Soybean gmbhlh300 gene and application thereof in improving yield of soybean under high-density planting and high-branching

Abstract

This invention relates to the field of genetic engineering technology, specifically to a soybean GmbHLH300 gene and its application in increasing soybean branch number and yield under dense planting conditions. The nucleotide sequence of the GmbHLH300 gene described in this invention is shown in SEQ ID NO:1, and the amino acid sequence of its encoded protein is shown in SEQ ID NO:2. This invention clones this gene into the PTF101 vector to construct an overexpression recombinant vector, and transforms soybeans using Agrobacterium EHA101-mediated transformation to achieve overexpression of the GmbHLH300 gene. Field dense planting trials show that overexpression of this gene can significantly increase the number of branches and stem diameter in soybeans under dense planting conditions, increase the number of pods and grains per plant, inhibit excessive plant growth, and ultimately significantly increase soybean yield under dense planting conditions. This invention provides important gene resources and technical approaches for molecular breeding of soybeans to tolerate high density and yield, and has good application value.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a soybean GmbHLH300 gene and its application in increasing the number of soybean branches and yield under dense planting. Background Technology

[0002] Soybean (Glycine max (L.) Merr.) is an important oilseed crop and source of plant protein globally, holding an irreplaceable strategic position in ensuring food security, optimizing dietary structure, and supporting agricultural economic development. With the continuous growth of the global population, the rigid reduction of arable land, and the increasing demand for vegetable oils and proteins due to the upgrading of residents' dietary structure, increasing soybean yield per unit area has become a core objective in soybean breeding and cultivation research. Soybean yield is jointly regulated by various agronomic traits, among which plant architecture, especially the number of effective branches, is a key factor determining the number of pods per plant, the number of grains per plant, and the final yield level.

[0003] Reasonable planting density is a classic cultivation regulation approach to improve the light energy utilization rate of the crop population and increase yield per unit area. For soybeans, appropriately increasing the planting density can expand the photosynthetic area of ​​the crop population and promote the accumulation of photosynthetic products, which is an effective measure to increase soybean yield. However, after the planting density is increased, the competition for light, water and mineral nutrients among individual plants is significantly intensified, which will induce a series of shade-avoidance adaptation responses in the plants, specifically manifested as excessive internode elongation, thin stems, and increased risk of lodging. Among these, the most prominent negative effect is a significant reduction in the number of branches per plant.

[0004] Branching in soybeans is a crucial foundation for the formation of yield organs. The yield per plant is comprised of pods from the main stem and pods from branches. The number of effective branches directly determines the number of pods and seeds per plant, making it a core plant type trait influencing the yield potential of individual soybean plants. Under dense planting conditions, apical dominance is enhanced, while light signal transduction pathways are altered, leading to inhibited axillary bud germination and hindered or even halted branching development. Ultimately, this results in a significant decrease in the number of effective branches per plant, preventing the full realization of yield potential. Traditional "increased yield through dense planting" strategies often fall into the dilemma of "increased population density, decreased yield per plant," highlighting an irreconcilable contradiction between high soybean yield potential and adaptability to dense planting. Therefore, identifying key gene resources that can maintain or promote branching development, stabilize and increase yield per plant under dense planting conditions, and using molecular breeding methods to synergistically improve soybean's tolerance to dense planting and yield traits, is a critical scientific issue that urgently needs to be addressed in high-yield soybean breeding.

[0005] The bHLH (basic helix-loop-helix) family is one of the largest transcription factor families in eukaryotes, widely involved in the regulation of plant growth, development, and stress responses. A typical characteristic of bHLH proteins is a conserved domain of approximately 60 amino acid residues, which can be divided into two functional regions: the N-terminal basic region, rich in basic amino acids, specifically recognizes and binds to cis-acting elements such as E-box (CANNTG) or G-box (CACGTG) in the DNA sequence; the C-terminal helical-loop-helical region regulates the activity and functional specificity of transcription factors by mediating the formation of homodimers or heterodimers.

[0006] In the model plant Arabidopsis thaliana, the function of the bHLH family has been systematically studied, confirming that its members are widely involved in biological processes such as photomorphogenesis, anthocyanin biosynthesis, trichome development, stamen development, and the transduction of various plant hormone signals. Among them, the PIFs (Phytochrome Interacting Factors) subfamily, as the core hub of light signal transduction, is a key factor regulating hypocotyl elongation and shade avoidance response; MYC2 is a core regulatory element of the jasmonic acid signaling pathway, mediating plant responses to biotic and abiotic stresses. In food crops, the functional study of bHLH genes is also progressing. For example, members of the OsPIL family in rice are involved in regulating plant type traits such as tillering angle and plant height; some bHLH genes in maize have been shown to be related to grain development and stress tolerance.

[0007] Compared to plants like Arabidopsis and rice, functional studies of the soybean bHLH transcription factor family remain fragmented, with the biological functions of most members still unclear. Currently, over 300 bHLH family members have been identified in the soybean genome, with systematic nomenclature from GmbHLH1 to GmbHLH300, but the reported functions of these members are very limited. Existing research indicates that some soybean bHLH genes are primarily involved in abiotic stress responses, such as GmbHLH25 and GmbHLH92, which are associated with stress tolerance; others are involved in secondary metabolic regulation, such as GmTT8, which participates in anthocyanin biosynthesis. To date, there are very few reports on the role of soybean bHLH family members in plant architecture and branching development regulation, and even fewer functional validations of bHLH genes regulating plant architecture and yield under dense planting stress.

[0008] Previous studies have shown that GmbHLH57 and GmbHLH300, belonging to the Ib subfamily, are key transcription factors regulating iron homeostasis in soybean. These two genes are mainly expressed in soybean roots and root nodules, and their expression is induced by iron deficiency stress. Overexpression of these genes can upregulate the expression levels of downstream iron uptake-related genes, increase iron accumulation in the plant, and thus improve soybean's tolerance to iron deficiency stress. Iron is an essential element for soybean symbiotic nitrogen fixation, and these genes affect the efficiency of symbiotic nitrogen fixation by regulating iron homeostasis, thereby influencing nitrogen assimilation and grain protein synthesis. However, no literature or patents currently disclose the function of the GmbHLH300 gene in regulating soybean branching development, adaptability to dense planting, and yield trait improvement; whether this gene participates in the regulation of soybean plant architecture and yield remains unknown.

[0009] In summary, current technologies lack comprehensive solutions for simultaneously increasing soybean branching number and yield under high-density planting conditions by regulating single genes or genetic modules. In particular, the pleiotropic and integrative regulatory role of the bHLH transcription factor family in the fine-grained regulation of soybean high-density planting adaptability and branching architecture remains a research gap. Developing and validating key genes with such functions is of significant theoretical and practical value for synergistically improving soybean tolerance to high-density planting and yield traits through genetic engineering, and for overcoming the technical bottleneck of increasing soybean yield under high-density planting conditions. Summary of the Invention

[0010] The purpose of this invention is to provide a soybean GmbHLH300 gene and its application in increasing the number of branches and yield in densely planted soybeans. This provides a novel research target for elucidating the molecular mechanism of plant type response and yield formation in densely planted soybeans, and also provides an important core gene resource for molecular breeding of soybeans that are tolerant to dense planting and high yield.

[0011] The objective of this invention is achieved through the following technical solution: This invention provides a soybean GmbHLH300 gene for increasing the number of soybean branches and yield under dense planting conditions. The nucleotide sequence of the GmbHLH300 gene is shown in SEQ ID NO:1.

[0012] The present invention also provides a protein encoded by the soybean GmbHLH300 gene, the amino acid sequence of which is shown in SEQ ID NO:2.

[0013] The present invention also provides a recombinant expression vector for increasing the number of branches and yield of soybean under dense planting conditions, wherein the recombinant expression vector contains the soybean GmbHLH300 gene.

[0014] Furthermore, the backbone of the recombinant expression vector is the PTF101 vector, and the GmbHLH300 gene is inserted into the multiple cloning site of the PTF101 vector, with the GmbHLH300 gene overexpressed by the 35S promoter.

[0015] The present invention also provides the application of the soybean GmbHLH300 gene, the protein, or the recombinant expression vector described herein in increasing the number of branches and yield of soybeans under dense planting conditions.

[0016] Furthermore, the improvement of soybean yield under dense planting conditions specifically refers to increasing the number of pods per plant, the number of grains per plant, and the yield per plant under dense planting conditions.

[0017] Furthermore, the application also includes enhancing the soybean's tolerance to dense planting, specifically by increasing stem diameter and inhibiting excessive plant growth under dense planting conditions.

[0018] Furthermore, overexpression of the soybean GmbHLH300 gene increases branch number and yield; the overexpression method includes the following steps: (1) The soybean GmbHLH300 gene was cloned into an overexpression vector to construct a recombinant overexpression vector; (2) The recombinant overexpression vector was transformed into Agrobacterium; (3) Use the transformed Agrobacterium to infect soybean seeds or explants to obtain transgenic plants or transgenic complex plants that overexpress the GmbHLH300 gene.

[0019] Furthermore, the overexpression vector in step (1) is the recombinant expression vector as described in claim 3 or 4.

[0020] Furthermore, the Agrobacterium mentioned in step (2) is Agrobacterium EHA101.

[0021] Beneficial effects: This invention is the first to demonstrate that the soybean GmbHLH300 gene can simultaneously regulate soybean branching development and yield performance under dense planting conditions. It fills the technical gap in the existing technology of lacking a single genetic module to synergistically improve soybean's adaptability and yield traits under dense planting conditions. It clarifies the multi-effect regulatory role of bHLH transcription factor family members in soybean dense planting architecture construction and fine regulation of branching. It provides a new research target for elucidating the molecular mechanism of soybean plant architecture response and yield formation under dense planting conditions, and also provides an important core gene resource for molecular breeding of soybean tolerating dense planting and high yield.

[0022] Overexpression of the GmbHLH300 gene in soybean can effectively promote the occurrence and development of effective branches under dense planting conditions, significantly increase the number of effective branches per plant, and alleviate the problems of suppressed axillary bud germination and reduced branching caused by increased interspecific competition, altered light signaling pathways, and enhanced apical dominance in high-density planting environments. At the same time, it can effectively increase plant stem diameter, inhibit excessive plant growth under dense planting conditions, optimize the overall plant structure of soybean, enhance the plant's adaptability to dense planting growth environment, reduce the risk of lodging in dense planting, and effectively solve the inherent contradiction of plant structure deterioration and reduced branching in traditional dense planting yield-increasing models.

[0023] Based on the improvement of plant type traits, overexpression of the GmbHLH300 gene can simultaneously increase the number of effective pods and grains per plant in soybeans under dense planting conditions, stabilize and improve the yield per plant, and achieve a synergistic improvement in the yield potential of individual soybeans and the light energy utilization rate of the population, ultimately achieving the goal of significantly increasing yield per unit area. After several years of field dense planting phenotypic identification and verification, the gene's regulatory effect on soybean branch number, stem diameter, and yield-related traits is stable and reliable. Furthermore, the higher the planting density, the more obvious the relative advantage of trait improvement, demonstrating excellent application value in large-scale field production.

[0024] This invention provides a recombinant vector for overexpressing the GmbHLH300 gene and a complete genetic transformation technology scheme. The overexpression recombinant vector is constructed using the PTF101 vector backbone, and soybean recipient material is transformed through Agrobacterium EHA101-mediated transformation. Transgenic soybean plants overexpressing the GmbHLH300 gene can be stably obtained. The technical path is mature and feasible, and can be directly applied to the breeding of high-yield, high-density-tolerant transgenic soybean varieties. It provides complete technical support for the synergistic improvement of soybean density tolerance and yield traits through genetic engineering, and has important theoretical guiding significance and practical application value for high-yield soybean breeding research and high-density cultivation production. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The image shows the results of detecting the relative expression patterns of the soybean GmbHLH300 gene in different tissues. Figure 2 The structural map of the GmbHLH300-PTF101 overexpression recombinant vector constructed in this invention; Figure 3This is an agarose gel electrophoresis image of a transgenic soybean candidate plant identified by genomic-level PCR using bar gene-specific primers; the left side of the image shows the DNA molecular weight marker, and the right side shows the lanes of PCR amplification products from different transgenic candidate lines. Figure 4 The figure shows the results of detecting the transcriptional expression level of the GmbHLH300 gene using real-time quantitative PCR technology. Figure 5 The field phenotypes of wild-type W82 and GmbHLH300OE-5# overexpression lines are compared at different planting densities. Among them: (a) is a visual comparison of the overall plant type and branching phenotype of soybean plants under the three planting densities; (b) is a visual comparison of the number of pods and yield per soybean plant under the three planting densities. Figure 6 The following are statistical results of agronomic and yield traits of wild-type W82 and GmbHLH300OE-5# overexpression lines under different planting densities: (a) is a bar chart of the number of branches per plant under different planting densities; (b) is a bar chart of the plant height per plant under different planting densities; (c) is a bar chart of the stem diameter per plant under different planting densities; (d) is a bar chart of the number of pods per plant under different planting densities; (e) is a bar chart of the number of seeds per plant under different planting densities; and (f) is a bar chart of the yield per plant under different planting densities. Detailed Implementation

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0030] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0031] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0032] The technical solution provided by the present invention will be described in detail below with reference to the embodiments. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional experimental conditions in the art or the conditions recommended in the reagent product instructions. Unless otherwise specified, the reagents, consumables, strains, carriers, and other materials used in the present invention can all be obtained through legitimate commercial channels.

[0033] Example 1: Tissue Expression Pattern Analysis of the Soybean GmbHLH300 Gene 1. Test materials The tested soybean variety was Williams 82 (Glycine max (L.) Merr.); the tested bacterial strain was Bradyrhizobium diazoefficiens strain USDA110, which was donated by Professor Cao Yangrong's research group at the College of Life Science and Technology, Huazhong Agricultural University.

[0034] 2. Test Methods (1) Soybean planting and inoculation treatment Soybean seeds were sown in vermiculite pre-soaked in a low-nitrogen nutrient solution and cultured conventionally for 7 days. Slow-growing soybean rhizobium USDA110 was inoculated into TY culture medium and cultured with shaking until the exponential growth phase (OD200). 600 =0.6~0.8), dilute the bacterial solution to OD using sterile ddH2O. 600 =0.08; 30 mL of diluted bacterial solution was poured over the roots of each soybean plant to complete the inoculation. Root, stem, leaf, and root nodule tissues were collected 21 days after inoculation, flower tissues were collected 60 days after inoculation, and pod and seed tissues were collected 80 days after inoculation. All samples were immediately flash-frozen in liquid nitrogen after collection and then transferred to a -80℃ freezer for storage.

[0035] (2) RNA extraction and quantitative PCR detection Total RNA was extracted from various tissue samples using TRIPure Reagent reagent, and the total RNA was reverse transcribed into cDNA using RNA reverse transcriptase. Primers for the specific expression detection of the GmbHLH300 gene were designed, with the sequences as follows: Upstream primer GmbHLH300-F: 5'-CCACAGACACAGATTGAACTTG-3' (SEQ ID NO:3) Downstream primer GmbHLH300-R: 5'-GTGAACGAAGTGAAGAAACCAA-3' (SEQ ID NO:4) Using cDNA from various tissues as templates, real-time quantitative PCR (qPCR) was performed using the primers described above to analyze the relative expression levels of the GmbHLH300 gene in different soybean tissues.

[0036] 3. Test Results The expression pattern of the GmbHLH300 gene in different soybean tissues is as follows: Figure 1 As shown. Figure 1 As shown, the expression level of the GmbHLH300 gene in root nodules, pods, and seeds is significantly higher than that in other tissues such as roots, stems, leaves, and flowers, suggesting that this gene may play an important biological role in soybean root nodule development, pod formation, and seed development. Based on this expression characteristic, further research will be conducted on the construction of overexpression vectors for the GmbHLH300 gene and the validation of its biological function.

[0037] Example 2 Construction of soybean GmbHLH300-PTF101 overexpression recombinant vector 1. Test materials PTF101 plant expression vector, Escherichia coli DH5α competent cells, Agrobacterium tumefaciens EHA101 competent cells, agarose gel DNA recovery kit, restriction endonucleases Hind III and BamH I, PCR amplification reagents, etc.

[0038] 2. Test Methods (1) Amplification primer design The coding sequence of the GmbHLH300 gene (SEQ ID NO:1) was obtained from the soybean genome database. Full-length amplification primers carrying restriction enzyme sites and protective bases were designed. The primer sequences are as follows (underlined sequences indicate the corresponding restriction enzyme sites and protective bases): Upstream primer GmbHLH300-101-F: 5'-GAATTCCCCGGGCTCGAGAAGCTTATGGTTGCTTTGTTTTCCC-3' (SEQ ID NO:5) Downstream primer GmbHLH300-101-R: 5'-CAGTTATCTAGATCCGGTGGATCCTTAGAAAATCCTTTGCTTCTCATA-3' (SEQ ID NO:6) (2) Amplification of the target gene fragment Total RNA was extracted from Williams 82 soybean leaves and reverse transcribed to obtain cDNA. Using the cDNA as a template, PCR amplification was performed using the primers described above to obtain the full-length coding region fragment of the GmbHLH300 gene.

[0039] (3) Recovery and purification of the target fragment The PCR amplification products were subjected to agarose gel electrophoresis. The gel block containing the target gene band was cut off under UV light and purified using an agarose gel DNA recovery kit to obtain the purified GmbHLH300 gene fragment.

[0040] (4) Vector digestion and homologous recombination ligation The PTF101 vector plasmid was extracted and double-digested with restriction endonucleases Hind III and BamH I to recover the linearized vector fragment. The purified GmbHLH300 target fragment was ligated into the linearized PTF101 vector via homologous recombination to construct a recombinant vector for the 35S promoter-driven overexpression of the GmbHLH300 genome.

[0041] (5) Transformation and identification of Escherichia coli The ligation product was transformed into E. coli DH5α competent cells, plated on LB agar plates containing the corresponding antibiotics, and incubated overnight at 37°C. Single colonies were picked for colony PCR verification, and positive clones were sent for sequencing verification. If the sequencing results were completely consistent with the target sequence, the recombinant vector was successfully constructed and named GmbHLH300-PTF101.

[0042] (6) Agrobacterium transformation The recombinant vector plasmid with correct sequencing was extracted, and Agrobacterium EHA101 competent cells were transformed using the freeze-thaw method. After PCR identification, the positive strain was stored in an ultra-low temperature freezer at -80℃ for later use.

[0043] 3. Test Results A successful recombinant overexpression vector of GmbHLH300-PTF101 was constructed. This vector, with a total length of 11893 bp, carries core components including a 35S promoter, a GmbHLH300 gene expression cassette, and a bar selection marker gene expression cassette. A complete map of the GmbHLH300-PTF101 recombinant vector is shown below. Figure 2 As shown.

[0044] Example 3 Molecular identification of soybeans transformed with recombinant vectors and transgenic plants 1. Test materials Mature soybean Williams 82 seeds, Agrobacterium EHA101 strain carrying the GmbHLH300-PTF101 recombinant vector, various plant tissue culture media, TRIPure Reagent reagent, DNA extraction reagents, etc.

[0045] 2. Test Methods (1) Soybean seed disinfection and germination Select plump, undamaged Williams 82 soybean seeds and disinfect them by chlorine fumigation for 10 hours. After disinfection, place the seeds in sterile water and incubate them in the dark at 26°C for 16 hours to allow the seeds to fully absorb water and germinate.

[0046] (2) Explant preparation Use a No. 15 scalpel blade to cut the epicotyl of the germinating seed, separate the two cotyledons along the midline of the two cotyledons, ensuring that each cotyledon has an intact growth point, and each seed can be used to prepare 2 independent explants.

[0047] (3) Agrobacterium infection and co-culture The prepared explants were placed in Erlenmeyer flasks at a ratio of 50 pieces / group, and 50 mL of Agrobacterium tumefaciens bacterial suspension resuspended in CCM liquid medium was added. The flasks were then incubated in the dark at 26°C and 90 rpm for 12 h on a shaker. After infection, the explants and bacterial suspension were poured onto autoclaved filter paper, and excess bacterial suspension on the surface of the explants was blotted dry. The explants were then dried in a clean bench. Subsequently, the explants were placed adaxially downwards in CCM solid medium culture dishes lined with filter paper, with 35 explants per dish. The dishes were incubated in the dark at 26°C for 4 days.

[0048] (4) Induction and subculturing of clustered shoots The co-cultured explants were transferred to shoot induction medium with the following formulation: Gamborg BasalSalt Mixture G768 (Phyto Tech) 3.1 g / L, sucrose (Shanghai Husheng Laboratory Equipment Co., Ltd.) 30 g / L, and MES (Sigma-Aldrich®) 0.59 g / L. The explants were cultured at 26°C in an incubator with 18 hours of light and 6 hours of darkness for 14 days. The resulting shoot clusters were then removed, and the explants were transferred to fresh shoot induction medium for subculture for 2 weeks.

[0049] (5) Bud elongation culture Explants with established meristems were harvested, and a new horizontal incision was made at the base of the growing point. The explants were then transferred to a shoot elongation medium. The elongation medium consisted of: MURASHIGE & SKOOG BAS SALT MIX M524 (PhytoTech) 4.4 g / L, sucrose 30 g / L, and MES 0.59 g / L. Every two weeks, the cultures were transferred to fresh elongation medium, with a new horizontal incision made at the base of the explant each time. After eight weeks of culture, clear differentiation of the elongated shoots began. Culture was continued for approximately four more weeks to obtain adventitious shoots of suitable length.

[0050] (6) Rooting culture and transplanting Healthy, elongated shoots approximately 5 cm in length were cut, and their bases were immersed in a 1 mg / mL IBA solution for 2 minutes. They were then transferred to a rooting medium for rooting culture. The rooting medium formula was: MURASHIGE & SKOOG BAS SALT MIX M5 242.2 g / L, sucrose 20 g / L, and MES 0.59 g / L. After two weeks of culture, once the adventitious shoots had developed sufficient roots, seedlings ready for transplanting were obtained. These seedlings were then transplanted into pots filled with potting soil, a mixture of vermiculite (Lingshou County Orchid, 3 mm particle size) and nutrient soil (Zhenjiang Peilei substrate) at a weight ratio of 3:1. After transplanting, the pots were covered with plastic bags to maintain moisture for 7 days. Afterward, the plastic bags were removed, and the plants were cultured at 26℃ under normal conditions.

[0051] (7) Molecular identification of transgenic plants: Leaf samples of transplanted and surviving plants were divided into two parts for genomic DNA extraction and total RNA extraction, respectively, and two-level molecular identification was carried out.

[0052] ① Genomic-level PCR identification: Genomic DNA was extracted from leaves and amplified by PCR using bar gene-specific primers. Positive transgenic plants were screened. The bar primer sequences are as follows: Upstream primer F: 5'-CTACATCGAGACAAGCACGGTCAA-3' (SEQ ID NO:7) Downstream primer R: 5'-AGAAACCCACGTCATGCCAGTTC-3' (SEQ ID NO:8) ② Transcriptional level qPCR identification: Leaves of plants that were positive by PCR were selected, and total RNA was extracted using TRIPure Reagent. After reverse transcription into cDNA, qPCR detection was performed using GmbHLH300 gene-specific detection primers (SEQ ID NO:3, SEQ ID NO:4) to verify the gene overexpression efficiency.

[0053] The overexpressing plants confirmed by molecular identification were cultured to maturity, and the harvested seeds were recorded as T0 generation seeds.

[0054] 3. Test Results The results of bar gene PCR identification are as follows: Figure 3 As shown. Figure 3 As shown, positive transgenic plants amplified the target band of the corresponding size, while wild-type W82 showed no corresponding band, confirming that the T-DNA fragment had been successfully integrated into the soybean genome. The qPCR expression level detection results are as follows: Figure 4 As shown. Figure 4 As shown, in the three transgenic lines GmbHLH300OE-1, GmbHLH300OE-3, and GmbHLH300OE-5, the relative expression level of the GmbHLH300 gene was significantly higher than that of the wild-type W82 plant, confirming the successful acquisition of soybean transgenic lines with stable overexpression of the GmbHLH300 gene.

[0055] Example 4: Identification of agronomic traits and yield of overexpressed soybean under dense field planting conditions 1. Test materials The test materials were stable T2 generation lines GmbHLH300OE-1#, GmbHLH300OE-3#, and GmbHLH300OE-5# overexpressing the GmbHLH300 gene, with wild-type Williams82 (W82) as the control material.

[0056] 2. Test Methods T0 generation seeds were cultured in a greenhouse. After screening for positive plants, T1 generation seeds were harvested. This process was continued until the T2 generation to obtain genetically stable homozygous lines for field trials. Field trials were conducted at the Gaocheng Dishang Experimental Station of the Hebei Provincial Grain and Oil Crops Research Institute. Sowing took place in early June each year, with harvesting in mid-October, and the trials were repeated for three consecutive years. Three planting density gradients were established, with a uniform row spacing of 50 cm. Low density: 30cm spacing between plants, equivalent to 67,000 plants per hectare; Medium density: 20cm spacing between plants, equivalent to 100,000 plants per hectare; High density: 10cm between plants, equivalent to 200,000 plants per hectare.

[0057] Conventional cultivation and management practices were adopted in the field. After the soybeans matured, representative plants with consistent growth were selected from each treatment, and agronomic and yield traits such as the number of branches, plant height, stem diameter, number of pods per plant, number of grains per plant, and yield per plant were investigated and statistically analyzed.

[0058] 3. Test Results Field plant phenotypes at different densities, such as Figure 5 As shown. Figure 5As shown in (a), the overall morphology and branching phenotype of wild-type W82 and GmbHLH300OE-5# plants in the field are compared at three plant spacings of 30cm, 20cm, and 10cm; Figure 5 As shown in Figure (b), this is a direct comparison of the number of pods per plant and the yield per plant at the corresponding densities. From the phenotypic photos, it can be clearly observed that as the planting density increases, the number of branches in the wild-type W82 plants decreases significantly, and the plant type tends to become leggy; while the GmbHLH300OE-5# overexpression line can maintain more effective branches under all density conditions, the plants are more robust overall, and the number of pods per plant is greater.

[0059] Statistical results of various agronomic and yield traits are as follows: Figure 6 As shown, the specific manifestation is as follows: (1) Number of branches: such as Figure 6 As shown in Figure (a), under the three planting densities, the number of branches of the GmbHLH300OE-5# line was significantly higher than that of the wild type W82. Moreover, the effect of increasing the number of branches was more prominent under high-density planting conditions, indicating that overexpression of GmbHLH300 can effectively alleviate the inhibitory effect of dense planting environment on soybean branching development.

[0060] (2) Plant height: such as Figure 6 As shown in (b), under the same planting density, the GmbHLH300OE-5# strain has a shorter plant height than the wild type W82. Under dense planting conditions, there is no obvious excessive growth, and the plant type is more suitable for high-density planting.

[0061] (3) Thick stem: such as Figure 6 As shown in (c), under low, medium, and high density conditions, the stem diameter of the GmbHLH300OE-5# strain was significantly greater than that of the wild type W82, indicating stronger stem support and reducing the risk of lodging under dense planting conditions.

[0062] (4) Number of pods per plant: such as Figure 6 As shown in (d), with increasing planting density, the number of pods per plant in wild-type W82 decreased significantly, while the GmbHLH300OE-5# line maintained a significantly higher number of pods per plant at all densities, providing sufficient storage capacity for yield formation.

[0063] (5) Number of seeds per plant: e.g. Figure 6 As shown in (e), under all three planting densities, the number of seeds per plant of the GmbHLH300OE-5# line was significantly higher than that of the wild type W82, with the difference reaching a significant level.

[0064] (6) Yield per plant: such as Figure 6As shown in (f), under low, medium and high density planting conditions, the yield per plant of the GmbHLH300OE-5# strain was significantly better than that of the wild type W82, and the higher the planting density, the more obvious the relative increase in yield.

[0065] The results of three years of repeated field trials show that overexpression of the GmbHLH300 gene can significantly optimize soybean plant architecture, effectively increase the number of effective branches, stem diameter, number of pods per plant and number of grains per plant under dense planting conditions, and ultimately achieve a significant increase in soybean yield under dense planting conditions. This provides important gene resources and technical approaches for high-yield soybean breeding that is tolerant to dense planting.

[0066] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A soybean GmbHLH300 gene for increasing the branch number of soybean and yield under dense planting conditions, characterized in that, The nucleotide sequence of the GmbHLH300 gene is shown in SEQ ID NO:

1.

2. The protein encoded by the soybean GmbHLH300 gene according to claim 1, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO:

2.

3. A recombinant expression vector for increasing the number of branches and yield in soybeans under dense planting conditions, characterized in that, The recombinant expression vector contains the soybean GmbHLH300 gene as described in claim 1.

4. The recombinant expression vector according to claim 3, characterized in that, The backbone of the recombinant expression vector is the PTF101 vector, and the GmbHLH300 gene is inserted into the multiple cloning site of the PTF101 vector, with the GmbHLH300 gene overexpressed by the 35S promoter.

5. The application of the soybean GmbHLH300 gene as described in claim 1, the protein as described in claim 2, or the recombinant expression vector as described in any one of claims 3-4 in increasing the number of branches and yield of soybeans under dense planting conditions.

6. The application according to claim 5, characterized in that, The improvement of soybean yield under dense planting conditions specifically refers to increasing the number of pods per plant, the number of grains per plant, and the yield per plant under dense planting conditions.

7. The application according to claim 5, characterized in that, The application also includes improving the ability of soybeans to tolerate dense planting, specifically by increasing stem diameter and inhibiting excessive plant growth under dense planting conditions.

8. The application according to claim 5, characterized in that, The overexpression of the soybean GmbHLH300 gene increases branch number and yield; the overexpression method includes the following steps: (1) The soybean GmbHLH300 gene was cloned into an overexpression vector to construct a recombinant overexpression vector; (2) The recombinant overexpression vector was transformed into Agrobacterium; (3) Use the transformed Agrobacterium to infect soybean seeds or explants to obtain transgenic plants or transgenic complex plants that overexpress the GmbHLH300 gene.

9. The application according to claim 8, characterized in that, The overexpression vector mentioned in step (1) is the recombinant expression vector described in claim 3 or 4.

10. The application according to claim 8, characterized in that, The Agrobacterium mentioned in step (2) is Agrobacterium EHA101.

Images