Application of GmERF13 gene in improvement of phosphorus content and yield of soybean seeds

Abstract

The invention discloses application of a GmERF13 gene in improvement of phosphorus content and yield of soybean seeds. Researches show that the GmERF13 gene has significant influence on the phosphorus content and yield of transgenic soybeans, and the GmERF13 gene responding to low phosphorus stress has the function of negatively regulating the phosphorus content and yield of soybean seeds; the identification of transgenic materials of an over-expression vector and a knockout vector shows that the over-expression of the GmERF13 gene can reduce the phosphorus content of plant seeds and the yield of a single plant; after the GmERF13 gene is knocked out, the yield of soybeans can be obviously increased, the number and weight of pods and seeds are increased, and meanwhile the phosphorus content of the seeds is increased. Therefore, the GmERF13 can be used as a target of gene editing, plays an important role in improving the soybean phosphorus efficiency and increasing the soybean yield, and provides an important gene resource for increasing the soybean yield.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of genetic engineering and plant breeding technology. More specifically, it relates to the application of GmERF13 gene in improving soybean grain phosphorus content and yield. BACKGROUND

[0002] Soybean (Glycine max) is one of the five cereals, and its edible oil and various bean products are widely used in people's daily life. In addition, feed made from soybean meal is also widely used in the livestock industry. Soybean is rich in oil, protein, carbohydrates, crude fiber, minerals, vitamins, and various bioactive substances such as soybean polypeptide, soybean isoflavone, and soybean saponin, making the soybean industry involve multiple fields such as oil, meat, eggs, and milk, and has important economic value. China has become the world's largest soybean importer, and the demand for soybean products is increasing. At present, China's soybean industry is facing severe challenges, so improving soybean yield is the main goal of soybean breeding.

[0003] Phosphorus is one of the essential nutrients for plant growth and development, and is very important for maintaining normal physiological and biochemical activities in plants. The diffusion coefficient of phosphorus in soil is low and the flowability is poor, which makes the level of available phosphorus in soil that can be directly absorbed and utilized by plants low. The lack of available phosphorus in soil has become one of the problems that limit global agricultural production. Therefore, improving crop phosphorus efficiency is an important measure to achieve green agriculture.

[0004] AP2 / ERF (APETALA2 / ethylene responsive factor) is a class of transcription factors unique to plants, characterized by containing an incomplete conserved peptide segment consisting of about 60 amino acid residues, i.e. AP2 / ERF domain. AP2 / ERF transcription factors have been reported to play an important role in biological stress defense, salt and drought stress resistance, flowering time, seed development and root growth. Although ERF gene family has been cloned and reported to be involved in root growth and development in Arabidopsis and rice, its research in leguminous crops is still less. Existing research shows that GmERF1 gene can affect soybean root development by regulating hormone levels under low phosphorus stress environment, thereby promoting the absorption of phosphorus by soybean, and is a potential candidate gene for developing low-phosphorus-tolerant crops (Ruiyang, Wang, Xiaoqian, Liu, et al. Transcription factors GmERF1 and GmWRKY6 synergistically regulate low phosphorus tolerance in soybean. [J]. Plant Physiol, 2023 / 03 / 19;192.). However, whether AP2 / ERF transcription factors affect the yield of phosphorus-regulated leguminous crops has not been reported, and there is still a lack of gene resources that can improve soybean yield and improve soybean phosphorus efficiency. SUMMARY

[0005] The technical problem to be solved by the present application is to overcome the shortcomings of existing gene resources for improving soybean yield and phosphorus efficiency, and to provide the application of GmERF13 gene in improving soybean grain phosphorus content and yield.

[0006] The purpose of the present application is to provide the application of GmERF13 gene in negatively regulating soybean grain phosphorus content and improving soybean yield.

[0007] Another purpose of the present application is to provide the application of a preparation lacking or knocking out the GmERF13 gene as shown in SEQ ID NO. 1.

[0008] Still another purpose of the present application is to provide a method for improving the yield of soybean plants.

[0009] Still another purpose of the present application is to provide a product for improving soybean yield or grain phosphorus content.

[0010] The above purposes of the present application are achieved by the following technical solutions:

[0011] The application shows that GmERF13 gene has a significant influence on the phosphorus content and yield of transgenic soybeans, and the GmERF13 gene responding to low-phosphorus stress has a function of negatively regulating the phosphorus content and yield of soybean grains; through the construction and identification of transgenic materials of the overexpression vector and the knockout vector, it is shown that overexpression of GmERF13 gene can reduce the phosphorus content and yield per plant of seeds; and knocking out GmERF13 gene can obviously improve the yield of soybeans, increase the number and weight of pods and seeds, and improve the phosphorus content of seeds. Therefore, GmERF13 can be used as a target for gene editing, and has an important role in improving the phosphorus efficiency and increasing the yield of soybeans, and provides an important genetic resource for improving the yield of soybeans.

[0012] Further, the nucleotide sequence of the GmERF13 gene is shown as SEQ ID NO. 1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2.

[0013] The application provides an application of the GmERF13 gene in negatively regulating the phosphorus content of soybean grains and improving the yield of soybeans.

[0014] The application provides an application of the GmERF13 gene in improving the phosphorus efficiency and increasing the yield of soybeans or in preparing a product for improving the phosphorus efficiency and increasing the yield of soybeans.

[0015] The application provides an application of the GmERF13 gene in cultivating high-yield soybean plants.

[0016] The application provides an application of the GmERF13 gene in constructing transgenic materials with high grain phosphorus content and high yield.

[0017] The application provides an application of a preparation for deleting or knocking out the GmERF13 gene shown as SEQ ID NO. 1 in increasing the yield of soybeans or in preparing a product for increasing the yield of soybeans.

[0018] The application provides an application of a preparation for deleting or knocking out the GmERF13 gene shown as SEQ ID NO. 1 in increasing the phosphorus content of soybean grains or in preparing a product for the phosphorus content of soybean grains.

[0019] The application provides an application of a preparation for deleting or knocking out the GmERF13 gene shown as SEQ ID NO. 1 in breeding for improving the yield of soybean plants.

[0020] Preferably, the preparation is a plasmid, a vector, or a recombinant bacterium for deleting or knocking out the GmERF13 gene shown as SEQ ID NO. 1.

[0021] The application provides a method for breeding soybean plants with improved yield, wherein a GmERF13 gene is knocked out in the soybean plants by a gene knockout technique, or a preparation with a deleted or knocked out GmERF13 gene, or a recombinant vector containing the knocked out GmERF13 gene or a recombinant bacterium thereof is introduced into the plants to obtain improved plants with high yield.

[0022] The application also provides a product for improving the yield or grain phosphorus content of soybeans, which contains a preparation with a deleted or knocked out GmERF13 gene, or a recombinant vector containing the knocked out GmERF13 gene or a recombinant bacterium thereof.

[0023] The application has the following beneficial effects:

[0024] The application discloses application of a GmERF13 gene in improving the phosphorus content and yield of soybean grains. It is found in the application that the GmERF13 gene responding to low-phosphorus stress has a function of negatively regulating the phosphorus content and yield of soybean grains. Through identification of transgenic materials of an overexpression vector and a knockout vector, it is shown that overexpression of the GmERF13 gene can reduce the phosphorus content of plant seeds and the yield per plant. After the GmERF13 gene is knocked out, the yield of soybeans can be obviously improved, the number and weight of pods and seeds are increased, and the phosphorus content of seeds is improved, which provides an important genetic resource for improving the yield of soybeans. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 For construction of the overexpression vector and the knockout expression vector and identification results of the transgenic lines (A: schematic diagram of the structure of the GmERF13 overexpression vector; B: herbicide resistance detection of the GmERF13 overexpression transgenic soybean leaves, the scale = 1.2 cm; C: gene PCR detection of the transgenic lines; D: relative expression amount of GmERF13 in wild type (WT) and overexpression lines (OX1 and OX2) roots, the data in the figure are the average value and standard error of 4 times of repetition; the asterisk * indicates a significant difference compared with WT (Student's t-test; "***", P < 0.001); E: schematic diagram of the structure of the GmERF13 knockout vector and the target sequence, sgRNA represents guide RNA, UTR represents non-coding region, the yellow square box represents exon, upstream / downstream: upstream / downstream region; F: herbicide resistance detection of the GmERF13 knockout transgenic soybean leaves, the scale = 1.2 cm; G: GmERF13 editing target point PCR detection; H: nucleotide sequence, peak chart and amino acid sequence of the CRISPR / Cas9 targeting region of wild type (WT) and two homozygous knockout lines (KO1 and KO2), the red horizontal line represents the deleted base, and the asterisk * represents a stop codon; WT is wild type YC03-3 soybean; OX, overexpression; KO, knockout).

[0026] Figure 2 Figure 7 is a result chart of histochemical localization analysis of GmERF13 (A: histochemical localization of GmERF13 in soybean leaves, flowers, young pods and seeds under different phosphorus treatments, scale = 2 cm; B: histochemical localization of GmERF13 in soybean root system, i: whole root system of soybean; ii: single root; iv: secondary lateral roots of the basal root; the upper right graph is a local enlarged view of the red arrow, and the lower right graph is a local enlarged view of the blue arrow, scale = 2 cm (whole root system) and 0.2 mm (locally enlarged root system); +P: 250 μM KH2PO4, -P: 5 μM KH2PO4).

[0027] Figure 3 Figure 6 is a result chart of the effect of overexpression or knockout of GmERF13 on soybean yield (A: field harvest phenotype of wild type and transgenic lines (upper graph, scale = 10 cm) and mature seed morphology (lower graph, scale = 1 cm); B: pod number; C: seed number; D: seed dry weight; E: seed phosphorus content; the data in the chart are the average and standard error of 8 repetitions, and different letters indicate significant differences between WT, OX and KO lines (Duncan, P < 0.05). DETAILED DESCRIPTION

[0028] The present application will be further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0029] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0030] The soybean germplasm YC03-3 used in the following examples is from the Root Center of South China Agricultural University.

[0031] Example 1 Construction of vector

[0032] 1. Construction of overexpression (pTF101s-GmERF13-OX) vector

[0033] According to the GmERF13 genomic sequence (nucleotide sequence as shown in SEQ ID NO. 1, the amino acid sequence of the encoded protein as shown in SEQ ID NO: 2), the primer software was used to design and synthesize the primer. The cDNA of soybean variety YC03-3 was used as a template, and the specific primers of the designed GmERF13-OX gene forward and reverse fragments were used: SEQ ID NO. 3: 5'- GCGAGCTCGGTACCCGGGATGTCCTCTTCCTCTTCCTCCTT-3', and SEQ ID NO. 4: 5'- CTCTAGAGGATCCCCGGGTCAGGAACTGAGAGTGTACCCAGTG-3', to amplify the gene CDS.

[0034] The reaction system (50 μL) was: 2 x phata max buffer 25 μL, 2 μL of forward and reverse primers (10 mM) respectively, 2 mM dNTPs 1 μL, Phanta Max Super-Fidelity DNA Polymerase (Novagen, China) 1 μL, cDNA template 2 μL, ddH2O 18 μL.

[0035] The PCR system was: 95 °C pre-denaturation for 3 min, 95 °C denaturation for 15 sec, 58 °C annealing for 30 s, 72 °C recombination for 1 min, 72 °C final extension for 5 min, wherein the denaturation to recombination was repeated for 30 times.

[0036] After amplification, the PCR product was subjected to gel electrophoresis, and the target band was recovered and purified according to the instructions of the agarose gel kit (Meiji Biological, China). And using Clone Express II One Step Cloning Kit recombination exchange kit (Novagen, China), the fragment was connected to the Sam I enzyme cutting site of linearized pTF101s plasmid, and the E. coli competent cell was transformed. After sequencing, the GmERF13-OX plasmid was extracted and transformed into Agrobacterium tumefaciens EHA101, and the overexpression pTF101s-GmERF13-OX vector (A) was obtained. Figure 1 A).

[0037] 2. Construction of knock-out (pGES201-GmERF13-KO) vector

[0038] The target site of GmERF13, i.e., sgRNA, was designed through a website TargetDesign (http: / / skl.scau.edu.cn / targetdesign / ), and was entrusted to GenScript Biotech Co., Ltd. for synthesis. The sequences of the forward and reverse oligonucleotide chains were as follows: SEQ ID NO. 5: 5'-TGGTCGGTGGTGATATGGGC-3'; SEQ ID NO. 6: 5'-GCCCATATCACCACCGACCA-3', and annealing into a double strand.

[0039] The annealing system (50 μL) was as follows: 5 μL of each of the forward and reverse primers (GmERF13-sgRNA-F / R, at a concentration of 10 μM), 5 μL of NaCl solution (at a final concentration of 100 mM), 5 μL of Tris-HCl solution (pH 7.4, at a final concentration of 50 mM), and ddH2O was added to make up 50 μL.

[0040] The annealing program was as follows: 95 °C for 4 min, RAMP 0.1 °C / s-95 °C gradient cooling to 16 °C for storage.

[0041] The annealed double strand was linked to the Bsa I enzyme digestion site of the linearized pGES201z vector, and was transferred into E. coli competent cells. After sequencing, the GmERF13-KO plasmid was extracted, and was transferred into Agrobacterium tumefaciens EHA105, to obtain the knock-out pGES201-GmERF13-KO vector Figure 1 E).

[0042] 3. Construction of GmERF13 fusion GUS expression (GmERF13-pTF102-GUS) vector

[0043] The forward and reverse specific primers of GmERF13-GUS-F / R gene were obtained by involving and synthesizing soybean YC03-3 genomic DNA as a template: SEQ ID NO. 7: 5'-CTATGACATGATTACGAATTCCATATCTAGACCTATTACCCGC-3', and SEQ ID NO. 8: 5'-GACTGACCTACCCGGGGATCC GAAATGAGTTGAGGATGAGGG-3', and a 1926 bp fragment of GmERF13 promoter was amplified.

[0044] The reaction system (50 μL) was: 2 x phata max buffer 25 μL, forward and reverse primers (10 mM) 2 μL each, 2 mM dNTPs 1 μL, Phanta Max Super-Fidelity DNA Polymerase 1 μL, DNA template 2 μL, 50 mM MgCl2 2 μL, ddH2O 16 μL.

[0045] The PCR system was: 95 °C pre-denaturation for 3 min, 95 °C denaturation for 15 sec, 58 °C annealing for 30 s, 72 °C recombination for 2 min, 72 °C final extension for 5 min, wherein the denaturation to recombination was repeated for 30 times.

[0046] After amplification, the PCR product was subjected to gel electrophoresis, and the target band was recovered and purified according to the instructions of the agarose gel kit. Then, the fragment was inserted into the BamH I enzyme cutting site of the linearized pTF102 plasmid using the Clone Express II One Step Cloning Kit recombination exchange kit, and was transferred into the E. coli competent cell. After sequencing, the GmERF13-GUS plasmid was extracted and transferred into Agrobacterium tumefaciens EHA101, and the GmERF13 fusion GUS expression vector was obtained.

[0047] Example 2 Transgenic material

[0048] 1. Obtaining of the whole transgenic soybean material

[0049] (1) Seed germination: select soybean YC03-3 seeds, sterilize in chlorine gas (100 mL sodium hypochlorite + 4.2 mL hydrochloric acid) for 12-14 h, after sterilization, the seeds are placed in an ultra-clean bench and blown for 30-60 min, and then sown in MS germination medium and cultured in a 28 °C constant temperature light incubator for 4-5 d;

[0050] (2) Bacterial liquid preparation: EHA101 Agrobacterium containing GmERF13-OX or GmERF13-GUS plasmid or EHA105 Agrobacterium containing GmERF13-KO plasmid was inoculated into 50 mL YEP liquid medium, and cultured at 28 °C constant temperature incubator for 12-14 h, and the bacterial colonies were collected by centrifugation at 6000 rpm, and suspended in CM liquid to OD650 of about 1.0;

[0051] (3) Cotyledon node infection and co-cultivation: The soybean cotyledon 5 cm from the hypocotyl was cut with a sterile scalpel, the cotyledon was vertically cut and the bud was removed; 7-8 cuts were made on the cotyledon node with a scalpel and the cotyledon was soaked in CM liquid medium for 30 min; finally, the explant was transferred to the co-cultivation solid medium (CM solid medium) with the cut surface downward, the culture dish was sealed with an airtight plastic wrap and transferred to a 24°C incubator for dark culture for 3 days;

[0052] (4) Induction of shoot: the explant cultured in the dark for 3 days was transferred to the liquid shoot induction medium (SI medium) and washed for 2-3 min, and then transferred to the SI solid medium containing herbicide and antibiotic, the hypocotyl and cotyledon of the explant were inserted into the medium, and the culture dish was sealed with a medical air-permeable tape; then the explant was transferred to a 24°C incubator for light culture (18 h light / 6 h dark) for two weeks, and then the elongated hypocotyl was cut and transferred to a new SI solid medium for continuous culture under the same conditions for two weeks;

[0053] (5) Induction of shoot elongation: the cotyledon part of the differentiated explant was cut and transferred to the shoot elongation medium (SE medium) for 2-8 weeks; the SE solid medium was replaced every two weeks;

[0054] (6) Induction of rooting: when the regenerated shoot grew to about 3-5 cm, it was cut from the explant and transferred to the rooting medium (RM medium) for continuous culture, and after about two weeks, when the roots on the stem grew to more than 3 cm, the regenerated plant was taken out from the RM medium and transplanted into a hydroponic bottle for continuous culture, and after four weeks, it was transferred to a greenhouse for soil culture until pod setting.

[0055] 2. Identification of transgenic whole soybean material

[0056] (1) Identification of transgenic plants driven by GmERF13 promoter: GUS staining solution was used to detect the root system or leaf of the transgenic plant, and any part of the tissue that could be colored by the GUS staining solution was a positive plant.

[0057] (2) Identification of overexpression GmERF13 transgenic lines: three methods of herbicide resistance detection, transgenic soybean PCR detection and quantitative PCR detection were used to identify the transgenic soybean. The specific method is as follows:

[0058] A young trifoliate leaf was selected, half of the leaf was evenly smeared with herbicide and marked, and after 2-3 days, the leaf reaction was observed, if the leaf smeared with herbicide had no obvious change compared with the leaf without herbicide, it was possible to be a positive plant, then the plant DNA was extracted and subjected to the next PCR verification;

[0059] The DNA of the extracted wild type and the transgenic plants to be detected is used as a template, and the gene fragment is amplified by using the forward and reverse primers pTF101s-JC-F / R (SEQ ID NO. 11: 5'-CATTTGGAGAGGACACGC-3', and SEQ ID NO. 12: 5'-CAACACATGAGCGAAACC-3') of the pTF101s vector and the GmERF13 gene primers GmERF13-OX-F / R (SEQ ID NO. 3 and SEQ ID NO. 4) in pairs. If a gene fragment of about 750 bp can be amplified, the next step of verification is continued;

[0060] Finally, the total RNA of the wild type and the transgenic plants to be detected is extracted, and after reverse transcription into cDNA, the expression amount of GmERF13 is detected by fluorescence quantitative PCR. If the expression amount of GmERF13 is significantly up-regulated compared with the wild type, it is a transgenic line with overexpression effect.

[0061] The herbicide resistance screening results show that the leaves of the wild type (WT) plant turn yellow after being coated with herbicide, and the leaves of the transgenic line have no obvious change Figure 1 B); the PCR amplification results show that the DNA of the transgenic line can amplify a fragment of about 750 bp, while the wild type DNA shows no amplification band Figure 1 C); and the qRT-PCR results show that the expression amount of GmERF13 in the two overexpression transgenic lines OX1 and OX2 is significantly increased compared with WT Figure 1 D), and these results show that the overexpression effect of GmERF13 in the transgenic line is significant.

[0062] (3) Identification of GmERF13 knock-out transgenic lines: three methods of herbicide resistance detection, PCR detection and Sanger sequencing are used to identify the transgenic soybean in turn. The specific method is as follows:

[0063] Select young trifoliate leaves, evenly coat half of the leaves with herbicide and mark them, and observe the leaves after 2-3 days;

[0064] The DNA of the extracted wild type and the transgenic plants to be detected is used as a template, and the gene fragment is amplified by using the forward and reverse primers pTF101s-JC-F / R (SEQ ID NO. 11: 5'-CATTTGGAGAGGACACGC-3', and SEQ ID NO. 12: 5'-CAACACATGAGCGAAACC-3') of the pTF101s vector and the GmERF13 gene primers GmERF13-OX-F / R (SEQ ID NO. 3 and SEQ ID NO. 4) in pairs. If a gene fragment of about 750 bp can be amplified, the next step of verification is continued;

[0065] The DNA of the transgenic plant to be detected was extracted as a template, and a GmERF13 gene primer GmERF13-Cas9-F / R was used to amplify a fragment (SEQ ID NO. 9: 5'-GAGCCACGTGATCAAATAACTCC-3', SEQ ID NO. 10: 5'-CTCGGCGGTTTCGAATGTA-3'), and the amplification product was sequenced by the Sanger method. If the base sequence has an insertion or deletion and causes a sense mutation of the amino acid, it is a successful gene knockout transgenic line.

[0066] The herbicide identification results show that the transgenic plant leaves have no response to the herbicide, and the wild type (WT) leaves are yellow and dry ( Figure 1 F) The PCR amplification results show that a fragment of about 500 bp is amplified from the DNA of the transgenic plant ( Figure 1 G), indicating that the GmERF13-KO vector is successfully integrated into the plant genome. Further, primers are designed around 200 bp before and after the target point and PCR amplification is performed, and the editing effect is verified by Sanger sequencing. The sequencing peak chart clearly shows a single base peak, and KO1 and KO2 lines are deleted by 1 bp and 2 bp, respectively, which leads to a frame shift and premature termination of the GmERF13 protein amino acid sequence ( Figure 1 H). These results confirm that we have successfully obtained a homozygous transgenic line of GmERF13 gene knockout.

[0067] Example 3 Functional analysis of GmERF13

[0068] The three transgenic soybean whole plant materials identified in Example 2 were cultured for the following experiments:

[0069] 1. GmERF13 tissue chemical localization analysis

[0070] Water culture experiment of GUS transgenic material: select several GUS transgenic material soybean seeds of uniform size, clean and full. Wash the wet quartz sand and put it in a suitable size bread box. When sowing, the hilum is downward, and one seed is sown every 1 cm. After sowing, cover the surface with 1-2 cm of quartz sand, and spray the surface of the quartz sand with an appropriate amount of water. Cover the surface of the bread box with plastic wrap. Germinate in a constant temperature and light culture room for 4-5 days. After the seeds germinate, select well-germinated and uniform seedlings and transfer them to different nitrogen and phosphorus concentrations of modified soybean full nutrient solution. Adjust the pH value (5.8-6.0) every 3 days with 0.1 mM KOH or H2SO4. The aeration device is set to 15 minutes / hour during the culture period. Harvest the leaves and roots after 12 days of transplanting and perform GUS staining; at the same time, harvest flowers, pods and seeds at R2, R4 and R5 stages, respectively, and perform GUS staining.

[0071] Utilizing Pro GmERF13 :GUS transgenic soybean whole plant lines, the histochemical localization results of GmERF13 under different phosphorus levels are shown in Figure 2 Compared with normal phosphorus treatment, low phosphorus significantly enhanced GUS staining of soybean aboveground parts, including leaves, flowers and young pods, while no obvious GUS staining was observed in seeds Figure 2 A). In the roots, including the main roots, basal roots and their secondary lateral roots, the whole root system showed enhanced GUS staining under low phosphorus stress Figure 2 B), indicating that low phosphorus stress enhanced the expression of GmERF13.

[0072] 2, the effect of overexpression or knockout of GmERF13 on soybean yield and seed phosphorus content

[0073] The overexpression lines OX1 and OX2 identified in Example 2, as well as the knockout homozygous lines KO1 and KO2, were subjected to routine culture, and the field harvest phenotype, yield and mature seed morphology were counted.

[0074] As shown in Figure 3 , the change in the expression level of GmERF13 significantly affected the yield per plant of soybean Figure 3 A), compared with WT, the number of pods per plant of OX1 and OX2 lines decreased by 21.6% and 28.3% respectively Figure 3 B), the number of seeds decreased by 22.3% and 24.2% respectively Figure 3 C), and the dry weight of seeds decreased by 22.7% and 23.5% respectively Figure 3 D); the number of pods per plant of KO1 and KO2 lines increased by 35.4% and 33.1% respectively, the number of seeds increased by 45.2% and 39.9% respectively, and the dry weight of seeds increased by 34.7% and 31.5% respectively. At the same time, the phosphorus content of seeds of OX1 and OX1 lines decreased by 26.9% and 24.2% respectively; KO1 and KO2 lines increased by 40.5% and 41.5% respectively Figure 3 E), these results indicated that GmERF13 regulated the phosphorus content of seeds and the yield per plant of soybean plants.

[0075] In conclusion, the application discloses application of the GmERF13 gene in improving phosphorus content and yield of soybean grains.

[0076] The above embodiment is a preferred embodiment of the application, but the embodiment of the application is not limited by the above embodiment, and any change, modification, substitution, combination, simplification, which does not deviate from the spirit and principle of the application, should be an equivalent replacement mode, and all are included in the protection scope of the application.

Claims

1. Use of GmERF13 gene as shown in SEQ ID NO. 1 in negative regulation of soybean seed phosphorus content and improvement of soybean yield.

2. Use of GmERF13 gene as shown in SEQ ID NO. 1 in improvement of soybean phosphorus efficiency and improvement of soybean yield or in preparation of products for improving soybean phosphorus efficiency and improving soybean yield.

3. Use of GmERF13 gene as shown in SEQ ID NO. 1 in breeding of high-yield soybean plants.

4. Use of GmERF13 gene as shown in SEQ ID NO. 1 in construction of transgenic materials with high seed phosphorus content and high yield.

5. Use of preparations for deletion or knockout of GmERF13 gene as shown in SEQ ID NO. 1 in improvement of soybean yield or in preparation of products for improving soybean yield.

6. Use of preparations for deletion or knockout of GmERF13 gene as shown in SEQ ID NO. 1 in improvement of soybean seed phosphorus content or in preparation of products for soybean seed phosphorus content.

7. Use of preparations for deletion or knockout of GmERF13 gene as shown in SEQ ID NO. 1 in breeding of soybean plants with improved yield.

8. Use according to any one of claims 5 to 7, characterized in that, The preparation is a plasmid, a vector, or a recombinant bacteria for deletion or knockout of GmERF13 gene as shown in SEQ ID NO.

1.

9. A method for breeding soybean plants for improved yield, characterized in that, The GmERF13 gene as shown in SEQ ID NO. 1 is knocked out by gene knockout technology in soybean plants, or the preparation for deletion or knockout of GmERF13 gene as shown in SEQ ID NO. 1, or a recombinant vector containing the knockout GmERF13 gene or a recombinant bacteria thereof is introduced into the plants to obtain improved plants with high yield.

10. A product for increasing soybean yield or grain phosphorus content, characterized by, The preparation contains deletion or knockout of GmERF13 gene as shown in SEQ ID NO. 1, or a recombinant vector containing the knockout GmERF13 gene or a recombinant bacteria thereof.

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