Application of soybean NAC transcription factor gene GmNAC4 in regulation and control of soybean grain size and / or drought resistance

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to application of a soybean NAC transcription factor gene GmNAC4 in regulation and control of soybean grain size and / or drought resistance. It is found that the soybean NAC transcription factor gene GmNAC4 has the dual functions of negatively regulating and controlling the grain size and positively regulating and controlling the drought resistance, expression of the soybean NAC transcription factor gene GmNAC4 is induced by dehydration stress, then a yield-stress resistance balanced molecular switch mechanism is revealed, a key target is provided for soybean molecular design and breeding, and the soybean NAC transcription factor gene GmNAC4 has a good application prospect. Through a space-time specific expression regulation or modular uncoupling strategy, collaborative improvement of high yield and drought resistance is realized, and the bottleneck of traditional breeding is broken through.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the soybean NAC transcription factor gene. GmNAC4 Applications in regulating soybean seed size and / or drought resistance, and applications of substances that regulate the expression of soybean NAC transcription factors in regulating soybean seed size or drought resistance. Background Technology

[0002] Soybeans Glycine max [L.] Merr. is an important crop and a major source of human oils and high-quality plant proteins. Therefore, it is of great significance to cultivate new high-yielding and stress-resistant soybean varieties.

[0003] Soybean seed size is determined by seed length, width, and thickness, and is one of the key factors contributing to soybean yield. This trait is a complex quantitative trait, and although several QTLs related to seed size have been located, the major genes and their regulatory networks remain poorly elucidated.

[0004] Drought is a key factor limiting soybean yield, potentially causing losses of up to 40%, and its impact is exacerbated by climate change. Soybeans are sensitive to water stress, especially during flowering and grain-filling stages. Drought affects the physiological and biochemical processes of soybeans, thereby reducing their yield and quality. Currently, progress in drought-resistant soybean breeding is slow, partly due to insufficient research on drought-responsive genes and their regulatory networks.

[0005] Seed size and drought resistance are both important factors affecting soybean yield. Currently reported genes regulating soybean seed size and drought resistance mostly operate independently; no key genes capable of simultaneously regulating both traits have been identified, which limits the synergistic improvement of high-yield and drought-resistant traits. Therefore, identifying genes regulating soybean seed size and drought resistance and elucidating their specific regulatory mechanisms is of great significance for breeding new high-yield and drought-resistant soybean varieties. Summary of the Invention

[0006] In view of the above-mentioned prior art, the purpose of this invention is to provide a soybean NAC transcription factor gene. GmNAC4 Applications in regulating soybean seed size and / or drought resistance, and applications of substances that regulate the expression of soybean NAC transcription factors in regulating soybean seed size or drought resistance.

[0007] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a soybean NAC transcription factor gene. GmNAC4 Application in regulating soybean seed size and / or drought resistance, the soybean NAC transcription factor gene GmNAC4 The nucleotide sequence is shown in SEQ ID NO.1; The application involves modifying the soybean NAC transcription factor gene. GmNAC4 Overexpression was used to obtain overexpressed plants to improve soybean drought resistance; or, the soybean NAC transcription factor gene was overexpressed. GmNAC4 Mutant plants were obtained by generating mutations to increase soybean seed size; the mutations were loss-of-function mutations.

[0008] Secondly, the present invention provides the application of substances that regulate the expression of soybean NAC transcription factors in regulating soybean seed size or drought resistance, wherein the soybean NAC transcription factors include the soybean NAC transcription factor protein GmNAC4.

[0009] In one optional embodiment, the soybean NAC transcription factor protein GmNAC4 comprises at least one of the following: A. A protein consisting of the amino acid sequence shown in SEQ ID NO.3; B. Proteins derived from sequence SEQ ID NO.2 that are related to soybean seed size or drought resistance, obtained by substitution and / or deletion and / or addition of one or more amino acid residues of the amino acid sequence shown in sequence SEQ ID NO.3.

[0010] In one alternative embodiment, the substance regulating soybean NAC transcription factor expression includes substances that silence or inhibit soybean NAC transcription factor expression, and the application includes its application in regulating soybean seed size.

[0011] In one optional implementation, the regulation of soybean seed size includes at least one of increasing soybean seed width, increasing soybean seed length, increasing soybean seed thickness, and increasing the weight of 100 soybean seeds.

[0012] In one optional embodiment, the substance that silences or inhibits the expression of soybean NAC transcription factors includes at least one of the following: I. Used for editing soybean NAC transcription factor genes GmNAC4 sgRNA; II. A gene editing vector containing the sgRNA; III. Transfect recombinant bacteria with the gene-editing vector described above.

[0013] In one alternative implementation, the method for editing the soybean NAC transcription factor gene... GmNAC4 sgRNAs include those used to activate the soybean NAC transcription factor gene. GmNAC4 sgRNAs that have undergone loss-of-function mutations.

[0014] In one optional implementation, the sgRNA includes a first target sequence and / or a second target sequence; wherein, The first target sequence is the inverse complementary sequence of positions 73 to 92 in SEQ ID NO.1; The second target sequence is the inverse complementary sequence of positions 219 to 238 in SEQ ID NO.1.

[0015] In one alternative embodiment, the substance regulating soybean NAC transcription factor expression includes a substance that causes soybean NAC transcription factor overexpression, and the application includes its application in regulating soybean drought resistance.

[0016] In one alternative implementation, the regulation of soybean drought resistance includes increasing soybean drought resistance, which is manifested as an increase in the survival rate of soybean plants and / or a reduction in water loss from detached leaves under soil drought conditions.

[0017] In one alternative embodiment, the substance that causes overexpression of soybean NAC transcription factor includes at least one of the following: 1) Soybean NAC transcription factor protein GmNAC4; 2) Nucleotides encoding the soybean NAC transcription factor protein GmNAC4; 3) Recombinant vectors, expression cassettes, transgenic cell lines, or recombinant bacteria containing nucleotides encoding the soybean NAC transcription factor protein GmNAC4.

[0018] In one alternative embodiment, the nucleotide encoding the soybean NAC transcription factor protein GmNAC4 comprises at least one of the following: a. The nucleotide sequence shown in SEQ ID NO.1; b. A nucleotide sequence that can hybridize with the nucleotide sequence shown in SEQ ID NO.1 under stringent conditions; c. A nucleotide sequence that has more than 90% homology with the nucleotide sequence shown in SEQ ID NO.1 and encodes a protein related to soybean drought resistance.

[0019] Preferably, the application is created using gene editing technology. GmNAC4 The sequence was edited gmnac4 Mutants can be used to increase soybean seed size, or overexpression techniques can be used to create mutants. GmNAC4 Overexpression of genetically modified soybeans to improve the drought resistance of soybeans.

[0020] Thirdly, the present invention provides a method for regulating soybean seed size, comprising the following steps: Silencing or suppressing soybean NAC transcription factor genes in soybean GmNAC4 The expression.

[0021] In one alternative implementation, the silencing or inhibition of the soybean NAC transcription factor gene in soybean is... GmNAC4 The expressions include: Through gene editing technology, the soybean NAC transcription factor gene was modified. GmNAC4 Loss-of-function mutations result in soybean plants with larger seeds.

[0022] In one optional implementation, the method for regulating soybean seed size includes the following steps: (1) Design two targeted knockout soybean NAC transcription factor genes based on the nucleotide sequence shown in SEQ ID NO.1. GmNAC4 The sgRNA was used to construct a CRISPR / Cas9 dual-target gene editing vector containing the sgRNA. This CRISPR / Cas9 gene editing vector was transformed into *Agrobacterium tumefaciens*, and the recombinant bacteria were then used to infect wild-type soybean. T0 generation transgenic seeds were harvested, and the soybean NAC transcription factor gene was screened and identified after planting. GmNAC4 Homozygous mutant plants with base sequence mutations at the site yield soybean plants with larger seeds.

[0023] Preferably, the gene target sequence of the sgRNA is shown in SEQ ID NO.4 and SEQ ID NO.5.

[0024] Preferably, the gene editing vector uses pGmEF1A2::SpCas9 as its backbone.

[0025] Preferably, the Agrobacterium tumefaciens is Agrobacterium EHA105.

[0026] Preferably, the wild-type soybean includes the Huachun 6 soybean variety.

[0027] Preferably, the increase in seed size manifests as an increase in seed width, seed length, seed thickness, and / or an increase in weight per 100 seeds.

[0028] Fourthly, the present invention provides a method for improving the drought resistance of soybeans, comprising the following steps: Increase soybean NAC transcription factor gene in soybean GmNAC4 The amount of expression; And / or, increase the activity of soybean NAC transcription factor protein GmNAC4 in soybean.

[0029] In one optional embodiment, the method for improving the drought resistance of soybeans includes the following steps: (1) Construct a structure containing the following as shown in SEQ ID NO.2 GmNAC4 Overexpression vectors of gene coding region sequences; (2) The overexpression vector from step (1) was transformed into Agrobacterium tumefaciens, and then the recombinant bacteria were used to infect wild-type soybeans. T0 generation transgenic seeds were harvested, and after planting, the seeds containing T-DNA and the soybean NAC transcription factor gene were screened and identified. GmNAC4 Overexpression of the gene or the protein activity of the soybean NAC transcription factor protein GmNAC4 can increase the expression level of the gene, thereby obtaining soybeans with enhanced drought resistance.

[0030] Preferably, the Agrobacterium tumefaciens is Agrobacterium EHA105.

[0031] Preferably, the wild-type soybean includes the Huachun 6 soybean variety.

[0032] Preferably, the enhanced drought resistance is manifested in increased plant survival rate and reduced water loss from detached leaves under drought treatment.

[0033] Fifthly, the present invention provides the application of the above method in soybean breeding.

[0034] Compared with the prior art, the present invention has the following beneficial effects: This invention discovered the soybean NAC transcription factor gene. GmNAC4 It possesses a dual function of "negatively regulating grain size and positively regulating drought resistance," and its expression is induced by dehydration stress. This discovery reveals the molecular switch mechanism of yield-resistance tradeoff, providing a key target for soybean molecular design breeding. Through spatiotemporally specific expression regulation or modular uncoupling strategies, it can achieve synergistic improvement of high yield and drought resistance, breaking through the bottleneck of traditional breeding.

[0035] Specifically, this invention relates to the soybean NAC transcription factor gene. GmNAC4 Applications in regulating soybean seed size and / or drought resistance, and applications of substances that regulate the expression of soybean NAC transcription factors in regulating soybean seed size or drought resistance, wherein the soybean NAC transcription factor is soybean NAC transcription factor. GmNAC4 Gene( Glyma.12G221500 The nucleotide sequence of its coding region is shown in SEQ ID NO: 2, and the amino acid sequence encoding the protein is shown in SEQ ID NO: 3. This invention utilizes CRISPR / Cas9 technology to create... gmnac4 The mutant was found to have larger seeds and increased 100-seed weight, but was more sensitive to soil drought stress and exhibited a faster rate of water loss from detached leaves; conversely, GmNAC4 Overexpression resulted in smaller seeds and a lower 100-seed weight in plants, but significantly enhanced drought resistance. The results indicate... GmNAC4 This invention reveals a gene that negatively regulates soybean seed size and positively regulates drought resistance, demonstrating its role in the trade-off between yield and stress resistance. It provides a key gene resource for soybean molecular design breeding, allowing for precise regulation of gene expression according to different breeding objectives, synergistically improving yield and stress resistance. Attached Figure Description

[0036] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0037] Figure 1 for GmNAC4 Target site mutation sequence illustration; (A) is GmNAC4 A diagram illustrating nucleotide sequence variations at target sites is provided, where straight lines represent introns, black boxes represent exons, and white boxes represent UTRs. The first and second target sites are the target sites for... GmNAC4 Guide RNA at the site, with bolded bases indicating PAM sites, "-" indicating deletion, and red bases indicating insertion; (B) is a diagram showing the amino acid sequence variations at the target site; Figure 2 for GmNAC4 Gene map of the overexpression vector pFGC5941-GmNAC4-eGFP; Figure 3 for GmNAC4 Image showing the identification results of overexpressing plants; (A) is GmNAC4 (B) is a graph showing the results of expression level analysis in overexpression plants; (B) is a graph showing the results of GmNAC4 protein expression level identification in overexpression plants, with Actin as the internal reference protein. Figure 4 for gmnac4 mutants and GmNAC4 Phenotypic diagram of overexpression plants; (A) wild-type Huachun 6 soybean, gmnac4 mutants and GmNAC4 (A) Grain length phenotypic diagram of overexpressing plants, scale bar 1 cm; (B) Grain length statistical results diagram; (C) Grain width phenotypic diagram, scale bar 1 cm; (D) Grain width statistical results diagram; (E) Grain thickness statistical results diagram; (F) 100-grain weight statistical results diagram; Figure 5 for GmNAC4 The results of the expression pattern analysis are shown in Figure (A). GmNAC4 The results of expression level analysis in the roots, stems and leaves of 10-day-old soybean seedlings; (B) is... GmNAC4 Figure showing the results of expression level analysis in 10-day-old soybean seedlings under natural dehydration treatment; Figure 6 for gmnac4Drought phenotype of mutants in soil; (A) is a drought phenotype of plants in soil, scale bar 10cm; (B) is a drought phenotype of 10-day-old wild-type Huachun 6 soybean and gmnac4 The survival rate of mutants after 10 days of soil drought treatment followed by 3 days of watering recovery is shown in Figure 1; (C) represents wild-type Huachun 6 soybean and gmnac4 Statistical results of water loss rate of detached leaves of mutants; Figure 7 for GmNAC4 Drought phenotypes of overexpressing plants in soil; (A) is a soil drought phenotype of the plants, scale bar 10 cm; (B) is a 10-day-old wild-type Huachun 6 soybean and GmNAC4 The survival rate of overexpressing plants after 10 days of soil drought treatment followed by 3 days of watering recovery is shown in Figure 1; (C) represents wild-type Huachun 6 soybean and GmNAC4 Statistical results of water loss rate in detached leaves of overexpressing plants; Figure 8 The following figures show the subcellular distribution and transcriptional activity analysis results of soybean transcription factor GmNAC4. (A) shows the subcellular localization results of GmNAC4 in Arabidopsis protoplasts. pA7-NLS-mCherry can express red fluorescent protein containing nuclear localization signals. The scale bar is 20 μm. (B) is a schematic diagram of the vector used in the GmNAC4 transcriptional activity analysis experiment. BD is an empty vector containing a GAL4 binding domain. HOS15 is a transcriptional repressor as a negative control. VP16 is a transcriptional activator as a positive control. 5×UAS represents five tandem GAL4 binding sites. CaMV minimal is the micropromoter sequence of cauliflower mosaic virus. LUC is the reporter gene encoding firefly luciferase. REN is the internal reference gene encoding renal luciferase. (C) is a GmNAC4 transcriptional activity analysis figure. The vertical axis represents the ratio between firefly luciferase activity and renal luciferase activity (LUC / REN). The LUC / REN ratio of the empty vector BD is 1. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0039] The following embodiments are provided to better understand the present invention, but are not intended to limit the invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods or selected according to the product instructions. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available. All quantitative experiments in the following embodiments were performed in triplicate, and the results were averaged.

[0040] Example 1: Obtaining the soybean NAC transcription factor GmNAC4 and its coding sequence

[0041] This invention cloned an NAC transcription factor gene from the soybean variety Huachun 6 (nationally approved soybean 2009012, hereinafter abbreviated as HC6). Glyma.12G221500 Named GmNAC4 (GeneBank accession number: NP_001238424). The full-length cDNA of this gene is 1491 bp, and its nucleotide sequence is shown in SEQ ID NO.1; the coding sequence (CDS) is 1038 bp in length, and its nucleotide sequence is shown in SEQ ID NO.2; this gene encodes a protein composed of 345 amino acid residues, and its amino acid sequence is shown in SEQ ID NO.3, with NAC domains located from position 15 to position 139 of this amino acid sequence.

[0042] Example 2: GmNAC4 Creation of gene-edited mutants

[0043] This invention utilizes CRISPR / Cas9-mediated gene editing technology to create two... gmnac4 mutant, i.e. gmnac4-1 and gmnac4-2 First, based on the above... GmNAC4 The cDNA sequence (SEQ ID NO.1) was used to design two specific targets. GmNAC4 The gene's guide RNA (sgRNA) was synthesized. The first target site was the inverse complementary sequence from positions 73 to 92 of SEQ ID NO. 1, and the second target site was the inverse complementary sequence from positions 219 to 238 of SEQ ID NO. 1. Target primers KO- were synthesized based on the sgRNA sequences. GmNAC4 -sg1F (SEQ ID NO.6), KO- GmNAC4 -sg1R (SEQ ID NO.7), KO- GmNAC4 -sg2F (SEQ ID NO.8) and KO- GmNAC4 -sg2R (SEQ ID NO. 9). Specifically, the two sgRNA sequences and their target primer sequences are shown in Tables 1 and 2 below.

[0044] Table 1 Target GmNAC4 sgRNA sequence information of the gene

[0045] Table 2 GmNAC4 Target primer sequence information

[0046] 1. Contains GmNAC4Construction of intermediate vectors for gene target sequences

[0047] Next, the first and second targets were loaded onto soybeans, respectively. GmU6 The expression of sgRNA is first carried out on the intermediate vectors GmpUC19-1 and GmpUC19-2, which drive sgRNA expression. First, restriction endonucleases are used... Bsa I-HFv2 (NEB, R3733) linearized the intermediate vectors GmpUC19-1 and GmpUC19-2, and then the target primer KO- GmNAC4 -1F / 1R and KO- GmNAC4 -2F / 2R annealing was used to form dsDNA. The annealed product of the first target primer was ligated to the GmpUC19-1 linearized vector using T4 DNA ligase, and the annealed product of the second target primer was ligated to the GmpUC19-2 linearized vector. The ligation products were transformed into *E. coli* DH5α competent cells. After plate culture, single clones were randomly picked and ligated using the universal primer M13R (5'-CAGGAAACAGCTATGACC-3', SEQ ID NO.10) and the target-specific primer KO- GmNAC4 -sg1R or KO- GmNAC4 Colony PCR was performed using -sg2R to screen for positive single clones. The obtained positive single clones were then subjected to Sanger sequencing to obtain the intermediate vector successfully loaded with the first and second target sites, namely KO- GmNAC4 -sg1 and KO- GmNAC4 -sg2.

[0048] 2. GmNAC4 Construction of dual-target gene knockout vectors

[0049] Based on the Golden Gate cloning principle, following the Golden Gate reaction system in Table 3 and the Goldengate reaction procedure in Table 4, the sgRNA expression cassettes of the two successfully constructed intermediate vectors were ligated to two vectors on the SpCas9 vector (pGmEF1A2::SpCas9) driven by the soybean transcription elongation factor EF1A2 promoter. Aar Between the restriction enzyme sites. The ligation product was transformed into *E. coli* DH5α competent cells. After plating, single clones were randomly picked and colony PCR was performed using universal primers M13F (5'-GTAAAACGACGGCCAGT-3', SEQ ID NO.11) and EF1A2-R (5'-GTAAAACGACGGCCAGT-3', SEQ ID NO.12) to screen for positive single clones. The obtained positive single clones were subjected to Sanger sequencing to obtain the contents of the restriction enzyme sites. GmNAC4 KO- gene dual-target sequence knockout vectorGmNAC4 .

[0050] Table 3 Golden Gate reaction system

[0051] Table 4 Golden Gate Reaction Procedure

[0052] 3. gmnac fou Creation of mutants

[0053] Agrobacterium-mediated genetic transformation of soybean cotyledonary nodes was employed, using Huachun 6 soybean as the genetic transformation recipient. First, chemical transformation was used to transform the above-mentioned... GmNAC4 Dual-target knockout vector KO- GmNAC4 Introduced into Agrobacterium tumefaciens EHA105 cells, yielding cells containing KO- GmNAC4 Agrobacterium colonies on the carrier. Plump and uniform Huachun 6 soybeans were sterilized with chlorine for 16–18 hours. Then, the sterilized seeds were placed in a laminar flow hood for 30 minutes to dissipate excess chlorine. The sterilized soybeans were soaked in an appropriate amount of sterile water for about 4 hours. Using a sterile scalpel, the soaked soybeans were cut in half along the hypocotyl, and excess radicles were removed. Next, the seeds were treated with a KO-containing... GmNAC4 Agrobacterium tumefaciens culture (OD) of the carrier 600 = 0.5–0.7) Infect the cut soybeans, remove the bacterial solution after 30 min, and air-dry the infected soybeans on sterile filter paper. Then, evenly inoculate them onto co-culture medium. After co-culturing in the dark at 28℃ for 3 days, remove the newly grown hypocotyl portion of the soybeans, leaving only 3–4 mm, and wash the soybeans sequentially with sterile water and sterile water containing 50 mg / L cephalosporin. After washing and drying, inoculate the soybeans onto induction medium and culture, changing the medium every half month, for 3 subcultures. Next, select soybeans with callus tissue, remove the soybeans, and inoculate the callus tissue onto elongation medium, changing the medium every half month, for 3–4 subcultures. When regenerated seedlings grow from the callus tissue, separate the regenerated seedlings from the callus tissue and inoculate them onto rooting medium until the seedlings root. Finally, cultivate the hardened seedlings in nutrient soil containing vermiculite and harvest the seeds.

[0054] Positive identification of T0 generation transgenic seedlings was performed using leaf DNA as a template. PCR amplification was conducted using pGmEF1A2::SpCas9 vector-specific primers CF3492 (SEQ ID NO.13) and CF3594 (SEQ ID NO.14) to detect whether the transgenic seedlings contained SpCas9. Simultaneously, primers M13F and KO- GmNAC4-sg2R was used to detect the presence of a target expression cassette. Seeds from plants containing both SpCas9 and the target expression cassette were collected and propagated. Based on this, the T1 generation plants were identified, and plants containing T-DNA were selected for use... GmNAC4 Site-specific primer V- GmNAC4 -F (SEQ ID NO.15) and V- GmNAC4 PCR amplification was performed using -R (SEQ ID NO.16), and the amplification products were detected by Sanger sequencing. GmNAC4 Whether sequence variations occurred at the site. The results showed that two individual plants in the T1 generation had... GmNAC4 The site may have been edited, so the seeds of the aforementioned single plants were collected and propagated to obtain T2 generation plants. Analysis of the T2 generation plants revealed two types of... GmNAC4 The mutation, of which mutation type 1, involves a 146 bp deletion between the first and second target sites, resulting in... GmNAC4 The loss of the start codon in a gene may affect its transcription and translation; mutation type 2 involves the insertion of 1 bp at both the first and second target sites, which may cause a frameshift mutation in the GmNAC4 protein and prematurely terminate translation, as detailed below. Figure 1 As shown. Subsequently, the two mutation types mentioned above will be named... gmnac4-1 and gmnac4-2 The seeds were harvested and planted to obtain homozygous mutants for higher generations. Specific information on the primers used for transgenic seedling detection is shown in Table 5 below.

[0055] Table 5 gmnac4 Mutant identification primer sequence information

[0056] Example 3: GmNAC4 Obtaining transgenic plants through overexpression

[0057] 1. GmNAC4 Construction of the overexpression vector pFGC5941-GmNAC4-eGFP

[0058] This invention also constructed a vector pFGC5941-GmNAC4-eGFP, which expresses GmNAC4 driven by the cauliflower mosaic virus promoter CaMV 35S and fused with enhanced green fluorescent protein (eGFP). Firstly, based on the cloned... GmNAC4 Based on the CDS sequence (excluding the stop codon), eGFP sequence, and multiple cloning site of the plant expression vector pFGC5941, specific primers were designed using the principle of homologous recombination. GmNAC4 -1F (SEQ ID NO.17), OE- GmNAC4 -1R (SEQ ID NO. 18), OE-GmNAC4 -2F (SEQ ID NO.19) and OE- GmNAC4 -2R (SEQ ID NO.20), the specific primer sequence is shown in Table 6 below.

[0059] Total RNA was extracted from the leaves of 10-day-old HC6 soybeans. Using 2 μg of the extracted total RNA as a template, a reaction system was prepared according to the instructions of TransCript One-Step gDNA Removal and cDNA Synthesis SuperMix (AT311-02). The mixture was incubated in a PCR instrument at 42℃ for 30 min and 85℃ for 5 sec for reverse transcription to obtain cDNA. Using this cDNA as a template, primers OE-... GmNAC4 -1F、OE- GmNAC4 -1R and the high-fidelity enzyme PrimeSTAR Max DNA Polymerase (TaKaRa, R045Q) were used to amplify fragment one (containing... GmNAC4 (Coding sequence); simultaneously, using pD1301s plasmid as a template, primer OE- GmNAC4 -2F and OE- GmNAC4 -2R amplification yielded fragment two (containing the eGFP coding sequence). Subsequently, using fragment one and fragment two as a common template, primer OE- GmNAC4 -1F and OE- GmNAC4 -2R was used for overlap PCR to finally obtain [the desired product]. GmNAC4 Fragment three of the -eGFP fusion coding sequence. Using restriction endonuclease... Xho I (NEB, R0146) and XmaThe pFGC5941 vector was double-digested using enzyme I (NEB, R0180) to remove the petunia chalcone synthase gene intron (chSA intron), and the 9909 bp linear vector fragment was then recovered. The PCR product fragment III was then recombined with the linearized vector in vitro using the ClonExpress II One Step Cloning Kit (Vazyme, C112-01). The recombinant product was transformed into *E. coli* DH5α competent cells, and after plating, single clones were randomly picked and colony PCR was performed using the universal primers pCaMV-F and peGFP-C-5 to preliminarily screen for positive single clones. Positive single clones were selected and Sanger sequencing was performed using primers pCaMV-F (SEQ ID NO.21), peGFP-N-3 (SEQ ID NO.22), and peGFP-C-5 (SEQ ID NO.23) to verify that the GmNAC4-eGFP fusion reading frame and ligation interface sequence were completely correct. The recombinant plasmid pFGC5941-GmNAC4-eGFP was finally obtained, and its gene map is shown below. Figure 2 As shown.

[0060] Table 6 Primer sequence information related to the construction of the pFGC5941-GmNAC4-eGFP vector.

[0061] 2. GmNAC4 Obtaining transgenic plants through overexpression

[0062] Referring to the soybean genetic transformation method in Example 2 of this invention, the pFGC5941-GmNAC4-eGFP expression vector was transformed into Huachun 6 soybean. After co-culture, induction, elongation, and rooting culture, T0 generation transgenic plants were obtained. Using its leaf genomic DNA as a template, specific primers pCaMV-F and OE- were used to transform the transgenic plants. GmNAC4 -2R was used for PCR amplification to screen for positive plants containing T-DNA. Positive plants were harvested and propagated to obtain higher-generation lines. After multiple generations of self-pollination and molecular identification, two stable genetic lines were finally obtained. [[ID=I19]]GmNAC4 Overexpression homozygous lines were named as follows: GmNAC4 OE1 and GmNAC4 OE2.

[0063] Extracted from 3-week-old babies GmNAC4 Total RNA was overexpressed from plant leaves and reverse transcribed into cDNA. This cDNA was then used as a template... GmNAC4 Gene-specific primers GmNAC4 -qRT-F (SEQ ID NO.24) and GmNAC4RT-qPCR analysis was performed using soybean endogenous gene GmEF-1α (SEQ ID NO.25) -qRT-R (SEQ ID NO.25). Glyma.17G186600 ) was used as an internal reference gene. The results were as follows: Figure 3 As shown in A, in the overexpression lines GmNAC4 The expression level was more than 40-fold higher than that of HC6. Furthermore, the expression of the GmNAC4-eGFP fusion protein was detected at the protein level using Western blotting. Extracts were taken from 3-week-old animals. GmNAC4 Total protein from the leaves of the overexpressing plants was separated by 5% stacking gel and 10% separating gel SDS-PAGE, and then transferred to a nitrocellulose membrane (Cytiva, 10600003) at room temperature for 40 min under constant voltage of 20 V using a semi-dry transfer instrument (Bio-Rad Trans-Blot SD). After transfer, the membrane was blocked with 5% skim milk at room temperature for 2 h, followed by incubation at room temperature for 1 h with mouse anti-GFP monoclonal antibody (clones 7.1 and 13.1, Roche, 11814460001, 1:5000 dilution). After washing the membrane three times with TBST, it was incubated at room temperature for 1 h with HRP-labeled Goat Anti-Rabbit Mouse IgG-HRP (Abmart, M21003, 1:10000 dilution) secondary antibody. Actin, the internal control, was detected using a mouse anti-plant ACTIN monoclonal antibody (BBI, D191048, 1:10000 dilution). After ECL chemiluminescence development, signals were acquired using a GE AI600 gel imaging system. Results are as follows: Figure 3 As shown in B, 31]]GmNAC4 The expression of GmNAC4-eGFP fusion protein was significantly increased in overexpressing plants, consistent with the results of RT-qPCR detection.

[0064] Table 7 RT-qPCR Primer Sequence List

[0065] Example 4: GmNAC4 The regulatory role of genes in soybean seed size

[0066] Wild-type HC6, gmnac4 mutant ( gmnac4-1 , gmnac4-2 ), GmNAC4 Overexpression lines ( GmNAC4 OE1 and GmNAC4(OE2) Plants were planted in an open field experimental plot with a plant spacing of 20 cm and a row spacing of 40 cm. For each line, 10 consecutive plants outside the edge row were randomly selected for phenotypic investigation. The experiment was repeated three times. After seed maturity, 30 mature, undamaged seeds were selected from each replicate to measure seed length, width, and thickness; another 100 seeds were weighed to determine the 100-seed weight, using a digital vernier caliper and a 0.1% electronic balance, respectively. Results are as follows: Figure 4 As shown, gmnac4 The mutant seeds were significantly longer, wider, and thicker than the wild type. Specifically, compared to HC6 seeds, gmnac4-1 and gmnac4-2 Seed length increased by 2.5% and 4.4%, respectively; seed width increased by 3.1% and 7.8%; and seed thickness increased by 3.7% and 5.4%. Correspondingly, gmnac4-1 and gmnac4-2 The weight of 100 grains increased by 16.7% and 23.4% respectively. Figure 4 ).on the contrary, GmNAC4 Overexpression lines ( GmNAC4 OE1 and GmNAC4 The seed length, width, thickness, and 100-seed weight of OE2 seeds were significantly lower than those of the wild type. These results indicate that... 49]]GmNAC4 The gene negatively regulates soybean seed size; inhibiting its expression can increase seed size and improve 100-seed weight, making it suitable for molecular breeding of high-yield soybean varieties.

[0067] Example 5: GmNAC4 Analysis of gene expression patterns under natural dehydration stress

[0068] To explore GmNAC4 The role of genes in soybean drought stress response was first analyzed in this invention. GmNAC4 Gene expression patterns in different tissues and organs. Total RNA was extracted from the roots, stems, and leaves of 10-day-old HC6 soybeans, and cDNA was obtained by reverse transcription. The cDNA obtained by reverse transcription was diluted 10-fold as a template for real-time quantitative PCR (RT-qPCR), and then the expression patterns of genes in the above soybean tissues were detected using the ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711-02) qPCR kit. GmNAC4 Gene expression levels, in GmEF-1α The qPCR primer sequences used as internal reference genes are shown in Table 7 of Example 3. The results are as follows: Figure 5 As shown, GmNAC4 The expression was observed in the roots, stems, and leaves of 10-day-old soybean seedlings, with the main expression found in the leaves and roots. Figure 5(A) Next, 10-day-old Huachun 6 soybean seedlings with good and uniform growth were selected. The vermiculite and potting soil around the roots were washed away, and the roots were dried with clean tissue paper. The seedlings were then placed on filter paper for natural dehydration (ambient temperature 28℃, relative humidity 60%). Whole Huachun 6 seedlings were collected at 0 h, 1 h, 3 h, 5 h, and 9 h of dehydration, with each sample containing 3 individual plants. These were wrapped in aluminum foil and flash-frozen in liquid nitrogen, then stored at –80℃. Total RNA was then extracted from these samples and detected using RT-qPCR. GmNAC4 Gene expression levels under natural dehydration stress, using soybean genes GmRLP24 ( Glyma.13G318800 As a reference gene for drought stress, the qPCR primer sequences are shown in Table 8 below. Results are as follows... Figure 5 As shown, GmNAC4 Gene expression is upregulated under natural dehydration stress, and this upregulation increases with increasing dehydration time. GmNAC4 The expression level gradually increased, and after 9 hours of dehydration treatment, GmNAC4 The expression level was nearly 11 times that at 0 h ( Figure 5 (B) in the text. This indicates that... GmNAC4 It may be involved in regulating the soybean drought stress response.

[0069] Table 8. Internal reference genes under drought stress GmRLP24 RT-qPCR primer sequence listing

[0070] Example 6: GmNAC4 Identification of drought resistance phenotypes in gene-edited mutants and overexpressing plants

[0071] one, gmnac4 It seems there might be a misspelling in line 82 where it says "gmnac fou", which I've left as is since it's part of the original text. Also, there's an "I19" in line 119 which might be a typo but is preserved as per the instructions. Identification of mutant soil drought phenotype

[0072] Next, this invention applies soil drought stress treatment to... gmnac4 The drought resistance of the mutants was evaluated. Nutrient soil with large clods removed was sterilized by drying in a 121℃ oven for 3 hours. The sterilized nutrient soil was then mixed with vermiculite at a 1:1 ratio. Approximately 250 g of the mixture was weighed into 130-gallon flowerpots, with 8 pots per tray. 4 L of water was added to each tray. After 48 hours, each flowerpot was weighed, and the soil moisture content at a depth of 20 cm was measured. Pots with relatively consistent soil moisture content (37% ± 5%) were selected for subsequent planting. Plump, uniformly sized wild-type Huachun 6 were selected. gmnac4-1 and gmnac4-2Forty-eight seeds of each mutant strain were planted in the aforementioned water-absorbed flowerpots, with six seeds evenly planted in each pot at a depth of 0.5 cm. The trays were covered and placed in an artificial climate chamber at 28°C, with a photoperiod of 16 h light / 8 h dark and a relative humidity of 60%. After 5 days, the lids were removed, and on the 10th day, 2 L of water was added to the trays. After approximately 3 weeks of cultivation, when the soil moisture content dropped to 15% ± 5%, the plant growth was photographed and recorded. Subsequently, the plants were subjected to drought treatment, and after 10 days, watering was resumed, with 4 L of water added to each tray to bring the soil moisture content to 47% ± 5%. The plant growth was photographed and recorded again after 3 days of recovery. The growth of Huachun 6 and... gmnac4-1 and gmnac4-2 Survival rate of mutant plants. The experiment was repeated three times, and the average survival rate from the three experiments was plotted. The results are shown below. Figure 6 As shown. The results show that, compared with the wild-type Huachun 6, gmnac4-1 and gmnac4-2 The mutant exhibited significantly reduced drought resistance, with its leaves almost completely wilting, while the control group still retained some tender green leaves. Figure 6 (A) Furthermore, the survival rate of HC6 after regeneration was 43.75%, while gmnac4-1 and gmnac4-2 The percentages of mutants were much lower than those of HC6, at only 8.33% and 15.28% ( Figure 6 (B) In summary, this shows that gmnac4 The mutant exhibits reduced tolerance to soil drought.

[0073] two, gmnac4 Statistics on water loss rate of detached leaves of mutants

[0074] Select 3-week-old HC6, gmnac4-1 and gmnac4-2 The water loss rate of the first trifoliate leaf of the mutant was measured (ambient temperature 25℃, relative humidity 40%–50%). Three seedlings of similar growth were selected from each line, and one trifoliate leaf was taken from each. The leaves were placed on pre-weighed weighing paper, and their weights were measured at 0 h, 15 min, 30 min, 45 min, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 5.5 h, and 6 h, and the data were recorded. The experiment was repeated three times, and the average water loss rate from the three experiments was plotted. The water loss rate was calculated as: (leaf weight at each time point - leaf weight at 0 h) / (leaf weight at 0 h - weight of weighing paper) × 100%. The results are shown below. Figure 6 As shown in C, after half an hour of natural water loss, gmnac4-1 and gmnac4-2The rate of water loss from detached leaves of the mutant was significantly faster than that of the wild-type HC6, consistent with the mutant's reduced drought tolerance phenotype. Figure 6 (C in the text). This indicates that the soybean NAC transcription factor gene... GmNAC4 Positive regulation of the drought stress response process in soybeans.

[0075] three, GmNAC4 Identification of drought-related phenotypes in overexpressing plants

[0076] Referring to the soil drought treatment method for mutant plants in the embodiments of the present invention, 3-week-old HC6, GmNAC4 OE1 and GmNAC4 OE2 plants were subjected to drought stress. After 10 days of drought, watering was resumed for 3 days. Results were as follows: Figure 7 As shown, the overexpression lines that experienced drought showed better growth and produced new leaves. GmNAC4 The survival rate of OE1 was 77.78%. GmNAC4 OE2 is 75% ( Figure 7 The values ​​of A and B in the sample were significantly higher than those of HC6 (43.75%). Furthermore, samples were taken from 3-week-old HC6... GmNAC4 OE1 and GmNAC4 The first trifoliate compound leaf of OE2 seedlings was analyzed for water loss rate in detached leaves. After 15 minutes of natural water loss, the water loss rate of detached leaves from the overexpression line was significantly slower than that of HC6. Figure 7 (C in the above results) Together, they indicate that... GmNAC4 This gene positively regulates the drought stress response process in soybeans. Overexpression of this gene can improve the drought resistance of soybeans and is suitable for the breeding of drought-resistant soybean varieties.

[0077] Example 7: Subcellular localization and transcriptional activity analysis of GmNAC4

[0078] I. Subcellular localization of GmNAC4 protein

[0079] according to GmNAC4 Based on the CDS sequence of the gene (excluding the stop codon) and the cloning site region of the plant expression vector pA7-YFP, specific primers pA7-GmNAC4-F (SEQ ID NO. 30) and pA7-GmNAC4-R (SEQ ID NO. 31) with homologous arms were designed. The primer sequences are shown in Table 9 below. Using the above pFGC5941-GmNAC4-eGFP plasmid as a template, PCR amplification was performed to obtain... GmNAC4 CDS fragments, using Bam H I-HF (NEB, R3136) and Spe The pA7-YFP vector was linearized using the I-HF (NEB, R3133) restriction endonuclease. Subsequently, homologous recombination technology was used to... GmNAC4 The CDS fragment was inserted into the pA7-YFP vector to construct the subcellular localization recombinant expression vector pA7-GmNAC4-YFP. The pA7-GmNAC4-YFP plasmid and the nuclear localization marker vector pA7-NLS-mcherry plasmid were co-transformed into Arabidopsis protoplasts using a PEG-mediated method, with protoplasts co-transformed with the empty pA7-YFP plasmid and pA7-NLS-mcherry serving as a control group. After incubation at 23℃ in the dark for 12–16 h, the fluorescence signal of the protoplasts was observed using a Zeiss LSM 900 laser confocal scanning microscope. The results are as follows: Figure 8 As shown, the fluorescence signal of the GmNAC4-YFP fusion protein is mainly concentrated in the nuclear region and highly overlaps with the signal of nuclear localization marker proteins. Figure 8 The A in the figure indicates that the GmNAC4 protein is located in the cell nucleus, consistent with its function as a transcription factor.

[0080] Table 9 Primer sequence information for pA7-GmNAC4-YFP vector construction

[0081] II. GmNAC4 Transcriptional Activity Analysis

[0082] This embodiment provides a method for detecting GmNAC4 transcriptional activity using a dual-luciferase reporter system. First, the... GmNAC4 The CDS sequence of the gene was inserted into the 35S-GAL4-BD vector to obtain the fusion expression vector GAL4-BD-GmNAC4. The CDS fragment was obtained by PCR amplification, and its sequence is shown in Table 10. Vector construction employed... Sal I (NEB, R3138) and Kpn I-HF (NEB, R3142) double digestion and homologous recombination techniques were used. Subsequently, GAL4-BD-GmNAC4, the reporter vector 35S-LUC (containing the firefly luciferase gene), and the internal control vector pTRL (containing the Renida luciferase gene) were co-transformed into Arabidopsis protoplasts. GAL4-BD empty vector, GAL4-BD-VP16 (activation control), and GAL4-BD-HOS15 (inhibition control) were used as control groups. The plasmid co-transformation combinations are shown in Table 11. Transformed protoplasts were cultured at 23℃ in the dark for 12–16 hours, and then the LUC and REN signal intensities were measured using the Dual-Luciferase Reporter Assay System (Promega, E1910), and the LUC / REN ratio was calculated. The results are shown in the figure; GmNAC4 significantly increased... LUC Gene expression levels, the LUC / REN ratio increased by approximately 2-fold. Figure 8 (C in the text). The above results indicate that GmNAC4 has transcriptional activation function in this system and can be used for the regulation of plant gene expression.

[0083] Table 10 Primer sequence information for constructing the GAL4-BD-GmNAC4 vector

[0084] Table 11. Plasmid co-transformation combinations for GmNAC4 transcriptional activity detection

[0085] The above description merely illustrates preferred embodiments of the present invention and is quite specific and detailed, but it should not be construed as limiting the scope of protection of this invention. It should be noted that those skilled in the art can make various modifications, improvements, and substitutions within the spirit and principles of this application, and these all fall within the scope of protection of this invention.

Claims

1. Soybean NAC transcription factor gene GmNAC4 Application in regulating soybean seed size and / or drought resistance, the soybean NAC transcription factor gene GmNAC4 The nucleotide sequence is shown in SEQ ID NO.1; The application involves modifying the soybean NAC transcription factor gene. GmNAC4 Overexpression was used to obtain overexpressed plants to improve soybean drought resistance; or, the soybean NAC transcription factor gene was overexpressed. GmNAC4 Mutant plants were obtained by generating mutations to increase soybean seed size; the mutations were loss-of-function mutations.

2. The application of substances that regulate the expression of soybean NAC transcription factors in regulating soybean seed size or drought resistance, wherein the soybean NAC transcription factors include the soybean NAC transcription factor protein GmNAC4.

3. The application according to claim 2, characterized in that, The soybean NAC transcription factor protein GmNAC4 includes at least one of the following: A. A protein consisting of the amino acid sequence shown in SEQ ID NO.3; B. Proteins derived from sequence SEQ ID NO.2 that are related to soybean seed size or drought resistance, obtained by substitution and / or deletion and / or addition of one or more amino acid residues of the amino acid sequence shown in sequence SEQ ID NO.

3.

4. The application according to claim 2 or 3, characterized in that, The substances that regulate the expression of soybean NAC transcription factors include substances that silence or inhibit the expression of soybean NAC transcription factors, and the application includes its application in regulating soybean seed size.

5. The application according to claim 4, characterized in that, The regulation of soybean seed size includes at least one of increasing soybean seed width, increasing soybean seed length, increasing soybean seed thickness, and increasing the weight of 100 soybean seeds.

6. The application according to claim 4, characterized in that, The substance that silences or inhibits the expression of soybean NAC transcription factors includes at least one of the following: I. Used for editing soybean NAC transcription factor genes GmNAC4 sgRNA; II. A gene editing vector containing the sgRNA; III. Transfect recombinant bacteria with the gene-editing vector described above.

7. The application according to claim 6, characterized in that, The gene used for editing soybean NAC transcription factor gene GmNAC4 sgRNAs include those used to activate the soybean NAC transcription factor gene. GmNAC4 sgRNAs that have undergone loss-of-function mutations.

8. The application according to claim 7, characterized in that, The sgRNA includes a first target sequence and / or a second target sequence; wherein... The first target sequence is the inverse complementary sequence of positions 73 to 92 in SEQ ID NO.1; The second target sequence is the inverse complementary sequence of positions 219 to 238 in SEQ ID NO.

1.

9. The application according to claim 2 or 3, characterized in that, The substances that regulate the expression of soybean NAC transcription factors include substances that cause overexpression of soybean NAC transcription factors, and the applications include applications in regulating soybean drought resistance.

10. The application according to claim 9, characterized in that, The regulation of soybean drought resistance includes increasing soybean drought resistance, which is manifested in increased soybean plant survival rate and / or reduced water loss from detached leaves under drought conditions.

11. The application according to claim 9, characterized in that, The substance that causes overexpression of soybean NAC transcription factor includes at least one of the following: 1) Soybean NAC transcription factor protein GmNAC4; 2) Nucleotides encoding the soybean NAC transcription factor protein GmNAC4; 3) Recombinant vectors, expression cassettes, transgenic cell lines, or recombinant bacteria containing nucleotides encoding the soybean NAC transcription factor protein GmNAC4.

12. The application according to claim 11, characterized in that, The nucleotide encoding the soybean NAC transcription factor protein GmNAC4 includes at least one of the following: a. The nucleotide sequence shown in SEQ ID NO.1; b. A nucleotide sequence that can hybridize with the nucleotide sequence shown in SEQ ID NO.1 under stringent conditions; c. A nucleotide sequence that has more than 90% homology with the nucleotide sequence shown in SEQ ID NO.1 and encodes a protein related to soybean drought resistance.

13. A method for regulating soybean seed size, characterized in that, Includes the following steps: Silencing or suppressing soybean NAC transcription factor genes in soybean GmNAC4 The expression.

14. The method according to claim 13, characterized in that, The silencing or inhibition of the soybean NAC transcription factor gene in soybean. GmNAC4 The expressions include: Through gene editing technology, the soybean NAC transcription factor gene was modified. GmNAC4 Loss-of-function mutations result in soybean plants with larger seeds.

15. A method for improving the drought resistance of soybeans, characterized in that, Includes the following steps: Increase soybean NAC transcription factor gene in soybean GmNAC4 The amount of expression; And / or, increase the activity of soybean NAC transcription factor protein GmNAC4 in soybean.

16. The application of the method according to any one of claims 13 to 15 in soybean breeding.

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