Genes for modulating soybean compact plant type and uses thereof

Abstract

The present application provides isolated regulatory genome sequences of legume plants, preferably Glycine, Pisum, Cicer plants, such as Glycine max, Pisum sativum, Vigna radiata, Vicia faba, Phaseolus vulgaris, Cicer arietinum, and uses thereof, as shown in SEQ ID NO: 3. The present application has important application value for developing close-planting soybeans, intercropped soybeans, etc., and has great value for optimizing the architecture of soybean plant type and improving the yield of soybeans per unit area related to theoretical and practical research.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to genes that regulate soybean density tolerance and their uses. Background Technology

[0002] Soybeans provide humans with abundant plant protein and oil, making them an important grain and oilseed crop. However, my country's long-term reliance on soybean imports remains unchanged, and the supply-demand gap in my country's soybean industry is widening year by year, with a significant difference in soybean yield per unit area compared to the international average. Therefore, given the reality of gradually decreasing arable land in my country, how to avoid competing with major food crops for land while meeting the changing dietary needs of the Chinese population for plant protein and increasing soybean yield per unit area is a crucial issue. Optimizing soybean planting density per unit area through the design of an ideal soybean plant architecture, thereby reducing light loss in the field, provides the biological basis for solving this problem.

[0003] As early as 1968, Donald proposed crop ideotype-guided breeding (Donald, 1968), which involves deeply understanding the molecular regulatory mechanisms of existing high-yielding plant traits and, based on this, optimizing the combination of various desirable traits to design crops with ideal plant architectures in order to achieve higher grain yields. During the "Green Revolution," planting semi-dwarf wheat and rice, sacrificing straw biomass, not only increased grain yields but also demonstrated excellent lodging resistance (Peng et al., 1999). Rice IPA1 (Ideal Plant Architecture 1) can reduce the number of tillers and increase the number of spikelet branches, thereby increasing yield (Jiao et al., 2010; Miura et al., 2010; Wang et al., 2018). Wheat TaCOL-B5 increases yield by 11.9% by increasing the number of spikelet branches and spikelet nodes (Zhang et al., 2022).

[0004] To achieve a green revolution in the soybean industry, an ideal soybean plant type has been proposed, characterized by suitability for high-density planting and appropriate plant height, shorter internode length, more nodes, fewer or no branches, moderate number of pods per node, high pod setting rate, high four-pod ratio, moderate 100-seed weight, small petiole angle, and short petiole (Liu et al., 2020). However, previous research on increasing soybean yield has primarily focused on higher seed weight and more pods, often resulting in a small "source" (source) and insufficient "sink" (sink), hindering breakthrough increases. Therefore, ensuring a balance between "source" and "sink" to provide reasonable spatial placement for lateral organs, optimizing plant structure, and improving overall light energy utilization efficiency in the field are crucial for increasing soybean yield. Soybean branching plant type design has emerged as an excellent approach to solving this problem.

[0005] Therefore, reducing light loss in the field and increasing soybean planting density requires considering a suitable branching structure as the primary consideration. Among the many traits affecting soybean yield, branching traits, including the length, number, and angle of branches with the main stem, influence ventilation and light utilization within the soybean plant population, making them crucial factors in constructing an ideal soybean plant architecture and impacting yield. Branch number is a significant agronomical trait affecting crop yield. In rice, a gramineous plant, reducing tiller number and increasing spikelet branch number ultimately leads to increased yield. In soybean, a dicotyledonous plant, branch number, a quantitative trait, is controlled by multiple genes with major and minor effects, and is also influenced by environmental factors such as planting density and light conditions, making research on soybean branch number traits particularly complex. Currently, there are no reports on genes related to soybean's tolerance to high-density plant architecture. Summary of the Invention

[0006] This invention, through functional studies of the soybean Ln gene, discovered that the natural variation of Ln, ln, significantly reduces the number of branches. Transgenic functional verification revealed that ln can shorten petioles and internodes, reduce plant height, and significantly reduce the number of branches to a single stem, resulting in an excellent dense-planting plant type. This not only lays a genetic foundation for elucidating the molecular mechanisms of soybean plant type shaping but also provides genetic resources for subsequently improving soybean yield per unit area.

[0007] Specifically, the present invention provides the following technical solutions:

[0008] On the one hand, the present invention provides isolated genomic sequences of leguminous plants, preferably plants of the genera *Glycine*, *Vicia*, and *Chickpea*, such as soybean, pea, mung bean, broad bean, black bean, and densely planted chickpea, characterized in that the genomic sequences are as shown in SEQ ID NO:3.

[0009] On the other hand, the present invention provides the use of ln in regulating the dense plant type of leguminous plants, preferably plants of the genera *Glycine*, *Vicia*, and *Chickpea*, such as soybean, pea, mung bean, broad bean, black bean, and chickpea, characterized in that the coding sequence of the ln is as shown in SEQ ID NO:1.

[0010] In some embodiments, the amino acid sequence of the ln is as shown in SEQ ID NO:2.

[0011] In some implementations, the genomic sequence of the ln is shown in SEQ ID NO:3.

[0012] In some implementations, the use takes one or more of the following forms:

[0013] a. Reduce the number of branches;

[0014] b. Reduce plant height;

[0015] c. Reduce the number of sections;

[0016] d. Shorten the intersegmental length;

[0017] e. Shorten the petiole length.

[0018] On the other hand, the present invention provides an expression vector, characterized in that the expression vector comprises the genomic sequence as described above.

[0019] In some embodiments, the expression vector has an antibiotic label and / or an anti-chemical reagent label.

[0020] On the other hand, the present invention provides a host cell, characterized in that the host cell contains the genome sequence or expression vector as described above.

[0021] On the other hand, the present invention provides a method for regulating the dense plant type of legumes, preferably plants of the genera *Glycine*, *Vicia*, and *Chickpea*, such as soybean, pea, mung bean, broad bean, black bean, and chickpea. The method is characterized by the step of introducing the expression vector or host cell as described above into legumes, preferably plants of the genera *Glycine*, *Vicia*, and *Chickpea*, such as soybean, pea, mung bean, broad bean, black bean, and chickpea plants, cells, or tissues.

[0022] In some implementations, the dense-tolerant plant type exhibits one or more of the following characteristics compared to the wild type or control plant: a. fewer branches; b. lower plant height; c. fewer nodes; d. shorter internode length; e. shorter petiole length, while still growing well at high density.

[0023] definition

[0024] High-density planting: Dongnong 50 is defined as planting 80-120 plants per square meter, for example, planting density of 5cm plant spacing and 50cm row spacing.

[0025] Low-density planting: Dongnong 50 is defined as planting 26-40 plants per square meter as low-density planting, for example, planting density of 15cm plant spacing and 50cm row spacing.

[0026] Beneficial effects

[0027] The soybean plant architecture shaping proteins, their encoding nucleic acid sequences, and their genomic nucleic acid sequences (including promoters) provided by this invention are all first discoveries of the applicant. Furthermore, phenotypic analysis of transgenic and wild-type plants has verified that expressing the plant architecture shaping proteins of this invention can significantly affect soybean plant architecture.

[0028] This invention will have significant theoretical and practical value for the construction of ideal soybean plant architecture, the breeding and creation of densely planted varieties, and related basic and applied research. Attached Figure Description

[0029] Figure 1 The results show that ln produces a compact plant architecture at low density, indicating that ln can shape a dense-plant-tolerant soybean architecture. Specifically, A shows that ln results in a compact plant architecture; B shows that ln significantly reduces the number of branches, plant height, and internode distance; C shows that ln shortens petioles; and D shows the relative expression level of ln in leaves in overexpressing lines.

[0030] Figure 2 The results show the performance of transgenic materials at different planting densities. A shows the performance of DN50 planted at a low density (15cm plant spacing; 50cm row spacing); B shows the performance of DN50 planted at a high density (5cm plant spacing; 50cm row spacing); C shows the performance of the ln line planted at a low density (15cm plant spacing; 50cm row spacing); and D shows the performance of the ln line planted at a high density (5cm plant spacing; 50cm row spacing).

[0031] Figure 3 The diagram shows the construction of the ln genome expression vector.

[0032] Figure 4 This study presents statistical results regarding plant height (n≥30), number of branches, number of nodes, petiole length, and internode length between ln transgenic materials and wild-type Dongnong 50 materials at low density. Results include sample size, sample mean, sample standard deviation, sample extreme values, and the significance level of differences between ln transgenic materials and wild-type Dongnong 50 materials. A one-tailed t-test was used for significance testing, with P < 0.01 and n = 30.

[0033] Figure 5This study shows the yield test of the wild-type Dongnong 50 transgenic material under different planting densities (LD: low density, 15cm plant spacing, 50cm row spacing; HD: high density, 5cm plant spacing, 50cm row spacing). A shows the yield per plant (n=30) under different planting densities; B shows the yield per acre (n=3) of each plot under different planting densities. One-way ANOVA and Tukey's multiple comparisons were used, with P<0.05 or P<0.01 as the minimum acceptable value. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0035] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the invention. In the quantitative experiments described below, all experiments were repeated three times, and the results were averaged to ensure statistical significance.

[0036] In the following examples, the transformation recipient was Dongnong 50 (DN50, Heishendou 2007022), which is commercially available. The vector pTF101.1 and Agrobacterium strain EHA101 were purchased from the China Plasmid Vector Strains Cell Gene Depository Center (Biovector Science Lab, Inc.).

[0037] Consumables such as the homologous recombination reagent kit were purchased from Novizan Biotechnology Co., Ltd. and Tiangen Biotech (Beijing) Co., Ltd.

[0038] Example 1: Discovery of the ln protein and its encoding gene

[0039] Previously, our research group identified the gene *ln* (Fang et al., 2013) controlling the pointed leaf and four-pod traits in soybean using map-based cloning. Its CDS sequence is shown in SEQ ID NO:1, and the encoding amino acid sequence is shown in SEQ ID NO:2. To further investigate the gene function of *ln*, we amplified approximately 8 kb of the full-length *ln* gene (SEQ ID NO:3) from the soybean cultivar DN50 (ln / ln). This amplified the gene, including a 2.6 kb upstream promoter, a 1.8 kb *ln* genomic DNA sequence, and a 3.2 kb downstream sequence (the present invention selects an existing genomic DNA sequence to more closely approximate the actual transcription and translation process in vivo). This amplified gene into DN50 significantly improved the soybean plant structure. The compact plant type, shorter petioles, lack of branching, and shortened internodes suggest suitability for dense planting, which helps increase yield per unit area.

[0040] Example 2: Functional Verification of the ln Protein

[0041] I. Construction of Recombinant Plasmids

[0042] Genomic DNA was extracted from Dongnong 50 (DN50) and used as a template to amplify the ln gene via KOD high-fidelity PCR. Then, homologous recombination was performed into the pTF101.1 vector to obtain the recombinant plasmid pTF101.1-pGmln-gGmln. The specific procedures are as follows:

[0043] 1. Extract DNA from the leaves of soybean variety DN50.

[0044] 2. Using the total DNA obtained in step 1 as a template, perform PCR amplification with primers consisting of F1 and R1 to obtain PCR amplification products.

[0045] F1:

[0046] 5'-ATGTTGACCTGCAGGCATGCAAGCTTGGCGTCAAACTTTACCGAT-3'(SEQ ID NO:4)

[0047] R1:

[0048] 5'-AACAGCTATGACATGATTACGAATTCACTTTTAAGCACACCTGACC-3'(SEQ ID NO:5)

[0049] 3. The pTF101.1 vector (purchased from the China Plasmid Vector Strains Cell Gene Preservation Center) was double-digested with restriction endonucleases Hind III and EcoRI, and the linearized vector of about 8 kb was recovered by 1% agarose gel electrophoresis.

[0050] 4. The PCR product from step 2 and the vector backbone from step 3 were ligated using homologous recombination to obtain the recombinant plasmid pTF101.1-pGmln-gGmln (its pattern can be found in...). Figure 3 ).

[0051] II. Obtaining ln-overexpressing transgenic plants

[0052] 1. The recombinant plasmid pTF101.1-pGmln-gGmln was introduced into Agrobacterium tumefaciens strain EHA101 (purchased from the China Plasmid Vector Strains Cell Gene Preservation Center) to obtain recombinant Agrobacterium.

[0053] 2. The recombinant Agrobacterium obtained in step 1 was used for soybean genetic transformation using the Agrobacterium cotyledonum infection transformation method (Li et al., 2017), and the recipient plant was transformed into DN50.

[0054] (1) Sterilization of soybean seeds: Select large, plump soybean seeds with smooth surfaces and no disease spots or scratches on the seed coat. Divide the seeds into petri dishes and place them in a desiccator with the lids off. Use chlorine fumigation sterilization for seed disinfection. Place a small beaker in the desiccator, add 100 mL of sodium hypochlorite, then add 5 mL of concentrated hydrochloric acid, and quickly cover the desiccator. Sterilize for 10-14 hours. After sterilization, remove the soybeans from the desiccator and blow them in a clean bench for more than 30 minutes to remove excess chlorine.

[0055] (2) Soybean seed imbibition germination: Sterilize the seeds with the hilum facing down and insert them into the germination medium. Place them in a dark environment at 23°C for 16-18 hours.

[0056] (3) Agrobacterium preparation: Take out the bacteria stored at -80℃ and add them to 5 mL of YEB medium containing rifampicin and kanamycin resistance. Incubate at 28℃ and 250 rpm for 24 h to recover. Then, take 200 μL of the recovered bacterial solution and add it to 200 mL of YEB medium containing rifampicin and kanamycin resistance. Incubate at 28℃ and 250 rpm until OD. 600 =0.6 Collect bacterial suspension. Centrifuge at 22℃, 5,000 rpm for 10 min, then resuspend the bacterial cells in liquid inoculation medium to OD. 600 =0.5, 22℃, 70rpm incubate for 0.5h for later use.

[0057] (4) Preparation of explants: Remove the seed coat of the immobilized seed, cut off the part of the hypocotyl away from the cotyledon with a scalpel, so that the remaining hypocotyl is about 2-3 mm, then divide the soybean seed in half, remove the embryo with a small iron brush, and the prepared explant is obtained.

[0058] (5) Infection of explants: Place the prepared explants into Agrobacterium suspension, ensuring the resuspension covers the explants, gently shake, and incubate for 0.5 h.

[0059] (6) Co-culture of explants with Agrobacterium: After infection, the excess bacterial solution of the explants is absorbed on sterile filter paper. A sterile filter paper is placed on the co-culture medium, and the dried explants are placed on the medium and cultured in the dark at 23°C for 3-5 days.

[0060] (7) Callus induction culture: After the explants were co-cultured with Agrobacterium, the explants were transferred into the callus induction culture medium. At this time, the explants were tilted at 45 degrees and the bottom was gently inserted into the culture medium. The culture was carried out at 23°C for 16 hours of light and 8 hours of darkness for 14 days.

[0061] (8) Bud induction culture: The induced callus was cut off from the explant and placed in the bud induction culture medium. It was cultured at 23℃ for 16h light / 8h dark for 14d. Every 14d, it was transferred to a new bud induction culture medium.

[0062] (9) Bud elongation culture: The explants that have induced new buds are transferred to the bud elongation culture medium. Every 2 weeks, they are transferred to a new bud elongation culture medium until the buds elongate to more than 3 cm.

[0063] (10) Rooting culture: Cut off the elongated buds and insert them into the rooting culture medium until the roots are longer than 3cm. Then harden the seedlings and transplant them to the greenhouse at 24℃, 16h light / 8h dark until maturity. During this period, spray with Basta and PCR to identify positive lines.

[0064] Table 1. Formulation of 1L germination medium

[0065]

[0066] Table 2. Formulation of 1L YEB medium

[0067]

[0068]

[0069] Table 3 Formulation of 1L Infection Solution

[0070]

[0071] Table 4. Formulation of 1L co-culture medium

[0072]

[0073]

[0074] Table 5. Formulation of 1L callus induction medium

[0075]

[0076] Table 6. Formulation of 1L bud induction medium

[0077]

[0078] Table 7. Formulation of 1L bud elongation medium

[0079]

[0080]

[0081] Table 8. Formulation of 1L rooting medium

[0082]

[0083] Soybean leaves were treated with 0.1% Basta herbicide (Coollab, CB2471-100mL). Plants showing no yellowing after 3 days were considered transgenic positive plants. T0 generation seeds were harvested. Subsequent T1 generation and subsequent generations of transgenic lines were sprayed with 0.1% Basta herbicide for screening. Successful ln overexpression transgenic plants were obtained after all lines developed resistance to Basta, resulting in three transgenic lines: ln-1, ln-2, and ln-3. Figure 1 ).

[0084] Total RNA was extracted and cDNA was reverse transcribed. The expression level of ln transcription was detected by quantitative real-time PCR. The RT-qPCR primers were designed as follows:

[0085] ln gene

[0086] F2:5'-GTGTTTGCCTCACATCATTTTTCC-3'(SEQ ID NO:6)

[0087] R2:5'-TTGTGCAGCAATGTTATGATCACA-3'(SEQ ID NO:7)

[0088] Internal reference gene ACTIN

[0089] F3:5'-CGGTGGTTTCTATCTTGGCATC-3'(SEQ ID NO:8)

[0090] R3:5'-GTCTTTCGCTTCAATAACCCTA-3'(SEQ ID NO:9)

[0091] The instrument model is Roche Light Cycler 480, and the reagent is... 480 SYBR Green IMaster, reaction system as follows (20 μL):

[0092] Table 9 RT-qPCR reaction system

[0093]

[0094] The RT-qPCR parameters are set as follows:

[0095] 95℃ hot start for 5 min; 95℃ denaturation for 10 s, 59℃ annealing for 10 s, 72℃ extension for 7 s, 45 cycles; after the amplification cycle, a melting curve was generated: 95℃ denaturation for 5 s, 65℃ for 1 min, 65℃ to 97℃, with a heating rate of 0.11℃ / s.

[0096] Each amplification reaction should be technically replicated at least three times. Using 2... -ΔΔCt The relative expression level is calculated using this method.

[0097] The results showed that in the overexpression lines, ln expression increased a hundredfold, and the expression level was significantly upregulated (see...). Figure 1 D).

[0098] Example 3: Statistical analysis of transgenic phenotypes in ln

[0099] This embodiment verifies that transgenic plants overexpressing the ln gene under the DN50 background exhibit a compact plant type, significantly reduced branching (single stem), significantly shorter plant height (reduced to 56.8%, 49.24%, and 46.04% of the original height), fewer nodes (reduced to 7.01%, 11.06%, and 11.37% of the original number), significantly shorter internodes (reduced to 39.94%, 38.84%, and 37.71% of the original length), and significantly shorter petioles (reduced to 13.3%, 10.35%, and 6.75% of the original length). Figure 1 AC, Figure 4 Furthermore, compared to low-density planting conditions (15cm plant spacing; 50cm row spacing), high-density sowing methods (5cm plant spacing; 50cm row spacing) still resulted in good performance for ln transgenic plants (see...). Figure 2 This example demonstrates that ln gene overexpression can significantly improve plant architecture, resulting in a more compact plant structure, and plays an important role in dense planting.

[0100] Therefore, we further compared the yields at different densities. The results showed that high-density planting reduced the yield per DN50 plant by 68.2%, while the yield per ln transgenic plant decreased by only 22.9%. Figure 5 A) suggests that ln transgenic plants can increase overall yield by increasing the number of plants per unit area. Furthermore, we identified plot yields at different planting densities, and the yield per acre comparison showed that ln transgenic plants significantly increased yield per acre. Under high-density planting conditions, ln transgenic plants yielded 38.5% more than DN50, and under low-density planting conditions, yield increased by 27.9% compared to DN50. Figure 5 B). This result further confirms that ln transgenic plants can increase soybean yield by increasing planting density, reducing light loss in the field, increasing light energy utilization per unit area, and ultimately increasing soybean yield.

[0101] sequence

[0102] SEQ ID NO:1 ln protein CDS sequence

[0103] ATGAGACCAGAACGAAACCCCTTACATCTTAACAATTTGCCCGATGAGTACTCTAGAGATGGCAAACAAGTCCTCGAAGACCATACCTCTTCATCCGGTTGCAGGAAAAAGAAAAGCGGCGGGAAGGATGGAAAAGACGAGTGTGGGAAGGTCTACGAGTGTAGATTTTGTTCCCTCAAGTTCTGCAAGTCTCAGGCTCTTGGGGGACACATGAACCGCCACCGCCAAGAGAGGGAAACGGAGACGCTGAACCAGGCTCGTCAACTGGTCTTTCGTTGTGATCATAACATTGCTGCACAAGGTGCCCCTCACTTAGGATGCTGCCAAACAATAGGAACGGGGGGTTATCATCCCTCAGGAGACCCAACAGTGCCTCTAAGATTCCCAAGATACTTCTCAGGTTCATCCTCAACTCACATGCCACCATCCCCGCCACCGCCGCCGCCACCGCAACGACCATACCTATACCCTTCACCTACGAGGCCAGTGTCATTTGGGTCATCACACTTCCCTCTCCAGCATGCAGTGAACGATTACTATGTGGGCCACGTGATGAGTGGTGGCAGCCACGGACACTATGTTGGAGGAGAGAGCACAAGGAGTTACACGTGCATTGGTGCACCGGTGGGGCAAGGTGGCGGATTCGCTGGTGGTAAGGAGGGGTCTGCAGTGCAGGAGGAAGGGTTGAGTACTTGGGGAAGGGGCTATTCAGGTGCACAGGATCGTTTGGATCCTCCCTCAGCGATCAATCGGTTTCAAGATGGTTTCTAA

[0104] SEQ ID NO: The amino acid sequence of ln protein

[0105] MRPERNPLHLNNLPDEYSRDGKQVLEDHTSSSGCRKKKSGGKDGKDECGKVYECRFCSLKFCKSQALGGHMNRHRQERETETLNQARQLVFRCDHNIAAQGAPHLGCCQTIGTGGYHPSGDPTVPLRF PRYFSGSSSTHMPPSPPPPPPPPQRPYLYPSPTRPVSFGSSHFPLQHAVNDYYVGHVMSGGSHGHYVGGESTRSYTCIGAPVGQGGGFAGGKEGSAVQEEGLSTWGRGYSGAQDRLDPPSAINRFQDGF

[0106] SEQ ID NO:3ln genome sequence (including promoter and 5'-UTR (underlined part), exons, introns, 3'-UTR sequence, downstream sequence)

[0107] TGGCGTCAAACTTTACCGATAAAAGAAGAAACTTTTGAATTGTTTATAAATAGTTAACTATACAATTAT AGACAGGGTAATTCGGTTTGGTTTCAAAGAATGATTTGATTTAATTTGGTTTGAAAGTTTTGCTAGTGGTATATTTA CTGTTGGTTCCCCGCCTCATGTATGTTTAGAGTTGAAAGTCGAAACCTCTTGAGATTGCATCATTTAAGCGCTTACA GTACCCCTCGGCTCAAATGAATTTGTCAGGAAACTTTTTTCCTCTAATACATATCACCATAATCTGATTTGTAGTAC CACGATCTGGAACAGATATTCTCCTCCCCACGTTTTTATCAATAGAATCCAACACTCCTTGTAAATTTGAAATATGA AAGTTTTAATACTTGATGTTATTTTGGTATTTCTCATGGTCTAAAACTCGATCTCATGTAATTTACAATCTTTGGCT TTGTTTGTTTCATTTCTTTGCCTAAAATATAATTAATTGACGGGTTTGATTTCATGCACTTTCTGTCACAAGGAAAG GTAACATTTAAAAGTTTTCAATTTTAAATTTTGACTGCTTGAAGGGAAGCTAGGTAGGAAGGCAACATGATGATGAA GACAATTTCCTCTACTGGTTTGTAAGTTTGCCTTGCTAGAGTTCTGAACATGGAAGAAATGTTTGTTGGATTTACTG CTTAAATTCGTGAAGGACCACTTGAAAAGTAATGATATATATACACACACTTGCAAGAAAAATTTCAGATAGGTGGG GCGCGGTCATTGCATGAATCTTTGGGTCTAGGTTTTGAAAATATCGAATGTGTCTCATATCATTTCCTTCATAATAC CAACCAGCTAGTCAAGGCCAGCTGCAGTAGTGTGGTCCATGAATATCACCTGATTTTAATGCCTAGCTAGCCCTCTT ATTAGGTTGTGTCTTCTTTCTCCTTTATATCCATTTCAGGTGTGTGGTGGGGGAAGCACTGATCAGCTTGTGGACAA CATCAGCCCCTCTAACTATTGTTTCTTTTTGCCATTTACACATATATGCTGCTCCAAATCAAGACCCAGGCCCTTAAT TACCAAGTAATTAAAACAGCTGGTAATTATTATTATTATTATTATTATTTCAACCGTTAAAGCTAGCTAGCTTTTTG ACCTGTACTAGTAGTATGTATATATACTTTTCTCTCAGCTTTAAACTAGATAGTAATCAATAACCCCCACCACTATT TTTTAAAAAAAAAATGTAAAACAGTTAATTGCTTCTGCATATTAATTTGGAGAACACTTTGTTTTACCTAGCTAACT AGTTAACTATCTTATAAAGGAATCACTCATTAGAGATGGAGCTATAGTGTTTGGATTCAAAGAAAACGCATCTAAAG CCAACAGAGAATATAATGATGAATGTGAGACATACATTAAGAAAATTCAAGTTCATATATTTCTCCACTTTTTTCAT GAAAAGGACTATGTATATGTAATCTATGAGATATAGCAACTTTCAAAGAATAAAACAACAAGTGGGGGGTCGAGTCT ATCTCAGCTTAGATTGCAAAACCCTAATGATAGATACTGATAGCAACCCTTCCTTGATGCAAGTCTAAGTCCCCT GGGTAGAATTTTACCCCCAGAAGGAGGCATTGAGCTATTGTGGACCATACACGAACTGTTCTCAAACATGCCCCCCC TCTTTAAGCACAATAACCACGCAAAATGTCTACGTACCCTATGCCTTGTCTTGCTTTCTTTCTGGTATAAATGTCAT CTTTCATTCTTAGGGCAACCTTTAATGCTCAAAGATTTGTACTGTACATTTGATGGACATCCTTTGTTGGTAAAGAT TTTCTTTTGAGATATTAATTATTTTTTTCAGTGTATTTGACTGATATTGTTTGAATATTTTTGGACCCTTTGT TGATAGCTCTAGCTAGGGCAAAACAAAAAAAACTAGATGCAGAAGTATCATATATCTATGCAACATCATATTTTTG CAATAAAATTATATAATAACTAGCATAAATGAAATAGTCATTTTTTTTATATATCTCTTCATCATATAATTCACTT TATCACATGTTTCTTTTTATCTCTTATTGTTGTATATATTTTATTATTAAATAAATTCTCTATAGATTACTAAA TAGGAATAATTAATACAAAAGCAACATGTTTCCGAATCACATATCTTTGCCCTTTTTCTAATTCTGTCCTATCATTTC CCAAGTCTCAACCAAACTAGATTAAGTTAAGCCCCCCCCCCGCCCCCCCCTCCACACTCACTCTCACACTCTCTTT TTTTAAAGTCACGAGACCCACTGTCCAATTATTCATTCAATTTTTGTGTGGGGGGCGGGGGGAGATAAGAGAGAGA GAGAGAGTGGCAGAGGAACTGATAGAGAACTTTCAAAGCTCTTTATCTTCTACCATCACTCAACCAGCCTCTATATT GCAGTCTCAAACTGAAAGTCTAAAATTTTTTGTAAAAAGCTTTTAGTTTTTCCTACCCCCACCCCATCTGAAAGAA AGAGTGTTTGCCTCACATCATTTTTCCCCTTTCTGTCTCTCTCTCTGTCGGTACC

[0108] GTATGCACCCATTACTTCCATGCAGCGATTCTCTTCCTATTTCTTCTTC

[0109] TTGTTCAACATTTATATATTTCATTTCTCAAATACTTGTTTTTCTGTGGG

[0110] TTCTTTAGCGTTTGTTAACGTTGTTTTTTCAAGGTATAACACATAATAT

[0111] TTGGGTACTCAGCATGACACTGTTTGATACTGCGATTTATTTGTGATA

[0112] ATATTCAATTCAGATGCTGCCAAACAATAGGAACGGGGGGTTATCATC

[0113] CCTCAGGAGACCCAACAGTGCCTCTAAGATTCCCAAGATACTTCTCA

[0114] GGTTCATCCTCAACTCACATGCCACCATCCCCGCCACCGCCGCCGCC

[0115] ACCGCAACGACCATACCTATACCCTTCACCTACGAGGCCAGTGTCAT

[0116] TTGGGTCATCACACTTCCCTCTCCAGCATGCAGTGAACGATTACTATG

[0117] TGGGCCACGTGATGAGTGGTGGCAGCCACGGACACTATGTTGGAGG

[0118] AGAGAGCACAAGGAGTTACACGTGCATTGGTGCACCGGTGGGGCAA

[0119] GGTGGCGGATTCGCTGGTGGTAAGGAGGGGTCTGCAGTGCAGGAGG

[0120] AAGGGTTGAGTACTTGGGGAAGGGGCTATTCAGGTGCACAGGATCG

[0121] TTTGGATCCTCCCTCAGCGATCAATCGGTTTCAAGATGGTTTCTAAAG

[0122] AGATGAGAGATTCTTTGTTTGAGTGGTTTTTGGTTTGTGTTATGTTTC

[0123] TGTTATGTTATGTGGTATCATCATAAAGACACGCAATCCAGAGAGAGA

[0124] GAGAGGTGGTTTTTGGTTTATCTACAGTAACCAGGGACAGCTTTTGA

[0125] GCTCATGGACACGCTCAGCTACTTTGGCTTGGAGTTGTGGGTGGGAT

[0126] TTCTTCTCAACATCGCCTTTTGTATTGGTAGCTAGCTAGCTAGCTTGG

[0127] ACAACCAGTTAGATTTGAACTGAAACTTTCCAAACTCCTTTTCCTTT

[0128] GCATGCCAAATTACAACCATTTTCCTTCTCAGTAATTTGTTCTTTCAAT

[0129] ATCCTTCTATTATATTACACTAGTAACTAGTAAGTAATATCTATCTCTGT

[0130] TACTATTTTATATATACATCCTTCTATTTTATATATGCCTGTTTTTGTGTT

[0131] TAATTTGCTATTCCAAAATCTCTAAAACCCCCCGTCACTCTCGATCGC

[0132] TCTCACTCTCTCTATCCAAACCAAAATTGGGATCTTCTAGTTTCTTGA

[0133] AATGTTTTATGGTCTCACTGTCACACTATGCTAGCACAATTAGGGTAA

[0134] TGACACAGAATTTTTGGAGTCCTCTTGCGAAAAAACCATCTTGCTGA

[0135] ATTAATGCTTCCGGAATAGGATATTCAAACAATCAAATTTGCCAAAAT

[0136] ATCTAATTTCTTCTTAAAGAAAAGCTTTATCCTCCCTCCCTATTTCTAC

[0137] GATATTGTAAATAATTATTTTAAAATGTTAAAATACAGATCGAGATTAT

[0138] TTTTAGCAGCAGACAGTGAATGCAGCTATATTTAATTTGCTTGGTACA

[0139] TACCGAACATAACTACTATCTAAGGAATAAAACACTCTTGCTGTCA

[0140] GCTCCAAGAGCTTCTACCAAATGCAGCAGCTTAGAGAAATATATACA

[0141] CAGCCATAACTAGCTCATGATCGTTCTTGAAACAGTAAAACACGTGT

[0142] AAATAATGAAATAATGAAACCACATTATTAGCATGATTTTTCTTTTCTT

[0143] TTCTCTTGTTTTCAATACTTTTATTTACCGACTAGTTCTCTCCTCTCTG

[0144] TTGGAAATGGATATATTTTTCTTTGTATATAGGTTGTGCTTCCTTTACG

[0145] CACAGTAAGTGAGGATTTTATTATAAGTAAATTATCCTCATAAAAAATA

[0146] TTTTAAAGGTATATTCTGGCTTGGCTTCTACTATTCCTATATATTTTT

[0147] GGTTCGTGTTATATAGTTTAGTTCGTGTTTTCACTCAAGGAGCATTAA

[0148] TTCCTTGACTCAACAAAGGCAAATCTGCTAAAACCATTAATAAGTGAT

[0149] GAAATTATTATTTTTCTAGGGATGGTAAAATGGTTTGTATCGTATGAA

[0150] AAGGAAAGGCGATGATTAGTGACTTAGTGGCAGTGTTTATGGTAGAT

[0151] CCAAATATTCAATGCAATAGCAACATTGGCTGATGCGGCACGAATT

[0152] CAGTGGCATGGTGATGATGGCAAGTAGTGACATCAGCAATTAAAGAT

[0153] GAAGATGGAAGAAGGTGGTGTAGTAAATGGCTGCAATAACAATTG

[0154] CTATACAGCAAAAGAATGTACGTAAATAATTTTTTTTTAATAGAATACAT

[0155] GTATTCTTTTCATTATATAATTATGCAAGAGTGATAATTATTATGGGCGA

[0156] TATATTTTTTCTTTAAAACAAATTATATATAAGTTGATTTACAAGTTTAGG

[0157] AGAAGAATAATGAGTACTTAGAATTTAATTAGGATTGATTTATGAA

[0158] AAATAGGTGTCTAAATTATAGATTATTGTCAAGAGTGAAATTTGAATT

[0159] TACATTTTGGCTTGGCTGGTAAAAGTAAAAAAGACACTACAAATTTT

[0160] GAATTTAACTCATTTAGTTTATTATATGACTTAATTGTATTTTAATGTTT

[0161] AATTCTATTTAAATAATTTTTTATATTATAAATTCAATATATTTAACAA

[0162] ATTCATGTTTGAAGAATAAAGTGAAATAGTGCCACAAGACCTATGGT

[0163] TCAGCATCTTTCTTTTCACTTGATCCATCTCGCTTTAATTAGAGTCATA

[0164] ATATATCTCTTCTGATCTTCTACTGCCTGCGGTACAAGTCAACACATA

[0165] GTTCAATAATTGTCATCTTAATGTTGGTTGACAAGTGAAGCATATATAT

[0166] GTCGAAATAGTTGGACTTATATTTTGCATAAGATCACAGCTTGTGGGA

[0167]

[0168] SEQ ID NO:4ln primer amplification sequence F1

[0169] ATGTTGACCTGCAGGCATGCAAGCTTGGCGTCAAACTTTACCGAT

[0170] SEQ ID NO:5ln primer amplification sequence R1

[0171] AACAGCTATGACATGATTACGAATTCACTTTTAAGCACACCTGACC

[0172] SEQ ID NO: 6ln Real-time PCR Primer F2

[0173] GTGTTTGCCTCACATCATTTTTCC

[0174] SEQ ID NO:7ln Real-time PCR Primer R2

[0175] TTGTGCAGCAATGTTATGATCACA

[0176] SEQ ID NO:8 Internal reference gene ACTIN real-time PCR primer F3

[0177] CGGTGGTTCTATCTTGGCATC

[0178] SEQ ID NO:9 Internal reference gene ACTIN real-time PCR primer R3

[0179] GTCTTTCGCTTCAATAACCCTA

[0180] References

[0181] Donald CM(1968)The breeding of crop ideotypes.Euphytica 17:385-403.

[0182] Fang C,Li WY,Li GQ,Wang Z,Zhou ZK,Ma YM,Shen YT,Li CC,Wu YS,Zhu BG,etal.(2013)Cloning of Ln gene through combined approach of map-based cloningand

[0183] association study in soybean.J Genet Genomics 40:93-96.

[0184] Jiao YQ,Wang YH,Xue DW,Wang J,Yan MX,Liu GF,Dong GJ,Zeng DL,Lu ZF,ZhuXD,et al.(2010)Regulation of OsSPL14 by OsmiR156 defines ideal plant

[0185] architecture in rice.Nat Genet 42:541-544.

[0186] Li SX,Cong YH,Liu YP,Wang TT,Shuai Q,Chen NN,Gai JY,Li Y(2017).Optimization of Agrobacterium-mediated transformation in soybean.Front PlantSci 8:246.Liu SL,Zhang M,Feng F,Tian ZX(2020)Toward a“green revolution”forsoybean.Mol

[0187] Plant 13:688-697.

[0188] Miura K,Ikeda M,Matsubara A,Song XJ,Ito M,Asano K,Matsuoka M,KitanoH,Ashikari M(2010)OsSPL14 promotes panicle branching and higher grainproductivity in rice.Nat Genet 42:545-549.

[0189] Peng J,Richards DE,Hartley NM,Murphy GP,Devos KM,et al.(1999)'Greenrevolution'genes encode mutant gibberellin response modulators.Nature 400:256-261.Wang J,Zhou L,Shi H,Chern M,Yu H,Yi H,He M,Yin JJ,Zhu XB,Li Y,et al.(2018)A single transcription factor promotes both yield and immunity inrice.Science 361:1026-1028.

[0190] Zhang XY,Jia HY,Li T,Wu JZ,Nagarajan R,Lei L,Powers C,Kan CC,Hua W,Liu

[0191] Z,et al.(2022)TaCol-B5 modifies spike architecture and enhances grainyield in wheat.

[0192] Science 376:180-183.

[0193] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The use of overexpression of the ln gene in regulating soybean dense-plant-tolerant architecture, characterized in that, The amino acid sequence encoded by the ln gene is shown in SEQ ID NO:2, and the dense-plant-tolerant type exhibits one or more of the following: a. Reduce the number of branches; b. Reduce plant height; c. Reduce the number of sections; d. Shorten the intersegmental length; e. Shorten the petiole length.

2. The use according to claim 1, characterized in that, The coding sequence of the ln gene is shown in SEQ ID NO:

1.

3. The use according to claim 1, characterized in that, The nucleotide sequence of the ln gene is shown in SEQ ID NO:

3.

4. The use of an expression vector containing the nucleotide sequence shown in SEQ ID NO:3 in regulating the plant density tolerance type of soybean, characterized in that, The dense-tolerant plant type exhibits one or more of the following characteristics: a. Reduce the number of branches; b. Reduce plant height; c. Reduce the number of sections; d. Shorten the intersegmental length; e. Shorten the petiole length.

5. The use according to claim 4, characterized in that, The expression vector is labeled with antibiotics and / or anti-chemical reagents.

6. Use of a host cell comprising the expression vector of claim 4 or 5 in regulating soybean density-tolerant plant architecture, characterized in that, The dense-tolerant plant type exhibits one or more of the following characteristics: a. Reduce the number of branches; b. Reduce plant height; c. Reduce the number of sections; d. Shorten the intersegmental length; e. Shorten the petiole length.

7. A method for regulating the dense-planting tolerance of soybeans, characterized in that, The method includes the step of introducing the expression vector of claim 4 or 5 or the host cell of claim 6 into a soybean plant or cell or tissue, wherein the dense-tolerant plant type exhibits one or more of the following characteristics relative to the control or wild type: a. reduced number of branches; b. reduced plant height; c. reduced number of nodes; d. shortened internode length; e. shortened petiole length.

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