Application of GmERF5 in regulating soybean seed size

Abstract

This application discloses the application of GmERF5 in regulating soybean seed size, belonging to the field of genetic engineering technology. The amino acid sequence of the protein encoded by the GmERF5 gene is shown in SEQ ID NO.1, the CDS sequence of the gene is shown in SEQ ID NO.2, and the full-length nucleotide sequence is shown in SEQ ID NO.3. This application has discovered and verified through a series of experiments that this gene or its encoded protein has a positive regulatory effect on soybean seed size. By increasing the expression level of this gene or its encoded protein, the weight, length, width, and cross-sectional area of ​​soybean seeds increase, thus providing a new approach for breeding high-yield soybeans.

Description

Technical Field

[0001] This application belongs to the field of genetic engineering technology, specifically relating to the application of GmERF5 or its encoded protein in regulating soybean seed size. Background Technology

[0002] Soybean (Glycine max) is one of the world's most important economic crops, and increasing soybean yield is a key focus of current breeding efforts. The main components of soybean yield are the number of plants per unit area, the number of seeds per plant, and the weight of 100 seeds. Seed size is not only a major agronomic trait in domestication and selection, but also the most direct indicator of soybean 100-seed weight; therefore, increasing soybean seed size is a crucial way to improve soybean yield.

[0003] Soybean seed size is a quantitative trait controlled by multiple genes and significantly influenced by the environment. Identified pathways regulating seed size include the ubiquitin-proteasome pathway, G protein signaling pathway, mitogen-activated protein kinase (MAPK) signaling pathway, HAIKU pathway, plant hormones, and transcription factors. Currently, the SoyBase genome database (https: / / www.soybase.org / ) records approximately 400 QTLs related to soybean seed size. Most QTLs are located in large chromosomal regions, making them less effective for crop improvement; only a few functional genes have been isolated from these QTLs. Therefore, identifying functional genes and their molecular regulatory mechanisms related to soybean seed size is crucial for improving soybean yield.

[0004] The APETALA2 / ERF family is a plant-specific transcription factor family containing a large number of members with at least one AP2 domain. These members are involved in regulating flower, embryo, and seed development in many crops, mediating seed size and weight, and playing a crucial role in seed yield. GmERF5, a member of the APETALA2 / ERF family, has been identified as being associated with soybean resistance to the pathogen Phytophthora sojae and resistance to abiotic stresses such as salt and drought. However, there are currently no reports of GmERF5 participating in the regulation of soybean seed size. Summary of the Invention

[0005] In view of this, the primary objective of this application is to provide the application of GmERF5 in regulating soybean seed size. In the early screening, the GmERF5 gene was obtained and a series of experiments were conducted to verify that GmERF5 is a positive regulator of soybean seed size and that soybean seed size is significantly correlated with the expression level of GmERF5 or its encoded protein.

[0006] To achieve the above objectives, this application adopts the following technical solution:

[0007] One aspect of this application discloses the application of GmERF5 in regulating soybean seed size, wherein the amino acid sequence of the protein encoded by the GmERF5 gene is shown in SEQ ID NO.1.

[0008] Another aspect of this application discloses a biological material containing nucleotide molecules that enhance the expression level of GmERF5 or its encoded protein as described in this application.

[0009] Another aspect of this application discloses the use of the gene GmERF5 or its encoded protein upregulated molecules, or biological materials, in at least one of the following:

[0010] (i) Increase soybean yield;

[0011] (ii) Producing high-yield soybeans;

[0012] (iii) Prepare products that increase soybean yield.

[0013] Another aspect of this application discloses a method for cultivating high-yield soybeans or increasing soybean yield, comprising:

[0014] Steps to increase the expression of GmERF5 or its encoded protein in soybean plants.

[0015] Another aspect of this application discloses a kit comprising the biological materials described in this application.

[0016] The beneficial effects of this application are:

[0017] This application experimentally verified that GmERF5 is a positive regulator of soybean seed size. Increasing the expression level of GmERF5 or its encoded protein can improve soybean yield, specifically manifested as increased seed weight, and / or increased seed length and width, and / or increased seed cross-sectional area. This application provides a novel method for increasing soybean yield, enabling the cultivation of high-yield soybeans through genetic engineering or artificial means. Attached Figure Description

[0018] Figure 1 In Embodiment 2 of this application erf5 Two knockout forms of mutant plants, erf5 -1 indicates a single target knockout within a 166bp coding region. erf5 -2 indicates the presence of dual-target knockout at 2bp and 30bp in the coding region.

[0019] Figure 2 For WT and erf5 Statistical analysis of mature seed morphology and paraffin section cell morphology of mutants; among which... Figure 2In the middle, 'a' indicates that the mature grains are significantly larger; Figure 2 In the middle b, the cell morphology of the seed paraffin section is shown, with a significantly increased cross-sectional area of ​​the cells. The scale bar length is 1000 μm. Figure 2 In the middle (c), cross-sectional analysis and cell size analysis of the grain section show that both the grain area and the grain cell area are significantly increased. express p <0.01.

[0020] Figure 3 To build GmERF5 Image of the pCAMBIA1305-EGFP vector in Arabidopsis thaliana -OE.

[0021] Figure 4 Phenotypic analysis of Arabidopsis plants overexpressing GmERF5; Figure 4 In the middle, 'a' indicates that overexpression of GmERF5 increases the height of Arabidopsis thaliana plants; Figure 4 In the middle b, the root length of Arabidopsis thaliana overexpressing GmERF5 on day 6 was not significantly different; Figure 4 In the middle c, the grains of Arabidopsis thaliana overexpressing GmERF5 were significantly larger; Figure 4 In the middle section, Arabidopsis thaliana overexpressing GmERF5 exhibited morphological characteristics including enlarged leaves; Figure 4 In the *E* group, overexpression of GmERF5 significantly increased the thousand-seed weight, seed length, and seed width of *Arabidopsis thaliana*. (ns) p >0.05; for p <0.05, for p <0.01. Detailed Implementation

[0022] The embodiments of this application will be clearly and completely described below. The technical solutions in the embodiments described below are exemplary and only possible technical implementations of this application, not all possible implementations. Those skilled in the art can combine the embodiments of this application to obtain other embodiments without creative effort, and these embodiments are also within the protection scope of this application.

[0023] The first aspect of this application discloses the application of the gene GmERF5, which regulates soybean seed size, in the regulation of soybean seed size. Through preliminary sampling combined with biological information analysis, this application precisely located the functional gene GmERF5 that regulates soybean seed size. The amino acid sequence of its encoded protein is shown in SEQ ID NO.1, the CDS sequence of this gene is shown in SEQ ID NO.2, and the full-length nucleotide sequence is shown in SEQ ID NO.3. The soybean reference genome version is... Glycine max Wm82.a6.v1.

[0024] It should be understood that in this application, GmERF5 can refer to either the corresponding gene or the protein it encodes. Those skilled in the art can understand its meaning in different contexts, which is not difficult for them.

[0025] In this application, GmERF5 was identified as a positive regulator of soybean seed size. Specifically, increasing the expression level of GmERF5 or its encoded protein can increase the weight of soybean seeds (e.g., 100-seed weight, 1000-seed weight, etc.), and / or increase seed width and length, and / or increase the cross-sectional area of ​​the seeds.

[0026] As described in this application, "expression level" can be the expression level of a gene (such as the expression level of mRNA) or the expression level of its encoded protein. The specific detection method can be a method known in the art or developed independently, such as PCR quantification or Western blotting, immunohistochemistry / immunocytochemistry, etc. Those skilled in the art have such capabilities, so they will not be described in detail here.

[0027] The second aspect of this application discloses a biological material containing nucleotide molecules that enhance the expression level of GmERF5 or its encoded protein.

[0028] In some examples, the “biological material” may be a recombinant vector obtained by inserting a nucleotide molecule of GmERF5 into an expression vector. By inserting GmERF5 as the target gene into the expression vector, a recombinant vector is formed. Under suitable conditions, the recombinant vector can achieve the expression of GmERF5, thereby achieving the large-scale expression of GmERF5.

[0029] Here, "expression vector" refers to an artificially constructed DNA molecule capable of carrying a target gene into a host cell and effectively expressing that gene using the host cell's transcription and translation systems. As a recombinant DNA construct, its specific type is not particularly limited; it can be of known or independently developed types in the art, such as plasmids or viruses. As a preferred example, the expression vector is a plasmid; in the specific example of this application, the expression vector is a standard universal vector.

[0030] In other examples, the term "biomaterial" refers to a transgenic cell line, which is a population of cells that, through genetic engineering techniques, stably integrates a foreign target gene (or a corresponding nucleic acid construct) into the host cell genome, or exists stably and heritably in the form of a foreign episome, and is capable of expressing the target protein. Typically, this is achieved by introducing a recombinant vector containing the target gene into host cells, followed by screening and culture.

[0031] In this application, "host cell" refers to any living cell capable of accepting exogenous nucleic acid constructs and supporting their replication, maintenance, and guidance of target gene expression, providing the necessary transcription, translation, post-translational modification, and energy metabolism systems for the recombinant vector. There is no particular limitation on the type of host cell; it can be any type of prokaryotic cell, eukaryotic cell, or animal cell. Specific examples of prokaryotic cells include, but are not limited to, *Escherichia coli* and *Bacillus subtilis*. Specific examples of eukaryotic cells include, but are not limited to, fungal cells such as *Saccharomyces cerevisiae*, *Pichia pastoris*, and *Komagataella phaffii*, as well as plant cells. Animal cells can be insect cells, mammalian cells, etc., which will not be specifically described here.

[0032] In this application, the process typically involves constructing and delivering nucleic acids containing the target gene into a host cell using physical, chemical, or biological methods, ultimately achieving the stable presence or integration of the target gene. Common methods used in this application include Agrobacterium-mediated transformation, but gene gun methods, electroporation, and others can also be employed. Those skilled in the art can choose appropriate techniques based on experimental objectives or research needs, and therefore there are no particular limitations.

[0033] The third aspect of this application discloses the use of upregulated molecules of the gene GmERF5, or biological materials, in at least one of the following:

[0034] (i) Increase soybean yield;

[0035] (ii) Producing high-yield soybeans;

[0036] (iii) Prepare products that increase soybean yield.

[0037] It is understood that the increase in soybean yield or high-yield soybeans mentioned in this application refers to an increase in the weight of soybean seeds, and / or an increase in the width and length of the seeds, and / or a larger cross-sectional area of ​​the seeds.

[0038] In this application, the term "upregulated molecule" refers to any chemical or biological factor capable of specifically enhancing the expression level or activity of the target gene GmERF5 or its encoded protein. It can be a plant hormone or its analogue, or a receptor agonist. In other examples, the upregulated molecule can be a specific transcription factor or its agonist. There are no particular limitations on the specific type, and the upregulated molecule can be determined experimentally.

[0039] The fourth aspect of this application discloses a method for cultivating high-yield soybeans or increasing soybean yield, comprising:

[0040] Steps to increase the expression of GmERF5 or its encoded protein in soybean plants.

[0041] Generally, methods well-known to those skilled in the art can be used. For example, as described above, after constructing the corresponding biomaterials, the biomaterials are introduced into the corresponding cells to achieve stable inheritance and expression of the target protein. This will not be elaborated further here.

[0042] The fifth aspect of this application discloses a reagent kit comprising the biological materials described in this application.

[0043] It is understood that the "reagent kit" of this application is an integrated solution product. In addition to the core reagents necessary to achieve the experimental objectives, it typically includes specialized consumables or reagents tailored to different technical requirements, as well as operating instructions. These operating instructions should at least specify the detailed operating steps. The reagent kit of this application can facilitate solutions for increasing soybean yield and breeding high-yield soybeans.

[0044] The present application will be further illustrated below with reference to specific embodiments. It should be noted that the specific embodiments below are for illustrative purposes only and do not limit the scope of the present application in any way.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application.

[0046] In addition, unless otherwise specified, methods without detailed conditions or steps are conventional methods, and the reagents and materials used are commercially available.

[0047] Example 1: GmERF5 Candidate gene discovery

[0048] In this embodiment, 146 soybean germplasm accessions grown in 14 environments across three major ecological regions of China were collected. These accessions were then identified using a genome-wide association study (GWAS), revealing significant correlations with soybean seed size traits. SW9-1Furthermore, by constructing a population of recombinant inbred lines (RILs), candidate genes were finely mapped. Combined with transcriptome sequencing and weighted correlation network analysis (WGCNA), key genes regulating grain size were screened. GmERF5 .

[0049] Example 2: erf5 Identification of knockout mutants and gene editing forms

[0050] 2.1 erf5 Construction of knockout mutants

[0051] In this embodiment, Williams 82 was used as the test soybean, and the well-known CRISPR / Cas9 gene editing technology was employed. Four target pairs were designed with Williams 82 as the background. GmERF5 Knockout, build erf5 mutant materials, respectively erf5-1 and erf5-2 The knockout targets are shown in Table 1:

[0052] Table 1 Knockout target sequences

[0053]

[0054] 2.2 erf5 Gene editing type identification

[0055] Extraction using CTAB method erf5 Leaf DNA from the knockout mutant was used as a template for cloning using the specific primers shown in Table 2. GmERF5 Gene sequence:

[0056] Table 2 Specific primers

[0057]

[0058] The amplification reaction was carried out using TAKARA's high-fidelity enzyme Primer STAR Max Premix (2×). The amplification reaction system and reaction program are shown in Tables 3 and 4, respectively.

[0059] Table 3 PCR reaction system

[0060]

[0061] Table 4 PCR reaction procedure

[0062]

[0063] The obtained PCR products were analyzed by agarose gel electrophoresis at a mass ratio of 1.5%. The PCR products were sent to a third-party institution, Sangon Biotech (Shanghai) Co., Ltd., for sequencing, and the gene editing mode was identified by sequence alignment using SnapGene.

[0064] The results are as follows Figure 1 As shown, erf5 Two knockout gene forms in mutant plants, among which, erf5 -1 indicates a single-target knockout of 166 bp in the coding region between positions 1 and 166. erf5 -2 indicates the presence of two target knockout sites with lengths of 30 bp and 2 bp between sites 29 and 58 and between sites 94 and 95 in the coding region, respectively.

[0065] Example 3: erf5 Phenotypic identification of knockout mutants

[0066] 3.1 Plant Culture

[0067] Soybean plants (including wild-type WT and wild-type soybeans) were cultivated using nutrient soil (black soil:vermiculite volume ratio of 3:1). erf5 The knockout mutant was planted in the soybean greenhouse of Anhui Agricultural University (20,000 Lux, 70% humidity, 28℃ / 8h during the day and 26℃ / 16h at night).

[0068] 3.2 Phenotypic Identification

[0069] Record the morphology of soybean plants at different stages; harvest the seeds after the plants mature and place them in a 40℃ oven for 3 hours to dehydrate; determine the weight of 100 seeds using a 0.01% balance.

[0070] Plant height, leaf area, grain length, and grain width were measured using ImageJ software; statistical analysis was performed using SPSS software; and tissue sections were analyzed using CaseViewer.

[0071] The steps for preparing paraffin slices are as follows:

[0072] (1) Fixation: Soybean leaves and seeds were fixed using FAA fixative. The materials were degassed and allowed to settle in the fixative before being refrigerated at 4°C overnight. The FAA fixative formula was (volume ratio) ethanol (70%): glacial acetic acid: formaldehyde = 18:1:1.

[0073] (2) Dehydration: First, prepare ethanol with different volume fractions of 30%, 50%, 70%, 80% and 95%. Then, wash the material with distilled water 3 times for 10 minutes each time. Soak the sample in the above volume fractions of ethanol for 30 minutes each time. Then wash it with anhydrous ethanol 3 times for 30 minutes each time (the second time, the anhydrous ethanol is replaced with toluidine blue staining solution).

[0074] (3) Clearing: Clear with xylene for 20 minutes; if dehydration is complete, the tissue will appear transparent; if dehydration is incomplete, there will be a white cloud-like appearance in the tissue, in which case the solution of anhydrous ethanol and xylene in a 1:1 mixture should be replaced. When using xylene for clearing, its evaporation and absorption of moisture from the air should be avoided, and it should be kept in an anhydrous state.

[0075] (4) Embedding and wax trimming: Prepare a small paper box for embedding (mark the box), place the material in the appropriate position and pour in liquid paraffin, let it cool and solidify. Then use a single-edged blade to cut off the excess paraffin around the wax block to make it a square of appropriate size, and trim the material to create a cut surface.

[0076] (5) Sectioning: Firmly attach the trimmed wax block to the wooden wax base, and then fix the material on the microtome. Adjust the blade to the appropriate position, turn the handle to cut a complete cross section, then adjust the section thickness to 8µm, continue slicing, cut continuous strips, take a glass slide, drop 1 drop of adhesive tablet on it, spread it evenly, attach the strip section to the glass slide, and then put it in an oven at 37℃ to dry for 3-4 days.

[0077] (6) Dewaxing, rehydration, staining and mounting: First soak in xylene for 30 min or place at 40℃ for 10 min, then soak in 1 / 2 xylene and 1 / 2 ethanol for 3 min, wash twice with anhydrous ethanol for 3 min each time, then use 95%, 80%, 70%, 50% and 30% ethanol for 3 min each time, stain with toluidine blue or fast green for 15 min each time, then use 30%, 50%, 70%, 80% and 95% ethanol for 30 s each time, then use anhydrous ethanol twice for 30 s each time, soak in 1 / 2 xylene and 1 / 2 ethanol for 3 min, then use xylene twice for 3 min each time, finally mount with mounting medium, remove xylene and dry.

[0078] (7) Microscopic examination: The prepared paraffin sections were observed, measured and photographed using an upright fluorescence digital microscope (ZEISS.Germany) and AutoCAD.

[0079] 3.3 Results Presentation

[0080] After testing, WT and erf5 The average 100-grain weights of -2 were 25.73±0.58g and 19.99±2.01g, respectively; WT and erf5 The particle lengths of -2 were 0.93±0.28 cm and 0.81±0.29 cm, respectively; WT and erf5 The particle widths of -2 were 0.81±0.21 cm and 0.73±0.34 cm, respectively. Compared to WT, erf5 The weight of 100 grains decreased by 22.31% at -2, while the grain length and width decreased by 12.90% and 9.88%, respectively, all of which were highly significant.p＜0.01 ). Figure 2 (a)

[0081] WT and erf5 The sliced ​​area of ​​the seeds at -2 mm² was 44.50 mm². 2 and 28.09mm 2 Compared to WT, erf5 -2% of the grain slice area decreased by 36.88%. Cytological analysis showed that, compared to WT, erf5- The cell area of ​​the seed slices in sample 2 decreased by 40.83%, which was highly significant. p<0.01 ). Figure 2 (b and c)

[0082] Example 4: GmERF5 -OE Arabidopsis thaliana construction and phenotypic identification

[0083] 4.1 Experimental Materials

[0084] Wild-type Arabidopsis thaliana (Col-0), expression vector pCAMBIA1305-EGFP, competent Escherichia coli cells Mach1-T1 and Agrobacterium tumefaciens GV3101.

[0085] 4.2 GmERF5 -OE Arabidopsis

[0086] (1) Vector primer design: The universal standard vector pCAMBIA1305-EGFP was selected (the pattern is shown in the figure). Figure 3 The Spe I and BamH I restriction sites in the d35S-EGFP region were designed for insertion. GmERF5 Coding region fragment. The complete sequence (starting and ending 20 bp upstream and downstream of the restriction site) and the target gene coding region sequence were uploaded to the Vazyme website to obtain primers with homologous arms. Primer specificity was then tested using NCBI.

[0087] Wild-type soybean leaf DNA extracted using the CTAB method was used as a template to amplify the target fragment. The PCR amplification and reaction procedures were the same as in Example 2. The vector primers are shown in Table 5.

[0088] Table 5 Specific primers

[0089]

[0090] (2) Double digestion of the vector: The pCAMBIA1305-EGFP vector was double-digested according to the system (37℃ / 4h, 80℃ / 20min) to obtain the double-digested products. The digestion system is shown in Table 6.

[0091] Table 6 Double enzyme digestion system

[0092]

[0093] (3) Homologous recombination: The purified DNA product and the double enzyme digestion product were subjected to homologous recombination using the ClonExpress II One Step Cloning Kit (Vazyme). The reaction system was prepared by dispersing each group on ice, mixing and centrifuging, and then reacting in a PCR instrument at 37°C for 30 min. The ligation product was immediately placed on ice to cool or stored in a freezer at -20°C. The recombination system is shown in Table 7.

[0094] Table 7 Homologous Recombination System

[0095]

[0096] (4) E. coli transformation: Mach-T1 competent cells were stored at -80°C and thawed on ice after removal. 1 μL of the above ligation product was added and the mixture was gently stirred at the bottom of the centrifuge tube. The mixture was placed on ice for 25 min and then transferred to a water bath for 42°C heat shock for 45 s. It was then quickly returned to ice and allowed to stand for 2 min. 700 μL of antibiotic-free LB liquid medium was added to the mixture in a pre-sterilized laminar flow hood. After mixing, the mixture was placed in a 37°C constant temperature shaker at 200 r / min for 1 h to recover. The centrifuge tubes were centrifuged at 5000 r / min for 1 min to collect bacteria. Approximately 100 μL of supernatant was collected in the laminar flow hood and gently mixed by pipetting. The mixture was then plated on LB solid medium containing Kans antibiotic. The plates were incubated upside down in a 37°C incubator for approximately 12 hours. Single colonies (round) were picked from a clean bench and placed in a 50 mL centrifuge tube containing 20 mL of LB broth containing Kans antibiotic. The tube was then incubated upside down in a 37°C shaker at 200 rpm for 9 hours. Positive colonies were detected using PCR and sequenced. The correct bacterial culture was then preserved (50% glycerol and bacterial culture were mixed 1:1 and stored at -80°C in a clean bench).

[0097] (5) Extraction of E. coli plasmids: The AxyPrep Plasmid Miniprep Kit was used for plasmid extraction. 4 mL of the above E. coli bacterial culture was added in batches to 2 mL sterile, enzyme-free centrifuge tubes. The tubes were centrifuged at 12000 rpm for 1 min, and the supernatant was discarded. 250 μL of Buffer S1 was added to the centrifuge tubes and mixed by pipetting. Then, 250 μL of Buffer S2 was added, and the tubes were inverted 4-6 times to fully lyse the bacteria until a clear solution was formed. 250 μL of Buffer S3 was added to the centrifuge tubes, and the tubes were inverted 4-6 times (until white flocculent material appeared). The mixture was then transferred to a centrifuge and centrifuged at 12000 rpm for 10 min.

[0098] (6) Agrobacterium competent cell transformation: Agrobacterium competent cells (GV3101) were stored at -80℃, then thawed on ice. 0.4 μL of recombinant plasmid was added and gently mixed. The mixture was then placed on ice for 5 min, frozen in liquid nitrogen for 5 min, placed in a 37℃ water bath for 5 min, and placed on ice for 5 min. 700 μL of antibiotic-free YEP culture medium was added to the mixture in a clean bench and cultured at 220 rpm for 2 h in a 28℃ constant temperature shaker. The cells were collected by centrifugation at 5000 rpm for 1 min in centrifuge tubes, transferred to a clean bench, and approximately 100 μL of supernatant was collected. The supernatant was gently mixed by pipetting and then plated onto YEP solid medium containing Kan and Rif antibiotics. The medium was inverted and cultured at 220 rpm for 20-24 h in a 28℃ constant temperature shaker. Positive colonies were detected and sequenced using bacterial culture PCR. The correct bacterial culture was preserved (50% glycerol and bacterial culture were mixed 1:1 and stored at -80℃ in a clean bench). The supernatant was transferred to a 2mL preparation tube and centrifuged at 12000 rpm for 1 min, discarding the filtrate. The preparation tube was returned to the centrifuge tube, 500 μL of Buffer W1 was added, and the mixture was centrifuged at 12000 rpm for 1 min, discarding the filtrate. The preparation tube was returned to the centrifuge tube, 700 μL of Buffer W2 was added, and the mixture was centrifuged at 12000 rpm for 1 min, discarding the filtrate; this process was repeated once. The preparation tube was returned to a 2mL centrifuge tube and centrifuged at 12000 rpm for 2 min. The preparation tube was transferred to a new 1.5mL centrifuge tube, 60 μL of ddH2O was added to the center of the membrane, and the mixture was incubated at room temperature for 2 min; then centrifuged at 12000 rpm for 1 min, discarding the preparation tube to obtain the target plasmid DNA. After measuring the concentration and mass using a micro spectrophotometer, store in a -20°C refrigerator.

[0099] (7) Arabidopsis thaliana cultivation: Wild-type Arabidopsis thaliana (Col-0) was cultivated using soil cultivation. Sterilized vermiculite and nutrient soil were mixed in a 3:1 ratio to form a substrate, which was then placed in a small flowerpot. Arabidopsis thaliana seeds were evenly scattered on the soil surface, covered with a film, and then punched holes in the film. The pot was placed in a 22℃ light incubator (16h light / 8h darkness, light intensity: 30000Lux, humidity: 60%) and transplanted after 7 days of growth.

[0100] (8) Flower infusion method: Transfer 1 mL of Agrobacterium tumefaciens culture containing the plant overexpression vector constructed above into a 15 mL centrifuge tube containing 2 mL of YEP culture medium (Kan, Rif), and incubate at 28℃ for 24 h in a constant temperature shaker at 220 r / min; after turbidity, transfer to a 50 mL centrifuge tube containing 10 mL of YEP culture medium (Kan, Rif), and incubate at 28℃ for 24 h in a constant temperature shaker at 220 r / min. Centrifuge the cultured bacterial solution at 5000 r / min for 10 min, collect the bacteria and discard the supernatant, wrap the centrifuge tube with aluminum foil to protect it from light; add the prepared infusion solution to adjust the OD to about 1.3. Select wild-type Arabidopsis thaliana with good growth and suitable growth stage, cut off the pods and flowering inflorescences for infection; soak the unflowered inflorescences in the prepared bacterial solution for 1 min, and transfer them to a dark environment for one day. Repeat the infusion 3 times, with an interval of one week between each infusion; water the plant the day before infusion.

[0101] (9) Identification of homozygous overexpressing Arabidopsis thaliana: Infected Arabidopsis thaliana seeds (T1) were collected, and positive Arabidopsis thaliana were screened using MS medium with hygromycin resistance. Plants with good growth after 7 days were identified as positive plants; they were then transplanted into prepared nutrient soil for cultivation. Seeds were harvested from each individual plant to screen for positive plants, and homozygous overexpressing lines were obtained by the T3 generation.

[0102] (10) Arabidopsis seed germination: Select dried Arabidopsis seeds, sterilize them in a clean bench, and proceed as follows. Place about 100 Arabidopsis seeds into a 1.5 mL centrifuge tube; add 12% sodium hypochlorite solution to the centrifuge tube, place it in a shaker to wash for 15 min, centrifuge for 1 s; discard the supernatant, add 1000 μL of sterilized ddH2O, place it in a shaker to wash for 3 min, centrifuge for 30 s, and repeat this step 7 times. Use a sterilized pipette tip to pick up Arabidopsis seeds and spot them individually on the streaked area of ​​MS medium; after completion, seal the plate with aluminum foil to protect it from light, invert it in a 4℃ refrigerator for vernalization for 3 days, transfer it to a plant tissue culture incubator after vernalization, and transplant it into nutrient soil after 7 days of growth.

[0103] 4.3 GmERF5 -OE Arabidopsis thaliana phenotypic identification

[0104] Phenotypic identification was performed according to section 3.2 of Example 3, and the results are as follows: Figure 4 As shown in the image.

[0105] In Arabidopsis thaliana, WT and GmERF5-OE The thousand-seed weights of the plants were 23.13±0.95 mg and 24.20±0.79 mg, respectively, compared to WT. GmERF5-OE The thousand-grain weight increased by 4.63%, which is a significant difference. p＜0.05 Compared to WT, GmERF5-OEThe particle length increased by 9.47%, which was highly significant. p＜0.01 ); compared to WT, GmERF5-OE The particle width increased by 6.24%, showing a significant difference. p＜0.05 ).

[0106] The above embodiments demonstrate that, GmERF5 It is a positive regulator of soybean seed size; GmERF5 Overexpression is expected to increase soybean yield.

[0107] It should be noted that this application is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments with the same structure and effect as the technical concept within the scope of this application are included in the technical scope of this application. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of this application, are also included in the scope of this application.

Claims

1. The application of GmERF5 in positively regulating soybean seed size, characterized in that, The amino acid sequence of the protein encoded by the GmERF5 gene is shown in SEQ ID NO.

1.

2. The application as described in claim 1, characterized in that, The CDS sequence of the GmERF5 gene is shown in SEQ ID NO.2, and the full-length nucleotide sequence is shown in SEQ ID NO.

3.

3. The application as described in claim 1 or 2, characterized in that, The positive regulation refers to increasing the expression level of GmERF5 or its encoded protein, thereby increasing the weight of soybean seeds, and / or increasing the seed width and length, and / or increasing the cross-sectional area of ​​the seeds.

4. The use of the gene GmERF5 as defined in any one of claims 1-3 in at least one of the following: (i) Increase soybean seed size; (ii) Preparation of large-grain soybeans; (iii) Prepare products that increase the size of soybean seeds.

5. The application as described in claim 4, characterized in that, The increase in soybean seed size or large-seed soybeans refers to an increase in the weight of soybean seeds, and / or an increase in the width and length of the seeds, and / or an increase in the cross-sectional area of ​​the seeds.

6. A method for cultivating large-seed soybeans or increasing soybean seed size, characterized in that, include: The step of increasing the expression of GmERF5 or its encoded protein as defined in any one of claims 1-3 in soybean plants.

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