Soybean-derived protein and application of related biological materials thereof in regulation and control of plant salt tolerance

Abstract

The invention discloses application of soybean-derived protein and related biological materials thereof in regulation and control of plant salt tolerance. According to the technical scheme protected by the invention, the soybean-derived protein is applied to regulation and control of plant salt tolerance, the name of the soybean-derived protein is GmATHB12, and the soybean-derived protein can be a protein of which the amino acid sequence is a sequence 2 in a sequence table. The gene GmATHB12 is cloned from the soybean variety Zihuang 34, the gene GmATHB12 is over-expressed in the soybean to obtain a transgenic GmATHB12 plant, the salt tolerance of the transgenic GmATHB12 plant is remarkably higher than that of a non-transgenic wild type, and the GmATHB12 protein and related biological materials have the effect of improving the salt tolerance and can be applied to plant breeding or variety breeding.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of soybean-derived proteins and related biomaterials in regulating plant salt tolerance. Background Technology

[0002] Soybeans are an important crop used for both grain, oil, and feed, and hold a vital strategic position in national economic development and in ensuring national food and oil security.

[0003] Salt stress is a major abiotic stress affecting global crop productivity. Currently, over 6% of the world's land area and approximately 20% of arable land are affected by salinization, and this area is continuously expanding. The effects of salt stress on plants can be divided into two main stages: osmotic stress and ion toxicity. Osmotic stress occurs in the early stages of salt stress in plants, where reduced root water potential makes water absorption difficult, inhibiting new leaf growth. Ion toxicity occurs when plants excessively absorb external salt ions (Na+) through an electrochemical gradient. + Cl - High salt content in soil can disrupt intracellular ion homeostasis, damage cell membrane structure, cause metabolic disorders, and severely affect the normal growth and development of plants. For crops, high soil salinity inhibits nutrient absorption by roots, suppresses photosynthesis, reduces photosynthetic products, and consequently causes flower abortion, decreased fruit set, and ultimately reduced crop yield.

[0004] Soybean is a moderately salt-tolerant plant; salt stress significantly inhibits soybean seed germination, vegetative growth, and reproductive growth. Soybean yield drops drastically when soil salinity reaches 5 Ds / m³. Therefore, identifying and studying soybean salt-tolerant genes is crucial for a deeper understanding of soybean's salt tolerance mechanisms and provides molecular tools for creating new salt-tolerant soybean germplasm and breeding moderately to severely salt-tolerant soybean varieties. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to regulate the salt tolerance of soybeans and / or how to improve the salt tolerance of soybeans and / or how to cultivate salt-tolerant soybean varieties.

[0006] To address the aforementioned technical problems, the present invention first provides any of the following applications of proteins: P1. Application of the protein in regulating plant salt tolerance P2. Application of the protein in improving plant salt tolerance. P3. Application of the aforementioned protein in plant breeding P4. Application of the protein in plant quality improvement; The protein may be one of the following: A1) The amino acid sequence is that of sequence 2 in the sequence listing; A2) Proteins derived from A1) or proteins with more than 80% identity and function to the amino acid sequence shown in Sequence 2 of the sequence listing, obtained by substitution and / or deletion and / or addition of amino acid residues. A3) is a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1) or A2).

[0007] In the above applications, the protein may be derived from soybeans.

[0008] The proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0009] In the aforementioned proteins, the protein tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, to facilitate the expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0010] In the above-mentioned proteins, identity refers to the identity of the amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences to calculate the identity value (%), then the identity value can be obtained.

[0011] In the aforementioned proteins, the 80% or more identity can be at least 81%, 82%, 85%, 86%, 88%, 90%, 91%, 92%, 95%, 96%, 98%, 99%, or 100% identity.

[0012] In the above applications, the plant may be any of the following: D1) Dicotyledons, D2) Leguminosae (family Fabaceae) D3) Leguminosae (family legumes) D4) Plants of the genus *Glycine*, D5) Soybeans.

[0013] To address the aforementioned technical problems, the present invention also provides any of the following applications of biomaterials related to the proteins described above: Q1. Application of the aforementioned biomaterials in regulating plant salt tolerance Q2. Application of the aforementioned biomaterials in improving plant salt tolerance Q3. Application of the aforementioned biomaterials in plant breeding. Q4. Application of the aforementioned biomaterials in plant quality improvement The biomaterial may be any of the following: B1) Nucleic acid molecules that encode the proteins described above; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2); B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2); B7) Transgenic plant organs containing the nucleic acid molecules described in B1), or transgenic plant organs containing the expression cassette described in B2); B8) Nucleic acid molecules that promote or enhance gene expression of the proteins described above; B9) Expression cassettes, recombinant vectors, recombinant microorganisms or transgenic plant cell lines containing the nucleic acid molecules described in B8).

[0014] In the above applications, the nucleic acid molecule described in B1) can be the gene encoding the protein as shown below: b1) A DNA molecule whose coding sequence is the nucleotide of sequence 1 in the sequence listing; b2) Nucleotides are DNA molecules that are sequence 1 in the sequence listing. b3) A cDNA or DNA molecule that hybridizes with the cDNA or DNA molecule defined in b2) and encodes a protein with the same function.

[0015] In the above applications, the plant may be any of the following: D1) Dicotyledons, D2) Leguminosae (family Fabaceae) D3) Leguminosae (family legumes) D4) Plants of the genus *Glycine*, D5) Soybeans.

[0016] The indicators for plant breeding include salt tolerance.

[0017] Furthermore, in the applications described above, the purpose of plant breeding includes cultivating salt-tolerant plants (with salt tolerance higher than that of the parent plants).

[0018] Furthermore, in the applications described above, the plant breeding includes increasing or enhancing or upregulating the expression of the gene encoding the protein in the target plant, and the purpose of the plant breeding includes cultivating salt-tolerant plants (with higher salt tolerance than the target plant).

[0019] In the aforementioned biological materials, the expression cassette containing nucleic acid molecules described in B2) refers to DNA capable of expressing the proteins described in the above applications in host cells. This DNA may include not only promoters that initiate transcription of protein-coding genes but also terminators that terminate transcription of protein-coding genes. Furthermore, the expression cassette may also include enhancer sequences. Promoters that can be used in this invention include, but are not limited to: constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters.

[0020] Recombinant expression vectors containing the protein-coding gene expression cassettes can be constructed using existing plant expression vectors. These plant expression vectors include binary Agrobacterium vectors and vectors suitable for plant microbombardment, such as pAHC25, pWMB123, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb (CAMBIA). The plant expression vectors may also contain the 3' untranslated region of the exogenous gene, i.e., containing a polyadenylated signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylated signal can guide the addition of polyadenylated nucleotides to the 3' end of the mRNA precursor, such as Agrobacterium crown gall tumor inducing (Ti) plasmid genes (e.g., carmine synthase genes). Nos The untranslated regions transcribed at the 3' end of plant genes (such as soybean storage protein genes) have similar functions. When constructing plant expression vectors using the genes of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent start codons, etc., but they must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence.

[0021] In the aforementioned biological materials, the recombinant microorganisms may specifically be yeast, bacteria, algae, and fungi.

[0022] To address the aforementioned technical problems, the present invention also provides a method for improving the salt tolerance of plants, comprising upregulating, enhancing, or increasing the activity of the proteins described above and / or the expression level of the genes encoding the proteins described above in the target plant, thereby improving the salt tolerance of the target plant.

[0023] In the above method, the upregulation, enhancement, or increase of the activity of the protein described above and / or the expression level of the gene encoding the protein described above in the target plant is achieved by introducing the gene encoding the protein described above into the target plant.

[0024] In the above methods, the plant and / or the target plant is any one of the following: D1) Dicotyledons, D2) Leguminosae (family Fabaceae) D3) Leguminosae (family legumes) D4) Plants of the genus *Glycine*, D5) Soybeans.

[0025] The proteins and / or biological materials described above are also within the scope of protection of this invention. This invention is the first to identify the function of soybean GmATHB12 protein in regulating soybean salt tolerance, and proposes the use of overexpression... GmATHB12 Gene-enhanced salt tolerance in soybeans. Experiments have shown that overexpression of the GmATHB12 protein in soybeans significantly improves salt tolerance compared to wild-type soybeans. This invention provides methods and a basis for the creation of new salt-tolerant soybean germplasm and for addressing salt-tolerant soybean breeding issues. Attached Figure Description

[0026] Figure 1 for GmATHB12 The relative expression levels of genes in response to salt stress treatment under different salt stress treatment durations.

[0027] Figure 2 for GmATHB12 Gene expression in different soybean tissues.

[0028] Figure 3 For the transfer GmATHB12 Overexpression lines GmATHB12 Expression levels were detected. WT was wild-type soybean W82, and OE1, OE2, and OE4 were three overexpressing genes. GmATHB12 The turn GmATHB12 Soybean lines with overexpressed genes.

[0029] Figure 4 For the transfer GmATHB12 Phenotypic diagram of overexpression lines of soybean under salt stress. CK is the result of 10 days of water treatment followed by transformation. GmATHB12Growth of overexpression lines OE1, OE2, OE4, and wild-type soybean. Salt was used after 10 days of salt treatment. GmATHB12 Growth of overexpression lines OE1, OE2, OE4 and wild-type soybean. In the figure, W82 represents wild-type soybean, and OE1, OE2, and OE4 represent the three overexpression lines. GmATHB12 of soybean strains.

[0030] Figure 5 For the transfer GmATHB12 Physiological data of overexpression lines of soybean and wild-type soybean after salt treatment and control treatment. A represents the physiological data of overexpression lines. GmATHB12 Comparison of fresh weight between overexpression-treated soybean lines and wild-type soybean. B represents the transgenic line. GmATHB12 Comparison of chlorophyll content in leaves of overexpression-promoted soybean lines and wild-type soybean. C represents the transgenic soybean line. GmATHB12 Comparison of survival rates between overexpression lines and wild-type soybean. In the figure, W82 represents wild-type soybean, and OE1, OE2, and OE4 represent three soybean transgenic lines. GmATHB12 Overexpression lines. Detailed Implementation

[0031] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0033] The following examples used SAS 8.0 statistical software to process the data, and the experimental results are expressed as mean ± standard deviation. Figure 1 The Student's t-test was used, and P < 0.01 (**) indicates a highly significant difference. Figure 2 , Figure 3 ,and Figure 5 Fisher's least significant difference (LSD) test (P<0.05) was used, with different letters indicating significant differences between different tissues or materials.

[0034] Example 1. GmATHB12 Gene cloning and expression detection The material used in this embodiment is the salt-tolerant soybean variety Qi Huang 34 (QH34). This variety is described in the literature "Hu JM, Zhuang YB, Li XC, et al. Time-series transcriptome comparison reveals the gene regulation network under salt stress in soybean (…). Glycine max ) roots. BMC The invention was disclosed in "Plant Biology, 2022, 322:157" and is available to the public from the Crop Research Institute of Shandong Academy of Agricultural Sciences. It is used only for the purpose of repeating this invention.

[0035] 1. Extraction of total RNA from different soybean tissues under salt stress conditions 1.1 Tissue sampling of soybeans under salt stress Select uniformly sized Qihuang 34 seeds, disinfect them with 10% (v / v) sodium hypochlorite, rinse them five times with deionized water, and sow them in quartz sand. After 3 days of growth, select uniform seedlings and transfer them to plastic baskets. Place the baskets in plastic pots containing 1 / 2 modified Hogland nutrient solution. Once the true leaves unfold, transfer them to 5 liters of 1 / 2 modified Hogland nutrient solution for cultivation. Maintain aeration for 24 hours using an aeration pump. Change the nutrient solution every 3 days, replacing it with full Hogland nutrient solution on the second change. After 6 days of cultivation, subject them to salt stress (NaCl) treatment. The NaCl concentrations were 0 mol / L and 0.15 mol / L, with 0 mol / L serving as the control and 0.15 mol / L as the salt stress treatment. Soybean roots from the control and salt stress treatments were collected at 1 h, 3 h, 6 h, 9 h, and 12 h after treatment and stored in liquid nitrogen for later use.

[0036] 1.2 Sampling of different tissues of soybean Sampling for expression detection in different tissue parts was performed using a soil culture method. Specifically, soybean Qihuang 34 seeds of uniform size were selected and sown in a 10 cm × 10 cm round plastic pot containing a mixture of vermiculite and peat moss (ratio 1:3). Roots, leaves, and stems were harvested 12 days after sowing. Flowers were harvested after the plants reached the flowering stage, and pods and seeds were harvested during the grain-filling stage. The samples were then stored in liquid nitrogen for later use.

[0037] All materials were cultured in the greenhouse of the Crop Research Institute of Shandong Academy of Agricultural Sciences, with a culture temperature of 26℃, a light exposure time of 14h, and a humidity of 60%.

[0038] 1.3 RNA Extraction RNA was extracted from salt-stressed samples and samples from different tissue sites using the FastPure Universal Plant Total RNA Isolation Kit (RC411) (Nanjing Novizan Biotechnology Co., Ltd.). After extraction, electrophoresis was used to detect whether the RNA had degraded. The obtained RNA was then reverse transcribed into cDNA using Novizan's HiScript III RTSuperMix for qPCR (+gDNA wiper) for later use.

[0039] 2. GmATHB12 Gene expression detection Using the cDNA obtained in step 1 as a template, PCR was performed on a Roche Lightcycler® 480 real-time PCR instrument using the ChamQ Universal SYBR qPCR Master Mix kit (Nanjing Novizan Biotechnology Co., Ltd.). Three biological replicates were set up for the experiment. GmActin As an internal control gene, detection GmATHB12 Relative gene expression levels. Primer sequences are as follows: q GmATHB12 -F: 5′-CCAATGGAGCCAGGTCAGAG-3′; q GmATHB12 -R: 5′-GATATTTCCATTGATGGGCTTGG-3′; q GmActin -F: 5′-CGGTGGTTTCTATCTTGGCATC-3′; q GmActin -R: 5′-GTCTTTCGCTTCAATAACCCTA-3′.

[0040] Test results as follows Figure 1 and Figure 2 As shown: Compared with the control (CK) (0 mol / L NaCl), salt stress treatment (0.15 mol / L NaCl) significantly induced root saturation at different time points (1 h, 3 h, 6 h, 9 h, and 12 h). GmATHB12 Gene expression was induced by salt treatment for 6 hours, resulting in the highest fold increase. Figure 1 (As shown).

[0041] By measuring different tissue parts (roots, stems, leaves, flowers, pods, and seeds) GmATHB12 Gene expression findings, GmATHB12 The expression level is higher in the stem and lower in the grain. Figure 2 (As shown).

[0042] 3. GmATHB12 Cloning of genes Using the cDNA obtained in step 1 as a template, primers GmATHB12-F: 5′-ATGGAATATACTTATTCAGC-3′ and GmATHB12-R: 5′-GGACCAGAAGTCCCACCATT-3′ were used for profiling. GmATHB12 Gene PCR amplification was performed using the 2×Phanta Flash Master Mix (Dye Plus) (P520) high-fidelity enzyme from Nanjing Novizan Biotechnology Co., Ltd. The obtained PCR products were detected by agarose gel electrophoresis and then extracted and recovered using a universal DNA purification and recovery kit from Tiangen Biotech Co., Ltd. Sequencing was then performed at Qingke Biotechnology Co., Ltd.

[0043] Sequencing results showed that the nucleotide sequence of the PCR-purified and recovered product was sequence 1 in the sequence listing. The gene of the DNA molecule whose CDS sequence was sequence 1 was named... GmATHB12 The full-length ORF sequence is 717 bp (positions 1-717 of sequence 1 in the sequence listing), encoding 239 amino acids. The protein is named GmATHB12, and the amino acid sequence of the GmATHB12 protein is sequence 2 in the sequence listing.

[0044] Sequence 1 is as follows (5'-3'): ATGGAATATACTTATTCAGCAGGTGTAGAGGCAGAAACTCACACAAGTAGCAGCACCACCCCATCAAGATCAAAGAAGAGAAACAACAACACAAGAAGGTTCAGTGATGAACAAATCAAATCATTGGAGACCATGTTTGAATCAGAGTCAAGGCTTGAGCCTAGAAAGAAGTTGCAGCTGGCCAGAGAGCTTGGATTGCAGCCAAGGCAAGTTGCTATATGGTTTCAGAACAAGAGGGCTAGGTGGAAGTCAAAGCAACTTGAGAGAGACTATGGCATACTCCAATCCAATTACAACAGTTTGGCTTCCCGTTTTGAAGCTCTTAAGAAGGAAAATCAAACATTACTAATTCAGCTGCAGAAGCTGAATCATCTAATGCAGAAGCCAATGGAGCCAGGTCAGAGATGCACACAAGTTGAAGCAGCAAACAGCATGGACAGTGAATCAGAAAATGGAGGCACCATGAAATGTGAAGCTGAGGGAAAGCCAAGCCCATCAATGGAAATATCAGAACATTTACTTGGTGTTCTGTCTGATGATGACACAAGCATAAAGGTGGAAGACTTTGGCCTAGAAGATGAACATGGCCTTCTGAATTTTGCTAAGCATGTTGATGGTTCCTTGACTTCACCAGAAGATTGGAATGCTTTTGAATCCAATGATCTATTAGGCCAATCAACCACTGATGATTACCAATGGTGGGACTTCTGGTCCTGA。

[0045] The sequence 2 is as follows: MEYTYSAGVEAETHTSSSTTPSRSKKRNNNTRRFSDEQIKSLETMFESESRLEPRKKLQLARELGLQPRQVAIWFQNKRARWKSKQLERDYGILQSNYNSLASRFEALKKENQTLLIQL QKLNHLMQKPMEPGQRCTQVEAANSMDSESENGGTMKCEAEGKPSPSMEISEHLLGVLSDDDTSIKVEDFGLEDEHGLLNFAKHVDGSLTSPEDWNAFESNDLLGQSTTDDYQWWDFWS.

[0046] Example 2. (Transfer) GmATHB12 Soybean Acquisition and Functional Verification The expression vector used in this embodiment is pCambia3301 (preserved in our laboratory, related literature: Liu Wei, Wang Yubin, Li Wei, et al. Overexpression of soybean isopropyl malate dehydrogenase gene GmIPMDH promotes flowering and growth of plants [J]. Acta Agronomica Sinica, 2024, 50(3):613-622. It is available to the public from the applicant and is only used to replicate this invention and not for other purposes); the Escherichia coli strain used is DH5α and the Agrobacterium strain is EHA105 purchased from Shanghai Weidi Biotechnology Co., Ltd., for vector construction and soybean genetic transformation.

[0047] 1. Transfer GmATHB12 Soybean harvesting 1.1. Construction of the recombinant expression vector pCambia3301-GmATHB12 (1) Using the PCR purified and recovered product obtained in Example 1 as a template, primers were used... GmATHB12 -BamHI-F:5'- CTCTCTCTCAAGCTTGGATCC ATGGAATATACTTATTCAGC-3', GmATHB12 -XbaI-R:5'- TGCCTGCAA GTCGACTCTAGA PCR amplification was performed using GGACCAGAAGTCCCACCATT-3' (the underlined part of the primer is the sequence on the vector pCambia3301). The resulting PCR product was named GmATHB12-3301. GmATHB12-3301 contains fragments of the vector sequence on pCambia3301 and BamHI and XbaI enzyme recognition sites at both ends.

[0048] (2) pCambia3301 was linearized by double restriction endonuclease digestion with BamHI and XbaI and then recovered. The recovered product was ligated with GmATHB12-3301 purified in (1) for homologous recombination (ClonExpressII One Step Cloning Kit (C113) purchased from Nanjing Novizan Biotechnology Co., Ltd.). The recombinant product was transformed into DH5α Escherichia coli and plated on LB resistant plates containing kanamycin (50 mg / L) and incubated overnight at 37°C. Single colonies from the LB plates were picked and inoculated into liquid medium with the same concentration of kanamycin and incubated overnight at 37°C with shaking at 200 rpm. The bacterial culture was identified by PCR using the full-length primers of the target gene (the identification primers were...). GmATHB12 -F: 5'-ATGGAATATACTTATTCAGC-3' and GmATHB12 -R: 5'-GGACCAGAAGTCCCACCATT-3'). The identified positive clones were sent to Qingdao Qingke Biotechnology Co., Ltd. for sequencing. Sequencing results were compared using DNAMAN, and clones with correct sequencing were selected for amplification culture. Plasmid extraction was performed using a plasmid extraction kit from Tiangen Biotech Co., Ltd., and the extracted positive recombinant vector plasmid was named pCambia3301- GmATHB12 Recombinant vector plasmid pCambia3301- GmATHB12 Contains the sequence shown in sequence 1 of the sequence list GmATHB12 The CDS sequence of the cDNA can express the sequence shown in sequence 2 of the sequence listing. GmATHB12 protein.

[0049] 1.2 Obtaining recombinant Agrobacterium Take 1 μg of the recombinant vector plasmid pCambia3301- prepared in step 1.1 above. GmATHB12 Competent cells of Agrobacterium EHA105 were transformed and cultured for two days at 28°C on YEP medium (containing 50 mg / L kanamycin and 50 mg / L rifampin, abbreviated as YEP+Kan+Rif). Single colonies were picked and placed in 5 ml sterile EP tubes, with 2 ml of YEP+Kan+Rif liquid medium added beforehand, and cultured overnight by shaking. Primers were then used... GmATHB12 -F: 5'-ATGGAATATACTTATTCAGC-3' and GmATHB12 -R: 5'-GGACCAGAAGTCCCACCATT-3' was used for PCR identification, and the positive bacterial solution obtained by PCR identification was named recombinant Agrobacterium EHA105 / pCambia3301- GmATHB12 The bacterial culture was stored at -80 ℃ for later use in genetic transformation experiments.

[0050] Recombinant Agrobacterium EHA105 / pCambia3301- GmATHB12 The CDS nucleotide sequence containing the cDNA from Example 1 is the gene of sequence 1 in the sequence listing. GmATHB12 .

[0051] 1.3. Transfer GmATHB12 Obtaining genetically modified soybeans (1) Preparation of recipient plants: Select about 100 plump, smooth and crack-free soybean Williams 82 seeds (these seeds are published in the following literature: Taifei Yu, Zehao Hou, Hailong Dong, et al. Soybean steroids improve crop abiotic stress tolerance and increase yield. Plant Biotechnology Journal, 2024, 1-15, doi: 10.1111 / pbi.14349, which can be obtained by the public from the Crop Research Institute of Shandong Academy of Agricultural Sciences and is only used to repeat the experiment of this invention), put them in Erlenmeyer flasks, soak them in 70% ethanol for 5 min in a clean bench, take 15% hydrogen peroxide to cover the seeds, put the Erlenmeyer flasks in a shaker, shake at 110 rpm for 12 min, pour off the hydrogen peroxide, wash 5 times with sterile dd H2O in a clean bench, add hormones and place at 4℃ for 12 h to obtain sterilized soybean seeds.

[0052] (2) Preparation of infecting bacterial solution: The recombinant Agrobacterium EHA105 / pCambia3301- prepared in step 1.2 and stored at -80℃ was infecting the bacterial solution. GmATHB12 Streak the culture in YEP solid medium supplemented with Kan (50 mg / L kanamycin) and Rif (50 mg / L rifampin). Pick a single colony and place it in a 5 ml sterile EP tube. Pre-activate the EP medium with YEP+Kan+Rif liquid culture at 28°C and shake at 220 rpm overnight. (Inoculate the activated bacterial culture at a 1:500 ratio into 50 mL of YEP liquid medium (containing Kan and Rif), and incubate at 28°C and 220 rpm until OD reaches [the desired concentration]. 600nm The OD value was set to 0.8. The recombinant Agrobacterium bacterial suspension was transferred to a 50 mL centrifuge tube and centrifuged at 5000 rpm for 10 min to enrich the bacterial cells. The cells were resuspended in culture medium, and the OD value was adjusted. 600nm The concentration was reduced to approximately 0.5, and the resulting inoculum was prepared for later use.

[0053] (3) Cotyledon node method for infecting soybeans: The sterilized soybean seeds obtained in step (1) are removed by scalpel in a clean bench and a cut is made at the growth point. The treated seeds are placed in a sterilized conical flask, the prepared bacterial inoculation solution is poured in, and the flask is placed in a shaker and inoculated at 110 rpm for 3 h.

[0054] (4) Co-culture: Discard the infection solution, dry the seeds and transfer them to the co-culture medium, and co-culture for 3 days in a sterile environment.

[0055] (5) Recovery culture: In a clean bench, transfer the co-cultured soybeans to recovery culture medium and recover culture for 4 days.

[0056] (6) Screening culture: After cutting off the roots of the soybeans in the recovery culture, they were inserted into the screening medium for screening culture.

[0057] (7) Elongation culture: Transfer the fresh surviving tissues after screening and culture to elongation culture medium for elongation culture until tissue culture seedlings grow.

[0058] (8) Rooting culture: The tissue culture seedlings were transferred to the prepared rooting culture medium for rooting culture until roots grew. After rooting, they were first transferred to vermiculite. After they grew well, they were transferred to a pot containing a mixture of vermiculite and peat moss (ratio of 1:3) to obtain transgenic T0 generation plants.

[0059] (9) Identification of positive seedlings: DNA was extracted from leaves of T0 generation plants for plant resistance gene detection. The primer sequences were Bar-F1: 5'-CCATCGTCAACCACTACATCGAGACA-3' and Bar-R1: 5'-CTTCAGCAGGTGGGTGTAGAGCGT-3'. Lines that detected the target band (product size 269bp) were cultured normally until maturity, and their seeds were harvested as positive T0 generation transgenic seeds. The positive T0 generation transgenic seeds were planted in flowerpots until the true leaves unfolded. Leaves were cut and tested positive using Bar test strips (Shanghai Youlong Biotechnology Co., Ltd., AA1032-LS). Positive plants were retained and seeded. The same method was used to continue identifying the next generation until the T3 generation transgenic seeds were harvested, thus obtaining the transgenic seedlings. GmATHB12 The T3 generation of homozygous lines OE1, OE2, and OE4 (also known as the three overexpression lines) are transgenic seeds. T3 generation transgenic seeds are the seeds of homozygous lines that no longer segregate, used for subsequent phenotypic testing.

[0060] 2. Turn GmATHB12 Salt stress phenotypes in soybeans 2.1 Transgenic plants GmATHB12 Detection of relative gene expression levels Select the transfer obtained in step 1 GmATHB12Homozygous T3 lines (OE1, OE2, and OE4) and control soybean William 82 plants (hereinafter referred to as W82) were cultured in a greenhouse under the same soil culture conditions as in Example 1. Twelve days after cultivation, RNA was extracted from the leaves of the transgenic soybeans and control plants, reverse transcribed into cDNA, and analyzed using Real-time RT-PCR. GmActin As an internal control gene, detection GmATHB12 Relative gene expression levels. Primer sequences are the same as in Example 1. Amplification... GmActin The primer is q GmActin -F:5′-CGGTGGTTTCTATCTTGGCATC-3′; q GmActin -R: 5′-GTCTTTCGCTTCAATAACCCTA-3′, amplification GmATHB12 The primer for the gene is q. GmATHB12 -F: 5′-CCAATGGAGCCAGGTCAGAG-3′; q GmATHB12 -R: 5′-GATATTTCCATTGATGGGCTTGG-3′.

[0061] Test results as follows Figure 3 As shown: Compared with control W82, the conversion... GmATHB12 In the T3 generation homozygous lines OE1, OE2, and OE4 GmATHB12 High gene expression levels indicate GmATHB12 The overexpression soybean line was successfully constructed.

[0062] 2.2 Detection of salt stress phenotypes and physiological assays in transgenic soybeans Wild-type control soybeans (W82) and genetically modified soybeans of uniform size were selected. GmATHB12 Seeds of homozygous T3 generation lines OE1, OE2, and OE4 were sown in 10 cm × 10 cm round plastic pots filled with a mixture of vermiculite and peat moss (1:3 ratio), with all pots containing the same soil weight. Each line was divided into 6 replicates, with 5 pots per replicate and 3 seeds per pot. Salt stress and normal watering treatments were established, and the procedures were identical for both treatments except for the liquid used in the first and second irrigations described below.

[0063] Salt stress treatment: After the plants were cultured at 26℃ with normal watering for 10 days, they were subjected to salt stress treatment for 10 days. During the salt stress treatment, they were irrigated twice with a 0.2 mol / L NaCl solution (composed of NaCl and a solvent, with water as the solvent). The first irrigation was recorded as day 0 of the salt treatment, and the second irrigation was 6 days after the first irrigation (i.e., day 6 of the salt treatment).

[0064] Normal watering treatment (CK): After culturing the plants at 26℃ with normal watering for 10 days, normal watering was continued for another 10 days. During the normal watering treatment, the above-mentioned solvent (water) was used for irrigation twice. The first irrigation was recorded as day 0 of the salt control treatment, and the second irrigation was 6 days after the first irrigation (recorded as day 6 of the salt control treatment). On day 10 of the salt stress treatment or normal watering treatment (i.e., day 4 after the second irrigation), the soybean phenotype was observed, the survival rate was recorded, and aboveground samples were taken to determine the fresh weight and chlorophyll content.

[0065] The results showed that under normal watering conditions, the growth of W82 and the three overexpression lines OE1, OE2, and OE4 was consistent. However, after 10 days of salt stress treatment, the leaves of W82 began to wilt, and the growth of the three overexpression lines was significantly better than that of W82. Figure 4 As shown). Statistical results showed that under normal conditions, there were no differences in fresh weight, leaf chlorophyll content, and survival rate among the four lines W82, OE1, OE2, and OE4; however, after salt stress treatment, the fresh weight of the three overexpression lines OE1, OE2, and OE4 (as shown). Figure 5 As shown in Figure A), chlorophyll content ( Figure 5 (as shown in B) and survival rate ( Figure 5 The values ​​shown in Figure C were significantly higher than those of the control W82. These results indicate that soybeans... GmATHB12 Overexpression of this gene can improve the salt tolerance of soybeans. Therefore, by regulating... GmATHB12 Protein expression can regulate the salt stress resistance of soybeans. This invention can be applied to the breeding and cultivation of salt-tolerant soybean varieties.

[0066] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Any of the following applications of proteins: P1. Application of the protein in regulating plant salt tolerance P2. Application of the protein in improving plant salt tolerance. P3. Application of the aforementioned protein in plant breeding P4. Application of the protein in plant quality improvement; The protein in question is the following: A1) The amino acid sequence is that of the protein in sequence 2 of the sequence listing; A2) Proteins derived from A1) or proteins with more than 80% identity and function to the amino acid sequence shown in Sequence 2 of the sequence listing, obtained by substitution and / or deletion and / or addition of amino acid residues. A3) is a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1) or A2).

2. The application according to claim 1, characterized in that: The protein is derived from soybeans.

3. The application according to claim 1 or 2, characterized in that: The plant is any one of the following: D1) Dicotyledons, D2) Leguminosae (family Fabaceae) D3) Leguminosae (family legumes) D4) Plants of the genus *Glycine*, D5) Soybeans.

4. Any of the following applications of biomaterials related to the protein described in claim 1 or 2: Q1. Application of the aforementioned biomaterials in regulating plant salt tolerance Q2. Application of the aforementioned biomaterials in improving plant salt tolerance Q3. Application of the aforementioned biomaterials in plant breeding. Q4. Application of the aforementioned biomaterials in plant quality improvement The biomaterial is any one of the following: B1) A nucleic acid molecule encoding the protein described in claim 1; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2); B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2); B7) Transgenic plant organs containing the nucleic acid molecules described in B1), or transgenic plant organs containing the expression cassette described in B2); B8) Nucleic acid molecules that promote or enhance gene expression of the protein described in claim 1; B9) Expression cassettes, recombinant vectors, recombinant microorganisms or transgenic plant cell lines containing the nucleic acid molecules described in B8).

5. The application according to claim 4, characterized in that: B1) The nucleic acid molecule is the gene encoding the protein shown below: b1) A DNA molecule whose coding sequence is the nucleotide of sequence 1 in the sequence listing; b2) Nucleotides are DNA molecules that are sequence 1 in the sequence listing. b3) A cDNA or DNA molecule that hybridizes with the cDNA or DNA molecule defined in b2) and encodes a protein with the same function.

6. The application according to claim 4 or 5, characterized in that: The plant is any one of the following: D1) Dicotyledons, D2) Leguminosae (family Fabaceae) D3) Leguminosae (family legumes) D4) Plants of the genus *Glycine*, D5) Soybeans.

7. A method for improving the salt tolerance of plants, comprising upregulating, enhancing or increasing the activity of the protein of claim 1 or / and the expression level of the gene encoding the protein of claim 1 in the target plant, thereby improving the salt tolerance of the target plant.

8. The method according to claim 7, characterized in that: The upregulation, enhancement, or increase of the activity of the protein of claim 1 or / and the expression level of the gene encoding the protein of claim 1 in the target plant is achieved by introducing the gene encoding the protein of claim 1 into the target plant.

9. The method according to claim 7 or 8, characterized in that: The plant and / or the target plant is any one of the following: D1) Dicotyledons, D2) Leguminosae (family Fabaceae) D3) Leguminosae (family legumes) D4) Plants of the genus *Glycine*, D5) Soybeans.

10. The protein of claim 1 and / or the biomaterial of claim 4 or 5.

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