Application of soybean citrate transporter in enhancing plant tolerance to iron deficiency stress

Abstract

This invention relates to the field of bioengineering technology and provides an application of soybean citrate transporter protein in enhancing plant tolerance to iron deficiency stress. Specifically, by mining and applying the gene encoding the soybean citrate transporter protein (as shown in SEQ ID NO:1), this invention solves the problem of limited growth and yield of soybeans in iron-deficient environments. Overexpression of this gene in soybeans significantly enhances their tolerance to iron deficiency stress. Stable genetic lines of soybeans overexpressing the gene for the soybean citrate transporter protein exhibit higher iron absorption and transport capacity under iron-deficient conditions by increasing the rate of citric acid efflux and the content of citric acid and iron in xylem sap, thereby significantly improving soybean tolerance to low-iron environments. Furthermore, it has been found to improve soybean yield and quality, providing strong technical support for expanding the planting area of ​​soybeans in iron-deficient soil areas.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to the application of a soybean citric acid transporter in enhancing the tolerance of plants to iron deficiency stress. Background Technology

[0002] Iron (Fe) is an essential micronutrient for almost all living organisms. In plants, it is necessary for various biological processes, including photosynthesis, respiration, and chlorophyll biosynthesis. Although iron may be abundant in the Earth's crust, its limited solubility in neutral and alkaline soils makes it poorly available to plants, leading to iron deficiency chlorosis (IDC) and yield loss in many crops, and in severe cases, death. This affects the yield and quality of soybeans, causing economic losses to agricultural production. Therefore, iron deficiency stress is one of the key factors limiting crop yield in agricultural production. Statistics show that about one-third of the world's arable land suffers from varying degrees of iron deficiency, especially in northern calcareous and saline-alkali soil regions. High soil pH causes iron to exist in a form that is difficult for plants to absorb, leading to iron deficiency chlorosis and severely restricting crop yield and quality. Therefore, improving the tolerance of plants to iron deficiency stress has become an important issue for sustainable agricultural development.

[0003] Soybean (Glycine max L.), belonging to the genus Glycine in the family Fabaceae, is one of the world's major economic crops and an important source of protein in livestock feed and human diets. As a crucial dual-purpose crop (grain, oil, and feed), soybean is an indispensable strategic resource in the global agricultural economy. In recent years, with the improvement of people's living standards, the demand for soybeans has been continuously increasing. Therefore, expanding the soybean planting area and improving its yield and quality are urgent tasks. With the deepening of genomic research on soybeans and other crops, plants have developed a series of complex mechanisms to cope with iron deficiency stress during long-term evolution. Among them, the reduction mechanism is a major adaptive strategy for dicotyledonous and non-grass monocotyledonous plants, involving root proton secretion, upregulation of iron reductase activity, and activation of iron transport proteins.

[0004] Recent studies have shown that specific gene families play a core regulatory role in this process. For example, the MATE (Multi-drug and Toxic Compound Extrusion) family of genes encodes transport proteins that can activate and absorb iron from the soil by secreting chelates such as citric acid. Although studies have reported the involvement of multiple MATE family members in plant responses to stresses such as aluminum toxicity and salinity, the molecular mechanisms by which specific MATE genes, especially in important economic crops such as soybeans, directly regulate iron deficiency stress tolerance remain unclear. This limits the ability to use key genes for molecular breeding. Given the current low soybean production capacity, low self-sufficiency rate, and heavy reliance on imports, increasing soybean yield and quality, and expanding planting area are issues of great concern. Traditional iron fertilizer application methods suffer from low utilization rates and high environmental risks; while they can temporarily alleviate iron deficiency symptoms, they are not a long-term solution. While molecular breeding technology provides a direction for solving iron deficiency stress, there are still shortcomings in identifying genes that can simultaneously improve soybean alkali tolerance, iron deficiency tolerance, and iron content. Summary of the Invention

[0005] The purpose of this invention is to provide an application of soybean citric acid transporter in enhancing plant tolerance to iron deficiency stress, thereby addressing the problems raised in the background art.

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

[0007] Application of soybean citrate transporter in enhancing plant tolerance to iron deficiency stress, wherein the nucleotide sequence of the gene encoding the soybean citrate transporter is shown in SEQ ID NO:1; and the plant is soybean.

[0008] Furthermore, the promoter nucleotide sequence upstream of the gene encoding the soybean citrate transporter is shown in SEQ ID NO:2.

[0009] Furthermore, the gene encoding the soybean citric acid transporter is used to enhance the soybean's ability to grow in iron-deficient soil; the iron-deficient soil is calcareous soil or soda saline-alkali soil.

[0010] Another object of the present invention is to provide a recombinant vector for enhancing the tolerance of plants to iron deficiency stress, which contains a gene encoding a soybean citrate transporter; the nucleotide sequence of the gene encoding the soybean citrate transporter is shown in SEQ ID NO:1; the plant is soybean; the recombinant vector is an overexpression vector of pro35S::GmMATE75; wherein the primer pair used to construct the overexpression vector of pro35S::GmMATE75 includes 101-MATE75-F and 101-MATE75-R, the nucleotide sequences of which are shown in SEQ ID NO:4 and SEQ ID NO:5, respectively.

[0011] Another object of the present invention is to provide a recombinant bacterium containing the above-mentioned recombinant vector; the recombinant bacterium is Escherichia coli or Agrobacterium for plant genetic transformation.

[0012] Another object of the present invention is to provide the application of the above-mentioned recombinant bacteria in enhancing the tolerance of plants to iron deficiency stress or exhibiting the iron deficiency response of plants, wherein the plant is soybean.

[0013] Furthermore, a method for enhancing the tolerance of plants to iron deficiency stress includes the following steps: transforming the gene encoding the soybean citrate transporter into plant cells via a recombinant vector, thereby overexpressing the gene encoding the soybean citrate transporter in the plant; and cultivating the transformed cells to obtain complete transgenic plants.

[0014] Another object of the present invention is to provide a soybean comprising a gene encoding an exogenously introduced soybean citrate transporter; the nucleotide sequence of the gene encoding the soybean citrate transporter is shown in SEQ ID NO:1.

[0015] This invention addresses the problem of limited soybean growth and yield in iron-deficient environments by identifying and applying the gene encoding the soybean citrate transporter, whose nucleotide sequence is shown in SEQ ID NO:1. Overexpression of this gene in soybean significantly enhances its tolerance to iron deficiency stress. This invention provides a key gene resource for molecular breeding: stable soybean lines overexpressing the gene encoding the soybean citrate transporter exhibit higher iron absorption and transport capacity under low iron conditions by increasing the efflux rate of citric acid and the citric acid and iron content in xylem sap, thereby significantly improving soybean tolerance to low iron environments. Furthermore, it has been found that this can improve soybean yield and quality, providing strong technical support for expanding the planting area of ​​soybeans in iron-deficient soil areas. Attached Figure Description

[0016] Figure 1The images show agarose gel electrophoresis diagram and gene structure diagram of the GmMATE75 gene after PCR amplification; where A is the agarose gel electrophoresis diagram of the GmMATE75 gene after PCR amplification; and B is the structure diagram of the GmMATE75 gene.

[0017] Figure 2 The diagram shows the vector construction of the GmMATE75 gene and its promoter; where A is the diagram of the construction of the PCAMBIA2301-pGmMATE75-GUS expression vector; and B is the diagram of the construction of the GmMATE75 gene in the soybean overexpression vector PTF101-35S-GmMATE75.

[0018] Figure 3 Analysis of the expression pattern of the GmMATE75 gene in root tissues under iron deficiency stress; where A represents low iron supply and B represents normal iron supply.

[0019] Figure 4 This is a comparison of GmMATE75 expression levels between wild-type soybean (WT) and overexpressing homozygous soybean lines (OE-GmMATE75) #5 and #19 after 7 days of iron-deficient treatment.

[0020] Figure 5 A comparison of glufosinate treatment phenotypes in wild-type soybean (WT) and overexpressing homozygous soybean lines (OE-GmMATE75) #5 and #19 after 7 days of iron-deficient treatment.

[0021] Figure 6 This is a comparison of the phenotypic results of soybean wild-type (WT) and overexpression homozygous soybean lines (OE-GmMATE75) #5 and #19 after 7 days of iron-deficient treatment.

[0022] Figure 7 A comparison of SPAD values ​​between wild-type soybean (WT) and overexpression homozygous soybean lines (OE-GmMATE75) #5 and #19 after 7 days of iron-deficient treatment.

[0023] Figure 8 This is a comparison of iron content in different parts of soybeans treated with iron deficiency for 7 days, showing the difference between wild-type soybean (WT) and overexpression homozygous soybean lines (OE-GmMATE75) #5 and #19.

[0024] Figure 9 A comparison of root citric acid efflux rates between wild-type soybean (WT) and overexpressing homozygous soybean line (OE-GmMATE75) #5 after 7 days of iron-deficient treatment.

[0025] Figure 10A comparison of FCR activity between wild-type soybean (WT) and overexpression homozygous soybean line (OE-GmMATE75) #5 after 7 days of iron-deficient treatment.

[0026] Figure 11 A comparison of citric acid efflux rates in the aboveground xylem of wild-type soybean (WT) and overexpression homozygous soybean line (OE-GmMATE75) #5 after 7 days of iron-deficient treatment.

[0027] Figure 12 A comparison of xylem iron content between wild-type soybean (WT) and overexpression homozygous soybean line (OE-GmMATE75) #5 after 7 days of iron-deficient treatment. Detailed Implementation

[0028] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0029] In one embodiment of the present invention, an application of soybean citrate transporter in enhancing the iron deficiency stress tolerance of plants (such as soybeans) is provided. The nucleotide sequence of the gene encoding soybean citrate transporter (named GmMATE75 gene) is shown in SEQ ID NO:1. The promoter nucleotide sequence upstream of the gene encoding soybean citrate transporter is shown in SEQ ID NO:2. This gene encoding soybean citrate transporter can enhance the ability of soybeans to grow in iron-deficient (low-iron) soils; iron-deficient soils are calcareous soils or soda saline-alkali soils, such as the saline-alkali soils of Baicheng area in Northeast China.

[0030] In another embodiment of the present invention, a recombinant vector is also provided, which is an overexpression vector of pro35S::GmMATE75, containing the encoding gene of the soybean citrate transporter described above; the primer pair for constructing the overexpression vector of pro35S::GmMATE75 includes 101-MATE75-F and 101-MATE75-R, whose nucleotide sequences are shown in SEQ ID NO:4 and SEQ ID NO:5, respectively.

[0031] In another embodiment of the present invention, a recombinant bacterium is also provided, comprising the above-described pro35S::GmMATE75 overexpression vector. This recombinant bacterium is selected from *Escherichia coli* DH5α, *Agrobacterium* GV3101, or *Agrobacterium* K599. *Escherichia coli* DH5α is used for cloning and preserving the overexpression vector, and *Agrobacterium* is used to mediate the transformation of the overexpression vector into soybean.

[0032] In another embodiment of the present invention, a method for enhancing the tolerance of plants to iron deficiency stress is also provided, comprising the following steps: transforming the gene encoding the soybean citrate transporter into plant cells using the overexpression vector pro35S::GmMATE75, thereby overexpressing the gene encoding the soybean citrate transporter in the plant; culturing the transformed cells to obtain complete plants, identifying single-copy homozygous soybean plants through multi-level identification; and using Agrobacterium GV3101-mediated cotyledonary node transformation of soybeans.

[0033] In another embodiment of the present invention, a soybean is also provided, which includes an exogenously introduced gene encoding a soybean citrate transporter; the nucleotide sequence of the gene encoding the soybean citrate transporter is shown in SEQ ID NO:1; the exogenous introduction method is: introduction via an overexpression vector of pro35S::GmMATE75.

[0034] In this invention, the GmMATE75 gene (systematically named Glyma.13G203000), which encodes the soybean GmMATE75 protein, is specifically used to address the core problem of limited soybean growth in iron-deficient environments, particularly in the saline-alkali soils of Baicheng, Northeast China. Experiments have shown that overexpression of the GmMATE75 gene in soybeans significantly enhances the plant's tolerance to iron deficiency stress. This invention overcomes the drawbacks of traditional iron fertilizer application, such as low utilization and high environmental risks, providing a key gene resource for molecular breeding: GmMATE75 effectively promotes iron absorption and utilization by plants through pathways such as regulating citric acid secretion. This not only improves the adaptability of soybeans in low-iron soils but also enhances their yield and quality, providing strong technical support for expanding the planting area of ​​soybeans in iron-deficient soils. This achievement lays the foundation for cultivating iron-tolerant crops, reducing agricultural dependence on chemical fertilizers, and promoting precision breeding, and is of great significance for alleviating soybean production shortages and improving self-sufficiency.

[0035] The following embodiments are implementation examples of the technical solution of the present invention in practical applications, but are not limited thereto. Unless otherwise specified, all materials and reagents involved are commercially available products; unless otherwise specified, all experimental methods used are conventional methods.

[0036] Example 1: Transcriptome data analysis of soybeans subjected to iron deficiency stress identified a key transporter protein, GmMATE75, regulating the citric acid metabolism pathway (the systematic name of its encoding gene, Glyma.13G203000). Sequence information was obtained from the National Center for Biotechnology Information (https: / / www.ncbi.nlm.nih.gov). The amino acid sequence of the transporter protein GmMATE75 is shown in SEQ ID NO:3, the nucleotide sequence of its encoding gene, GmMATE75, is shown in SEQ ID NO:1, and the upstream promoter nucleotide sequence is shown in SEQ ID NO:2. A structural diagram of the GmMATE75 gene was also constructed. Figure 1 As shown in Figure B. In the experiment, total RNA was extracted from the roots of wild-type soybean Williams 82 after iron deficiency treatment, and the cDNA of the GmMATE75 gene was obtained by PCR amplification. The PCR reaction system is shown in Table 1, the reaction procedure in Table 2, and the primers used in Table 3. The PCR amplification products were identified by agarose gel electrophoresis, and the results are shown in Figure B. Figure 1 As shown in Figure A, a clear band is visible at approximately 1788 bp, and the sequencing results of the amplified product are consistent with SEQ ID NO:1. This invention provides the first identification and cloning of a longer transcript or major functional transcript of this gene (i.e., the 1788 bp version); this new sequence contains previously unknown additional functional domains or key amino acid modules; this newly discovered, longer gene sequence (1788 bp) exhibits additional functions not previously revealed by shorter sequences (such as 1288 bp).

[0037] Table 1

[0038]

[0039] Table 2

[0040]

[0041] Table 3

[0042]

[0043] Example 2: Construction of the soybean GmMATE75 gene overexpression vector PTF101-35S-GmMATE75 (pro35S::GmMATE75). A schematic diagram of the vector construction is shown below. Figure 2 B. The specific method is as follows:

[0044] When constructing the GmMATE75 gene into a plant expression vector, a 35S enhancing promoter (used in this example) or other enhancing promoters or inducible promoters can be added before its transcription initiation nucleotide. Using root cDNA from wild-type soybean Williams 82 as a template, the GmMATE75 cDNA fragment was amplified using the high-fidelity enzyme KOD Plus Neo from Toyobo. The amplification primers were 101-MATE75-F and 101-MATE75-R. The amplification products were identified by agarose gel electrophoresis (see...). Figure 1 Following step A), the gel was excised and recovered according to the instructions of the TIANGEN ordinary gel extraction kit. The recovered cDNA fragments were then constructed downstream of the 35S promoter of the linearized PTF101-35-NOS vector, which had been digested with XbaI and SacI restriction endonucleases. The ligation process was performed according to the instructions for the 2×Seamless Cloning Mix. The ligation products were transformed into *E. coli* DH5α competent cells and incubated upside down at 37°C for 16 h. Single clones were selected and cultured at 37°C and 200 rpm for 12 h. Positive clones were identified by bacterial PCR and then sequenced for comparison. The successfully sequenced bacterial culture was added to an equal volume of 50% glycerol and stored at -80℃ for a long period of time. 100µL of the above-stored bacterial culture was added to 100mL of LB liquid medium containing spectinomycin (Spec) and cultured at 37℃ and 200rpm for 12h. The plasmid was extracted according to the instructions of the TIANGEN plasmid miniprep kit to obtain the overexpression vector pro35S::GmMATE75, named PTF101-35S-GmMATE75.

[0045] The overexpression vector pro35S::GmMATE75 constructed above was transformed into Agrobacterium GV3101. In this example, Agrobacterium-mediated transformation was used, and the specific method is as follows: 40 µL of Agrobacterium competent cells (aseptic operation) were slowly thawed on ice, and 2 µL of extracted plasmid (about 200 ng) was added. The cells were then incubated on ice for 5 min; frozen in liquid nitrogen for 5 min; heat-shocked at 37°C for 5 min; 1 mL of LB liquid medium was added, and the cells were incubated at 200 rpm and 28°C for 2 h; the incubated bacterial culture was spread on LB solid plates containing the corresponding antibiotics (rifampicin Rif and spectinomycin Spec), and incubated upside down at 28°C for 2 days. Single clones were selected for colony PCR identification. After positive PCR identification, an equal volume of 50% glycerol was added to the bacterial culture, and it was stored at -80°C for long-term storage.

[0046] The overexpression vector of pro35S::GmMATE75 was transformed into soybean: Using an Agrobacterium-mediated cotyledonary node genetic transformation system, the overexpression vector of pro35S::GmMATE75 was transformed into soybean. After screening for positive plants, they were propagated in a laboratory greenhouse and then at a transgenic seed propagation base the following year. Foliar spraying with 25 μg / mL glufosinate solution was used to identify positive plants. Positive T0 generation transgenic plants were initially obtained through PCR screening. Before conducting iron deficiency experiments, each seedling underwent glufosinate spraying verification and expression level verification. After confirming the plants as positive, subsequent experiments were carried out.

[0047] Example 3: Constructing the vector PCAMBIA2301-pGmMATE75-GUS, which tags the upstream promoter pGmMATE75 of the soybean GmMATE75 gene. A schematic diagram of the vector is shown below. Figure 2 A in the example. The specific method is as follows:

[0048] When constructing the promoter approximately 1500 bp upstream of the GmMATE75 gene into a plant expression vector, a GUS tag (used in this example) can be added after its transcription termination nucleotide. Using leaf DNA from wild-type soybean Williams 82 as a template, the cDNA fragment of GmMATE75 was amplified using the high-fidelity enzyme KOD Plus Neo from Toyobo. The amplification primers were pGmMATE75-GUS-F and pGmMATE75-GUS-R. Following the steps described above, the successfully sequenced bacterial culture was added to an equal volume of 50% glycerol and stored at -80°C for an extended period. 100 µL of the stored bacterial culture was added to 100 mL of LB liquid medium containing kanamycin and cultured at 37°C and 200 rpm for 12 h. Plasmids were extracted according to the instructions of the TIANGEN plasmid miniprep kit to obtain a vector with the pGmMATE75::GUS tag, named PCAMBIA2301-pGmMATE75-GUS.

[0049] The pGmMATE75::GUS-tagged vector constructed above was transformed into Agrobacterium K599. In this example, Agrobacterium-mediated transformation was used, and the specific method is as follows: 40 µL of Agrobacterium competent cells (aseptic operation) were slowly thawed on ice, and 2 µL of extracted plasmid (approximately 200 ng) was added. The cells were then incubated on ice for 5 min; frozen in liquid nitrogen for 5 min; heat-shocked at 37°C for 5 min; 1 mL of LB liquid medium was added, and the cells were incubated at 200 rpm and 28°C for 2 h; the incubated bacterial culture was spread onto LB solid plates containing the corresponding antibiotics (rifampicin Rif and spectinomycin Spec), and incubated upside down at 28°C for 2 days. Single clones were selected for colony PCR identification. After positive PCR identification, an equal volume of 50% glycerol was added to the bacterial culture, and it was stored at -80°C for long-term preservation.

[0050] Transform soybeans with the pGmMATE75::GUS-tagged vector: A portion of Agrobacterium was cultured and activated, then extensively spread onto fresh LB solid medium containing the corresponding antibiotic. The medium was sealed and incubated upside down at 28°C for 2-3 days. Soybean seeds (Williams 82) were sterilized and germinated in the dark for 5 days. The 5-day-old seedlings were removed from the soil and washed thoroughly with distilled water. A small amount of Agrobacterium was picked up with a syringe needle and used to infect the hypocotyl region of the soybean seedlings. The seedlings were then placed in modified Hoaglands nutrient solution for further culture. For 10-15 days after infection, the hypocotyl was kept moist by spraying water daily, and the taproot was pruned appropriately to promote root growth. Once a large number of roots had emerged from the hypocotyl, transgenic root development was identified using GUS staining solution (pCAMBIA2301 vector). Negative roots were removed, resulting in composite plants with normal aboveground parts and transgenic roots in the underground parts.

[0051] Example 4: The expression pattern of the GmMATE75 gene in the transgenic rooted composite plants obtained in Example 3 was observed by staining. Figure 3 As shown in the figure. The results showed that under normal iron supply conditions, the expression of this gene was specifically localized in the root stele; while under low iron stress conditions, its expression range expanded to the entire root system, and the expression intensity in the stele was further upregulated. This expression pattern, regulated by the iron environment, suggests that the GmMATE75 gene may play a key role in the systemic regulation of the root's adaptive response to iron deficiency stress by altering its expression site and intensity.

[0052] Example 5: The positive T0 generation transgenic plants obtained by PCR screening in Example 2 were self-pollinated and propagated. The expression level of the target gene was detected in the T2 or T3 generation lines by qRT-PCR. Figure 4 ), and used herbicide resistance to screen homozygous single plants ( Figure 5Ultimately, two genetically independent, homozygous overexpression lines with stable GmMATE75 gene expression were obtained, named OE#5 and OE#19, respectively. These results indicate that the GmMATE75 gene can be successfully and efficiently transcribed and expressed in soybean overexpression lines. Soybean lines overexpressing GmMATE75 showed greater tolerance to iron deficiency than wild-type (WT) soybeans under low-iron hydroponic conditions (e.g., ...). Figures 6-12 As shown); In this embodiment of the invention, through Agrobacterium-mediated genetic transformation, transgenic lines #5 and #19 were obtained that stably overexpress the GmMATE75 gene in the model soybean variety Williams 82. Under strictly controlled low-iron hydroponic conditions, the overexpressing lines showed significantly enhanced iron deficiency tolerance compared to the wild type. Specific physiological evidence includes: milder chlorosis symptoms in the terminal leaves (…). Figure 6 ), leaf SPAD value (e.g.) Figure 7 Higher chlorophyll content, higher root iron reductase (FCR) activity, and stronger citric acid efflux rate (e.g.) Figure 9 and Figure 10 The iron and citric acid content in the xylem sap also increased significantly (e.g. Figure 11 and Figure 12 This ultimately leads to a significant increase in iron content in the aboveground parts (e.g. Figure 8 These results demonstrate for the first time at the whole-plant level that overexpression of the GmMATE75 gene can systematically enhance the iron deficiency tolerance of soybean. Its mechanism of action is likely through strengthening the activation (citric acid secretion) and reduction (increased FCR activity) of insoluble iron in the rhizosphere, thereby improving the iron nutritional status within the plant. This discovery not only provides a key gene for elucidating the molecular mechanism of iron deficiency tolerance in soybean, but also lays a solid foundation for utilizing this gene resource to breed new soybean varieties adapted to low-iron stress soils such as calcareous and saline-alkali soils.

[0053] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification.

Claims

1. The application of soybean citrate transporter in enhancing plant tolerance to iron deficiency stress, characterized in that, The nucleotide sequence of the gene encoding the soybean citric acid transporter is shown in SEQ ID NO:1; the plant is soybean.

2. The application according to claim 1, characterized in that, The promoter nucleotide sequence upstream of the gene encoding the soybean citrate transporter is shown in SEQ ID NO:

2.

3. The application according to claim 1, characterized in that, The gene encoding the soybean citric acid transporter is used to enhance the ability of soybeans to grow in iron-deficient soils; the iron-deficient soils are calcareous soils or soda saline-alkali soils.

4. A recombinant vector, characterized in that, The recombinant vector is used to enhance the tolerance of plants to iron deficiency stress and contains the encoding gene of soybean citrate transporter; the nucleotide sequence of the encoding gene of soybean citrate transporter is shown in SEQ ID NO:1; the plant is soybean; the recombinant vector is an overexpression vector of pro35S::GmMATE75; wherein, the primer pair used to construct the overexpression vector of pro35S::GmMATE75 includes 101-MATE75-F and 101-MATE75-R, the nucleotide sequences of which are shown in SEQ ID NO:4 and SEQ ID NO:5, respectively.

5. A recombinant bacterium, characterized in that, The recombinant bacteria include the recombinant vector of claim 4; the recombinant bacteria are *Escherichia coli* or *Agrobacterium* used for plant genetic transformation.

6. The application of the recombinant vector as described in claim 4, or the recombinant bacteria as described in claim 5, in enhancing plant tolerance to iron deficiency stress or demonstrating plant iron deficiency responses, characterized in that... The plant in question is soybean.

7. The application according to claim 6, characterized in that, A method for enhancing the tolerance of plants to iron deficiency stress includes the following steps: transforming the gene encoding the soybean citrate transporter into plant cells via a recombinant vector, thereby overexpressing the gene encoding the soybean citrate transporter in the plant; and cultivating the transformed cells to obtain complete transgenic plants.

8. Soybeans, characterized in that, The soybean contains a gene encoding an exogenously introduced soybean citrate transporter; the nucleotide sequence of the gene encoding the soybean citrate transporter is shown in SEQ ID NO:1.

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