Soybean coding gene gmrwos1 and application thereof in synergistically regulating soybean grain weight and oil content

Abstract

The application discloses a soybean coding gene GmRWOS1 and application thereof in synergistically regulating soybean kernel weight and oil content. The protein provided by the application is as follows: (a), (b) or (c): (a) a protein consisting of the amino acid sequence shown in SEQ ID NO. 1; (b) a protein derived from SEQ ID NO. 1 by substitution, deletion and / or addition of one or more amino acid residues and related to plant seed kernel weight and oil content; and (c) a protein consisting of an amino acid sequence having at least 75% homology with the amino acid sequence shown in SEQ ID NO. 1 and related to plant seed kernel weight and oil content. The regulating protein and the coding gene thereof have important significance in improving plant seed kernel weight and oil content and cultivating high-yield and high-oil plant varieties.

Description

Technical Field

[0001] This invention relates to the field of biotechnology and to the application of plant-encoding genes, particularly to the soybean-encoding gene GmRWOS1 and its synergistic regulation of soybean grain weight and oil content. Background Technology

[0002] my country is the origin of soybeans and the world's largest consumer and importer of soybeans. In 2022, soybean imports reached 91.08 million tons, with an import value of approximately US$50 billion. One of the reasons for the imbalance between domestic soybean supply and demand is the low yield per unit area of ​​soybeans in my country. Currently, the soybean varieties promoted for planting in my country have low oil content, mostly between 17% and 20%. According to the national standard for soybeans (GB 1352-2009), the quality indicators for high-oil soybeans are as follows: crude fat content ≥22.0% is classified as Grade 1 high-oil soybeans, crude fat content ≥21.0% as Grade 2, and crude fat content ≥20.0% as Grade 3.

[0003] Given the current imbalance between domestic soybean supply and demand, leveraging the exponentially growing genomic data and utilizing recombination and variation accumulated over thousands of generations of population evolution to clone key genes regulating soybean yield and quality, and then breeding high-yielding and high-quality soybean varieties through gene aggregation, is an important approach to addressing the current soybean supply-demand imbalance. Researchers have cloned several grain weight regulating genes through forward genetics, including PP2C-1, GmKIX8-1, GmST05, and ST1. For example, the GmKIX8-1 gene affects cell division by regulating the expression of the cell cycle regulator GmCYCD3;1-10, thereby regulating soybean grain weight. Furthermore, several important genes regulating lipid synthesis have been identified in soybeans, including GmMYB73, GmZF351, and GmWRI1. For instance, Hu et al. found that GmZF351 can activate the expression of lipid biosynthesis-related genes GmBCCP2, GmKASIII, GmDGAT1, and GmOLEO2, enhance the transcriptional activity of WRI1, and positively regulate lipid biosynthesis, thereby promoting lipid accumulation. Transcriptome association analysis of soybeans revealed that gene expression regulates grain weight and oil content in both synergistic and antagonistic ways. In the process of breeding superior soybean varieties, aggregating and synergistically regulating multiple yield- and quality-related phenotypes is more conducive to simultaneously improving soybean yield and quality.

[0004] Although genes regulating single traits such as soybean grain weight or oil content have been widely reported, few studies have elucidated key genes that synergistically regulate multiple traits, including grain weight and oil content. In this study, we validated a gene encoding a potassium-dependent sodium pump protein, GmRWOS1, which synergistically regulates soybean seed shape, grain weight, and oil content. Using the high-oil, high-yield soybean variety Williams 82 as a background, we knocked out and overexpressed the GmRWOS1 gene. Compared to the wild-type (Williams 82) control, knocking out GmRWOS1 significantly increased 100-grain weight by approximately 8.80% and oil content by approximately 6.92%. The average oil content of the knockout lines reached 22.047%, higher than most domestically cultivated soybean varieties. Furthermore, single-plant yield statistics showed a significant 13.82% increase in yield per plant in the knockout lines. In actual production, the combined regulatory effects of this gene on the two economic traits can be expected to significantly increase economic benefits while maintaining constant field production costs. The proteins and their encoding genes involved in this invention have broad application prospects in the breeding of high-oil and high-yield soybean varieties. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to synergistically regulate soybean grain weight and oil content.

[0006] To address the aforementioned technical problems, this invention first provides the encoding gene GmRWOS1 and its applications.

[0007] The protein provided by this invention, named GmRWOS1, is a coding gene derived from soybean (Glycine max (L.) Merrill), and is the application of any one of the following substances (a), (b), or (c) in soybean grain weight and oil content:

[0008] (a) A protein consisting of the amino acid sequence shown in SEQ ID NO.1;

[0009] (b) Proteins derived from SEQ ID NO.1 with one or more amino acid residues substituted and / or deleted and / or added, and which are related to plant grain weight and oil content;

[0010] (c) A protein consisting of an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the amino acid sequence shown in SEQ ID NO.1 and relating to plant grain weight and oil content.

[0011] SEQ ID NO.1 consists of 359 amino acid residues.

[0012] The substitutions and / or deletions and / or additions in (b) above may be caused by natural variation or artificial mutagenesis.

[0013] The proteins mentioned in (a), (b) or (c) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0014] Meanwhile, the gene GmRWOS1 encoding the protein is also within the scope of protection of this invention.

[0015] The gene may be a DNA molecule as follows (1) or (2) or (3):

[0016] (1) The DNA molecule shown in SEQ ID NO.2;

[0017] (2) DNA molecules that hybridize with the DNA sequence defined in (1) under strict conditions and encode proteins related to plant grain weight and oil content;

[0018] (3) A DNA molecule that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the DNA sequence defined in (1) and encodes a protein related to plant grain weight and oil content.

[0019] SEQ ID NO.2 consists of 1,077 nucleotides, all of which are coding sequences for the GmRWOS1 protein.

[0020] In addition, recombinant vectors containing the gene, knockout vectors targeting the gene, expression cassettes, transgenic cell lines or recombinant bacteria are all within the scope of protection of this invention.

[0021] In an embodiment of the present invention, a CRISPR / Cas9 knockout vector is used to replace the sequence between the SmaI / HindIII restriction sites of the knockout vector with an sgRNA sequence module designed for the gene GmRWOS1, thereby obtaining the knockout recombinant vector CRISPR / Cas9-GmRWOS1. The specific method is described in Example 3.

[0022] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes that can be expressed in plants, encoding enzymes that produce color changes or luminescent compounds (GUS genes, luciferase genes, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes). Examples include the nptII gene for resistance to kanamycin and related antibiotics, the bar gene for resistance to the herbicide phosphinic acid, the hph gene for resistance to the antibiotic hygromycin, the dhfr gene for resistance to methatrexate, the EPSPS gene for resistance to glyphosate, and the mannose-6-phosphate isomerase gene that provides the ability to metabolize mannose.

[0023] Primer pairs that amplify the full length of the gene or any fragment thereof are also within the scope of protection of this invention.

[0024] The primer pair may specifically be as follows (Ⅰ) or (Ⅱ):

[0025] (I) A primer pair consisting of the DNA shown in SEQ ID NO.3 and the DNA shown in SEQ ID NO.4;

[0026] (II) Primer pair consisting of the DNA shown in SEQ ID NO.5 and the DNA shown in SEQ ID NO.6.

[0027] This invention also protects a method for cultivating transgenic plants, which involves introducing a gene-editing vector into a target plant to obtain a transgenic plant with a higher oil content than the target plant. The transgenic plant is understood not only to include the first-generation transgenic plant obtained by transforming the target plant with the gene-editing vector, but also its progeny. For transgenic plants, the gene can be propagated within the species, or it can be transferred into other varieties of the same species using conventional breeding techniques, particularly commercial varieties. Introducing the gene-editing vector into the target plant inhibits the synthesis of the target protein in the plant, thereby improving the grain weight and oil content traits of the target plant.

[0028] The gene can be modified as follows before being introduced into the host to achieve better expression:

[0029] 1) Optimize according to actual needs to ensure that the knockout vector works efficiently and accurately; select different PAM types corresponding to Cas proteins from different bacterial species according to the experimental design to improve the accuracy of sgRNA; the knockout site should be located in the coding sequence (CDS) region and preferably at the front end of the protein or in an important functional domain; select targets with high editing efficiency and no off-target effects; increasing the number of sgRNAs to achieve effective knockout of a single gene or multiple genes is also a common practice in experimental design.

[0030] 2) In the promoter regions of the Cas gene region and the CRISPR unit, select promoters suitable for efficient expression in the corresponding crops to achieve a more effective knockout effect; the promoters may include constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-selective, and tissue-specific promoters; the selection of promoters will vary with the expression time and space requirements, and also depends on the target species; for example, tissue or organ-specific expression promoters, depending on the stage of the recipient's development; although it has been shown that many promoters derived from dicotyledons are functional in monocotyledons and vice versa, ideally, dicotyledonous promoters should be selected for expression in dicotyledons, and monocotyledonous promoters should be selected for expression in monocotyledons;

[0031] 3) Linking with a suitable transcription terminator can also improve the knockout efficiency of the gene of the present invention; for example, tml from CaMV, E9 from rbcS; any available terminator known to function in plants can be linked with the gene of the present invention.

[0032] 4) Introduce enhancer sequences, such as intron sequences (e.g., derived from Adhl and Bronzel) and viral leader sequences (e.g., derived from TMV, MCMV, and AMV).

[0033] In practice, the gene of this invention can also be targeted to specific cells. This can be achieved using existing techniques in the field. For example, by fusing a target gene sequence derived from a target organelle with the gene sequence of this invention and then introducing it into plant cells, targeting can be achieved.

[0034] The gene can be introduced into the target plant via the recombinant vector. The recombinant vector carrying the gene can be used to transform plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electrocoagulation, and Agrobacterium-mediated transformation, and the transformed plant tissues can be cultured into plants. The target plant can be either a monocotyledonous or dicotyledonous plant. The target plant can be a legume (such as soybean, birdsfoot, alfalfa, and water clover), an oilseed crop (such as rapeseed, sunflower, and corn), or an oilseed tree species. The dicotyledonous plant can be Arabidopsis thaliana, such as the Colombian ecotype Arabidopsis thaliana.

[0035] The application of the gene GmRWOS1 in the synergistic regulation of soybean grain weight and oil content is characterized by the fact that knocking out or reducing or inhibiting the expression of this gene can simultaneously increase soybean grain weight and oil content. Compared with regulating only a single trait, this can more efficiently improve the economic benefits of soybean crops.

[0036] This invention relates to the application of the GmRWOS1 gene knockout vector, expression cassette, transgenic cell line, or recombinant bacteria in increasing soybean grain weight and oil content. The oil content refers to the total oil content within the seed.

[0037] Beneficial effects:

[0038] This invention utilizes the CRISPR / Cas9 knockout system to knock out the gene GmRWOS1 in soybean, obtaining transgenic soybean lines with the gene knocked out, and performing phenotypic statistics and comparisons between wild-type controls and transgenic lines.

[0039] Experimental results showed that in small-scale field replicates, knocking out the GmRWOS1 gene significantly increased soybean grain weight and oil content, with average 100-grain weight and average oil content reaching 18.34 g and 22.047%, respectively. Compared to the wild-type control, the 100-grain weight increased significantly by approximately 8.80%, and the oil content increased by approximately 6.92%. Furthermore, single-plant yield statistics showed that the knockout line exhibited a significant 13.82% increase in yield per plant. In actual production, the combined regulatory effects of this gene on the two economic traits are expected to result in a significant increase in economic benefits while maintaining constant field production costs. The protein and its encoding gene involved in this invention have broad application prospects in the cultivation of high-oil-content plants. Attached Figure Description

[0040] Figure 1 Amplification of the coding nucleic acid sequence of the gene GmRWOS1 (left) and detection of the knockout vector CRISPR / Cas9-GmRWOS1 (right). Lane 1 represents the amplified GmRWOS1 fragment, and lane 2 represents the successfully constructed fragment of the knockout vector CRISPR / Cas9-GmRWOS1.

[0041] Figure 2 Expression levels of the gene GmRWOS1 in different soybean tissues.

[0042] Figure 3 Schematic diagram of the plant knockout vector CRISPR / Cas9-GmRWOS1.

[0043] Figure 4 Schematic diagram of the plant overexpression vector pFGC5941-GmRWOS1.

[0044] Figure 5 Molecular identification of soybean knockout line gmrwos1.

[0045] Figure 6 Molecular identification of soybean overexpression line GmRWOS1-OX1 / 2.

[0046] Figure 7 Plant types of control line, knockout line gmrwos1 and overexpression line GmRWOS1-OX1 / 2.

[0047] Figure 8 Differences in grain weight and oil content among control line, knockout line gmrwos1 and overexpression line GmRWOS1-OX1 / 2.

[0048] Figure 9 Differences in yield per plant among control line, knockout line gmrwos1, and overexpression line GmRWOS1-OX1 / 2. Detailed Implementation

[0049] The following examples are provided to help to better understand the present invention, but are not intended to limit the invention.

[0050] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0051] Unless otherwise specified, all experimental materials used in the following examples were commercially available. All primers used were synthesized by Beijing Qingke Biotechnology Co., Ltd.; the transgenic transformation process was performed by Jiangsu Weimi Biotechnology Co., Ltd. Unless otherwise specified, all percentages in the following examples refer to mass percentages. T2 generation represents seeds produced by self-pollination of T1 generation and the plants grown from them; T3 generation represents seeds produced by self-pollination of T2 generation and the plants grown from them.

[0052] The experimental materials used in the following examples were: Williams 82 (Wm82) soybean seeds, purchased from Jiangsu Weimi Biotechnology Co., Ltd.; the construction of the CRISPR / Cas9 recombinant vector, Agrobacterium transformation, and transgenic experimental procedures were all completed by Jiangsu Weimi Biotechnology Co., Ltd.

[0053] Example 1: Cloning of the soybean coding gene GmRWOS1

[0054] I. Designing specific primers for cloning genes

[0055] The start position of the gene Glyma.12G064800 on the genome was found on the SoyBase website as Gm12:4759743-4766201, and the genomic sequence of this gene was obtained. To amplify the coding region sequence of this gene, primers were designed at the start and stop points, with the sequences as follows:

[0056] GmRWOS1-F1:5'-ATGTCCGAACCTCATTCATCG-3' (SEQ ID NO.3);

[0057] GmRWOS1-R1:5'-CTATTGTGATTTTGGCTTGGCAATC-3' (SEQ ID NO.4);

[0058] II. Genomic sequence and coding sequence of the cloned gene GmRWOS1

[0059] DNA was extracted from soybean Williams 82 leaves using the CTAB method. Using the DNA as a template, the genomic sequence of the gene was amplified using high-purity thermostable DNA polymerase (Novizan, Jiangsu, China) and primer pair GmRWOS1-F1 / R1 (SEQ ID NO.3 and SEQ ID NO.4).

[0060] RNA was extracted from soybean Williams 82 leaves using the TriZol method and reverse transcribed to obtain the cDNA sequence of the gene. Using the cDNA as a template, the coding sequence of the gene was amplified using primers GmRWOS1-F1 / R1 (SEQ ID NO.3 and SEQ ID NO.4). Agarose gel electrophoresis was used to detect whether the amplified gene fragment conformed to the sequence size (…). Figure 1 The amplified sequence was used for subsequent experiments. The amplified sequence was then sent to the company (Qingke, Beijing, China) for Sanger sequencing to verify the correctness of the cloned sequence.

[0061] Example 2: Functional and tissue expression characteristics of the soybean encoding gene GmRWOS1

[0062] I. Function of the encoding gene GmRWOS1

[0063] Based on phenotypic data of soybean seed weight and oil content, genome-wide association analysis was performed on hundreds of natural soybean populations, identifying the candidate gene GmRWOS1, which is highly associated with seed weight and oil content. Therefore, a correlation study was conducted on the phenotypes of this gene in relation to seed weight and oil content.

[0064] II. Expression characteristics of GmRWOS1 in different organs of soybean

[0065] Analysis of transcriptome libraries from different tissues of soybean variety Williams 82 revealed differences in the expression levels of the gene GmRWOS1 across various tissues. Figure 2 Comparative analysis revealed that GmRWOS1 expression levels were relatively high in organs related to fruit setting, such as flowers and seeds, while expression levels were relatively low in organs related to vegetative growth, such as roots, stems, and leaves. Tissue expression level analysis showed that the gene was expressed in all tissues, with higher expression levels in seeds. This suggests that the gene is involved in many plant developmental processes and may be involved in soybean grain development. Therefore, a correlation study was conducted between this gene and grain weight and oil content phenotypes.

[0066] Example 3: Construction of GmRWOS1 plant overexpression and knockout vectors

[0067] I. Construction of GmRWOS1 plant knockout vector

[0068] Based on the Glyma.12G064800(GmRWOS1) cDNA sequence information retrieved from SoyBase, and using the Huazhong Agricultural University CRISPR-P 2.0 website (http: / / crispr.hzau.edu.cn / CRISPR2 / ), different types of PAM sites corresponding to different types of CRISPR systems in soybean crops were selected to screen for reference target sequences. Based on the off-target rate parameter and GC content, two specific sgRNAs with the lowest off-target rate and suitable GC content were selected, and their sequences are as follows:

[0069] GmRWOS1-sgRNA1:5'-GTAGAAAACCTATCCTTGACTGG-3'(SEQ ID NO.5);

[0070] GmRWOS1-sgRNA2:5'-AATGTTTTCTGGTATGAGTCAGG-3' (SEQ ID NO. 6). GmRWOS1-sgRNA1 is located on the third exon of the GmRWOS1 gene, and GmRWOS1-sgRNA2 is located on the fifth exon of the GmRWOS1 gene. The target sequence and direct repeat (DR) sequences were arranged alternately, and the expression of this module was driven by the U6 promoter, forming a CRISPR unit. A CRISPR unit containing the specific sgRNA sequence (SEQ ID NO. 11) was obtained through gene synthesis (performed by Nanjing Genscript Biotech Co., Ltd.). The CRISPR unit was then used to replace the sequence between the SmaI / HindIII restriction sites in the CRISPR / Cas9 knockout vector, ultimately yielding the corresponding knockout vector CRISPR / Cas9-GmRWOS1 (SEQ ID NO. 11). Figure 1 and Figure 3 ).

[0071] II. Construction of GmRWOS1 plant overexpression vector

[0072] 1. RNA was extracted from the leaves of soybean variety Williams 82 and reverse transcribed into cDNA.

[0073] 2. Design specific primer pairs containing BamHI and NcoI linker sequences as follows:

[0074] GmRWOS1-F2: 5'-tacatttacaattaccatggATGTCCGAACCTCATTCATCG-3' (SEQ IDNO.7);

[0075] GmRWOS1-R2: 5'-ctctagactcacctaggatccCTATTGTGATTTTGGCTTGGCAATC-3' (SEQ ID NO. 8).

[0076] 3. Using the cDNA from step 1 as a template, perform PCR with the specific primer pair from step 2, and recover the PCR product.

[0077] 4. Digest the PCR product with restriction endonucleases BamHI and NcoI, and recover the digested product.

[0078] 5. The pFGC5941 vector was digested with restriction endonucleases BamHI and NcoI, and the vector backbone was recovered.

[0079] 6. Ligate the enzyme digestion product from step 4 with the vector backbone from step 5 to obtain the ligation product.

[0080] 7. Sequencing of the ligation product revealed the recombinant expression vector pFGC5941-GmRWOS1 (the original plasmid was pFGC5941, with the DNA shown in Sequence Listing 2 inserted between the BamHI and NcoI restriction sites after the CaMV 35S promoter). The recombinant expression vector pFGC5941-GmRWOS1 is shown below. Figure 4 As shown.

[0081] Example 4: Obtaining and identifying GmRWOS1 transgenic soybeans

[0082] I. Identification of Transformed Soybeans and Genetically Modified Plants

[0083] 1. The recombinant expression vectors CRISPR / Cas9-GmRWOS1 and pFGC5941-GmRWOS1 were introduced into Agrobacterium EHA101 to obtain recombinant Agrobacterium.

[0084] 2. By infecting the axillary meristem of the cotyledonary node with Agrobacterium, the recombinant Agrobacterium was transferred into Williams82. After the growth stabilized, it was transferred to nutrient soil for culture. At the same time, the transformation efficiency was identified, and the positive T0 plants were harvested and propagated.

[0085] 3. Harvest T1 generation individual plants, sow seeds from each individual plant separately, and continue screening to observe the segregation of T2 generation. Repeat this process until T3 generation to obtain the genetically stable homozygous knockout line gmrwos1 and homozygous overexpression line mRWOS1-OX1 / 2.

[0086] II. Molecular Identification of GmRWOS1 Transgenic Plants

[0087] 1. Molecular identification of soybean knockout line (gmrwos1) transgenic GmRWOS1 gene

[0088] DNA was extracted from leaves of the T3 generation homozygous line (gmrwos1) and control plants at stage V2. The sequences were obtained using pre-designed specific primers as follows:

[0089] GmRWOS1-F3: 5'-TCCTAGAAGGGGGAGGATGT-3' (SEQ ID NO.9);

[0090] GmRWOS1-R3: 5'-ACCAGTGACGAAAGACCATACA-3' (SEQ ID NO. 10);

[0091] Using DNA as a template, PCR amplification was performed, yielding an amplification product of approximately 400 bp. Sequencing revealed varying degrees of knockout at the target site in the transgenic lines, leading to premature termination of translation of the coding gene. Specific knockout details and sequencing results are as follows: Figure 5 As shown.

[0092] 2. Molecular identification of soybean overexpression line transgenic with GmRWOS1 gene (GmRWOS1-OX1 / 2)

[0093] RNA was extracted from leaves of the T3 generation homozygous line (GmRWOS1-OX1 / 2) and control plants at stage V2, and reverse transcribed into cDNA. The cDNA was then used as a template for RT-PCR identification using primer pairs composed of GmRWOS1-F4 and GmRWOS1-R4. The results are shown in the table below. Figure 6 The primer sequences used are as follows:

[0094] GmRWOS1-F4: 5'-AAGATGGCATTTCCTCCCGCA-3' (SEQ ID NO. 12);

[0095] GmRWOS1-R4: 5'-GGCTCCAAAGTTGAACAATCCA-3' (SEQ ID NO. 13);

[0096] III. Phenotypic Analysis of GmRWOS1 Transgenic Soybeans

[0097] The experimental samples are as follows: T3 generation soybean knockout line transgenic to GmRWOS1 (gmrwos1), T3 generation soybean overexpression line transgenic to GmRWOS1 (GmRWOS1-OX1 / 2), and control line Williams 82.

[0098] 1. Determination of particle weight phenotype

[0099] When both transgenic and control lines were planted in the same field environment, no significant differences were observed in plant type, number of branches, and other agronomic traits between the transgenic and control lines. Figure 7 These materials were harvested individually and placed in an oven at 45℃ for 48 hours for complete drying. The number of soybeans per plant, seed length, seed width, and 100-seed weight were statistically analyzed using an intelligent seed analysis system (Zhejiang Top Cloud Agriculture Technology Co., Ltd.).

[0100] 2. Determination of seed oil content

[0101] Oil content refers to the percentage of total fatty acids by mass in the seed.

[0102] The method for determining oil content is as follows: After thorough grinding of the seeds, weigh 100 mg, add 500 μL of isopropanol solution, mix thoroughly, and centrifuge overnight at 37 degrees Celsius; centrifuge at 3000 rpm for 3 minutes, and collect the supernatant into a new centrifuge tube (pre-weigh the centrifuge tube weight W0); add another 500 μL of isopropanol to the remaining powder, mix thoroughly, centrifuge at 3000 rpm for 3 minutes, collect the supernatant into the same centrifuge tube, and then place the centrifuge tube in a fume hood (24 h) to allow the isopropanol to completely evaporate; finally, weigh the centrifuge tube again (W1). The change in weight of the centrifuge tube before and after centrifugation is the weight of extracted lipids (W1-W0).

[0103] 3. Phenotypic analysis of soybeans transgenic with the GmRWOS1 gene

[0104] Phenotypic data analysis of transgenic soybean materials and control lines revealed that the average 100-seed weight and average oil content of the GmRWOS1 knockout transgenic lines reached 18.34 g and 22.047%, respectively. Compared with superior domestic soybean varieties, the oil content was significantly higher, and the 100-seed weight was also within the range of superior varieties. Compared with the experimental control lines, the 100-seed weight increased significantly by approximately 8.80%, and the oil content increased by approximately 6.92%, verifying the function of this gene in synergistically regulating seed weight and oil content. Figure 8 Meanwhile, we found that, under the same field conditions and production processes, knocking out the GmRWOS1 gene significantly increased soybean yield per plant by approximately 13.82%. Figure 9 Overexpression of the GmRWOS1 gene revealed that, compared to the control lines, the overexpressing lines exhibited a phenotype characterized by smaller grain length and width, reduced grain weight, and decreased oil content. Figure 8 ).

[0105] Transgenic experiments involving the knockout and overexpression of the GmRWOS1 gene demonstrated that the GmRWOS1 gene is related to soybean seed development and participates in the regulation of seed weight and oil content formation. Among them, knocking out the GmRWOS1 gene can significantly increase both seed weight and oil content, thereby increasing the yield per soybean plant and exhibiting superior oil content compared to excellent domestic soybean varieties.

Claims

1. Genes GmRWOS1 Its application in regulating soybean grain weight and oil content is characterized by... Knocking out or reducing / inhibiting the expression of this gene can increase soybean grain weight and oil content. GmRWOS1 The nucleotide sequence is shown in SEQ ID NO.

2.

2. Genes GmRWOS1 The application of knockout carriers in improving soybean grain weight and oil content is characterized by, The aforementioned gene GmRWOS1 The nucleotide sequence is shown in SEQ ID NO.2.

Images