Application of soybean GmHY1 gene in negative regulation of soybean single plant grain number and / or pod number

Abstract

This application discloses the application of the soybean GmHY1 gene in negatively regulating the number of seeds and / or pods per soybean plant, belonging to the field of genetic engineering technology. This application experimentally verifies that the soybean GmHY1 gene with gene ID Glyma.16G167600.1.p or its encoded protein has the function of negatively regulating the number of seeds and / or pods per soybean plant. Reducing the expression or activity of the GmHY1 gene or its encoded protein can significantly increase the number of seeds and / or pods per soybean plant, which is of great significance for breeding high-yield soybeans or increasing soybean yield.

Description

Technical Field

[0001] This application belongs to the field of genetic engineering technology, specifically relating to the application of the soybean GmHY1 gene in negatively regulating the number of seeds and / or pods per soybean plant. Background Technology

[0002] As a globally important oilseed crop and source of plant protein, the genetic improvement of soybean yield traits has always been a core focus of breeding efforts. The number of seeds per plant, a key factor in soybean yield, is directly determined by the number of pods per plant and the number of seeds per pod. Therefore, elucidating the molecular mechanisms regulating the number of pods and seeds per soybean plant, and identifying related functional genes, is of significant theoretical and practical value for breeding high-yielding soybean varieties and ensuring food security.

[0003] Currently, molecular biology research on soybean yield-related traits has made some progress. Researchers have used techniques such as quantitative trait locus (QTL) mapping and genome-wide association analysis (GWAS) to identify multiple genetic loci and candidate genes associated with the number of seeds and pods per soybean plant. Meanwhile, the crucial role of transcription factors in regulating plant growth, development, and yield trait formation has been widely recognized. Among them, the triple-helix transcription factor family is a class of widely distributed and evolutionarily conserved regulatory proteins in plants, characterized by a DNA-binding domain composed of three tandem α-helices. The 64 known members of the triple-helix family in higher plants have been systematically classified into five subfamilies: GT-1, GT-2, GTγ, SH4, and SIP1, and play important regulatory functions in light response, stress tolerance, and organ development.

[0004] However, the systematic identification and biological function research of members of the soybean triple-helix transcription factor family are still relatively lagging behind, especially regarding whether these family members participate in regulating key yield traits such as the number of seeds per plant and the number of pods, with extremely limited discovery of relevant functional genes. Therefore, in-depth research on members of the soybean triple-helix transcription factor family, and the screening and identification of functional genes that can regulate the number of seeds per plant and the number of pods, is of great practical significance for elucidating the molecular regulatory network of soybean yield formation and creating new high-yielding germplasm. Summary of the Invention

[0005] In view of this, the primary objective of this application is to provide the application of the soybean GmHY1 gene in the negative regulation of the number of seeds and / or pods per soybean plant. This application verifies through relevant experiments that the soybean GmHY1 gene has a negative regulatory effect on the number of seeds and / or pods per soybean plant. By reducing the expression or activity of the GmHY1 gene or its encoded protein, the number of seeds and / or pods per soybean plant can be significantly increased, which is of great significance for breeding high-yield soybeans.

[0006] To achieve the above objectives, this application adopts the following technical solution: One aspect of this application discloses the application of the soybean GmHY1 gene or its encoded protein in the negative regulation of the number of seeds and / or pods per soybean plant, wherein the gene ID of the soybean GmHY1 gene is Glyma.16G167600.1.p, and the soybean reference genome version is Wm82.a4.v1.

[0007] Another aspect of this application discloses the use of the soybean GmHY1 gene in at least one of the following: (1) Increase the number of seeds per soybean plant and / or the number of pods per soybean plant; (2) To prepare soybeans with a high number of grains per plant and / or a high number of pods per plant; (3) Prepare products that increase the number of grains per soybean plant and / or the number of pods per soybean plant.

[0008] Another aspect of this application discloses a method for cultivating soybeans with a high number of seeds per plant and / or a high number of pods per plant, or for increasing the number of seeds per soybean plant and / or the number of pods per plant, comprising: The step of reducing the expression or activity of the GmHY1 gene or its encoded protein as described in this application in soybean plants.

[0009] This application has at least the following beneficial effects: The GmHY1 gene or its encoded protein provided in this application has a significant negative regulatory effect on the number of seeds and pods per soybean plant. By silencing, knocking out, or editing the gene of GmHY1 or its encoded protein, the expression level of GmHY1 or its encoded protein is significantly downregulated or its expression activity is significantly reduced, resulting in a significant increase in the number of seeds and pods per soybean plant. The new discovery of the function of the GmHY1 gene or its encoded protein in this application is crucial for high-yield soybean breeding. Attached Figure Description

[0010] Figure 1 This is a representation of the GmHY1 gene knockout form.

[0011] Figure 2 The map shows the GmHY1 overexpression vector.

[0012] Figure 3 The changes in plant architecture of soybean plants were compared between wild-type (WT), GmHY1 knockout (gmhy1), and GmHY1 overexpressing plants (OE). Figure 3 In the image, 'a' represents a plant 15 days after sowing in a greenhouse. Figure 3 In the middle, b represents a fully mature plant in the field. Figure 3 In Figure a, 'c' represents the soybean plant height corresponding to the height shown in Figure a. Figure 3 In the middle, d represents the soybean plant height corresponding to graph b.

[0013] Figure 4 The study investigated changes in protein and oil content, as well as the number of grains per plant and the number of pods, in wild-type (WT), GmHY1 knockout (gmhy1), and GmHY1 overexpressing (OE) soybeans. Figure 4 In the figure, 'a' represents the change in protein content. Figure 4 b represents the change in fat content. Figure 4 c represents the change in the number of grains per plant. Figure 4 In the figure, d represents the change in the number of pods.

[0014] In the figure, ns indicates no statistically significant difference. This indicates that p < 0.05. This indicates that p < 0.01. This means p < 0.001. Detailed Implementation

[0015] The embodiments of this application will be clearly and completely described below. The technical solutions in the embodiments described below are exemplary and only possible technical implementations of this application, not all possible implementations. Those skilled in the art can combine the embodiments of this application to obtain other embodiments without creative effort, and these embodiments are also within the protection scope of this application.

[0016] This application first discloses a novel function of a soybean gene GmHY1 (gene ID: Glyma.16G167600.1.p, soybean reference genome version Wm82.a4.v1) and its encoded protein. This application experimentally verifies that this gene has a negative regulatory effect on soybean yield traits; specifically, reducing the expression or activity of this gene or its encoded protein can significantly increase the number of seeds and / or pods per soybean plant.

[0017] In this application, for ease of description, the names of genes and proteins are used interchangeably. For example, GmHY1 can refer to a gene or the protein encoded by that gene. Those skilled in the art will understand that they represent different substances in different descriptive contexts. For instance, in this application, when GmHY1 is described as a protein, it refers to the protein with the amino acid sequence SEQ ID NO.1. When GmHY1 is described as a gene, it refers to the gene with the CDS sequence SEQ ID NO.2 or the full-length nucleotide sequence SEQ ID NO.3.

[0018] In practical applications, the phenotypic traits of soybean plants can be directionally altered by intervening in the expression level or biological activity of the soybean GmHY1 gene or its encoded protein. Specifically, negative regulation refers to the enhancement or increase of the regulated traits when the function of the GmHY1 gene or its encoded protein is weakened, inhibited, or eliminated. In practical applications, the expression of GmHY1 in soybean plants can be artificially and directionally reduced or its activity inhibited. In this application, the effect of negative regulation is specifically manifested as follows: compared to unregulated wild-type or control soybean plants, soybean plants with weakened GmHY1 gene function after regulation show a statistically significant increase in the number of grains per plant and / or the number of pods per plant. The number of grains per plant refers to the total number of fully plump grains harvested from a mature soybean plant, and the number of pods per plant refers to the total number of viable pods on a soybean plant. These two traits are key agronomic traits constituting soybean yield per plant; therefore, the discovery of this gene function in this application is of great significance for breeding high-yield soybeans or increasing soybean yield.

[0019] Furthermore, any known means in the art can be used to reduce expression levels or activity, without particular limitation. For example, gene editing technologies (such as the CRISPR / Cas9 system) can be used to introduce insertion or deletion mutations into the coding region or key regulatory region of the GmHY1 gene, thereby causing premature frameshifting or translation termination, preventing the production of a functional, complete protein. Another example is the use of RNA interference technology to introduce expression vectors capable of transcribing double-stranded RNA that forms hairpin structures into soybean cells, utilizing the plant's RNA silencing mechanism to specifically degrade the mRNA of the GmHY1 gene, thus significantly reducing its transcript abundance. Yet another example is the use of chemical mutagenesis (such as ethyl methanesulfonate, EMS, etc.) or physical mutagenesis (such as gamma ray radiation) to treat soybean seeds, allowing for the selection of individuals in the progeny population with increased seed and pod numbers due to loss-of-function mutations in the GmHY1 gene.

[0020] It should be understood that, regardless of the specific technical means used, the effect is clear and consistent: compared to the original soybean material without any intervention (such as wild-type soybean varieties), the number of seeds per plant and / or the number of effective pods per plant are significantly increased in soybean plants after negative regulation treatment.

[0021] The novel gene functions revealed in this application can be applied in the following aspects: In some specific examples, this could involve increasing the number of seeds per soybean plant and / or the number of pods per plant. In other specific examples, it could involve producing soybeans with a high number of seeds per plant and / or a high number of pods per plant. Specifically, this involves using the GmHY1 gene as a molecular marker or manipulation target to create or screen new soybean germplasm or varieties with high yield potential. Typically, the genetic engineering, gene editing, or marker-assisted selection breeding techniques described above can be used to directionally obtain soybean plants with weakened or absent GmHY1 gene function, thereby producing genetically stable, high-yielding new soybean materials.

[0022] Based on the above applications, this application further provides a specific method for cultivating high-yield soybeans or increasing soybean yield. The core step of this method is to reduce the expression or activity of the GmHY1 gene or its encoded protein as described above. Specifically, this can be at any stage of soybean growth and development, such as during seed germination, seedling stage, flowering stage, or pod-setting stage. The selected target tissue can be any part of the soybean, such as roots, stems, leaves, flowers, or developing seeds. As long as the final effect is to reduce the function of the GmHY1 gene in the soybean plant as a whole or in a local area (such as reproductive organs), the goal of increasing the number of seeds per plant and / or the number of pods can be achieved.

[0023] In some specific implementations, the GmHY1 gene is knocked out using CRISPR / Cas9 gene editing technology. The specific steps can be referred to existing technologies, and no specific limitations are made here.

[0024] Furthermore, in other specific examples, GmHY1 can be a product for increasing the number of seeds and / or pods per soybean plant. It should be understood that "product" here is a broad concept, referring to any formulation or kit that achieves its core mechanism of action by reducing the function of the GmHY1 gene, thereby increasing the number of seeds and / or pods per soybean plant. The product should at least include reagents that reduce the expression level or activity of the GmHY1 gene or its encoded protein.

[0025] The reagent is not specifically limited and can be any reagent in the art capable of specifically reducing the expression or activity of the GmHY1 gene or its encoded protein. In some specific examples, the reagent can be a specific antibody against the GmHY1 gene-encoded protein. When such an antibody is introduced into soybean cells, it can specifically bind to the GmHY1 protein, thereby blocking the protein's active site or promoting its degradation, achieving a functional inhibitory effect. For another example, the reagent can be a small molecule compound that can specifically bind to the GmHY1 protein and inhibit its activity as a transcription factor, enzyme, or signal transduction component. This application does not provide exhaustive examples.

[0026] In other specific examples, the core ingredient in the product is biomaterials that knock down or silence the GmHY1 gene.

[0027] In this application, knockdown generally refers to partially suppressing gene expression, while silencing refers to almost completely suppressing gene expression. The biomaterials referred to are materials capable of interfering with the expression of the target gene GmHY1 at the molecular level.

[0028] As specific examples, biological materials capable of knocking down or silencing the GmHY1 gene include, but are not limited to: DNA constructs expressing short hairpin RNA targeting the GmHY1 gene, recombinant vectors expressing precursor sequences of artificial microRNAs targeting the GmHY1 gene, DNA sequences capable of being transcribed to form antisense RNA targeting GmHY1 gene mRNA, or recombinant transgenic cell lines. Introducing these biological materials into soybean plants can continuously and stably reduce the transcriptional level of the GmHY1 gene.

[0029] Typical examples of biological materials used in this application include recombinant vectors or transgenic cell lines, but are not limited to these.

[0030] The recombinant vector refers to a circular or linear DNA molecule formed by artificially inserting a foreign DNA fragment (e.g., the aforementioned DNA sequence capable of forming hairpin RNA) into a backbone DNA molecule capable of replication and / or expression in host cells. Transforming soybean explants with such a recombinant vector via Agrobacterium-mediated transformation yields transgenic soybean plants with a stably knocked-down GmHY1 gene. There are no particular limitations on the choice of plant expression vector; for example, pCAMBIA3301 is widely used due to its high efficiency in Agrobacterium-mediated plant transformation and the ease of screening marker genes. Other vector series such as pBI121 and pGreen can also be selected in practical applications, and this application does not impose any limitations on this.

[0031] The aforementioned transgenic cell line refers to a cell population that can be cultured in vitro, in which the above-mentioned recombinant vector or its functional elements have been integrated into its genome through genetic transformation technology and can be stably inherited.

[0032] It is understandable that introducing the above-mentioned biological materials into soybean plants in a manner known in the art to knock down or silence the GmHY1 gene is a conventional method in the art, and therefore will not be described in detail here.

[0033] The present application will be further illustrated below with reference to specific embodiments. It should be noted that the specific embodiments below are for illustrative purposes only and do not limit the scope of the present application in any way.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application.

[0035] In addition, unless otherwise specified, methods without detailed conditions or steps are conventional methods, and the reagents and materials used are commercially available.

[0036] Example 1: Discovery of the GmHY1 gene In previous research, the applicant screened a gene that significantly regulates nitrogen adaptation in soybeans by analyzing the low-nitrogen transcriptome. Through field and greenhouse cultivation, the applicant found that this gene significantly regulates the number of seeds per soybean plant and the number of pods. This gene was named GmHY1 (Glycine max High Yield 1), with the gene ID Glyma.16G167600.1.p and the soybean reference genome version Wm82.a4.v1. For details, please refer to the website https: / / phytozomenext.jgi.doe.gov / info / Gmax_Wm82_a4_v1.

[0037] The amino acid sequence of the protein encoded by the GmHY1 gene is shown in SEQ ID NO.1, the CDS sequence is shown in SEQ ID NO.2, and the full-length nucleotide sequence is shown in SEQ ID NO.3.

[0038] Example 2: GmHY1 knockout material—gmhy1 mutant The tested soybean variety was Williams 82. Using CRISPR / Cas9 gene editing technology, GmHY1 was knocked out using Williams 82 as the target, resulting in the GmHY1 knockout mutant. The tested soybeans were provided by the Anhui Agricultural Soybean Molecular Breeding Research Group. The knockout target sites are shown in Table 1. Table 1 Target knockout sequences

[0039] Using the DNA extracted from the leaves of the gmhy1 mutant by the CTAB method as a template, the GmHY1 gene sequence was cloned using the specific primers in Table 2: Table 2 Primer Information

[0040] PCR amplification was performed using Vazyme's 2 × Rapid Taq Master Mix according to the reaction system in Table 3 and the reaction procedure in Table 4: Table 3 PCR reaction system

[0041] Table 4 PCR reaction procedure

[0042] The obtained PCR products were detected by agarose gel electrophoresis at a mass percentage of 1.0%.

[0043] The PCR products were sent to Hefei Youkang Biotechnology, a third-party commissioned agency, for sequencing, and the gene editing method was identified by sequence alignment using SnapGene.

[0044] pass Figure 1 It can be seen that the GmHY1 gene is missing a 76bp fragment in the gmhy1 mutant, indicating the successful construction of the knockout material.

[0045] Example 3: Construction of GmHY1 overexpression material OE Overexpression vectors were constructed using homologous recombination and the Golden Gate seamless cloning method (see Bird, JE, Marles-Wright, J., & Giachino, A. (2022). A User's Guide to Golden Gate Cloning Methods and Standards. ACS Synthetic Biology, 11(11). https: / / doi.org / 10.1021 / acssynbio.2c00355). The CDS region nucleotide sequence (SEQ ID NO.2) of GmHY1 was ligated into the plant expression vector pCAMBIA1305-d35SGFP (vector map is shown in...). Figure 3 The recombinant plasmid was then transferred into Agrobacterium to obtain recombinant Agrobacterium. The recombinant Agrobacterium was then transformed according to the cotyledon node transformation method (for details, please refer to Zhao Xiaowen, Wu Fangfang, Di Shaokang, et al. Agrobacterium-mediated genetic transformation technology process and key points of soybean cotyledon node [J]. Soybean Science, 2011, 30(03):362-368.) to obtain T0 generation plants.

[0046] Example 4: Identification of OE phenotype in gmhy1 mutant and overexpression materials Soybeans (including wild-type WT, knockout gmhy1, and overexpression OE) were mixed with vermiculite at a ratio of 2:1 and planted in a greenhouse at Anhui Agricultural University (16000 Lux, 70% humidity, 28℃ / 12h during the day and 26℃ / 12h at night). Simultaneously, they were planted at the experimental base of the Northern Anhui Experimental Station of Anhui Agricultural University in June 2025, and the phenotypic and yield indicators at each maturity stage were recorded.

[0047] The results are as follows Figure 3 As shown, on the 16th day of greenhouse growth, the plant heights of the mutant, wild-type, and overexpression materials reached 14.11 cm, 21.30 cm, and 26.62 cm, respectively. Compared with the wild-type, the plant heights of the mutant and overexpression materials decreased by 33.76% and increased by 24.98%, respectively. Figure 3 (a and c); at full maturity in the field, the plant heights of the mutant, wild-type, and overexpression material reached 78.73 cm, 86.97 cm, and 103.61 cm, respectively. Compared with the wild-type, the plant heights of the mutant and overexpression material decreased by 9.47% and increased by 19.13%, respectively. Figure 3 (b and d in the middle).

[0048] The results are as follows Figure 4 As shown, at full maturity in the field, there were no significant differences in protein and lipid content between the mutant and the overexpression material compared to the wild type. Figure 4 The number of individual plants in mutant, wild-type, and overexpression materials were 568.33, 427.00, and 310.33, respectively. Compared with the wild-type, the number of individual plants in mutant and overexpression materials increased by 33.10% and decreased by 27.32%, respectively. The number of pods in mutant, wild-type, and overexpression materials were 205.67, 155.67, and 108.67, respectively. Compared with the wild-type, the number of pods in mutant and overexpression materials increased by 32.12% and decreased by 30.19%, respectively. Figure 4 (c and d).

[0049] The above examples demonstrate that inhibiting the expression of GmHY1 can significantly increase the number of grains and pods per soybean plant.

[0050] It should be noted that this application is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments with the same structure and effect as the technical concept within the scope of this application are included in the technical scope of this application. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of this application, are also included in the scope of this application.

Claims

1. The application of the soybean GmHY1 gene or its encoded protein in the negative regulation of soybean seed number and / or pod number per plant, characterized in that, The gene ID of the soybean GmHY1 gene is Glyma.16G167600.1.p, and the soybean reference genome version is Wm82.a4.v1.

2. The application as described in claim 1, characterized in that, The amino acid sequence of the protein encoded by the soybean GmHY1 gene is shown in SEQ ID NO.

1.

3. The application as described in claim 1, characterized in that, The CDS sequence of the soybean GmHY1 gene is shown in SEQ ID NO.2, and the full-length nucleotide sequence is shown in SEQ ID NO.

3.

4. The application as described in claim 1, characterized in that, The negative regulation refers to reducing the expression level or activity of GmHY1 or its encoded protein, thereby increasing the number of seeds per soybean plant and / or the number of pods per plant.

5. The use of the soybean GmHY1 gene as defined in any one of claims 1-4 in at least one of the following: (1) Increase the number of seeds per soybean plant and / or the number of pods per soybean plant; (2) To prepare soybeans with a high number of grains per plant and / or a high number of pods per plant; (3) Prepare products that increase the number of grains per soybean plant and / or the number of pods per soybean plant.

6. The application as described in claim 5, characterized in that, The product is a formulation or a reagent kit.

7. The application as described in claim 5, characterized in that, The product contains reagents that reduce the expression level or activity of GmHY1 or its encoded protein; or, contains biological material that knocks down or silences GmHY1.

8. The application as described in claim 7, characterized in that, The biological material is a recombinant vector or a transgenic cell line.

9. A method for cultivating soybeans with a high number of seeds per plant and / or a high number of pods per plant, or for increasing the number of seeds per soybean plant and / or the number of pods per plant, characterized in that, include: The step of reducing the expression or activity of the GmHY1 gene or its encoded protein as defined in any one of claims 1-4 in soybean plants.

10. The method as described in claim 9, characterized in that, The expression or activity of the GmHY1 gene or its encoded protein is reduced by knocking out GmHY1 using CRISPR / Cas9 gene editing technology.

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