Method for improving salt tolerance of soybeans

Abstract

The present invention relates to the technical field of plant genetic engineering, and specifically relates to a method for improving the salt tolerance of soybeans. Provided is a GmZF392 protein capable of significantly improving the salt tolerance of a plant. The GmZF392 protein can not only effectively improve the salt tolerance of the plant, but also significantly increase the oil and fatty acid content of plant seeds. The oil and fatty acid content in the seeds of a constructed transgenic line overexpressing GmZF392 is significantly increased. Moreover, the salt tolerance of the transgenic line is significantly higher than that of a control line.

Description

A method to improve the salt tolerance of soybeans Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to a method for improving the salt tolerance of soybeans. Background Technology

[0002] The process by which plants respond to abiotic stress is jointly regulated by multiple signaling pathways, and the receptor proteins, regulatory proteins, signaling molecules and effector proteins involved in these pathways have become a hot research topic in recent years.

[0003] Regarding the regulation of salt tolerance in plants, researchers proposed a Ca2+ signal transduction mechanism using Arabidopsis thaliana as a model. 2+ The salt-dependent SOS signaling pathway comprises three components: SOS1, SOS2, and SOS3. Under salt stress, cytoplasmic calcium... 2+ The level rose sharply in a short period of time, Ca 2+ The binding protein SOS3 binds to calcium in the cytoplasm. 2+ It also activates serine / threonine protein kinase SOS2, which is recruited to the plasma membrane and phosphorylates Na+. + / K + The serine site of the antitransporter SOS1 relieves the autoinhibitory activity of SOS1 and promotes Na+ transport. + On the one hand, SOS2 indirectly activates K through phosphorylation. + The transport protein AKT1 promotes K + Entering the cytoplasm to maintain cytoplasmic Na + / K + Balance. In recent years, this signaling pathway has been continuously enriched and improved, and has also been confirmed in other plants such as rice and maize. The phosphatidylinositol (GPI) signaling pathway is another important salt tolerance signaling pathway, including inositol triphosphate IP3 and phosphatidic acid PA, which mediate the transmission and response of stress signals (Guo et al., J of Genet Genomics, 2024, 51: 16-34).

[0004] Transcription factors, by binding to specific DNA fragments in the promoter regions of target genes, regulate the expression levels of those genes and are crucial components in plant responses to abiotic stress. Transcription factors can act as downstream components of signal transduction, directly regulating the expression levels of effector proteins, or as regulators of signal transduction, modulating the expression of key components. The AP2 / ERF transcription factor DREB has been reported to positively regulate stress tolerance. The applicant has previously identified several stress-related transcription factors in rice, wheat, cotton, and soybean, including OsVTE1, OsSIK1, TaWRKY1, TaWRKY19, GhGT23, GmNAC1, GmWRKY54, GmWRKY27, GmMYB73, and GmPHD6. Overexpression of these genes in rice, Arabidopsis thaliana, and soybean improved the stress tolerance of transgenic plants.

[0005] The discovery of salt tolerance-related genes is of great significance for the breeding of salt-tolerant materials. Typically, salt-tolerant materials are bred based on known salt tolerance-related genes through transgenic technology, marker-assisted selection, or superior allelic screening.

[0006] However, overexpression of some identified genes that positively regulate salt tolerance increases salt tolerance but decreases other agronomic traits. For example, overexpression of soybean microRNA1 72a increases the salt tolerance of transgenic plants, but reduces yield under normal conditions. Moreover, overexpression or gene editing of most identified salt tolerance-related genes only improves salt tolerance and fails to improve other agronomic traits; for instance, overexpression of GmMYB27 only increases salt tolerance. Summary of the Invention

[0007] One objective of this invention is to provide the application of GmZF392 in regulating plant salt tolerance and a method for improving plant salt tolerance. Another objective of this invention is to provide the application of GmZF392 in regulating plant salt tolerance as well as oil and / or fatty acid content.

[0008] Previous studies identified the key gene GmZF392, which regulates seed lipid accumulation, in transcriptome analysis of high-oil / low-oil soybean materials. The expression of this gene gradually increases during seed development, synchronized with lipid accumulation. Transgenic experiments showed that overexpression of this gene in Arabidopsis and soybean increased fatty acid content in transgenic seeds (Lu et al., New Phytologist, 2021, 231: 661-678); moreover, previous studies also indicated that GmZF392 is a seed-specific transcriptional activator. Further research showed that GmZF392 gene expression is very low in roots under normal conditions. However, this invention unexpectedly found that GmZF392 expression increases under salt stress, meaning that GmZF392 expression is induced by salt stress. Salt tolerance analysis of transgenic soybeans overexpressing GmZF392 showed that their salt tolerance was significantly higher than that of the recipient control. Therefore, GmZF392 not only regulates the lipid and fatty acid content in seeds but also simultaneously regulates the salt tolerance of plants. GmZF392, as one of the very few reported transcription factors that can simultaneously improve plant salt tolerance and agronomic traits (seed oil content), has important application value for the breeding of plants such as soybeans.

[0009] Specifically, the present invention provides the following technical solutions.

[0010] In a first aspect, the present invention provides any one of the following applications of the GmZF392 protein, a nucleic acid molecule encoding the GmZF392 protein, or a biological material containing said nucleic acid molecule, or a substance regulating the expression level or activity of said GmZF392 protein:

[0011] (1) Regulating plant salt tolerance;

[0012] (2) Prepare products for regulating plant salt tolerance;

[0013] (3) Prepare plants with enhanced salt tolerance;

[0014] (4) Plant breeding, the goal of which is to cultivate plants with enhanced salt tolerance.

[0015] The functional verification experiment of GmZF392 showed that the transgenic lines overexpressing GmZF392 exhibited significantly improved growth under salt stress conditions compared to the control lines, and the pod-setting rate was also significantly higher.

[0016] In a second aspect, the present invention provides any one of the following applications of the GmZF392 protein, a nucleic acid molecule encoding the GmZF392 protein, or a biological material containing said nucleic acid molecule, or a substance regulating the expression level or activity of said GmZF392 protein:

[0017] (1) Regulate plant salt tolerance as well as oil and / or fatty acid content;

[0018] (2) Prepare products for regulating plant salt tolerance as well as oil and / or fatty acid content;

[0019] (3) Prepare plants with enhanced salt tolerance and increased oil and / or fatty acid content;

[0020] (4) Plant breeding, the goal of which is to cultivate plants with enhanced salt tolerance and increased oil and / or fatty acid content.

[0021] This invention verifies through experiments that transgenic lines overexpressing GmZF392 not only have significantly improved salt tolerance, but also significantly increased oil and fatty acid content in seeds and leaves.

[0022] In this invention, the GmZF392 protein positively regulates salt tolerance and the content of lipids and fatty acids.

[0023] The applications of the first and second aspects mentioned above include: causing the plant to express the GmZF392 protein, or enhancing the expression level and / or activity of the GmZF392 protein in the plant.

[0024] In this context, enabling the plant to express the GmZF392 protein is generally applicable when the plant itself does not express the GmZF392 protein; for example, for plants other than soybeans, the gene encoding the GmZF392 protein is introduced into the plant to enable the plant to express the GmZF392 protein.

[0025] Enhancing the expression level and / or activity of GmZF392 protein in the plant involves modifying the GmZF392 protein, its encoding gene, or regulatory elements to modify its transcription, translation, protein structure, etc., thereby enhancing the expression level of GmZF392 protein and / or its activity as a transcriptional regulatory factor (including its ability to bind to target DNA sequences).

[0026] In this invention, there are no special limitations on the technical means to achieve increased expression levels and / or activity. For example, commonly used genetic engineering techniques and gene editing methods can be used to modify the protein, gene, or its regulatory elements to increase the protein's expression levels and / or activity.

[0027] For example, the enhancement of the expression level and / or activity of the GmZF392 protein can be achieved by any one or more of the following methods (1) to (4):

[0028] (1) Increase the copy number of the gene encoding the GmZF392 protein;

[0029] (2) Replace the transcriptional and / or translational regulatory elements of the gene encoding the GmZF392 protein with more active elements;

[0030] (3) Mutate the nucleotide sequence of the gene encoding the GmZF392 protein;

[0031] (4) Mutate the amino acid sequence of the GmZF392 protein.

[0032] The above-mentioned increase in gene copy number can be achieved by increasing the copy number of the gene on the chromosome, or by introducing a foreign plasmid containing the gene.

[0033] The transcriptional and translational regulatory elements mentioned above include promoters, enhancers, untranslated sequences, terminators, etc.

[0034] The mutations in the nucleotide sequences described above include deletions, insertions, or substitutions of one or more nucleotides.

[0035] The above-mentioned mutations in the amino acid sequence include deletions, insertions, or substitutions of one or more amino acids.

[0036] In this invention, the plant is an angiosperm; preferably a dicotyledonous plant; more preferably a legume; and even more preferably soybean.

[0037] In this invention, the GmZF392 protein has any of the following amino acid sequences:

[0038] (1) The amino acid sequence as shown in SEQ ID NO.1;

[0039] (2) An amino acid sequence of a protein with the same function obtained by substituting, inserting or deleting one or more amino acids of the amino acid sequence shown in SEQ ID NO.1.

[0040] (3) An amino acid sequence having at least 80% homology with the amino acid sequence shown in SEQ ID NO.1; the homology is preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0041] In this invention, the nucleic acid molecule encoding the GmZF392 protein has any of the following nucleotide sequences:

[0042] (1) The nucleotide sequence shown in SEQ ID NO.2;

[0043] (2) A nucleotide sequence that is complementary to or homologous to the sequence shown in SEQ ID NO.2 and encodes a protein with the same function;

[0044] (3) A nucleotide sequence encoding the same function protein obtained by substituting, inserting or deleting one or more nucleotides of the nucleotide sequence shown in SEQ ID NO.2.

[0045] The nucleic acid molecule can be DNA or RNA.

[0046] In this invention, the biomaterial includes any one of the following:

[0047] (1) An expression cassette containing a nucleic acid molecule encoding the GmZF392 protein;

[0048] (2) A vector containing a nucleic acid molecule encoding the GmZF392 protein or the expression cassette described in (1);

[0049] (3) Microorganisms containing a nucleic acid molecule encoding the GmZF392 protein or the expression cassette described in (1) or the vector described in (2);

[0050] (4) A plant cell line containing a nucleic acid molecule encoding the GmZF392 protein or the expression cassette described in (1) or the vector described in (2);

[0051] (5) A plant tissue, plant organ or plant containing a nucleic acid molecule encoding the GmZF392 protein or the expression cassette described in (1) or the vector described in (2).

[0052] The expression cassette is a recombinant nucleic acid molecule for expressing the GmZF392 protein, obtained by linking the nucleic acid molecule to a promoter. The promoter may be a constitutive promoter, a tissue-, organ-, or developmentally specific promoter, or an inducible promoter, etc.

[0053] The vectors include plasmids, viruses, artificial chromosomes, transposons, etc.

[0054] The microorganisms mentioned include, but are not limited to, Escherichia coli and Agrobacterium.

[0055] The plant is an angiosperm; preferably a dicotyledonous plant; more preferably a legume; and even more preferably soybean.

[0056] In this invention, the substances that regulate the expression level or activity of the GmZF392 protein include any substances that can enhance the expression level or activity of the GmZF392 protein. The substances can be proteins, DNA or RNA, including but not limited to enhancers, transcription factors, activating proteins, etc.

[0057] In this invention, the fatty acid is selected from at least one of linolenic acid, linoleic acid, oleic acid, palmitic acid and stearic acid.

[0058] The oil and / or fatty acid content refers to the oil and / or fatty acid content of plant seeds or leaves.

[0059] The oil content is preferably the total oil or total fatty acid content of the seeds or leaves.

[0060] Thirdly, the present invention provides a method for enhancing the salt tolerance of plants, the method comprising: causing the plant to express the GmZF392 protein, or enhancing the expression level and / or activity of the GmZF392 protein in the plant.

[0061] Fourthly, the present invention provides a method for cultivating salt-tolerant plants, the method comprising: causing the plant to express the GmZF392 protein, or enhancing the expression level and / or activity of the GmZF392 protein in the plant.

[0062] Fifthly, the present invention provides a method for improving the salt tolerance of plants and the content of oils and / or fatty acids, the method comprising: causing the plant to express the GmZF392 protein, or enhancing the expression level and / or activity of the GmZF392 protein in the plant.

[0063] In a sixth aspect, the present invention provides a method for cultivating plants with enhanced salt tolerance and increased oil and / or fatty acid content, the method comprising: causing the plant to express the GmZF392 protein, or enhancing the expression level and / or activity of the GmZF392 protein in the plant.

[0064] The alternative methods for enabling plants to express the GmZF392 protein and enhancing the expression level and / or activity of the GmZF392 protein in the plants described in the third to sixth aspects above may refer to the applications described in the first to second aspects above.

[0065] As one embodiment of the present invention, the method includes: introducing a nucleic acid molecule encoding the GmZF392 protein into the plant.

[0066] Specifically, the method includes: introducing the gene encoding the GmZF392 protein into the plant. Preferably, the gene encoding the GmZF392 protein is placed in a plant expression vector to obtain a recombinant vector, and the recombinant vector is introduced into the plant. The plant expression vector includes, but is not limited to, pGWB412, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, and pCAMBIA1300.

[0067] Methods for introducing genes or vectors into plants can be achieved using conventional methods in the field, including but not limited to Agrobacterium-mediated transformation, microinjection, and electroporation.

[0068] In the above method, the plant is an angiosperm; preferably a dicotyledonous plant; more preferably a legume; and even more preferably soybean.

[0069] In the above method, the GmZF392 protein has any of the following amino acid sequences:

[0070] (1) The amino acid sequence as shown in SEQ ID NO.1;

[0071] (2) An amino acid sequence of a protein with the same function obtained by substituting, inserting or deleting one or more amino acids of the amino acid sequence shown in SEQ ID NO.1.

[0072] (3) An amino acid sequence having at least 80% homology with the amino acid sequence shown in SEQ ID NO.1; the homology is preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0073] In the above method, the fatty acid is selected from at least one of linolenic acid, linoleic acid, oleic acid, palmitic acid and stearic acid.

[0074] The oil and / or fatty acid content refers to the oil and / or fatty acid content of plant seeds or leaves.

[0075] The preferred oil content is the total oil or total fatty acid content of the seeds or leaves.

[0076] In a seventh aspect, the present invention provides a transgenic plant in which the expression level and / or activity of the GmZF392 protein is enhanced; the salt tolerance of the transgenic plant is enhanced due to the enhanced expression level and / or activity of the GmZF392 protein, or the salt tolerance and oil and / or fatty acid content of the transgenic plant are increased due to the enhanced expression level and / or activity of the GmZF392 protein.

[0077] Preferably, the transgenic plant is an angiosperm; more preferably, a dicotyledonous plant; more preferably, a legume; and even more preferably, a transgenic soybean.

[0078] In one embodiment of the invention, a transgenic soybean overexpressing GmZF392 is provided.

[0079] Eighthly, the present invention provides a plant seed derived from the genetically modified plant described above.

[0080] In a ninth aspect, the present invention provides a transgenic plant with enhanced salt tolerance, wherein the expression level and / or activity of the GmZF392 protein in the transgenic plant is enhanced; the salt tolerance of the transgenic plant is enhanced due to the enhanced expression level and / or activity of the GmZF392 protein.

[0081] Preferably, the transgenic plant is an angiosperm; more preferably, a dicotyledonous plant; more preferably, a legume; and even more preferably, a transgenic soybean.

[0082] In a tenth aspect, the present invention provides a transgenic plant with enhanced salt tolerance and increased oil and / or fatty acid content, wherein the expression level and / or activity of GmZF392 protein in the transgenic plant is enhanced; the salt tolerance and oil and / or fatty acid content of the transgenic plant are increased due to the enhanced expression level and / or activity of GmZF392 protein.

[0083] Preferably, the transgenic plant is an angiosperm; more preferably, a dicotyledonous plant; more preferably, a legume; and even more preferably, a transgenic soybean.

[0084] The beneficial effects of this invention include at least the following: This invention provides the GmZF392 protein, which can significantly improve plant salt tolerance. Transgenic lines overexpressing GmZF392 show significantly improved growth under salt stress conditions compared to control lines, with a significantly higher pod-setting rate. Furthermore, the GmZF392 protein not only effectively improves plant salt tolerance but also significantly increases the oil and fatty acid content of plant seeds. Agronomic trait testing results of the GmZF392-overexpressing homozygous transgenic soybean lines constructed according to this invention show that yield traits, including 100-seed weight, number of pods per plant, and yield per plant, are not significantly different from the control, but the oil and fatty acid content in the seeds are significantly higher than the control; moreover, the salt tolerance of the transgenic lines is significantly higher than the control. The discovery of these novel functions of the GmZF392 protein provides an effective gene resource for breeding plants with improved salt tolerance and oil content, and has important application value for plant breeding. Attached Figure Description

[0085] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0086] Figure 1 shows the molecular detection of transgenic plants overexpressing GmZF392 and recipient controls in Example 3 of the present invention, where JACK is the recipient control, Null is the empty vector control, and OE is the transgenic plant overexpressing GmZF392.

[0087] Figure 2 shows the growth and yield traits of the control and overexpressing GmZF392 transgenic plants in Example 3 of the present invention, where JACK is the recipient control, Null is the empty vector control, and OE is the overexpressing GmZF392 transgenic plant.

[0088] Figure 3 shows the growth of the control and transgenic plants in soil containing 0.5% NaCl in Example 3 of the present invention (2023-7-outdoor), where JACK is the recipient control and OE is the transgenic plant overexpressing GmZF392.

[0089] Figure 4 shows the growth of the control and transgenic plants in soil containing 0.5% NaCl in Example 3 of the present invention (2023-9-outdoor), where JACK is the recipient control and OE is the transgenic plant overexpressing GmZF392.

[0090] Figure 5 shows the relative pod setting rate of the control and transgenic plants in Example 3 of this invention growing in saline / normal soil. The soil contained 0.5% NaCl (2023-11-outdoor). JACK is the recipient control, and OE is the transgenic plant overexpressing GmZF392.

[0091] Figure 6 shows the detection of oil and fatty acid content in seeds and leaves of control and transgenic lines in Example 3 of this invention (Long Lu, et al., New Phytologist (2021), 231: 661-678), where JACK is the recipient control, Null is the control of empty vector transgenic plants, and OE is the transgenic plant overexpressing GmZF392; in each comparison unit of the upper right and lower right figures, the bars are arranged from left to right as JACK, Null, OE-12, OE-17, and OE-26. Detailed Implementation

[0092] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0093] Example 1: Obtaining the target gene GmZF392 and constructing the overexpression vector

[0094] Total RNA was extracted from Heinong 44 (HN44) seedlings, and the RNA was reverse transcribed using reverse transcriptase to synthesize cDNA.

[0095] Based on the full-length cDNA sequence information of GmZF392 in the soybean genome sequence from PlantGDB, primers were designed, and the primer sequences are as follows:

[0096] GmZF392-up: 5'-ATGAGCAGTGTCTGTGCCA-3' (SEQ ID NO. 3);

[0097] GmZF392-dp: 5'-CATCAGCAACTCGTTCACC-3' (SEQ ID NO. 4).

[0098] Using HN44 cDNA as a template, PCR amplification was performed using GmZF392-up and GmZF392-dp primers, yielding a PCR product of approximately 1 kb. Sequencing revealed that the PCR product was 1179 bp in length and possessed the nucleotide sequence shown in SEQ ID NO.2. The gene represented by this nucleotide sequence is GmZF392, and the protein encoded by this gene is named GmZF392. The amino acid sequence of this protein is shown in SEQ ID NO.1. Gene cloning was performed using the Gateway system provided by Invitrogen. The 3′-T overhang of the vector was used to directly ligate the PCR product amplified by Taq polymerase. The gene was ligated into the cloning vector using the principle of Taq cloning. 8 / GW / TOPO. Both the TOPO vector and the overexpression vector pGWB412 contain recombination sites attL1 and attL2. The TOPO vector, which connects the target gene, and the overexpression vector pGWB412 undergo LR recombination under the action of recombinase. Finally, the target gene GmZF392 was successfully constructed into the overexpression vector pGWB412 and named pGWB412-GmZF392.

[0099] Example 2: Obtaining GmZF392 soybeans

[0100] 1. Obtaining recombinant Agrobacterium

[0101] The recombinant vector pGWB412-GmZF392 containing GmZF392 obtained in Example 1 was introduced into Agrobacterium GV3101 by electroporation. The recombinant Agrobacterium was picked and named GV3101 / GmZF392.

[0102] 2. Obtaining plants overexpressing GmZF392

[0103] Recombinant Agrobacterium GV3101 / GmZF392 was cultured to the logarithmic growth phase, and then transformed into the soybean recipient Glycine max (L.) Merr.cv Jack using the cotyledonary node transformation method. Seeds were harvested after cultivation. The seeds were sown in vermiculite for growth. Soybean leaves were treated with 0.1% Roundup (glyphosate); plants showing no yellowing reaction after 3 days were considered transgenic positive plants.

[0104] Molecular detection was performed on the above-mentioned positive plants. RNA was extracted from transgenic plant seedlings, and cDNA was obtained by reverse transcription. The primers used were 5'-TCTCGCCACCAGTATCACCAT-3' (SEQ ID NO.5) and 5'-CTTTAGCACCAGAAACAGGAGAAC-3' (SEQ ID NO.6) for Real-Time PCR identification. The soybean GmTubulin gene was used as an internal control, and the primers used were Primer-TF and Primer-TR. Specifically, Primer-TF was 5'-AACTCCATTTCGTCCATTCCTTC-3' (SEQ ID NO.7), and Primer-TR was 5'-TTGAGTGGATTCCCAACAACG-3' (SEQ ID NO.8). The recipient Jack and empty vector transgenic lines were used as controls. The experiment was repeated three times, and the results were taken as mean ± standard deviation. Three lines with different GmZF392 expression levels, OE-12, OE-17, and OE-26, were selected for further phenotypic analysis. The above three lines were propagated to the T3 generation. Each single plant in each generation was treated with 0.1% Roundup (glyphosate) on soybean leaves. Plants that did not show yellowing after 3 days were tested to determine if they were transgenic positive plants. Negative plants were eliminated to obtain pure transgenic lines that did not segregate in their offspring.

[0105] Example 3: Detection of yield and salt tolerance traits in transgenic soybeans

[0106] 1. Molecular detection of plants overexpressing GmZF392

[0107] Figure 1 shows the relative expression levels of GmZF392 in control and transgenic soybeans, indicating that the target gene GmZF392 has been expressed in transgenic soybeans.

[0108] 2. Detection of growth and yield traits in GmZF392 overexpressing plants and receptor controls

[0109] The growth of the greenhouse potted control and the transgenic lines showed no significant changes in the transgenic plant type (Figure 2A). The control and transgenic lines were planted at Pingxifu Farm in Changping District, Beijing, and the seed size, number of pods per plant, and seed weight per plant were compared after harvest. Phenotypic (Figure 2B, C) and statistical (Figure 2D) results showed that overexpression of GmZF392 did not significantly affect the 100-seed weight, number of pods per plant, or seed weight per plant (Long Lu, et al., New Phytologist (2021), 231: 661-678).

[0110] 3. Salt tolerance test of GmZF392 overexpressing plants and recipient controls

[0111] Salt tolerance was tested outdoors. Pure lines of OE-12, OE-17, and OE-26, along with control seeds, were sown in pots. The control group was watered, while the experimental group had its soil salinity (excluding the original soil salinity) adjusted to 0.5% with NaCl. Specifically, the soil was dried thoroughly and weighed (35 kg). 0.175 kg of NaCl was dissolved in 35 kg of water (0.5%), and the solution was poured into the soil, allowing it to permeate and mix evenly. After weighing the pots with soil, control and transgenic line seeds were directly sown in the same pot, with four plants per pot for each control and transgenic line, four replicates. The moisture content was maintained at 70-75% during growth. Greenhouse conditions: 16h:8h light duration (daytime:nighttime), temperature: 30-37℃ during the day and 25-28℃ at night. The growth status was observed at each stage.

[0112] The results showed that the control and GmZF392 overexpression lines grew similarly in normal soil (Figure 3), while the three GmZF392 overexpression lines in soil containing 0.5% NaCl showed significantly better growth in both the early and late stages than the control plants (Figures 3 and 4). Furthermore, the pod-setting rate of both the control and GmZF392 overexpression lines decreased significantly in soil containing 0.5% NaCl. The pod-setting rate of the control Jack in saline soil was approximately 20% of that under normal conditions, while OE-12, OE-17, and OE-26 were approximately 81%, 68%, and 88% of that under normal conditions, respectively, showing a highly significant increase compared to the pod-setting rate under salt stress (Figure 5). These results indicate that the transgenic lines suffered significantly less damage from salt stress than the control, suggesting that the transgenic lines had significantly higher salt tolerance than the control.

[0113] 4. Detection of oil and fatty acid content in seeds and leaves of control and transgenic lines

[0114] After harvesting control and transgenic plants in the field, the fatty acid content in seeds and leaves was measured.

[0115] After the seeds / leaves are dried, the fatty acid content is measured. Thoroughly dry the seeds / leaves to be tested, grind them into powder, and add 10 mg to a 2 mL screw-cap centrifuge tube. Weigh four aliquots of each sample. Add 10 μL of 17:0 fatty acid (Sigma, 51610) at 10 mg / mL as an internal standard. Add 1 mL of methanol solution containing 2.5% concentrated sulfuric acid, and incubate in an 85°C water bath for 1 hour, shaking several times during incubation. After natural cooling, transfer 500 μL of the supernatant to a new tube, add 600 μL of 0.9% NaCl solution and 300 μL of n-hexane, vortex for a few minutes, centrifuge at 4000 rpm for 10 minutes, and transfer the supernatant to a new tube. Incubate overnight in a fume hood to allow the n-hexane to evaporate completely, then add 50 μL of ethyl acetate to dissolve the methylated fatty acids. The relative contents of each component in the methylated fatty acid sample were determined by gas chromatography-mass spectrometry (Perkin-Elmer Turbomass). The relative contents of each component were then compared with an added 17:0 internal standard to obtain the relative content (Shen, B., et al., The homeobox gene GLABRA2 affects seed oil content in Arabidopsis, Plant Mol. Biol., 60:377-387, 2006.). The experiment was repeated three times, and the results were taken as mean ± standard deviation.

[0116] The results of fatty acid content detection are shown in Figure 6. The oil content in the seeds of 5 out of the 7 transgenic lines was significantly or extremely significantly higher than that in the control, and the oil content in the leaves of the transgenic lines was significantly higher than that in the control or the average value was higher than that in the control.

[0117] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention. Industrial applicability

[0118] The GmZF392 protein provided by this invention not only effectively improves the salt tolerance of plants but also significantly increases the oil and fatty acid content of plant seeds. Transgenic lines overexpressing GmZF392 showed significantly improved growth under salt stress conditions compared to the control lines, with a substantial increase in pod setting rate. As one of the few transcription factors capable of simultaneously improving both salt tolerance and agronomic traits in plants, GmZF392 has significant application value in plant breeding.

Claims

1. Any one of the following applications of the GmZF392 protein, the nucleic acid molecule encoding the GmZF392 protein, or biological material containing said nucleic acid molecule, or a substance regulating the expression level or activity of the GmZF392 protein: (1) Regulating plant salt tolerance; (2) Prepare products for regulating plant salt tolerance; (3) Prepare plants with enhanced salt tolerance; (4) Plant breeding, the goal of which is to cultivate plants with enhanced salt tolerance.

2. Any one of the following applications of the GmZF392 protein, the nucleic acid molecule encoding the GmZF392 protein, or biological material containing said nucleic acid molecule, or a substance regulating the expression level or activity of the GmZF392 protein: (1) Regulate plant salt tolerance as well as oil and / or fatty acid content; (2) Prepare products for regulating plant salt tolerance as well as oil and / or fatty acid content; (3) Prepare plants with enhanced salt tolerance and increased oil and / or fatty acid content; (4) Plant breeding, the goal of which is to cultivate plants with enhanced salt tolerance and increased oil and / or fatty acid content.

3. The application according to claim 1 or 2, characterized in that, The applications include: causing the plant to express the GmZF392 protein, or enhancing the expression level and / or activity of the GmZF392 protein in the plant.

4. The application according to claim 1 or 2, characterized in that, The plant is an angiosperm; preferably a dicotyledonous plant; more preferably a legume; and even more preferably soybean.

5. The application according to claim 1 or 2, characterized in that, The GmZF392 protein has any of the following amino acid sequences: (1) The amino acid sequence as shown in SEQ ID NO.1; (2) An amino acid sequence of a protein with the same function obtained by substituting, inserting or deleting one or more amino acids of the amino acid sequence shown in SEQ ID NO.

1. (3) An amino acid sequence having at least 80% homology with the amino acid sequence shown in SEQ ID NO.1; the homology is preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

6. The application according to claim 1 or 2, characterized in that, The biomaterial includes any one of the following: (1) An expression cassette containing a nucleic acid molecule encoding the GmZF392 protein; (2) A vector containing a nucleic acid molecule encoding the GmZF392 protein or the expression cassette described in (1); (3) Microorganisms containing a nucleic acid molecule encoding the GmZF392 protein or the expression cassette described in (1) or the vector described in (2); (4) A plant cell line containing a nucleic acid molecule encoding the GmZF392 protein or the expression cassette described in (1) or the vector described in (2); (5) A plant tissue, plant organ or plant containing a nucleic acid molecule encoding the GmZF392 protein or the expression cassette described in (1) or the vector described in (2).

7. The application according to claim 2, characterized in that, The fatty acid is selected from at least one of linolenic acid, linoleic acid, oleic acid, palmitic acid, and stearic acid.

8. The application according to claim 2, characterized in that, The oil and / or fatty acid content refers to the oil and / or fatty acid content of plant seeds or leaves.

9. A method for enhancing the salt tolerance of plants, characterized in that, The method includes: causing the plant to express the GmZF392 protein, or enhancing the expression level and / or activity of the GmZF392 protein in the plant.

10. A method for cultivating salt-tolerant plants, characterized in that, The method includes: causing the plant to express the GmZF392 protein, or enhancing the expression level and / or activity of the GmZF392 protein in the plant.

11. A method for improving the salt tolerance and oil and / or fatty acid content of plants, characterized in that, The method includes: causing the plant to express the GmZF392 protein, or enhancing the expression level and / or activity of the GmZF392 protein in the plant.

12. A method for cultivating plants with enhanced salt tolerance and increased oil and / or fatty acid content, characterized in that, The method includes: causing the plant to express the GmZF392 protein, or enhancing the expression level and / or activity of the GmZF392 protein in the plant.

13. The method according to any one of claims 9 to 12, characterized in that, The method includes introducing a nucleic acid molecule encoding the GmZF392 protein into the plant.

14. The method according to any one of claims 9 to 12, characterized in that, The plant is an angiosperm; preferably a dicotyledonous plant; more preferably a legume; and even more preferably soybean.

15. The method according to any one of claims 9 to 12, characterized in that, The GmZF392 protein has any of the following amino acid sequences: (1) The amino acid sequence as shown in SEQ ID NO.1; (2) An amino acid sequence of a protein with the same function obtained by substituting, inserting or deleting one or more amino acids of the amino acid sequence shown in SEQ ID NO.

1. (3) An amino acid sequence having at least 80% homology with the amino acid sequence shown in SEQ ID NO.1; the homology is preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

16. The method according to claim 11 or 12, characterized in that, The fatty acid is selected from at least one of linolenic acid, linoleic acid, oleic acid, palmitic acid, and stearic acid.

17. The method according to claim 11 or 12, characterized in that, The oil and / or fatty acid content refers to the oil and / or fatty acid content of plant seeds or leaves.

18. A transgenic plant with enhanced salt tolerance, characterized in that, The expression level and / or activity of the GmZF392 protein in the transgenic plant are enhanced; the salt tolerance of the transgenic plant is enhanced due to the enhanced expression level and / or activity of the GmZF392 protein.

19. A transgenic plant with enhanced salt tolerance and increased oil and / or fatty acid content, characterized in that, The expression level and / or activity of GmZF392 protein in the transgenic plant are enhanced; the salt tolerance and oil and / or fatty acid content of the transgenic plant are increased due to the enhanced expression level and / or activity of GmZF392 protein.

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