Use of gmgid1-2 gene in regulation of soybean plant architecture, nitrogen use efficiency, high yield and high oil content

Abstract

The use of a soybean GmGID1-2 gene in the regulation of soybean plant architecture, nitrogen use efficiency, high yield and high oil content. The GmGID1-2 gene has a CDS sequence as shown in SEQ ID NO: 1. The knockout of the GmGID1-2 gene can improve plant architecture (reducing plant height, and increasing branch number, node number of main stem, and pod number), increase stem diameter and stem strength, enhance nitrogen fixation in root nodules, and improve yield and seed oil content. For the pleiotropic gene GmGID1-2, by means of gene editing technology, two new mutations are generated in GmGID1-2, resulting in premature termination of a protein. The knockout of the GmGID1-2 gene simultaneously realizes the improvement of multiple traits in crops such as soybean.

Description

Application of GmGID1-2 gene in regulating soybean plant type, nitrogen use efficiency and high yield and high oil

[0001] This application claims priority to the Chinese patent application No. 202410616141X filed on May 17, 2024, and titled “Application of GmGID1-2 gene in regulating soybean plant type, nitrogen use efficiency and high yield and high oil”, the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present application belongs to the field of agricultural genetic engineering, and relates to the application of soybean GmGID1-2 gene in regulating plant type, nitrogen use efficiency and high yield and high oil of crops and plants such as soybean. BACKGROUND

[0003] Plant height is one of the important agronomic traits for agricultural production improvement. Rice and wheat have realized great green revolution by reducing plant height, improving resistance to lodging and increasing close planting (Peng et al., 1999 [1] ; Khush 1999 [2] ; Evenson et al., 2003 [3] ). Soybean originated in China, is an important food crop in the world, and is the main source of oil in the world (Graham et al., 2003 [4] ). Compared with rice, wheat and corn, the yield of soybean is far behind. The yield per mu of rice, wheat and corn can easily exceed 1000 kg, but the yield per mu of soybean is less than half of that. Rice, wheat and other crops produce semi-dwarf plants through mutants such as sd1 (Spielmeyer et al., 2002 [5] ) and Rht1 (Gale et al., 1985 [6] ), which promote the increase of tiller number under high-fertilizer and close planting conditions to achieve yield increase (Wu et al., 2020 [7] ). As a pod crop, soybean differs from rice and wheat, which are single ear top crops, in that it needs sufficient node number and branch number to ensure a certain number of pods and seeds (Liu et al., 2020 [8] ). Therefore, improving soybean plant type, in addition to appropriately reducing plant height and increasing resistance to lodging, increasing main stem node number, branch number, pod number per plant and seed number per pod has an important influence on increasing soybean yield per plant.

[0004] Although the green revolution has greatly improved the yield of rice, wheat and other crops, semi-dwarf genes have inhibited the use efficiency of inorganic nitrogen fertilizer by plants (Swarbreck et al., 2019 [9]). Therefore, in recent years, how to realize green and sustainable development of agriculture, low input and high output has become the core of modern agricultural development (Li et al., 2018

[0010] ; Wu et al., 2020 [7] ; Liu et al., 2021

[0011] ). As a representative crop of symbiotic nitrogen fixation between legumes and rhizobia, soybean biological nitrogen fixation can reduce the application of chemical fertilizers and help achieve green and sustainable development of agriculture.

[0005] References:

[0006] 1. Peng J, Richards DE, Hartley NM, et al.,. (1999). 'Green revolution' genes encode mutant gibberellin response modulators [J]. Nature 400: 256-261.

[0007] 2. Khush GS. (1999). Green revolution: preparing for the 21st century [J]. Genome 42: 646-655.

[0008] 3. Evenson RE, Gollin D. (2003). Assessing the impact of the green revolution, 1960 to 2000 [J]. Science 300: 758-762.

[0009] 4. Graham PH, Vance CP. (2003). Legumes: importance and constraints to greater use [J]. Plant Physiol 131: 872-877.

[0010] 5. Spielemeyer W, Ellis MH, Chandler PM. (2002). Semidwarf (sd-1), "green revolution" rice, contains a defective gibberellin 20-oxidase gene [J]. Proc Natl Acad Sci U S A 99: 9043-9048.

[0011] 6. Gale MD, Youssefian S, Russell GE. (1985). Dwarfing genes in wheat[J].

[0012] 7. Wu K, Wang S, Song W, et al.,. (2020). Enhanced sustainable green revolution yield via nitrogen-responsive chromatin modulation in rice[J]. Science 367.

[0013] 8. Liu S, Zhang M, Feng F, et al.,. (2020). Toward a "Green Revolution" for Soybean[J]. Mol Plant 13: 688-697.

[0014] 9. Swarbreck SM, Wang M, Wang Y, et al.,. (2019). A roadmap for lowering crop nitrogen requirement[J]. Trends Plant Sci 24: 892-904.

[0015] 10. Li S, Tian Y, Wu K, et al.,. (2018). Modulating plant growth-metabolism coordination for sustainable agriculture[J]. Nature 560: 595-600.

[0016] 11. Liu Y, Wang H, Jiang Z, et al.,. (2021). Genomic basis of geographical adaptation to soil nitrogen in rice[J]. Nature 590: 600-605.

[0017] SUMMARY

[0018] The purpose of the present application is to overcome the above-mentioned deficiencies in the prior art, and to provide the application of soybean GmGID1-2 gene in regulating soybean plant type, nitrogen use efficiency and high yield and high oil

[0019] Another purpose of the present application is to provide a mutated GmGID1-2 gene and its application.

[0020] The purpose of the present application can be achieved by the following technical solutions:

[0021] The application of GmGID1-2 gene in soybean variety improvement, the CDS sequence of GmGID1-2 gene is shown as SEQ ID NO. 1, knocking out GmGID1-2 gene can improve plant type, increase stem strength, enhance nodule nitrogen fixation, and increase yield and seed oil content.

[0022] In the present application, the nucleotide sequence shown in SEQ ID NO. 1 is specifically:

[0023] In the present application, the amino acid sequence of the protein encoded by the GmGID1-2 gene CDS sequence is shown as SEQ ID NO. 2, specifically:

[0024] As a preferred embodiment of the present application, the improved plant type includes reducing soybean plant height, increasing stem strength, significantly increasing the number of branches, stem nodes, pods, and grains of soybean.

[0025] As a further preferred embodiment of the present application, the number of pods is the number of pods per plant and / or the maximum number of pods per cluster.

[0026] As a further preferred embodiment of the present application, the number of grains is the number of grains per plant and / or the number of grains per pod.

[0027] As a preferred embodiment of the present application, the method for knocking out GmGID1-2 gene is gene editing technology or gene silencing VIGS technology.

[0028] As a further preferred embodiment of the present application, the sgRNA used for knocking out GmGID1-2 gene by gene editing technology is selected from sgRNA-1 or sgRNA-2, wherein the sense strand of sgRNA-1 is shown as SEQ ID NO. 3 or SEQ ID NO. 4, and the sense strand of sgRNA-2 is shown as SEQ ID NO. 7 or SEQ ID NO. 8.

[0029] The application of GmGID1-2 gene editing system in improving plant type, increasing stem strength, enhancing nodule nitrogen fixation, and increasing yield and seed oil content.

[0030] As a preferred embodiment of the present application, the application of GmGID1-2 gene editing system in reducing soybean plant height, increasing stem strength, significantly increasing the number of branches, stem nodes, pods, grains, and grains per plant, increasing soybean yield and seed oil content, promoting soybean root growth and soybean nodule symbiosis, and improving biological nitrogen fixation ability.

[0031] As a further preferred embodiment of the present application, the pod number is the number of pods per plant and / or the maximum number of pods per cluster.

[0032] As a further preferred embodiment of the present application, the pod number is the number of pods per plant and / or the maximum number of pods per cluster.

[0033] As a preferred embodiment of the present application, the gene editing system contains sgRNA of GmGID1-2 gene, and the sgRNA is selected from sgRNA-1 or sgRNA-2, wherein the sense strand of sgRNA-1 is shown in SEQ ID NO. 3 or SEQ ID NO. 4, and the sense strand of sgRNA-2 is shown in SEQ ID NO. 7 or SEQ ID NO. 8.

[0034] The mutated GmGID1-2 gene has a CDS sequence shown in SEQ ID NO. 5 or SEQ ID NO. 9.

[0035] The mutated GmGID1-2 gene described in the present application is used in soybean variety improvement. The sequence of the mutated GmGID1-2 gene causes the premature termination of protein translation in soybean, the protein sequence is shortened, and the mutated GmGID1-2 gene can improve plant type, increase stem strength, nitrogen use efficiency, nodule nitrogen fixation, and increase yield and seed oil content. Preferably, the soybean plant height is reduced, the stem strength is increased, the number of branches, the number of nodes on the main stem, the number of pods, the number of grains per plant, and the grain weight per plant are significantly increased, the yield and seed oil content of soybean are improved, the growth of soybean root system is promoted, the symbiosis of soybean root nodule is improved, and the biological nitrogen fixation ability is improved. Beneficial effects:

[0036] The present application discovers a one-factor-multiple-effect gene GmGID1-2, and two new mutations of GmGID1-2 are produced by gene editing technology to cause premature termination of protein. Knocking out the GmGID1-2 gene can simultaneously improve the following multiple traits:

[0037] (1) Knocking out the GmGID1-2 gene improves plant type, reduces soybean plant height, and significantly increases stem diameter (Figure 5); significantly improves the stem strength in the field of soybean (Figure 6); increases the number of branches, the number of nodes on the main stem, the number of pods per plant (including the number of three-pod and four-pod), the number of grains per plant, and the number of grains per pod (Figure 7), and the maximum number of pods per cluster (Figure 8), which are important factors for increasing the yield of soybean.

[0038] (2) Knocking out the GmGID1-2 gene improves the grain weight per plant and yield of soybean, and increases the seed oil content of soybean. Gene editing of GmGID1-2 shows that knocking out the gene can improve the grain weight per plant and yield of soybean (Figure 9); and increase the seed oil content of soybean (Figure 10).

[0039] (3) Knockout of GmGID1-2 gene promotes soybean root growth and nodule symbiosis, and improves the biological nitrogen fixation ability of soybean

[0040] Gene editing of GmGID1-2 found that knockout of the gene can significantly promote soybean root growth, such as root fresh weight, root dry weight, and nodule symbiosis, such as nodule number and nodule dry weight (Figure 11). Significantly improve the biological nitrogen fixation ability of soybean, including nitrogenase activity, acylurea content and nitrogen content (Figure 12).

[0041] (4) Overexpression of GmGID1-2 gene improves the absorption of inorganic nitrogen fertilizer by soybean

[0042] Overexpression of GmGID1-2 gene can significantly improve the absorption rate of inorganic nitrogen fertilizer by soybean plants under low nitrogen and high nitrogen conditions (Figure 13).

[0043] In addition, similar improvements to the gene, or its homologous genes or gibberellin signaling pathway-related genes, especially knockout materials created by gene editing of GmGID1-2, have important value in improving the plant type of soybean and other crops and plants, improving yield, seed oil content and biological nitrogen fixation. Using gene editing technology or other technologies (such as gene silencing: VIGS, etc.) to edit other sites of GmGID1-2 gene to achieve knockout or silencing and achieve loss of function, theoretically, the same results as this application can be obtained. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1. pCAMBIA3301 vector map;

[0045] Figure 2. Editing site and editing of GmGID1-2 gene in GmGID1-2 knockout lines;

[0046] Figure 3. Verification results of GmGID1-2 overexpression transgenic lines; Note: GmGID1-2 overexpression transgenic lines were obtained by GaMV35S strong promoter; Data are mean ± SE, n = 3, statistical analysis significance was performed using two-tailed Student's t-test; **P<0.01, ***P<0.001, ****P<0.0001;

[0047] Figure 4. Obtaining of GmGID1-2 gene edited soybean lines; (A) Editing GmGID1-2 gene produces two knockout lines GmGID1-2 KO-1 (+1bp) and GmGID1-2 KO-2 (-11bp), the recipient material is Williams82, and the figure is the sequencing peak result of the new mutant gene produced after editing GmGID1-2; (B) GmGID1-2KO The mutation site, GmGID1-2 KO The mutation sites are highlighted in red; (C) Control materials Williams 82 and gene-edited line GmGID1-2 KO-1 GmGID1-2 KO-2 Protein 3D structure prediction (https: / / swissmodel.expasy.org) and terminator location;

[0048] Figure 5. Knocking out the GmGID1-2 gene reduces soybean plant height and increases stem diameter, while overexpression of the GmGID1-2 gene increases soybean plant height and decreases stem diameter; (A) Williams 82, GmGID1-2 OE and GmGID1-2 KO The morphology of the strain at the first and third compound leaves stage, scale bar is 5cm; (B~C) Williams 82, GmGID1-2 OE and GmGID1-2 KO The plant height (B) and stem diameter (C) data of the line are the mean ± SE (n=3). At the α level of 0.05, one-way ANOVA and Duncan's multiple test were used to determine statistical significance.

[0049] Figure 6. Knocking out the GmGID1-2 gene increases soybean stem strength; (A-B) Soybean stem (A) and stem strength (B) in Hefei (summer 2023); Scale bar is 1 cm; Data are mean ± SE (n=18), and statistical significance was determined using the two-tailed Student's t-test; ****P<0.0001;

[0050] Figure 7. Knocking out the GmGID1-2 gene increases the number of soybean branches, main stem nodes, number of pods per plant (including three- and four-pod pods), number of seeds per plant, and number of seeds per pod; (A) Hefei (Summer 2023) Williams 82 and GmGID1-2 KO Morphology of the strains at maturity, scale bar 10cm; (B~G)Williams 82 and GmGID1-2 KO The plant type and yield-related traits of the line were compared, including the number of branches (B), the number of nodes on the main stem (C), the number of grains per plant (D), the number of pods per plant (E), the average number of grains per pod (F), and the number of single, two-, three-, and four-pod pods per plant (G). Data are presented as mean ± SE, n = 18. Statistical significance was analyzed using the two-tailed Student's t-test. NS indicates no significance, ***P < 0.001, ****P < 0.0001.

[0051] Figure 8. Knocking out GmGID1-2 gene increases the maximum cluster number of soybean; (A-B) The maximum cluster phenotype (A) and the maximum cluster number (B) per plant in Hefei (summer 2023); the scale bar is 2 cm; n = 18; yellow arrows indicate the maximum cluster of soybean; data are means ± SE (n = 18); statistical significance was determined using a two-tailed Student's t-test; ***P < 0.001, ****P < 0.0001;

[0052] Figure 9. Knocking out GmGID1-2 gene increases the grain weight per plant and the plot yield of soybean; (A) Grain weight per plant of soybean in Hefei summer 2023, data are means ± SE, n = 18; (B) Plot yield of soybean in Hefei summer 2023, data are means ± SE, n = 3; statistical significance was determined using a two-tailed Student's t-test; *P < 0.05, ****P < 0.0001;

[0053] Figure 10. Knocking out GmGID1-2 gene increases the seed oil content of soybean; Seed oil content of soybean in Hefei field; data are means ± SE (n = 18), statistical significance was determined using a two-tailed Student's t-test; ***P < 0.001;

[0054] Figure 11. Knocking out GmGID1-2 gene promotes root growth and nodule symbiosis of soybean; (A) Root system (scale bar is 5 cm) and nodule (scale bar is 0.5 cm) phenotype after 28 days of inoculation with rhizobia; (B-E) Root fresh weight (B), root dry weight (C), nodule number (D), nodule dry weight (E); data are means ± SE, n = 4, statistical significance was determined using one-way ANOVA and Duncan's multiple range test at the 0.05 level of significance;

[0055] Figure 12. Knocking out GmGID1-2 gene improves the biological nitrogen fixation capacity of soybean; Statistical analysis of allantoin content (A), nitrogenase activity (B) and nitrogen content (C); data are means ± SE (n = 4), statistical significance was determined using one-way ANOVA and Duncan's multiple range test at the 0.05 level of significance;

[0056] Figure 13. Overexpression of GmGID1-2 gene improves the absorption of inorganic nitrogen fertilizer by soybean; Statistical analysis of N-NO3 15 NO3 - under the conditions of 0.125 mM and 1.25 mM 15 N-NO3 -Absorption rate; data are means ± SE (n = 3), statistical significance was determined using a two-tailed Student's t-test; *P < 0.05, **P < 0.01. DETAILED DESCRIPTION

[0057] Terminology:

[0058] GmGID1-2: GID1 is the abbreviation of GIBBERELLIN INSENSITIVE DWARF1, GmGID1-2 is one of the soybean GID1 family in this patent application.

[0059] Gene: A gene is the entire nucleotide sequence required to produce a polypeptide chain or functional RNA.

[0060] Promoter: A promoter is a DNA sequence recognized by RNA polymerase that binds and initiates transcription, most often located upstream of the 5' end of a structural gene.

[0061] Overexpression: Also known as overexpression, refers to the expression (transcription) of a gene higher than the normal amount of expression, resulting in a large amount of mRNA and the corresponding protein product.

[0062] Gene editing: refers to the process of modifying specific targets in the genome of an organism using technologies such as CRISPR-Cas9. Efficient and precise implementation of gene insertion, deletion or replacement, thereby changing its genetic information and phenotypic characteristics.

[0063] sgRNA: short for small guide RNA. It is part of the CRISPR-Cas9 system, used to specifically recognize the sequence of the target gene, usually 20 bp (excluding linker).

[0064] NCBI: National Center for Biotechnology Information.

[0065] PCR: short for Polymerase Chain Reaction.

[0066] Example 1 Vector construction

[0067] 1. Overexpression vector construction

[0068] In this experiment, the overexpression vector for stable transformation of soybean is pCAMBIA3301 (Figure 1), in which the promoter is derived from the pBA002 vector, and the GaMV35S promoter from the Cauliflower mosaic virus is used; containing GUS (b-glucuroidase), Bar reporter gene.

[0069] (1) First, select the appropriate double enzyme cutting site (double enzyme cutting site Asc I and Pac I), design the one-step cloning primer of GmGID1-2 gene by software (CE Design V1.03) as follows:

[0070] F: tctagaggatctcgaggcgcgccATGGCTGGCAGCAACCAA (SEQ ID NO. 11);

[0071] R: attcgagctcactagttaattaaTTAACAGTCAGAATCAGAATTGACAAAG (SEQ ID NO. 12).

[0072] PCR amplification of the target sequence was performed, and the PCR amplification product was purified and recovered by a purification kit (Takara, Japan), obtaining the purified product of the target insertion sequence;

[0073] (2) Second, the E. coli storing the pCAMBIA3301 vector plasmid was expanded in LB medium with Kana (Kanamycin) resistance, and the pCAMBIA3301 vector plasmid was obtained by a plasmid extraction kit (Axygen, China). The vector plasmid was double enzyme cut by restriction enzymes (New England Biolabs, NEB), and the enzyme cut plasmid was purified and recovered, obtaining a linearized vector. The double enzyme cutting system is as follows:

[0074] (The enzyme cutting system was reacted at 37°C for 30 min)

[0075] (3) Finally, the recombination of the target sequence and the linearized vector was completed by using a homologous recombination enzyme (Vazyme, China), and the system was as follows:

[0076] (The recombination system was reacted at 37°C for 30 min)

[0077] The constructed recombinant plasmid was transformed into EHA105 Agrobacterium, which was used for stable transformation of soybean (the method is as follows).

[0078] 2. Gene editing vector construction

[0079] This example uses the CRISPR-Cas9 system with Bas1 as the enzyme cutting site. The sgRNA is recombined into the pGES201 vector (Figure 2) by using T4 ligase to construct the gene editing vector. The vector construction method is as follows:

[0080] (1) Design of sgRNA:

[0081] The CDS of GmGID1-2 gene was designed using the website (http: / / cbi.hzau.edu.cn / CRISPR2 / ), and a restriction site adapter was added and synthesized;

[0082] Sense oligonucleotide F: sgRNA-1: 5'-GGATTGTGGTGTCTGTGAACTACCGG-3' (SEQ ID NO. 3)

[0083] Antisense oligonucleotide R: sgRNA-1: 5'-AAACCCGGTAGTTCACAGACACCACA-3' (SEQ ID NO. 4)

[0084] Sense oligonucleotide F: sgRNA-2: 5'-GGATTGGCGGTCACCGGAGCATCGG-3' (SEQ ID NO. 7)

[0085] Antisense oligonucleotide R: sgRNA-2: 5'-AAACCCGATGCTCCGGTGACCGCCCA-3' (SEQ ID NO. 8)

[0086] (2) Annealing of oligonucleotides to double-stranded:

[0087] Sense oligonucleotide (F primer 10 mM) 5 μl

[0088] Antisense oligonucleotide (R primer 10 mM) 5 μl

[0089] NaCl (100 mM final concentration)

[0090] Tris-Cl pH 7.4 (50 mM final concentration)

[0091] Add water to 50 μl

[0092] 95℃ 4min

[0093] Gradient cooling (RAMP) 0.1℃ / S (95℃ to 16℃)

[0094] Keep 16℃

[0095] (3) Linearization of the vector:

[0096] BsaI enzyme digestion 1 μg pGES201 vector (resistance kana)

[0097] (4) Ligation transformation

[0098] The constructed recombinant plasmid was transformed into K599 Agrobacterium rhizogenes for cotyledon node root identification. The recombinant plasmid with editing effect was transferred into EHA105 Agrobacterium for stable transformation of soybean variety Williams 82. The method is as follows:

[0099] The EHA105 bacteria carrying the sgRNA vector were streaked in YEB plates containing Rif and Kana resistance, and single colonies were picked after 2 days of incubation in 3 ml YEB containing Rif and Kana. The bacteria were collected for soybean explant infection.

[0100] Full and high-activity soybean seeds were selected, and the seed coat surface was wiped with 75% ethanol with a cotton gauze to remove dust. After sterilization of the selected seeds by chlorine sterilization, the soybean seeds were soaked in sterile water for 16-24 h. The soybean seeds were cut into two explants along the embryo axis using a surgical knife and tweezers in a clean bench.

[0101] The explants were incubated in the resuspended infection solution for 30 min, and then the infected explants were cultured in co-culture solid medium (CCM) for 5 d (16 h / 8 h, 23°C). Then, they were transferred to bud induction solid medium (SIM) for 14 d (16 h / 8 h, 23°C), which required 2 periods of culture. Then, they were transferred to bud elongation solid medium (SEM) for 14 d (16 h / 8 h, 23°C), which required 4 periods of culture. The elongated seedlings in this process were identified, and the positive T0 plants were transferred to rooting medium (GM) for rooting, then transplanted and acclimated to obtain overexpression positive plants. The homozygous plants were used for subsequent related experiments.

[0102] Soybean stable transformation medium formula:

[0103] 3. Obtaining of GmGID1-2 overexpression transgenic lines

[0104] In the background of soybean variety Williams 82, three transgenic lines overexpressing GmGID1-2 gene were obtained by pCAMBIA3301 overexpression vector, and were named GmGID1-2 OE-1 , GmGID1-2 OE-2 and GmGID1-2 OE-3 , respectively. Compared with Williams 82, the overexpression up-regulation multiples were 106, 125 and 153 times, respectively (Figure 3).

[0105] 4. Obtaining of GmGID1-2 gene editing soybean lines

[0106] In the background of soybean variety Williams 82, the GmGID1-2 gene was edited by CRISPR-Cas9 technology, an insertion or deletion mutation (InDel) was generated in the GmGID1-2 gene, and two new mutant genes of GmGID1-2 were obtained. Compared with the original GmGID1-2 gene, 1bp was inserted into GID1-2-KO1(+1bp), and the corresponding mutant gene sequence is shown as SEQ ID NO. 5, specifically:

[0107] The amino acid sequence of the protein encoded by GID1-2-KO1(+1bp) is shown as SEQ ID NO. 6, specifically

[0108] 11bp was deleted in GID1-2-KO2(-11bp), and the corresponding mutant gene sequence is shown as SEQ ID NO. 9, specifically:

[0109] The amino acid sequence of the protein encoded by GID1-2-KO2(-11bp) is shown as SEQ ID NO. 10, specifically:

[0110] which leads to premature termination of translation of GmGID1-2, and two corresponding GmGID1-2 knockout lines are obtained, named GmGID1-2 KO-1 and GmGID1-2 KO-2 (FIG. 4).

[0111] Example 2

[0112] The following investigates the biological functions of the mutant strains and overexpression strains constructed in Example 1

[0113] 1. Knockout of GmGID1-2 gene reduces soybean plant height and increases stem diameter

[0114] Through the identification of transgenic strains, it is found that overexpression of GmGID1-2 gene significantly promotes soybean plant elongation, and the stem diameter is significantly thinned. Knockout of GmGID1-2 gene significantly reduces soybean plant height, and the stem diameter is significantly thickened (FIG. 5).

[0115] 2. Knockout of GmGID1-2 gene increases soybean stem strength

[0116] Analysis of soybeans planted in summer field plots in 2023 found that the stem strength of GmGID1-2 KO lines in the field was significantly higher than that of Williams 82 (FIG. 6).

[0117] 3. Knocking out GmGID1-2 gene increases soybean branch number, main stem node number, pod number per plant (including three-pod and four-pod number), seed number per plant and seed number per pod

[0118] Statistical analysis of soybean branch number, main stem node number, pod number and seed number in summer field trials in 2023 found that GmGID1-2 KO The branch number, main stem node number, pod number per plant, seed number per plant, seed number per pod, three-pod number and four-pod number of the strain were significantly higher than those of the control Williams 82 (Figure 7).

[0119] 4. Knocking out GmGID1-2 gene increases the maximum cluster pod number of soybean

[0120] Analysis of soybeans planted in summer fields in 2023 found that GmGID1-2 KO The strain had more pods on the soybean internodes, and statistical analysis of the maximum cluster pod number per plant found that GmGID1-2 KO The maximum cluster pod number of the strain was significantly higher than that of Williams 82 (Figure 8).

[0121] 5. Knocking out GmGID1-2 gene increases soybean seed weight per plant and field plot yield

[0122] Analysis of soybeans planted in summer field plots in 2023 found that GmGID1-2 KO The seed weight per plant and field plot yield of the strain were significantly higher than those of Williams 82 (Figure 9).

[0123] 6. Knocking out GmGID1-2 gene increases soybean seed oil content

[0124] Using a near-infrared whole-grain analyzer (Foss, Denmark), the oil content of mature soybean seeds harvested in summer fields in 2023 was determined, and it was found that the oil content of GmGID1-2 KO The seed oil content of the strain was significantly higher than that of the control Williams 82 (Figure 10).

[0125] 7. Knocking out GmGID1-2 gene promotes soybean root growth and rhizobial symbiosis

[0126] Soybean is a typical representative of biological nitrogen fixation, so we analyzed the biological nitrogen fixation of GmGID1-2 KO The strain, and 28 days after inoculation with rhizobium (USDA110), we found that the GmGID1-2 KO The strain significantly increased root system (dry weight and fresh weight), nodule number and nodule dry weight (Figure 11).

[0127] 8. Knocking out GmGID1-2 gene improves the biological nitrogen fixation ability of soybean

[0128] Meanwhile, we detected the biological nitrogen fixation of GmGID1-2 KO and found that the acetylene content, nitrogenase activity and nitrogen content of GmGID1-2 KO were significantly higher than those of Williams 82 (Figure 12). This indicates that GmGID1-2 KO has higher biological nitrogen fixation ability.

[0129] 9. Overexpression of GmGID1-2 gene improves soybean inorganic nitrogen fertilizer absorption

[0130] Through overexpression of GmGID1-2 gene by 35S promoter, it is found that the function enhancement can significantly improve the absorption rate of soybean plants to inorganic nitrogen fertilizer under low nitrogen and high nitrogen environment (Figure 13).

[0131] The above are preferred embodiments and corresponding examples of the present application. It should be noted that, for those skilled in the art, without departing from the inventive concept of the present application, a number of modifications and improvements can be made, including but not limited to the adjustment of proportions, processes, amounts and reaction vessels, such as the use of continuous flow reactors, which are within the scope of protection of the present application. The above are preferred embodiments and corresponding examples of the present application. It should be noted that, for those skilled in the art, without departing from the inventive concept of the present application, a number of modifications and improvements can be made, including but not limited to the adjustment of proportions, processes, amounts, which are within the scope of protection of the present application.

Claims

1. Application of GmGID1-2 gene in soybean variety improvement, characterized in that, The CDS sequence of the GmGID1-2 gene is shown as SEQ ID NO. 1; the application includes knocking out or silencing the GmGID1-2 gene to improve plant type, increase stem strength, enhance nodule nitrogen fixation, promote soybean root growth, and increase yield and seed oil content; or includes overexpressing the GmGID1-2 gene to significantly improve the absorption of inorganic nitrogen fertilizer by soybeans.

2. Use according to claim 1, characterized in that, The improved plant type includes reducing soybean plant height, increasing stem strength, increasing soybean stem diameter, and significantly increasing the number of branches, stem nodes, pods, and grains of soybeans.

3. Use according to claim 2, characterized in that, The pod number is the number of pods per plant and / or the maximum number of pods per cluster.

4. Use according to claim 2, characterized in that, The grain number is the number of grains per plant and / or the number of grains per pod.

5. The use according to claim 1, characterized in that, The method for knocking out the GmGID1-2 gene is gene editing technology or gene silencing VIGS technology.

6. Use according to claim 5, characterized in that, The sgRNA used for knocking out the GmGID1-2 gene by the gene editing technology is selected from sgRNA-1 or sgRNA-2, wherein the sense strand and the antisense strand of sgRNA-1 are shown as SEQ ID NO. 3 or SEQ ID NO. 4, and the sense strand and the antisense strand of sgRNA-2 are shown as SEQ ID NO. 7 or SEQ ID NO.

8.

7. A gene editing system for the GmGID1-2 gene in improving plant type, increasing stem strength, enhancing nodule nitrogen fixation, promoting soybean root growth, and increasing yield and seed oil content; the CDS sequence of the GmGID1-2 gene is shown as SEQ ID NO.

1.

8. Use according to claim 7, characterized in that, The improved plant type includes reducing soybean plant height, increasing soybean stem diameter, and increasing the number of branches, stem nodes, pods, and grains of soybeans. The increased yield includes increasing the grain weight per plant and / or the field plot yield. The enhanced nodule nitrogen fixation includes promoting soybean nodule symbiosis and / or increasing the biological nitrogen fixation capacity of soybeans.

9. Use according to claim 7, characterized in that, The gene editing system contains sgRNA of the GmGID1-2 gene, and the sgRNA is selected from sgRNA-1 or sgRNA-2, wherein the sense strand of sgRNA-1 is shown as SEQ ID NO. 3 or SEQ ID NO. 4, and the sense strand of sgRNA-2 is shown as SEQ ID NO. 7 or SEQ ID NO.

8.

10. A mutated GmGIDl-2 gene characterized in that, The CDS sequence is shown as SEQ ID NO. 5 or SEQ ID NO.

9.

11. Use of a mutated GmGIDl-2 gene according to claim 10 for soybean variety improvement, characterized in that, The mutated GmGID1-2 gene sequence causes the premature termination of protein translation and the shortening of protein sequence in soybeans; the mutated GmGID1-2 gene can improve plant type, increase stem strength, promote soybean root growth, enhance nodule nitrogen fixation, increase yield, and increase seed oil content; The improved plant type includes reducing soybean plant height, increasing soybean stem diameter, and increasing the number of branches, stem nodes, pods, and grains of soybeans. The increased yield includes increasing the grain weight per plant or the field plot yield. The enhanced nodule nitrogen fixation includes promoting soybean nodule symbiosis or increasing the biological nitrogen fixation capacity of soybeans.

12. Use according to claim 11, characterized in that, The pod number includes the number of pods per plant and / or the maximum number of pods per cluster; the number of pods per plant includes the number of three-pod pods and / or the number of four-pod pods.

13. The use according to claim 11, characterized in that, The grain number includes single plant grain number and / or grain number per pod.

14. The protein encoded by the mutated GmGIDl-2 gene of claim 10, characterized in that, The amino acid sequence is shown in SEQ ID NO. 6 or SEQ ID NO.

10.

15. The protein of claim 14 in the application of soybean variety improvement.

16. Use of a recombinant plasmid in the improvement of soybean varieties, characterized in that, The recombinant plasmid is inserted with a GmGID1-2 gene, a homologous gene of the GmGID1-2 gene or a gibberellin signal pathway related gene of the GmGID1-2 gene; the CDS sequence of the GmGID1-2 gene is shown in SEQ ID NO.

1.

17. Use according to claim 16, characterized in that, The backbone plasmid of the recombinant plasmid comprises pCAMBIA3301.

18. A method of soybean improvement, characterized by, The method comprises the following steps: knocking out or silencing a GmGID1-2 gene, a homologous gene of the GmGID1-2 gene or a gibberellin signal pathway related gene of the GmGID1-2 gene in a soybean recipient plant to obtain a knockout or silencing line; or, overexpressing a GmGID1-2 gene, a homologous gene of the GmGID1-2 gene or a gibberellin signal pathway related gene of the GmGID1-2 gene to obtain an overexpression transgenic line.

19. The application of a homologous gene of a GmGID1-2 gene or a gibberellin signal pathway related gene of a GmGID1-2 gene in soybean variety improvement.

20. The application of a gene editing system of a homologous gene of a GmGID1-2 gene or a gibberellin signal pathway related gene of a GmGID1-2 gene in improving plant type, increasing stem strength, enhancing nodule nitrogen fixation, promoting soybean root growth, and increasing yield and seed oil content.

21. The application of a protein encoded by a GmGID1-2 gene CDS sequence in soybean variety improvement; the amino acid sequence of the protein is shown in SEQ ID NO.

2.

22. The application of a protein encoded by a GmGID1-2 gene CDS sequence in improving plant type, increasing stem strength, enhancing nodule nitrogen fixation, promoting soybean root growth, and increasing yield and seed oil content; the amino acid sequence of the protein is shown in SEQ ID NO. 2.

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