Application of soybean GmDof13.3 gene in regulation and control of soybean flowering time and plant height growth

Abstract

The invention discloses application of a soybean GmDof13.3 gene in regulation and control of flowering time and plant height growth of soybeans, and belongs to the technical field of gene engineering and molecular breeding. The CDS sequence of the soybean GmDof13.3 gene is as shown in SEQ ID NO. 1 (sequence identifier number 1). According to the invention, the important function of the soybean GmDof13.3 gene in the aspect of regulating and controlling the flowering time and the plant height is confirmed for the first time. The gene is knocked out through a CRISPR / Cas9 technology, the flowering time of soybeans under the conditions of long sunshine and field can be obviously promoted to be advanced, and the plant height is effectively increased. The discovery fills up the research blank of the soybean DOF family gene in the field of flowering and plant height regulation and control, and provides a new key target for analyzing the molecular mechanism of soybean photoperiod response and growth and development.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and molecular breeding technology, and in particular to the application of a soybean GmDof13.3 gene in regulating soybean flowering time and plant height growth. Background Technology

[0002] Transcription factors play a central role in the molecular regulatory network of plant growth, development, and stress responses. Among them, the DOF (DNA binding with one finger) family, a plant-specific transcription factor family, is named for its conserved single zinc finger DNA-binding domain. Its members perform important regulatory functions in various plant physiological processes. In soybean, research on DOF family genes has largely focused on abiotic stress responses. Numerous studies have confirmed that some transcripts of this family participate in the regulation of soybean tolerance to stresses such as drought, salinity, and low temperature, providing important targets for improving soybean stress resistance through genetic engineering. However, as a typical short-day crop, flowering time and plant height are key agronomic traits determining soybean's adaptability and yield. Elucidating the related regulatory mechanisms is crucial for soybean molecular breeding, but current research on the function of soybean DOF family genes in regulating these core agronomic traits remains relatively scarce.

[0003] In model plants such as Arabidopsis thaliana and rice, functional studies of the DOF family of genes have expanded to the field of growth and development regulation. In Arabidopsis, several DOF members have been confirmed to participate in the photoperiod response pathway, regulating flowering time by binding to the promoter regions of downstream flowering-related genes, revealing the important role of DOF genes in the regulation of plant reproductive growth. This finding suggests that DOF family genes may exhibit functional conservation across different plants, and their regulatory network may encompass both stress responses and extend to core growth and development processes. However, gene function is heterogeneous among species, and findings from model plants cannot be directly extrapolated to soybean. Whether there are members in the soybean DOF family involved in regulating flowering time and plant height, and their specific regulatory mechanisms, require targeted research and verification. Currently, a systematic understanding of this research has not yet been formed.

[0004] A review of existing technical literature revealed a lack of functional studies on GmDof13.3, a specific member of the soybean DOF family. There are no reports on the gene's basic function, and even fewer studies documenting its association with agronomic traits such as flowering time and plant height. This research gap is primarily due to the large size of the soybean genome and the numerous members in the DOF family. The specificity of gene functions makes it difficult to screen for target genes regulating specific agronomic traits. Furthermore, compared to stress resistance studies, functional verification of genes related to flowering and plant height regulation requires a more refined phenotypic identification system, increasing the complexity of the research. These issues result in a lack of DOF gene targets for flowering time and plant height regulation in soybean molecular breeding, limiting the application of precise gene editing technologies to improve soybean adaptability and yield. Summary of the Invention

[0005] The purpose of this invention is to provide an application of the soybean GmDof13.3 gene in regulating soybean flowering time and plant height, thereby addressing the problems existing in the prior art. This invention reveals for the first time that knocking out the soybean GmDof13.3 gene can significantly promote earlier flowering and increase plant height, filling the functional gap in the regulation of flowering and plant height by this gene. This discovery provides a precise gene editing target for soybean molecular breeding, which can be directly used to cultivate new early-flowering, tall varieties, and has significant application value in improving soybean adaptability and breeding efficiency.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides the application of knocking out the soybean GmDof13.3 gene in any of the following:

[0008] (1) Promotes earlier flowering time of soybeans;

[0009] (2) Cultivating genetically modified soybeans with earlier flowering time;

[0010] (3) Promotes soybean plant height growth;

[0011] (4) Cultivating transgenic soybeans with higher plant height;

[0012] The CDS sequence of the soybean GmDof13.3 gene is shown in SEQ ID NO.1.

[0013] This invention also provides the use of inhibiting the expression of soybean GmDof13.3 protein in any of the following:

[0014] (1) Promotes earlier flowering time of soybeans;

[0015] (2) Cultivating genetically modified soybeans with earlier flowering time;

[0016] (3) Promotes soybean plant height growth;

[0017] (4) Cultivating transgenic soybeans with higher plant height;

[0018] The amino acid sequence of the soybean GmDof13.3 protein is shown in SEQ ID NO.2.

[0019] The present invention also provides the use of the recombinant vector for knocking out the soybean GmDof13.3 gene in any of the following:

[0020] (1) Promotes earlier flowering time of soybeans;

[0021] (2) Cultivating genetically modified soybeans with earlier flowering time;

[0022] (3) Promotes soybean plant height growth;

[0023] (4) Cultivate transgenic soybeans with higher plant height.

[0024] The present invention also provides the use of recombinant microorganisms containing the recombinant vector in any of the following:

[0025] (1) Promotes earlier flowering time of soybeans;

[0026] (2) Cultivating genetically modified soybeans with earlier flowering time;

[0027] (3) Promotes soybean plant height growth;

[0028] (4) Cultivate transgenic soybeans with higher plant height.

[0029] Optionally, the soybean GmDof13.3 gene in soybeans may be mutated using gene editing technology, resulting in the loss of function of the soybean GmDof13.3 gene expression protein, so as to advance the daytime flowering time of the soybeans and / or increase the plant height of the soybeans.

[0030] Optionally, methods for causing loss of function of the soybean GmDof13.3 gene expression protein include deletion, insertion, or mutation of bases on the target sequence.

[0031] The present invention also provides a method for advancing the flowering time of soybeans, comprising the following steps: editing the soybean GmDof13.3 gene in soybeans to render the function of the protein expressed by the soybean GmDof13.3 gene lost, thereby advancing the flowering time of soybeans; the CDS sequence of the soybean GmDof13.3 gene is shown in SEQ ID NO.1.

[0032] The present invention also provides a method for cultivating transgenic soybeans with advanced flowering time, comprising the following steps: gene editing of the soybean GmDof13.3 gene in the cotyledons of soybean to render the function of the protein expressed by the soybean GmDof13.3 gene lost; and then cultivating the soybeans to obtain transgenic soybeans with advanced flowering time; the CDS sequence of the soybean GmDof13.3 gene is shown in SEQ ID NO.1.

[0033] The present invention also provides a method for promoting soybean plant height growth, comprising the following steps: gene editing of the soybean GmDof13.3 gene in soybean to render the function of the protein expressed by the soybean GmDof13.3 gene lost, thereby promoting the plant height growth of soybean; the CDS sequence of the soybean GmDof13.3 gene is shown in SEQ ID NO.1.

[0034] The present invention also provides a method for cultivating transgenic soybeans with greater plant height, comprising the following steps: gene editing of the soybean GmDof13.3 gene in the cotyledons of soybean to render the function of the protein expressed by the soybean GmDof13.3 gene lost; and then cultivating the soybeans to obtain transgenic soybeans with greater plant height; the CDS sequence of the soybean GmDof13.3 gene is shown in SEQ ID NO.1.

[0035] The present invention discloses the following technical effects:

[0036] This invention is the first to demonstrate the important function of the soybean GmDof13.3 gene in regulating flowering time and plant height. Knocking out this gene using CRISPR / Cas9 technology significantly promoted earlier flowering in soybeans under long-day and field conditions, and effectively increased plant height. This discovery fills a research gap in the regulation of flowering and plant height in soybean DOF family genes, and provides a new key target for elucidating the molecular mechanisms of soybean photoperiod response and growth development.

[0037] Based on the above functions, this invention provides precise and efficient gene editing targets for soybean molecular breeding. By knocking out or inhibiting the expression of the GmDof13.3 gene or its protein, it can be directly applied to cultivate new transgenic soybean germplasm with early flowering and tall stalks, which helps to optimize the planting adaptability, light energy utilization efficiency and potential yield of soybeans, and has important practical application value for promoting soybean variety improvement and molecular design breeding. Attached Figure Description

[0038] 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.

[0039] Figure 1 The amino acid sequences of Dof proteins in different species are compared; underlined regions represent Dof domains; triangles represent four conserved Cys residues.

[0040] Figure 2 For predicting the hydrophobicity of the GmDof13.3 protein;

[0041] Figure 3 For the prediction of the transmembrane region of GmDof13.3;

[0042] Figure 4 Analysis of phosphorylation sites of GmDof13.3 protein;

[0043] Figure 5 The secondary structure of the GmDof13.3 protein;

[0044] Figure 6 Predicting the tertiary structure of the GmDof13.3 protein;

[0045] Figure 7 This is the phylogenetic tree of GmDof13.3;

[0046] Figure 8 Analysis of GmDof13.3 promoter elements;

[0047] Figure 9 The expression of the GmDof13.3 gene under long-day and short-day treatments is shown; where L represents long-day and S represents short-day.

[0048] Figure 10 The expression of GmDof13.3 under different hormone treatments with GA3 (A) and MeJA (B);

[0049] Figure 11 The expression of GmDof13.3 in different tissues;

[0050] Figure 12 Subcellular localization of the GmDof13.3 gene;

[0051] Figure 13 The structural diagram of the recombinant plasmid GmDof13.3-pGES401;

[0052] Figure 14The gene structure of the target site for the dof13.3 gene knockout mutant;

[0053] Figure 15 The expression of GmDof13.3 in transgenic soybeans;

[0054] Figure 16 Phenotypes of T3 generation wild-type and dof13.3 gene knockout lines under long-day conditions; where A is the inflorescence of wild-type and dof13.3 gene knockout lines; B is the overall phenotype of wild-type and dof13.3 gene knockout lines.

[0055] Figure 17 Phenotypes of field T3 generation wild-type and dof13.3 gene knockout lines. Detailed Implementation

[0056] 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.

[0057] 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. Any stated value or intermediate value within a stated range, as well as each smaller range between 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] Example 1: Bioinformatics analysis of the GmDof13.3 gene

[0062] 1. Experimental Methods

[0063] First, the coding region, amino acid sequence, and promoter sequence of the GmDof13.3 gene were located in Phytozome. Conserved domains of the GmDof13.3 protein were analyzed using NCBI, SMART, and Pfam online databases; isoelectric point and molecular weight were analyzed using Expasy online databases; phosphorylation sites were predicted using the online tool NetPhos; transmembrane structure was analyzed using TMHMM online databases; secondary structure was predicted using the SOPMA database; tertiary structure was predicted using MODBASE and SWISS MODEL online databases; homologous genes were retrieved from NCBI and Phytozome databases, and a phylogenetic tree of homologous genes was constructed using MEGA X software; cis-regulatory elements in the promoter region of the GmDof13.3 gene were analyzed using PlantCARE and TSSP websites.

[0064] 2. Experimental Results

[0065] 2.1 GmDof13.3 Protein Domain Analysis

[0066] The GmDof13.3 gene is located on soybean chromosome 13, with a total length of 1823 bp, including a 987 bp CDS region, as shown in SEQ ID NO.1. The encoded amino acid sequence is shown in SEQ ID NO.2.

[0067] SEQ ID NO.1 (GmDof13.3 gene CDS sequence):

[0068] ATGATAAAAAAAACAAACACACACTACACTAAGTTCAGATCACAGAGAGACAAAGATATGGAACAAGAGGGAGAAAAAGGGAGAGAGGAGAAAAGACAAATTCAACAACAGCAACCTCCTCCTCAGCAGCATCAGAAATGTCCACGCTGCGATTCCATGAACACCAAGTTCTGCTACTTCAACAACTATAGCCTCTCGCAGCCTCGTCATTTCTGCAAAGCGTGTAAAAGGTACTGGACACTCGGTGGAACCTTCAGGAACATACCCGTTGGTGGCGGTTCCCGCAAAGTGAAGCGTGGCAAAACAAATTCTCCATCTTCTTCTTCTTCTTCTTCTTCTTCTTGTTCAAATTTGCTCTCACAGCCGCAGCAGAATCTGTTGATGAGGCCTTCTCCACCACCACTCACTACTAATACTATGGTTCAGTCAACTAGTCCTTATTATTATAATCTTGGTGTTGGTGGAAATGGGTATCTCTCTTTTCATTCTTCTCTGAACAACAACACACCATCACAGCCTTCCGATCAGTATCTGAAAGTTGGTGGTGGTGATCATGTTGCTGGTTCTTCTAATATTTCTCCTCTTGTGTCTGGCTTCAATAACGCTGCTAGTTATTCGCTTCCACCGCGATTCCACCACCAGCAGCAGCAACAATCTATGCATCCTGCACAACAACAACAACAACGTTTGATTAACATTCCATCAAACATGGCTAGTAGTAGCAGTGATGTTTCTCATTCTGGTGTTCGCCCGCAAAGCTTGATCAACAATGTTTCAACAAGTACAGACCATAATGTTTCAACAAGTACAAACCATAGGGCTATTACTACTTCTGATGCTTCTTTGTGGAGTGCTGCTACTATCAACGCCACTTCCATCGGTGGAAACTCTGATCAGAACAATGTCGTCAAAGGGAGTTCTTCTTCTTCTTCTTCTTTGGTCCCAAATCTTTGGGTTCATCGCCCTGGGTTTGGTCCTCCTCAATAA。

[0069] SEQ ID NO.2:

[0070] MIKKTNTHYTKFRSQRDKDMEQEGEKGREEKRQIQQQQPPPQQHQKCPRCDSMNTKFCYFNNYSLSQPRHFCKACKRYWTLGGTFRNIPVGGGSRKVKRGKTNSPSSSSSSSSSCSNLLSQPQQNLLMRPSPPPLTTNTMVQSTSPYYYNLGVGGNGYLSFHSSL NNNTPSQPSDQYLKVGGGDHVAGSSNISPLVSGFNNAASYSLPPRFHHQQQQQSMHPAQQQQQRLINIPSNMASSSSDVSHSGVRPQSLINNVSTSTDHNVSTSTNHRAITTSDASLWSAATINATSIGGNSDQNNVVKGSSSSSSLVPNLWVHRPGFGPPQ*.

[0071] Analyze the domains of GmDof13.3 using online tools NCBI and SMART. Figure 1 A highly conserved fragment (zf-Dof) consisting of 53 amino acids was discovered. This fragment contains a CX2CX21CX2C motif and four conserved Cys residues. It is a conserved domain unique to the Dof (DNA binding with one finger) family of plant-specific transcription factors, proving that GmDof13.3 belongs to the soybean Dof transcription factor family.

[0072] 2.2 GmDof13.3 Analysis of protein physicochemical properties

[0073] Analysis using ExPASy's ProtParam tool showed that the GmDof13.3 protein has a relative molecular weight of 35.93 kDa, a theoretical isoelectric point of 9.87, an aliphatic coefficient of 49.91, and a total average hydrophilicity (GRAVY) of -0.899, classifying it as a hydrophilic protein. Figure 2 The prediction results of transmembrane helices indicate that the GmDof13.3 protein lacks transmembrane structures. Figure 3 ).

[0074] 2.3 Analysis of GmDof13.3 protein phosphorylation sites

[0075] NetPhos was used to predict the phosphorylation sites of GmDof13.3. The results showed that the amino acid sequence of GmDof13.3 contains multiple phosphorylation sites, including 42 serine, 11 threonine, and 6 tyrosine active sites. Figure 4 ).

[0076] 2.4 GmDof13.3 Protein Structure Analysis

[0077] The secondary structure of the GmDof13.3 protein was analyzed using the SOPMA website, and the results are as follows: Figure 5 The secondary structure of the encoded protein is characterized by 11.59% α-helices, 63.72% random coils, 21.34% extended strands, and 3.35% beta turns. The tertiary structure of the GmDof13.3 protein was analyzed using the MODBASE online tool as follows: Figure 6 As shown.

[0078] 2.5 Construction of the soybean GmDof13.3 phylogenetic tree

[0079] Download the amino acid sequence encoded by the GmDof13.3 gene from NCBI, select nine species with high homology to the GmDof13.3 protein, and construct a phylogenetic tree using the MEGA11 software package. Figure 7 The results showed that species with high homology clustered in two clades, and were in the same clade as soybean, Arabidopsis thaliana, cucumber, rice, wheat, tomato, and maize, with Arabidopsis showing high homology.

[0080] 2.6 Analysis of GmDof13.3 promoter elements

[0081] The portion of the GmDof13.3 gene promoter sequence beyond 2 kb from the start codon was inserted into the TSSP-predicted transcription start site. PlantCARE and the TSSP website were used to analyze the cis-regulatory elements in the GmDof13.3 gene promoter region. The results revealed multiple light-related cis-regulatory elements in the GmDof13.3 gene promoter sequence, at sites such as... Figure 8 As shown in the figure, this indicates that the gene GmDof13.3 may be involved in the photoperiodic signal transduction pathway.

[0082] Example 2: Analysis of the expression pattern of the GmDof13.3 gene

[0083] 1. Experimental Methods

[0084] 1.1 Expression analysis of GmDof13.3 gene under long and short day conditions

[0085] Soybeans Willams82 were heated at 25°C with 250 µmol m 2 s 1 In a white-light incubator, after the first trifoliate leaf of the Williams82 soybean seedlings had fully unfolded, the seedlings were subjected to long-day (16 h / 8 h light / dark) and short-day (8 h / 16 h light / dark) treatments for 15 days. Taking the moment the lights were turned on as ZT0 h, samples were taken at seven points: ZT0 h, 4 h, 8 h, 12 h, 16 h, 20 h, and 24 h to extract RNA for quantitative analysis.

[0086] 1.2 Analysis of expression patterns under different hormones

[0087] Soybean variety Williams82 was planted in small pots (vermiculite: nutrient soil = 1:1, v / v) and cultured in a light incubator at 24℃ under a 16-h light / 8-h dark incubator. After the first trifoliate leaf was fully expanded, soybean seedlings with uniform growth were treated as follows. Using normal growth treated with distilled water as a control, leaves were collected at 0, 0.5, 1, 3, 6, 9, 12, 24, and 48 hours and flash-frozen in liquid nitrogen. GA3 (100 μmol / L) and MeJA (450 μmol / L) were prepared and sprayed on soybean leaves. Leaves at 0, 0.5, 1, 3, 6, 9, 12, 24, and 48 hours were collected and stored at -80℃ for analysis of the expression pattern of the GmDof13.3 gene under different hormones.

[0088] 1.3 Analysis of Expression Patterns in Different Organizations

[0089] Soybean Willams82 was planted in small pots (vermiculite: nutrient soil = 1:1, v / v) and cultured in a light incubator at 24℃ under 16 h light / 8 h dark conditions. After the second trifoliate leaf was fully expanded, the roots, stems and leaves of the soybean were collected. After the plant flowered, the flowers, pods and fruits were collected. RNA was extracted from the above samples for tissue-specific expression pattern analysis of the GmDof13.3 gene.

[0090] 1.4 RNA extraction from Williams82 leaves

[0091] Total RNA was extracted using Trizol.

[0092] (1) Take 200mg of fresh leaves of Williams82 in a centrifuge tube and grind them into powder in liquid nitrogen using a steel ball of appropriate size and a grinding tool;

[0093] (2) Add 1 mL Trizol to the centrifuge tube, shake vigorously, and let stand for 5 min; then add 200 µL CHCl3, vortex, let stand at 4 °C for 5 min, and then at 12000 rpm for 15 min.

[0094] (3) Transfer the supernatant to a new tube, add 600 µL of C3H8O and mix well. Let stand at 4°C for 15 min, then at 12000 rpm for 10 min. Discard the supernatant.

[0095] (4) Add 1 mL of 75% C2H5OH to the centrifuge tube, centrifuge at 12000 rpm for 5 min, and then discard the supernatant;

[0096] (5) Centrifuge at 12000 rpm for 2 min, dry the RNA precipitate for 5-10 min, add 30-50 µL of DEPC water to the centrifuge tube to dissolve the RNA, and store at -80℃.

[0097] 1.5 cDNA Synthesis

[0098] (1) Add the reaction solution (Table 1) to the centrifuge tube:

[0099] Table 1 cDNA reaction system

[0100]

[0101] (2. Gently pipette to mix, incubate at 42°C for 2 min, and place on ice after the reaction is complete;)

[0102] (3) Prepare 2× Master Mix, and then add the (1) centrifuge tube to the system (Table 2).

[0103] Table 2 2×Master Mix reaction system

[0104]

[0105] (4) The above mixture was heated at 37°C for 15 min; at 85°C for 5 Ses; after the reaction was completed, it was stored at -20°C for later use.

[0106] 1.6 Quantitative Real-Time PCR

[0107] Based on the GmDof13.3 sequence (Glyma.13G177600) on Phytozome, primers for real-time PCR were designed (Table 3). The amplification system and amplification program are shown in Tables 4 and 5, respectively.

[0108] Table 3 GmDof13.3 Quantitative PCR Primers

[0109]

[0110] Table 4 Quantitative PCR reaction system

[0111]

[0112] Table 5 PCR reaction procedure

[0113]

[0114] 2. Experimental Results

[0115] 2.1 Expression patterns of GmDof13.3 under different day lengths

[0116] Real-time RT-PCR analysis was used to analyze the changes in the mRNA transcriptional abundance of the GmDof13.3 gene in soybean leaves over 24 hours under long and short day conditions. Figure 9 The transcriptional abundance of GmDof13.3 under long-day conditions was significantly higher than that under short-day conditions, with the highest abundance observed at 8 hours under long-day conditions, while the transcriptional level of GmDof13.3 did not change significantly under short-day conditions. This indicates that GmDof13.3 expression in Williams82 is induced by long-day conditions.

[0117] 2.2 Analysis of the expression patterns of GmDof13.3 under different hormones

[0118] To investigate the expression pattern of GmDof13.3 under different hormones, soybeans were treated with GA3 (100 μmol / L) and MeJA (450 μmol / L), respectively. The changes in GmDof13.3 expression levels under GA3 (100 μmol / L) and MeJA (450 μmol / L) treatments were analyzed using quantitative real-time PCR (qRT-PCR). Figure 10 As shown, the expression level of GmDof13.3 increased significantly after exogenous application of GA3 and MeJA, and the expression peaks appeared at 6 h and 3 h, respectively, indicating that GmDof13.3 was induced by GA3 and MeJA.

[0119] 2.3 Analysis of the expression patterns of GmDof13.3 in different tissues

[0120] RNA was extracted from the roots, stems, flowers, pods, and fruits of seedlings at the three-leaf stage. cDNA was obtained through reverse transcription and analyzed by real-time quantitative PCR (qRT-PCR). The results are as follows: Figure 11As shown, GmDof13.3 is expressed in roots, stems, flowers, pods and fruits, but its expression in tissues is specific, with relatively high expression levels in leaves and roots.

[0121] To investigate the subcellular localization of GmDof13.3, this invention constructed an expression vector (35S::GFP-GmDof13.3) fusing GmDof13.3 with green fluorescent protein (GFP), and transformed this fusion protein into tobacco leaves using Agrobacterium-mediated transformation. The green fluorescence signal in tobacco mesophyll cells was observed using fluorescence microscopy. The results showed that when expressed using the 35S::GFP vector, GFP fluorescence was uniformly distributed throughout the cell. However, the 35S::GFP-GmDof13.3 fusion protein specifically localized to the nucleus of tobacco mesophyll cells. Figure 12 These results indicate that GmDof13.3 is a nuclear localized gene.

[0122] Example 3: Construction and phenotypic analysis of GmDof13.3 gene knockout mutant

[0123] 1. Construction of the plant gene knockout vector GmDof13.3-pGES401

[0124] (1) Design sgRNA target sequences for the GmDof13.3 gene using the CRISPR 2.0 online tool.

[0125] sgRNA (GmDof13.3-1): TACTGATCGGAAGGCTGTGATGG, SEQ ID NO.13;

[0126] sgRNA (GmDof13.3-2): ACTTTCAGATACTGATCGGAAGG, SEQ ID NO.14;

[0127] sgRNA (GmDof13.3-3): GTATTAGTAGTGAGTGGTGGTGG, SEQ ID NO.15;

[0128] sgRNA (GmDof13.3-4): ATCTTGGTGTTGGTGGAAATGGG, SEQ ID NO.16;

[0129] (2) Design primers GmDof13.3-cas9-F and GmDof13.3-cas9-R, with the following specific sequences:

[0130] GmDof13.3-cas9-F: CAGTGGTCTCATGCAGTATTAGTAGTGAGTTGTGGGTTTCAGAG, SEQ IDNO.7;

[0131] GmDof13.3-cas9-R:CAGTGGTCTCATGCAGTATTAGTAGTGAGTTGTGGGTTTCAGAG; SEQ ID NO.8.

[0132] (3) Using soybean cDNA as a template, amplification was performed using Biorun Pfu PCR Mix. The reaction system (50 μL) consisted of: 1 μL template, 2 μL each of forward and reverse primers, 25 μL Biorun Pfu PCR Mix, and ddH2O to a final volume of 50 μL. The program was: 94℃ for 5 min; 94℃ for 30 sec, 50℃ for 45 sec, 72℃ for 34 sec, for 30 cycles; and a final extension at 72℃ for 30 min.

[0133] Gel recovery: The PCR products were extracted from the gel after agarose gel electrophoresis to obtain the sgRNA template fragment (rDNAT1).

[0134] (4) Enzyme digestion and ligation: The pGES401 vector was digested with Eco31I and ligated with the rDNA T1 fragment using T4 ligase. The reaction system (20 μL) consisted of: 4 μL vector, 4 μL fragment, 2 μL 10× Buffer, 1 μL Eco31I, 1 μL T4 ligase, and 8 μL ddH2O. The program was: 37℃ for 20 min; 37℃ for 10 min, 20℃ for 10 min, for 5 cycles; 80℃ for 2 min.

[0135] (5) Transformation and identification: The ligation product was transformed into Escherichia coli DH5α, plated on kanamycin-resistant LB agar plates, and incubated at 37℃ for 12 h. Single colonies were picked for bacterial PCR identification (primers STU-TEST-3F / STU-TEST-4R). Plasmids were extracted from bacterial colonies that were correctly sequenced and named GmDof13.3-pGES401. Figure 13 ).

[0136] STU-TEST-3F: CTACGAGACTAGGATCGATCTC, SEQ ID NO.9;

[0137] STU-TEST-4R: GGCTCGTATGTTGTGTGG, SEQ ID NO. 10.

[0138] 2. Agrobacterium-mediated transformation and soybean genetic transformation

[0139] The recombinant plasmid GmDof13.3-pGES401 was transformed into Agrobacterium EHA105 competent cells, plated on YEP plates containing the corresponding antibiotics, and cultured at 28°C for 2 days.

[0140] Genetic transformation of soybean cotyledonary nodes:

[0141] (1) Soybean seeds (Williams82) were surface sterilized (96 mL NaClO + 6 mL concentrated HCl, 16 h), washed with sterile water and then incubated in the dark for 16 h.

[0142] (2) Cut off the cotyledon nodes and soak them in OD. 600 In a culture of Agrobacterium tumefaciens with a concentration of ≈1.0, incubate for 30 min, then aspirate and co-culture in CCM medium (containing acetylsuccinone) for 4-5 days.

[0143] (3) Transfer to screening medium (containing 120 mg / L glufosinate and 500 mg / L cephalosporin) and culture for 10 days.

[0144] (4) Transfer the resistant shoots to elongation medium (SEM) for subculture, and then transfer them to rooting medium (containing 0.5 mg / LIBA) for 15-20 days.

[0145] (5) After hardening off, the seedlings were transplanted into peat soil to obtain T0 generation transgenic plants.

[0146] 3. Identification of genetically modified soybeans

[0147] Screening for glufosinate resistance: Leaves of T0 generation seedlings were smeared with 120 mg / L glufosinate and observed after 3 days. The leaves of resistant plants did not wilt.

[0148] PCR identification: Genomic DNA was extracted from leaves (CTAB method), and PCR detection was performed using target-specific primers.

[0149] Crispr-cas-GmDof13.3-F: 5'-TCCACCAACCTTGTTTCATAGG-3', SEQ ID NO.11;

[0150] Crispr-cas-GmDof13.3-R: 5'-ACATCACTGCTACTACTAGC-3', SEQ ID NO. 12.

[0151] Sequencing verification: Sequencing of the PCR products showed that the lines dof13.3-14, dof13.3-15, and dof13.3-55 had base deletions at the target site. Figure 14 And it can stably pass on to the T3 generation.

[0152] qRT-PCR validation: Under long-day conditions, the expression level of GmDof13.3 in the mutant was significantly lower than that in the wild type. Figure 15 ).

[0153] 4. Determination of growth period and agronomic traits of genetically modified soybeans

[0154] Potted plants were grown at Anhui University of Science and Technology. T3 generation material was removed and sown in plastic pots with a diameter of 16.5 cm and a height of 17 cm. The pots were filled with 16 cm of soil, covered with 0.5 cm of soil, and placed in a greenhouse with labels inserted. Photoperiod treatment was applied when the first trifoliate leaf was fully expanded. The experiment was evaluated using a PowerPoint presentation, followed by a flowering period survey. The greenhouse lights were set to be turned on at 8:00 AM and turned off at 12:00 AM, with a LD treatment light length of 16 hours (normally placed in the greenhouse). Normal management and pest and disease control were implemented for the plants during the experiment.

[0155] In 2025, field planting was conducted at Anhui University of Science and Technology. Summer sowing was carried out on June 23rd. T3 generation materials were knocked out and sown in the field with a row spacing of 30cm, a plant spacing of 8cm, and a sowing depth of 3-5cm. Field natural environmental conditions were observed, and labels were inserted. Flowering time was investigated after the first trifoliate compound leaf had fully unfolded, as determined by PPT.

[0156] The soybean reproductive stages R1, R2, R3, R4, R5, R6, R7, and R8 were recorded according to Fehr's soybean developmental standards (the soybean plant closest to the tag was recorded as the first plant, and plants were recorded in clockwise order). R1 ​​refers to the first open flower at any node on the main stem; R2 refers to one open flower at one of the top two nodes on the main stem, with fully developed leaves; R3 refers to a pod 3 / 16 inch long, with fully developed leaves; R4 refers to a pod 3 / 4 inch long; R5 refers to a seed 1 / 8 inch long in one of the four top nodes on the main stem, with fully developed leaves; R6 refers to a pod with plump green seeds in one of the four top nodes on the main stem, with fully developed leaves; R7 refers to a normal pod on the main stem that has reached its mature pod color; R8 refers to 95% of the pods being fully mature. Data was recorded for 5 plants per line, recording the number of days from the appearance of stage R1 to R8. After harvest, the soybean plant height, number of branches, number of nodes, total number of pods, number of grains per plant, and weight of 100 grains were investigated.

[0157] The flowering time statistics are shown in Tables 6 and 7. The statistics indicate that under long-day conditions, wild-type soybean flowers significantly later than the three dof13.3 transgenic lines. Wild-type lines required an average of 36.6 days to flower under LD conditions, but the dof13.3-55, dof13.3-14, and dof13.3-15 lines required averages of 34.20 days, 32.40 days, and 32.40 days, respectively (phenotypes are shown in Tables 6 and 7). Figure 16In the field, all three showed the same trend: the wild-type lines took an average of 39.0 days to flower, while the dof13.3-55, dof13.3-14, and dof13.3-15 lines took an average of 37.20 days, 36.20 days, and 34.60 days to flower, respectively (see phenotypic table). Figure 17 The above results indicate that the dof13.3 gene can significantly promote flowering and maturation of transgenic soybeans under both LD and field planting conditions.

[0158] Table 6. Flowering time data of soybean lines knocked out under long-day treatment (dof13.3).

[0159]

[0160] Table 7. Field data on flowering time of soybean plants with dof13.3 knockout.

[0161]

[0162] To explore the agronomic traits of transgenic dof13.3 plants under long-day conditions and in the field, and the effects between wild-type and homozygous transgenic lines, T3 generation homozygous transgenic plants and wild-type Williams82 plants were cultured. The investigation revealed that under long-day and field conditions, the transgenic lines did not differ significantly from wild-type plants in yield-related traits. However, regarding plant height, under long-day conditions and in the field, the three T3 generation transgenic lines were taller than wild-type plants (Tables 8 and 9).

[0163] Table 8. Agronomic traits of transgenic soybeans under long-day treatment (dof13.3).

[0164]

[0165] Note: * represents P<0.5, ** represents P<0.01.

[0166] Table 9. Agronomic traits of transgenic soybeans (dof13.3) in the field.

[0167]

[0168] Note: * represents P<0.5, ** represents P<0.01.

[0169] The above examples demonstrate that knocking out the GmDof13.3 gene can significantly promote soybean flowering and increase plant height, indicating that this gene plays an important role in regulating soybean flowering time and plant height, and can be used for soybean molecular breeding.

[0170] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of knocking out the soybean GmDof13.3 gene in any of the following: (1) Promotes earlier flowering time of soybeans; (2) Cultivating genetically modified soybeans with earlier flowering time; (3) Promotes soybean plant height growth; (4) Cultivating transgenic soybeans with higher plant height; in, The CDS sequence of the soybean GmDof13.3 gene is shown in SEQ ID NO.

1.

2. The application of inhibiting the expression of soybean GmDof13.3 protein in any of the following: (1) Promotes earlier flowering time of soybeans; (2) Cultivating genetically modified soybeans with earlier flowering time; (3) Promotes soybean plant height growth; (4) Cultivating transgenic soybeans with higher plant height; in, The amino acid sequence of the soybean GmDof13.3 protein is shown in SEQ ID NO.

2.

3. The use of the recombinant vector for knocking out the soybean GmDof13.3 gene as described in claim 1 in any of the following: (1) Promotes earlier flowering time of soybeans; (2) Cultivating genetically modified soybeans with earlier flowering time; (3) Promotes soybean plant height growth; (4) Cultivate transgenic soybeans with higher plant height.

4. The use of the recombinant microorganism containing the recombinant vector of claim 3 in any of the following: (1) Promotes earlier flowering time of soybeans; (2) Cultivating genetically modified soybeans with earlier flowering time; (3) Promotes soybean plant height growth; (4) Cultivate transgenic soybeans with higher plant height.

5. The application as described in any one of claims 1-4, characterized in that, The soybean GmDof13.3 gene in soybeans is mutated using gene editing technology, resulting in the loss of function of the soybean GmDof13.3 gene expression protein, thereby advancing the daytime flowering time of the soybeans and / or increasing the plant height of the soybeans.

6. The application as described in claim 5, characterized in that, Methods that result in loss of function of the soybean GmDof13.3 gene expression protein include deletion, insertion, or mutation of bases on the target sequence.

7. A method for advancing the flowering time of soybeans, characterized in that, The method includes the following steps: gene editing of the soybean GmDof13.3 gene in soybeans to render the function of the soybean GmDof13.3 gene protein lost, thereby promoting the earlier flowering time of the soybean; the CDS sequence of the soybean GmDof13.3 gene is shown in SEQ ID NO.

1.

8. A method for cultivating transgenic soybeans with advanced flowering time, characterized in that, Includes the following steps: The soybean GmDof13.3 gene was edited in the cotyledons of soybean to render the function of the soybean GmDof13.3 gene protein expression function lost; the soybean was then bred to obtain a transgenic soybean with an advanced flowering time; the CDS sequence of the soybean GmDof13.3 gene is shown in SEQ ID NO.

1.

9. A method for promoting soybean plant height growth, characterized in that, The method includes the following steps: gene editing of the soybean GmDof13.3 gene in soybeans to render the function of the soybean GmDof13.3 gene protein expression function lost, thereby promoting the plant height growth of soybeans; the CDS sequence of the soybean GmDof13.3 gene is shown in SEQ ID NO.

1.

10. A method for cultivating transgenic soybeans with higher plant height, characterized in that, Includes the following steps: The soybean GmDof13.3 gene was edited in the cotyledons of soybean to render the function of the soybean GmDof13.3 gene protein expression function lost; the soybean was then bred to obtain transgenic soybeans with higher plant height; the CDS sequence of the soybean GmDof13.3 gene is shown in SEQ ID NO.1.

Images