S139-F: CATGAGCATATGAATGTATAAGGG;

[0049] S139-R: CCTTAATTTACCGTTTGCCG;

[0050] S188-F: AAGACCGGAAAAACCAGGTC;

[0051] S188-R: TGCCTCTCTCTCTCTCTTTT.

[0052] 3. Candidate gene identification

[0053] Sequence alignment analysis of 12 ORFs from wild-type and sms1 mutants revealed a single SNP difference in the coding region of ORF5, while the coding regions of the other ORFs showed no difference. This nucleotide variation in the ORF5 coding region resulted in the change of amino acid position 204 from glycine (G) to aspartic acid (D) in the encoded protein (e.g., ...). Figure 2 (as shown in C in the diagram). Therefore, ORF5 (Glyma.10G022000) was identified as a candidate gene for GmSMS1, which encodes a component of a histone methylation complex: the polycomb protein EED. The nucleotide sequence of the GmSMS1 gene is shown in SEQ ID NO: 1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO: 2.

[0054] SEQ ID NO.1:

[0055]

[0056] SEQ ID NO.2:

[0057] MVGETAATGKSVGLGLGCDPVVGSLACSKKREYRVTNRLQEGKRPLYAVIFNFIDSRYFNVFATVGGNRVTVYQCLEGGVIAVLQSYVDEDKDESFYTVSWACNVDGTPFVVAGGINGVMRVIDVGSEKIHKSFVGHGDSVNEIRTQTLKPSLVISASKDESVRLWNVHTGICILIFAGAGGHRNEVLSVD FHPSDIYRIASCGMDNTVKIWSMKEFWTYVEKSFTWTDLPSKFPTKYVQFPVYNASVHLNYVDCNRWLGDFILSKSVDNEIILWEPKVKEQTPGEGVVDILQKYPVPECDIWFIKFSCDFHFNLAAVGNREGKIFVWELQSSPPVLVARLSHPQSKSPIRQTATSFDGSTILSCCEDGTIWRWDDVTNSPD.

[0058] Example 2: Construction of GmSMS1 gene knockout vector

[0059] 1. Knockout target design and vector construction

[0060] Two knockout target sites were designed in the exon regions of the GmSMS1 genome to construct a dual-target knockout vector. Knockout primers were designed for each of the two target sites, and the primer sequences are shown in Table 1 below.

[0061] Table 1 Primer sequences for gene knockout vector construction

[0062] GmSMS1-Cas9-1F AATAGTCAGCAACAAGGTTCGAACAAAGCACCAGTGGTCTAG GmSMS1-Cas9-1R TCTATACGCCGTCATTTTCATGCACCAGCCGGGAATCGAA GmSMS1-Cas9-2F TGAAAATGACGGCGTATAGAGTTTTAGAGCTAGAAATAGC GmSMS1-Cas9-2R GCTATTTCTAGCTCTAAAACATCGCCTTCAAGAGGGAAAGTGCACCAGCCGGGAATCGAA

[0063] PCR amplification was performed using PGTR as a template. The amplification system and procedure are shown in Table 2 below. After amplification, the amplification products were detected by agarose gel electrophoresis. The size of the first target fragment was 118 bp, and the size of the second target fragment was 213 bp. The PCR products of both targets were recovered.

[0064] Table 2 PCR amplification system and procedure

[0065]

[0066] The V129 vector was digested with the restriction endonuclease BbsⅠ. The digested vector was then ligated with the two target fragments using homologous recombination, and the reaction was carried out at 50°C for 1 hour. The ligation product was transformed into *E. coli* DH5α competent cells, incubated on ice for 30 min, heat-shocked at 42°C for 90 s, and then incubated on ice for 2 min. Subsequently, 300 μL of LB liquid medium was added, and the cells were incubated at 37°C for 40 min. After incubation, the cells were evenly spread on solid LB medium containing ampicillin and cultured overnight. The next day, single clones were picked from the medium and sequenced using primers LjU6-seq-F (GCTGAGGAGACTTGTGCTAT) and PMB1-seq-R (CTTTATAGTCCTGTCGGGTTTCGC).

[0067] 2. Construction of knockout expression vectors

[0068] The V135 knockout vector was digested with restriction endonucleases KpnⅠ and XbaⅠ. The correctly sequenced recombinant V129 vector was also double-digested with KpnⅠ and XbaⅠ to obtain a target fragment of approximately 700 bp. The digested V135 vector and the target fragment were ligated using T4 ligase. The ligation product was transformed into E. coli DH5α competent cells, incubated on ice for 30 min, heat-shocked at 42℃ for 90 s, then incubated on ice for 2 min. Subsequently, 300 μL of LB liquid medium was added, and the cells were incubated at 37℃ for 40 min. After incubation, the cells were evenly spread on solid LB medium containing spectinomycin and cultured overnight. The next day, single clones were picked from the medium and sequenced using primers LjU6-F (CTAGAGGTAGCTCTGTGCCTC) and P8ML-seq-R1 (GGGAAACGACAATCTGATC) for verification.

[0069] Example 3: Obtaining and phenotypic analysis of GmSMS1 transgenic knockout soybeans (decreased 100-grain weight of GmSMS1 transgenic knockout soybean recipient material)

[0070] 1. Obtaining genetically modified materials

[0071] Gene knockout was performed using the soybean variety Zhongji 602 as the recipient material. Genotyping was conducted on the T0 generation plants obtained from the transgenic strain: genomic DNA was extracted from leaves of each T0 generation plant, and PCR amplification was performed using transgenic detection primers. The PCR products were then sequenced and analyzed. The results showed that the knockout-positive plants exhibited Indel differences in the GmSMS1 sequence compared to the recipient material, indicating that the GmSMS1 gene had been successfully edited.

[0072] Seeds were harvested and sown from positive T0 generation knockout soybean plants to obtain T1 generation knockout mutant plants. DNA was extracted from leaves of the T1 generation mutants, and PCR amplification and product sequencing were performed using the same transgene detection primers. The results showed that the genotype was consistent with that of the T0 generation plants, but there were indel differences in the sequence with the recipient material. Seeds were harvested from homozygous and stably inherited T1 generation knockout mutant plants for T2 generation sowing and phenotypic identification.

[0073] 2. DNA extraction for positive identification of genetically modified soybeans

[0074] The leaf samples to be identified were placed in 2 mL centrifuge tubes, and genomic DNA was extracted using the classic CTAB method. The specific steps are as follows:

[0075] Take the preserved leaves and place them into numbered 2 mL centrifuge tubes. Add one steel ball to each tube. Immerse the centrifuge tubes in liquid nitrogen and grind them for 30 s using a grinder. Add 800 µL of CTAB extraction solution (2% CTAB, with 2% β-mercaptoethanol added before use) preheated at 65℃. Mix thoroughly and incubate at 65℃ for 45 min, inverting the tubes to mix every 10 min. After the water bath, add 5 µL of RNase A and incubate at 37℃ for 1 h.

[0076] Remove the centrifuge tube, add 800 µL of phenol:chloroform (volume ratio 25:24), mix thoroughly, and centrifuge at 4 ℃ and 12000 rpm for 10 min; transfer the supernatant to a new 2 mL centrifuge tube, add 600 µL of chloroform, mix by inversion, and centrifuge at 4 ℃ and 12000 rpm for 10 min.

[0077] Transfer the supernatant to a new centrifuge tube, add 500 µL of pre-chilled isopropanol, mix well, and incubate at -20 ℃ for 1 h to precipitate DNA; centrifuge at 4 ℃ and 12000 rpm for 10 min, and discard the supernatant; wash the precipitate 2–3 times with 75% ethanol; air dry the DNA precipitate at room temperature, and dissolve it in an appropriate amount of ddH2O.

[0078] Finally, DNA integrity was detected by 1% agarose gel electrophoresis, and DNA concentration was determined using NanoDrop One. The DNA was diluted to a working concentration of 50 ng / µL and stored at -20 ℃ for later use.

[0079] 3. Phenotypic Analysis

[0080] Knockout mutant families CR1 and CR2, with two different editing types, were selected and sown in the field in June, and cultivated and managed under normal natural conditions. After the plants matured, the 100-seed weight, seed length, and seed width of each individual plant were measured, using the recipient material Zhongji 602 as a control. The results are as follows: Figure 3As shown, compared with Zhongji 602 (ZJ602), the knockout mutants CR1 and CR2 showed significantly reduced seed weight, seed length, and seed width. The average seed weight per 100 seeds was 16.95 g for ZJ602, 12.35 g for CR1, and 13.58 g for CR2. Compared with ZJ602, the seed weight per 100 seeds of CR1 and CR2 decreased by 27% and 20%, respectively, and the differences were statistically significant.

[0081] In summary, through the above embodiments of the present invention, a stable genetic mutant sms1 with a significantly reduced 100-grain weight was obtained through EMS mutagenesis. The target gene was precisely located to a 99.6 kb region using BSA and map-based cloning techniques. Sequence alignment uniquely identified ORF5 (Glyma.10G022000), which encodes the histone methylation complex component EED, as the candidate gene GmSMS1. This gene in the mutant exhibits a single-base variation leading to the substitution of glycine for aspartic acid. Furthermore, CRISPR / Cas9 technology was used to perform targeted knockout of this gene in the soybean variety Zhongji 602, obtaining two homozygous mutant families, CR1 and CR2, with two independent editing types. Their 100-grain weight was significantly reduced by 27% and 20% respectively compared to the recipient control, and the grain length and width were also reduced synchronously. The experimental evidence obtained by cross-validation between the above-mentioned forward genetics (natural mutants) and reverse genetics (gene editing) reveals for the first time the positive regulatory function of the gene encoding the histone methylation complex component in soybean on 100-grain weight. Moreover, this regulatory mechanism is different from the previously reported pathways of cytochrome P450, lipid metabolism, sugar transport, or photosynthesis, providing a brand-new genetic resource and theoretical basis for high-yield molecular breeding of soybean.

[0082] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A gene regulating 100-grain weight in soybeans, characterized by: The gene is GmSMS1, derived from soybean, and is characterized in that the gene is a DNA molecule of the following type (1) or (2): (1) A DNA molecule with the sequence shown in SEQ ID NO.1; (2) DNA molecules that have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with the DNA sequence in (1) and encode a protein associated with the 100-grain weight of a plant.

2. The protein encoded by the soybean 100-seed weight regulatory gene GmSMS1 according to claim 1, characterized in that, The following proteins are examples: (a) A protein consisting of an amino acid sequence as shown in SEQ ID NO.2; (b) A protein derived from the sequence described in SEQ ID NO.2 by substitution and / or deletion and / or addition of one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.2, and which is related to the 100-grain weight of a plant.

3. The protein according to claim 2, characterized in that, The substitution and / or deletion and / or addition of one or more amino acid residues shall be the substitution and / or deletion and / or addition of no more than 10 amino acid residues.

4. A recombinant vector, expression cassette, transgenic cell line, or recombinant bacteria containing the gene described in claim 1.

5. The recombinant vector according to claim 4, characterized in that, The recombinant vector is a recombinant vector obtained by inserting the editing site of the gene described in claim 1 into the KpnⅠ and XbaⅠ recognition sites of the V135 vector.

6. Primers for amplifying the full length of the gene or any fragment thereof as described in claim 1.

7. The application of the soybean 100-seed weight regulating gene GmSMS1 as described in claim 1 in the cultivation of transgenic plants with high 100-seed weight, characterized in that, The gene is introduced into the target plant to obtain a transgenic plant; compared with the target plant, the transgenic plant has a higher 100-seed weight.

8. The application according to claim 7, characterized in that, The gene is introduced into the target plant using the recombinant vector as described in claim 4 or 5.

9. The application according to claim 7 or 8, characterized in that, The target plant is a dicotyledonous or monocotyledonous plant, specifically soybean.

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