Soybean drought stress tolerance gene and application thereof

Abstract

The invention belongs to the technical field of agricultural biology, and provides a soybean drought stress tolerance gene and application thereof, and the nucleotide sequence of the gene comprises a CDS sequence as shown in SEQ ID NO: 1; the nucleotide sequence has at least 90% of homology with the sequence shown in SEQ ID NO: 1 and is used for coding a protein with a drought stress response function; one or more nucleotides are substituted, deleted or inserted in the sequence as shown in SEQ ID NO: 1, and a nucleotide sequence of a protein with a drought stress response function is coded; according to the application disclosed by the invention, the direct association between the soybean gene Gm12g076200 and the drought stress tolerance is clarified, and the deletion of the soybean gene Gm12g076200 causes a soybean drought sensitive phenotype, so that the research blank of the gene on the stress response function is filled; the deletion of the gene can significantly reduce the tolerance of the soybean to drought stress, and the expression quantity is significantly increased under the drought stress, so that a key candidate gene is provided for research of a soybean drought-resistant molecular mechanism.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically a gene for soybean drought tolerance and its application. Background Technology

[0002] Soybean is an important global food, oilseed, and feed crop. Its growth and development are susceptible to abiotic stresses such as drought and high temperatures. Drought stress, in particular, can lead to a significant decrease in soybean yield, severely restricting agricultural production efficiency. Therefore, identifying key genes in soybeans that regulate drought stress tolerance, elucidating their molecular mechanisms, and applying them to molecular breeding to cultivate drought-resistant varieties is an important research direction in the field of agricultural biotechnology.

[0003] Plants have developed complex stress response mechanisms through long-term evolution, with transcription factors (TFs) playing a central regulatory role. The ARF family of transcription factors, as plant-specific transcription regulators, participates in auxin signaling pathway-mediated growth, development, and stress responses by binding to the Auxin response element in downstream gene promoters. Previous studies have shown that the functions of ARF family genes are closely related to plant organogenesis and stress tolerance; however, the specific functions of its members in soybean, especially in drought stress response, have not been fully elucidated.

[0004] Current research on soybean ARF family genes largely focuses on a few known members and their roles in growth and development, with limited studies on their association with drought stress tolerance. Furthermore, although some ARF family genes have been found to contain conserved Auxin-response and B3 domains, the specific relationships between these domains and drought stress regulation, as well as the molecular mechanisms of downstream gene regulatory networks, remain unclear, lacking direct functional validation data.

[0005] In terms of gene function verification technology, traditional methods rely on stable genetic transformation, which is time-consuming and inefficient. While gene editing technologies (such as CRISPR / Cas9) have made progress in soybean applications, the technical system for designing efficient targets for specific genes and combining them with rapid transformation systems (such as hairy root transformation) to achieve functional verification is still immature, resulting in low efficiency in identifying the function of drought-resistant genes in soybeans. In addition, existing studies on the expression patterns of drought-related genes are mostly limited to single stresses or tissues, lacking systematic research on the synergistic responses to multiple stresses (such as drought and heat stress) and tissue-specific expression, making it difficult to support the breeding needs for synergistic improvement under multiple adversities.

[0006] To this end, those skilled in the art have proposed a gene for soybean drought tolerance and its application, aiming to discover and verify ARF family genes in soybean that are directly related to drought tolerance, clarify their functions, domain roles and expression regulation patterns, and influence the drought resistance of soybean by regulating the expression of this gene, thereby providing key target genes and application methods for soybean drought resistance molecular breeding. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a gene for soybean drought tolerance and its application, thereby resolving the issues raised in the background section.

[0008] According to a first aspect of this disclosure, a gene for soybean drought tolerance is provided, the nucleotide sequence of which includes:

[0009] The CDS sequence shown in SEQ ID NO:1;

[0010] A nucleotide sequence that has at least 90% homology with the sequence shown in SEQ ID NO:1 and encodes a protein with a drought stress response function;

[0011] A nucleotide sequence in which one or more nucleotides are substituted, deleted, or inserted in the sequence shown in SEQ ID NO:1, and which encodes a protein having a drought stress response function.

[0012] The amino acid sequence of the protein encoded by the gene includes:

[0013] The amino acid sequence encoded by the CDS sequence shown in SEQ ID NO:1;

[0014] Proteins that have at least 85% homology to the amino acid sequence and possess conserved Auxin-response and B3 domains;

[0015] The amino acid sequence is substituted, deleted, or inserted with one or more amino acid residues to form a functional protein that is more sensitive to drought stress.

[0016] According to a second aspect of this disclosure, a recombinant vector is proposed, characterized in that it comprises the gene described in the first aspect, and a promoter connected upstream of the gene and a terminator connected downstream of the gene;

[0017] The vector is a plant expression vector, using the pCAMBIO1300-GFP vector. The gene is ligated into a linearized vector that has been double-digested with XbaI and BamHI via a one-step recombination method.

[0018] According to a third aspect of this disclosure, a host cell is provided comprising the recombinant vector described in the second aspect, wherein the host cell is an Agrobacterium cell, specifically Agrobacterium GV3101.

[0019] According to the fourth aspect of this disclosure, a method for improving the drought stress tolerance of soybeans is proposed, comprising the following steps:

[0020] S1. The gene is introduced into soybean cells via the recombinant vector; the introduction is achieved through Agrobacterium-mediated genetic transformation, using the pCAMBIA1300-GFP vector containing a 35S promoter and a GFP reporter gene.

[0021] S2. Cultivate the soybean cells to obtain transgenic soybean plants.

[0022] Preferably, the introduction method is Agrobacterium-mediated transformation, using a hairy root transformation system. The culture conditions for the transformed plants are: light intensity 200 μmol photons / m². -2 s -1 The conditions were: 16 hours of light, 28°C; 8 hours of darkness, 24°C; and 60% humidity.

[0023] According to a fifth aspect of this disclosure, a molecular marker for identifying drought stress tolerance in soybean is proposed, said molecular marker being designed based on the sequence of said gene, and the primer pair comprising:

[0024] P1:5'-CGGAGCTAGCTCTAGAATGAAGGAAGGTGAAAAGGTATTGGA-3';

[0025] P2:5'-TGCTCACCATGGATCCAGCAAATATGCTCAGAGGGCCT-3'.

[0026] According to a sixth aspect of this disclosure, an application of a gene for regulating soybean drought tolerance is proposed, wherein the gene editing comprises a crRNA array linked by a DR sequence; the target sequence includes:

[0027] gDNA1:5'-AGGTATTGGATCCGCAGCTA-3';

[0028] gDNA2:5'-ACAGGGTCACGCCGAACACG-3';

[0029] The vector contains a crRNA array based on the target sequence.

[0030] According to the seventh aspect of this disclosure, an application of a soybean mutant in drought-resistant breeding is proposed, said mutant containing a homozygous deletion mutation of said gene, said mutation causing the plant to have significantly lower fresh weight and chlorophyll content under drought stress than the wild type, and these values ​​are irreversible after rehydration.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 1. This invention is the first to clearly demonstrate the direct association between the soybean gene Gm12g076200 and drought stress tolerance. Its deletion leads to a drought-sensitive phenotype in soybeans, namely a significant decrease in fresh weight and chlorophyll content that cannot be recovered after rehydration, filling a research gap in the function of this gene in stress response. By regulating the expression of this gene, the drought resistance of soybeans can be affected, and it is shown that the deletion of this gene significantly reduces the tolerance of soybeans to drought stress, and its expression level is significantly upregulated under drought stress. This provides a key candidate gene for the study of the molecular mechanism of soybean drought resistance and can be directly used as a target gene for drought-resistant breeding.

[0033] 2. This invention demonstrates that the gene belongs to the ARF transcription factor family and contains conserved Auxin-response and B3 domains. Its functional verification provides data support for elucidating the transcriptional regulatory mechanism of ARF family genes in soybean stress response and for downstream gene network mining, which helps to reveal the molecular pathways of soybean drought resistance.

[0034] 3. This invention can quickly verify the function of the gene by constructing a mutant of the gene; by utilizing its specific primers and expression patterns, molecular markers can be developed for the screening and breeding of drought-resistant soybean varieties, shortening the breeding cycle and improving breeding efficiency. Attached Figure Description

[0035] Figure 1 This is a bioinformatics analysis diagram of the target gene Gm12g076200 in leguminous plants and the model plant Arabidopsis thaliana; wherein Figure 1 A is the phylogenetic tree analysis diagram. Figure 1 B is the conservative structural domain analysis diagram;

[0036] Figure 2 This is a diagram illustrating the protein localization and gene expression analysis of the present invention; wherein... Figure 2 A is a subcellular localization map of the protein encoded by Gm12g076200. Figure 2 B is a transcriptional abundance diagram of the Gm12g076200 gene in different tissue sites; Figure 2 C represents the transcriptional abundance of the Gm12g076200 gene after different abiotic stress treatments;

[0037] Figure 3 This is a verification diagram of the Gm12g076200 gene-edited homozygous material of the present invention; wherein... Figure 3 Figure A shows the analysis of the first-generation sequencing results of target 1 and target 2 in gene editing material line 1; Figure 3 B is a graph showing the analysis of the first-generation sequencing results of target 1 and target 2 in gene editing material line 2; Figure 3 C is a graph showing the analysis of the first-generation sequencing results of target sites 1 and 2 in line 3 of the gene editing material;

[0038] Figure 4 The figures show phenotypic diagrams and statistical data of the response to drought stress after deletion of the Gm12g076200 gene according to the present invention; wherein Figure 4 A shows the plant phenotypes of wild-type soybean and Gm12g076200 gene-deleted soybean before drought, after drought, and after rehydration. Figure 4 B is a statistical analysis chart of the fresh weight of plants before drought; Figure 4 C is a statistical analysis chart of the fresh weight of plants after drought. Figure 4 D is a statistical analysis chart of the fresh weight of plants after rehydration. Figure 4 E is a statistical analysis graph of leaf chlorophyll content before drought. Figure 4 F is a statistical analysis graph of leaf chlorophyll content after drought. Figure 4 G is a statistical analysis chart of the chlorophyll content of leaves after rehydration. Detailed Implementation

[0039] The embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention.

[0040] like Figure 1 As shown in Figure A, the specific experimental steps are as follows: 1. Align and download the homologous sequence of soybean Gm12g076200 from the phytozome database; 2. Convert to Fasta format, ensuring that the sequence ID contains species information and the format is correct; 3. Perform multiple sequence alignment of the gene sequence using the ClustalW method in MEGA v7.0; 4. Generate the alignment results of the multiple sequences and present them in Fasta format; 5. Construct a phylogenetic tree using the neighbor-joining method in MEGA v7.0; 6. Select the phylogenetic tree toolkit, beautify the format of the phylogenetic tree, add color annotations for different species, and finally export the phylogenetic tree in PDF format.

[0041] The results showed that soybean Gm12g076200 belongs to the ARF transcription factor family. Due to genome duplication during evolution, it has four homologous genes, and its similarity to ARF10 (At4g28350) in the model plant Arabidopsis thaliana is high. This indicates that the conservation of this gene in legumes provides a reference for its application in drought-resistant breeding of other legume crops (such as alfalfa and peas). The scope of drought-resistant targets can be expanded through homologous gene mining. The existence of its homologous genes provides a basis for elucidating the network mechanism of the ARF family's synergistic regulation of drought response, and can develop drought-resistant breeding strategies with multiple gene superposition.

[0042] like Figure 1 As shown in B, the conserved domain analysis was performed. The specific experimental steps were as follows: 1. Align and download the homologous sequence of soybean Gm12g076200 from the Phytozome database and convert it to Fasta format; 2. Enter the NCBI database, select the Concerned domain option, submit the protein sequence, obtain the NCBIDBB file, and download the data results in hitdata format; 3. Open the TBtools software analysis tool, select the Visualize domain pattern under the Graphics option; 4. Enter the gene protein sequence and hitdata file into the specified options as prompted, click start, and perform conserved domain analysis; 5. Under the results option, select to output the analysis results in PDF format, and you can modify the font and color later.

[0043] The results showed that these homologous genes belong to the ARF TFs family and all contain conserved Auxin-response and B3 domains, providing strong data support for the analysis and mining of their downstream transcriptional regulation and gene regulation networks. By designing small molecule activators that mimic the Auxin-response domain, the function of this gene can be enhanced under drought stress, improving drought resistance without the need for transgenics.

[0044] like Figure 2 As shown in Figure A, the specific experimental steps are as follows: 1. Download the CDS sequence (972bp) of Gm12g076200 from the phytozome database; 2. Design primers using the Snapgene online software:

[0045] P1:5'-CGGAGCTAGCTCTAGAATGAAGGAAGGTGAAAAGGTATTGGA-3';

[0046] P2:5'-TGCTCACCATGGATCCAGCAAATATGCTCAGAGGGCCT-3';

[0047] 3. PCR amplification using KOD high-fidelity enzyme was performed, and the target fragment amplified by PCR was ligated into the linearized vector pCAMBIO1300-GFP (the linearized vector was obtained after double digestion with XbaI and BamHI) using a one-step recombination method; 4. The successfully constructed vector was introduced into GV3101 Agrobacterium competent cells via chemical transformation after first-generation sequencing, and cultured in K+G double antibiotic resistant solid medium at 28°C for 2-3 days in an inverted incubator; 5. In a clean bench, single colonies were picked from the solid medium with a toothpick and transferred to 20 ml of K+G double antibiotic resistant liquid medium for concentrated culture; 6. The OD of the bacterial culture was measured using a spectrophotometer. 600 1. Terminate the culture; 6. Collect the cultured cells using an ultra-high-speed centrifuge and resuspend the cells in sterilized ddH2O to ensure the OD of the cells. 600 7. Using a 1 mL syringe, slowly inject the resuspended bacterial solution into tobacco mesophyll cells and incubate in the dark for 1 day; 8. Use a laser confocal microscope (ZEISS 980) to detect the subcellular localization of the target gene under a 488 nm excitation light source. (The empty vector injection was the negative control, and the successfully constructed vector injection was the experimental group).

[0048] Experimental results: The gene is located in the cell nucleus, emitting green fluorescence, and co-localizes with DAPI in the nucleus, emitting cyan fluorescence, as shown by the arrows in the figure. However, the subcellular localization of the empty vector is concentrated in the cell membrane and nucleus. Subcellular protein localization experiments confirmed that Gm12g076200 is located in the cell nucleus and co-localizes with DAPI. Therefore, based on nuclear localization characteristics, "nuclear-targeted" gene editing vectors can be designed to improve editing efficiency. Furthermore, by utilizing its fusion expression with GFP, a monitoring system for the dynamic changes of this protein in the cell nucleus under drought stress can be developed to assess the drought resistance status of plants in real time and provide molecular markers for drought early warning in the field.

[0049] KOD PCR amplification system:

[0050]

[0051] KOD PCR amplification program:

[0052] Pre-denaturation: 94℃ for 2 min

[0053] Repeat (40 times):

[0054] - Transformation: 98℃ for 10 seconds

[0055] Annealing: 68℃ for 30 seconds

[0056] -Extended range: 68℃ 60sec / kb

[0057] Final extension: 68℃ for 5 minutes

[0058] Save: 16℃∞

[0059] like Figure 2 As shown in B, the specific experimental steps are as follows: 1. Using the soybean variety Williams 82 (W82) as the research object, its seeds were germinated on sterilized vermiculite. 2. The soybean slow-growing rhizobium USDA110 inoculum was taken out from a -80℃ freezer and, using a spreader, was streaked in a zigzag pattern onto a solid culture medium containing Chl antibiotics in a laminar flow hood. The culture was then incubated at 28℃ for 4-5 days. 3. After single colonies had grown, single colonies were picked from the solid culture medium using a toothpick in a laminar flow hood and transferred to 200 ml of Chl antibiotic-resistant liquid culture medium for concentrated cultivation. 4. The OD of the bacterial culture was measured using a spectrophotometer. 600 5. Collect the cultured cells using an ultracentrifuge and resuspend them in sterilized ddH2O to ensure the OD value of the cells is 0.5. 600 6. After germination, when the true leaves of the seedlings are fully expanded, inoculate them with USDA110 rhizobium. 7. The inoculation amount per seedling is 10 ml. 8. On day 20 (20 dpi) after inoculation, collect tissues from soybean stems, shoot tips, true leaves, trifoliate leaves, roots, and root nodules. 9. Use liquid nitrogen for cryogenic grinding to break up the plant tissues. 10. Use RNA-easy extraction reagent (Vazyme, #r701-02) to extract total RNA samples from each tissue. 11. Perform reverse transcription PCR using the HiScript III first-strand cDNA synthesis kit (Vazyme, #r323-01). 12. Use ChamQ SYBR qPCR premix (Vazyme, #q311-02) to perform real-time quantitative qRT-PCR to detect the expression level of the target gene Gm12g076200. 13. In qRT-PCR analysis, transcriptional levels of different genes were standardized using GmActin11 transcriptional levels as a reference. Each experiment was performed in triplicate, and the values ​​given represent mean ± standard deviation. 14. qRT-PCR primers were designed using the Primer3 Plus online website:

[0060] qGmActin11-P1:5'-CCGGTCGTGACCTCACTGATTTCT-3';

[0061] qGmActin11-P2:5'-CATCAGGCAACTCGTAGCTCTTCTCG-3';

[0062] qGm12g076200-P1:5'-GACAATTTTCCCGCGTCTGG-3';

[0063] qGm12g076200-P2:5'-CCTCCGAATCCCAACACACA-3';

[0064] Tissue expression profiling revealed high expression of this gene in roots and root nodules, suggesting its central role in the underground stress response.

[0065] like Figure 2 As shown in Figure C, the specific experimental steps are as follows: 1. Using the soybean variety Williams 82 (W82) as the research object, its seeds were germinated on sterilized vermiculite. 2. The USDA110 inoculum was taken out from a -80℃ freezer and, in a laminar flow hood, the bacterial solution was streaked in a zigzag pattern onto a solid culture medium containing Chl antibiotics using a spreader. The culture was then incubated at 28℃ for 4-5 days. 3. After single colonies had grown, single colonies were picked from the solid culture medium using a toothpick in a laminar flow hood and transferred to 200 ml of Chl antibiotic-resistant liquid culture medium for concentrated cultivation. 4. The OD of the bacterial solution was measured using a spectrophotometer. 600 5. Collect the cultured cells using an ultracentrifuge and resuspend them in sterilized ddH2O to ensure the OD value of the cells is 0.5. 600 6. After germination, when the true leaves of the seedlings are fully expanded, inoculate them with USDA110 rhizobium. 7. The inoculation amount per seedling is 10 ml. 8. On day 10 (10 dpi) after inoculation, subject them to various stress treatments, including: normal conditions as control, heat stress (42°C incubator), drought stress, cold stress (4°C refrigerator), water flooding stress, and salt stress (250 mM sodium chloride aqueous solution). 9. Subsequently, take the aboveground leaves and extract total RNA samples from each tissue using RNA-easy extraction reagent (Vazyme, #r701-02). 10. Perform reverse transcription PCR using the HiScript III first-strand cDNA synthesis kit (Vazyme, #r323-01). 11. The expression level of the target gene Gm12g076200 was detected by real-time quantitative qRT-PCR using ChamQ SYBR qPCR premix (Vazyme, #q311-02). 12. In the qRT-PCR analysis, the transcriptional levels of different genes were standardized using the transcriptional level of GmActin11 as a reference. Each experiment was performed in triplicate, and the values ​​given represent the mean ± standard deviation.

[0066] Experimental results: Compared with normal environment, heat stress and drought stress significantly induced the expression of the target gene Gm12g076200, suggesting that it may be involved in regulating these stress processes, thereby affecting plant growth and development. This demonstrates that drought and heat stress significantly induce its expression, clarifying its regulatory potential in combined stress. These results support the following application scenarios:

[0067] By using root-specific promoters (such as the root hair-specific promoter RHE1) to drive the overexpression of this gene, only the drought resistance of the roots is enhanced, avoiding the growth redundancy caused by expression throughout the whole plant;

[0068] In the major soybean producing areas of North and Northwest China, where drought and high temperatures are frequent, overexpression of this gene can simultaneously enhance tolerance to both stresses and reduce yield reduction caused by extreme weather.

[0069] Based on its rapid upregulation of expression under drought stress, the drought resistance potential of plants can be identified at the seedling stage using the rapid detection method of qRT-PCR, thus shortening the breeding and screening cycle.

[0070] qRT-PCR system:

[0071] Reaction system (10 μL, SYBR Green)

[0072] 2×SYBR Green Master Mix 5μL

[0073] Forward primer (10 μM) 0.3 μM 0.3 μL

[0074] Reverse primer (10 μM) 0.3 μM 0.3 μL

[0075] 10-50 ng cDNA template 2 μL

[0076] RNase-free water to a final volume of 10 μL

[0077] Technical replication: Three biological replicates and three technical replicates were set up for each sample to reduce experimental error.

[0078] qRT-PCR system:

[0079] Pre-denaturation: 95℃ for 3 min

[0080] Repeat (40 times):

[0081] - Denaturation: 95℃ for 10 seconds

[0082] Annealing: 55-60℃ for 30 seconds

[0083] -Extension: 72℃ 30sec

[0084] Melting curve analysis:

[0085] Temperature range: -60°C to 95°C, increase by 0.5°C in increments, hold for 5 seconds.

[0086] Save: 4℃∞

[0087] like Figure 3 As shown, the specific experimental steps are as follows: 1. To verify the function of Gm12g076200, target design was performed based on CRISPR-Cpf1 genome-directed editing technology. The CDS sequence of Gm12g076200 was input, and TTTN was selected. 2. Since the result given by the website is 20bp in length, it is necessary to extend the 3' end by 2bp to make the crRNA length 22bp. A target with a suitable position and high score was selected, and the crRNA array was further designed. 3. DR sequences were added to both the 5' and 3' ends of the selected crRNA, and the arrangement and synthesis were performed to replace the crRNA array part in the original vector. After sequencing verification, the new CRISPR / LbCpf1 vector was finally obtained. 4. The efficiency of the target was verified through the hairy root transformation system. Using the soybean variety Williams82 (W82) as the research object, its seeds were germinated on sterilized vermiculite for 4 days. 7. Using a sterilized blade, the stem was obliquely cut at 45 degrees 1-2 cm away from the cotyledons. 8. Dip the cut end into the bacterial membrane of the target carrier and gently insert it into sterilized vermiculite. Incubate in the dark for 1 day, then incubate normally (light intensity 200 μmol photonsm). -2 s -1 (16 hours of light, 28℃; 8 hours of darkness, 24℃; 60% humidity). 9. Use a handheld fluorescence spectrometer to detect positive plants and compare the fluorescence intensity and plant phenotype between the experimental and control groups. 10. Select a highly efficient fusion vector and perform genetic transformation to obtain gene-edited plants. 11. Identify homozygous target editing in the three obtained mutant materials; sequencing results are as follows: Figure 3 As shown in AC.

[0088] Experimental results: Through first-generation sequencing alignment, 79 bp of gDNA1 was deleted in l in1, and gDNA2 was inserted into it. Figure 3 A). lin2 has a 71bp deletion in gDNA1 and crosses into gDNA2 ( Figure 3 B). The gDNA1 in l in3 has a single base C insertion at position 295, followed by a 10 bp deletion, but the gDNA2 target site is not edited. Figure 3 C). Furthermore, the sequencing peaks all show single peaks, indicating that the material is homozygous and usable. The target information is as follows:

[0089] gDNA15'-AGGTATTGGATCCGCAGCTA-3';

[0090] gDNA25'-ACAGGGTCACGCCGAACACG-3';

[0091] The target sequencing primers are:

[0092] CXP1:5'-CCAGTCTCGTGCTTGTACTTTG-3';

[0093] CXP2:5'-CAGATGATGAACCTGAACCTGAAC-3'.

[0094] like Figure 4 As shown, the specific experimental steps are as follows: 1. Soak and wash the seeds of wild-type (Williams 82, W82) and gene-edited homozygous materials in 75% alcohol for 10 minutes, then wash them three times with sterile ddH2O for 5 minutes each time. 2. Germinate the washed seeds on sterile vermiculite. 3. After germination and full unfolding of true leaves, inoculate with USDA110 rhizobium. 4. Start drought treatment on the 10th day after inoculation (20 dpi) and record the phenotype before treatment. On the 7th day of drought treatment, the plants reached the drought tolerance limit and the phenotype was recorded; subsequently, different plants were rehydrated, and the phenotype was recorded on the 5th day of rehydration. Figure 4 A). 5. Weigh the fresh weight of the plants using an electronic balance (Figure BD). 6. Measure the chlorophyll content using a chlorophyll meter (Figure EG).

[0095] Experimental results: Compared with the wild type, the fresh weight of plants after the target gene deletion was significantly reduced in the phenotype before drought treatment. Figure 4 B), and the chlorophyll content decreased ( Figure 4 E). Following drought stress, it was found that the deletion of the target gene resulted in extreme sensitivity to drought stress, and the plant fresh weight ( Figure 4 C) and chlorophyll content ( Figure 4 F) decreased significantly; and it was found that even rehydration could not restore the fresh weight of the plant caused by the loss of the target gene. Figure 4 D) and chlorophyll content ( Figure 4 G) A significantly reduced phenotype. This indicates that the drought-susceptibility phenotype of the mutant can serve as a "negative control" to evaluate the effects of other drought-resistant genes or regulatory measures, improving screening accuracy. The mutant can also be used as a tool to elucidate the molecular mechanisms by which ARF family genes regulate drought response through the auxin signaling pathway, and to identify key downstream target genes. Furthermore, based on the mutant's molecular markers (such as target deletion fragments), loss-of-function allelic variants of the gene in natural populations can be rapidly identified using PCR detection methods, allowing for the screening of materials with poor drought resistance for reverse validation.

[0096] In summary, deletion of the target gene Gm12g076200 reduces the tolerance of soybeans to drought stress. By regulating the expression level of the gene sequence, it increases the survival rate of soybeans under drought stress, suggesting that this gene may be an important candidate target for regulating soybeans' resistance to drought stress.

[0097] The knockout mutant of the target gene Gm12g076200 showed reduced stomatal conductance, decreased transpiration rate, and improved water use efficiency under drought stress, indicating a significant enhancement in its water management capacity.

[0098] The feasible operation involved transforming soybean cotyledonary nodes with a recombinant vector that drove gene overexpression using the CaMV35S promoter. The transgenic plants showed increased net photosynthetic rate and decreased transpiration rate under drought stress. By constructing a root-specific promoter-driven expression vector, the gene was specifically and highly expressed in the root system, increasing root dry weight and the number of lateral roots, thereby enhancing water absorption capacity.

[0099] Compared with traditional hybridization breeding, gene editing technology shortens the cycle of introducing drought-resistant traits and can precisely regulate specific functional domains. It has been proven effective in multiple species such as soybean, corn, and Arabidopsis thaliana, breaking through the limitations of single crops and showing its potential for cross-species application, while reducing the input of pesticides and water resources.

[0100] This invention protects the drought-resistant function of the target gene Gm12g076200, as well as variant sequences with nucleotide sequences encoding the same functional protein and possessing ≥90% homology, including functional sequences modified by substitution, deletion, or insertion. It also protects recombinant vectors, including the pCAMBIO1300-GFP recombinant vector containing this gene and its derivative vectors. Drought-resistant varieties bred using this invention can increase soybean yield per acre in drought years and reduce irrigation water and fertilizer application, as drought-resistant varieties have lower fertilizer requirements under stress, thus reducing agricultural non-point source pollution. Through systematic functional verification experiments, the core role of the Gm12g076200 gene in the soybean drought stress response was clarified; its deletion leads to drought sensitivity and irreversible rehydration, while overexpression enhances drought resistance. The gene sequence, encoded protein, technical methods, and application scenarios constitute a complete protection system, with application potential spanning molecular breeding research and development, agricultural production in arid regions, and ecological environmental protection. Its patent protection can effectively enhance the key targets and technologies for soybean drought-resistant molecular breeding, and has significant strategic value for improving the resilience of my country's soybean industry and promoting sustainable agricultural development.

[0101] It should be understood that numerous specific implementation decisions can be made during the development of any actual implementation method, and in any engineering or design project. Such development efforts may be complex and time-consuming, but for those of ordinary skill in the art who benefit from this disclosure, the development effort will be a routine work of design, manufacturing, and production without requiring much experimentation.

[0102] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A gene for soybean tolerance to drought stress, characterized in that, The nucleotide sequence of the gene includes: The CDS sequence shown in SEQ ID NO:1; A nucleotide sequence that has at least 90% homology with the sequence shown in SEQ ID NO:1 and encodes a protein with a drought stress response function; A nucleotide sequence in which one or more nucleotides are substituted, deleted, or inserted in the sequence shown in SEQ ID NO:1, and which encodes a protein having a drought stress response function.

2. The gene for soybean drought tolerance as described in claim 1, characterized in that: The amino acid sequence of the protein encoded by the gene includes: The amino acid sequence encoded by the CDS sequence shown in SEQ ID NO:1; Proteins that have at least 85% homology to the amino acid sequence and possess conserved Auxin-response and B3 domains; The amino acid sequence is substituted, deleted, or inserted with one or more amino acid residues to form a functional protein that is more sensitive to drought stress.

3. A recombinant vector, characterized in that: It comprises the gene as described in claim 1 or 2, and a promoter connected upstream of the gene and a terminator connected downstream of the gene; The vector is a plant expression vector, using the pCAMBIO1300-GFP vector. The gene is ligated into a linearized vector that has been double-digested with XbaI and BamHI via a one-step recombination method.

4. A host cell, characterized in that, The recombinant vector of claim 3 is contained in the host cell being Agrobacterium cell, specifically Agrobacterium GV3101.

5. A method for improving the drought stress tolerance of soybeans, characterized in that, Includes the following steps: S1. The gene is introduced into soybean cells via the recombinant vector; the introduction is achieved through Agrobacterium-mediated genetic transformation, and the vector used is the pCAMBIA1300-GFP vector containing a 35S promoter and a GFP reporter gene. S2. Cultivate the soybean cells to obtain transgenic soybean plants.

6. The method for improving soybean's tolerance to drought stress as described in claim 5, characterized in that: The introduction method is Agrobacterium-mediated transformation, using a hairy root transformation system. The culture conditions for the transformed plants are: light intensity 200 μmol photons / m². -2 s -1 The conditions were: 16 hours of light, 28°C; 8 hours of darkness, 24°C; and 60% humidity.

7. A molecular marker for identifying drought stress tolerance in soybeans, characterized in that, The molecular markers are designed based on the sequence of the gene, and the primer pairs include: P1:5'-CGGAGCTAGCTCTAGAATGAAGGAAGGTGAAAAGGTATTGGA-3'; P2:5'-TGCTCACCATGGATCCAGCAAATATGCTCAGAGGGCCT-3'.

8. The application of a gene for drought tolerance in soybeans in regulating soybean drought resistance, characterized by: The gene editing comprises a crRNA array linked by DR sequences; the target sequence includes: gDNA1:5'-AGGTATTGGATCCGCAGCTA-3'; gDNA2:5'-ACAGGGTCACGCCGAACACG-3'; The vector contains a crRNA array based on the target sequence.

9. The application of a soybean mutant in drought-resistant breeding, characterized in that, The mutant contains a homozygous deletion mutation of the gene, which causes the plant to have significantly lower fresh weight and chlorophyll content under drought stress than the wild type, and these values ​​are irreversible after rehydration.

Images