Zostera marina magnesium ion transporter gene ZosmaMGT10, recombinant vector and application

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a zostera marina magnesium ion transporter gene ZosmaMGT10, a recombinant vector and application. The CDS full length of the gene is 1239 bp, 412 amino acids are encoded, and the gene has a conservative CorA structural domain. It is proved for the first time that ZosmaMGT10 protein has Mg < 2 + > transport activity and can be specifically combined with Mg < 2 + > through E299, E300 and E303 residues, abscisic acid can remarkably induce expression of the ZosmaMGT10 protein in zostera marina leaves, the Mg < 2 + > absorption capacity of zostera marina is enhanced, and then plant stress resistance is improved. The gene ZosmaMGT10 provided by the invention can be used as a key candidate gene, is used for cultivating marine plant varieties with high Mg < 2 + > absorption efficiency and strong stress resistance, and provides a new tool and a theoretical basis for ecological restoration of coastal zones and breeding of stress-tolerant crops.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to a magnesium ion transporter gene ZosmaMGT10 from Algae macrocarpa, a recombinant vector, and its applications. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Large-leaved seaweed ( Zostera Marina *Macrophyllaria media* is an important submerged seagrass in temperate shallow seas, playing a vital role in stabilizing sediments and maintaining marine ecological balance. However, its habitat is increasingly deteriorating due to tidal changes, reduced freshwater input, and human activities, leading to the degradation of global *Macrophyllaria media* beds. Abscisic acid (ABA) is a key hormone in plant responses to stress, regulating physiological processes such as ion transport to enhance resilience, but its mechanism of action in marine submerged plants remains unclear. Magnesium ions (Mg...) 2+ Magnesium (Mg) is an essential nutrient element for plants, participating in important processes such as chlorophyll synthesis and enzyme activity regulation. The plant MGT (Magnesium Transporter) gene family is responsible for Mg. 2+ Transmembrane transport of Mg2+ has been studied in terrestrial plants such as Arabidopsis thaliana, but in marine submerged plants like *Echinochloa crus-galli*, it remains to be seen whether MGT family members are regulated by ABA and how they mediate Mg2+ transport. 2+ Absorption to enhance stress resistance has not yet been reported.

[0004] Current technologies lack systematic research on the expression patterns and functional characteristics of the MGT gene family in *Leptochloa macrophylla* under ABA stress, and in particular, cannot identify the regulatory mechanisms of Mg in the ABA signaling pathway. 2+ The lack of core target genes for transport has limited the elucidation of the stress resistance molecular mechanisms of *Echinochloa macrophylla* and the utilization of related gene resources. Summary of the Invention

[0005] This invention addresses the shortcomings of existing technologies by providing a *ZosmaMGT10* magnesium ion transporter gene from *Alternaria macrocarpa*, a recombinant vector, and its applications.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A magnesium ion transporter gene from *Zosma*, named ZosmaMGT10, has a full-length CDS of 1239 bp, and its specific base sequence is shown in SEQ ID NO.1. This gene encodes a protein containing 412 amino acids with a molecular weight (MW) of 46.47 kDa, a theoretical isoelectric point (pI) of 6.30, a hydrophilicity coefficient (GRAVY) of -0.033, and an instability index of 46.12. The protein contains a highly conserved C-terminal CorA domain and two transmembrane regions at the C-terminus. Subcellular localization results show that it is located in the cell membrane and cytoplasm.

[0007] This invention constructs a recombinant vector containing the magnesium ion transporter gene ZosmaMGT10 from Algae macrophylla. The recombinant vector includes a prokaryotic expression vector, a eukaryotic expression vector, and a yeast expression vector, as detailed below: The prokaryotic expression vector PGEX4T-1::ZosmaMGT10 was used for the expression of the first intracellular protein of ZosmaMGT10, and the expression of the first intracellular protein of ZosmaMGT10 and its mutants was detected. 2+ The binding ability; the eukaryotic expression vector pBI121-EGFP-2::ZosmaMGT10 is used for subcellular localization of ZosmaMGT10; the yeast expression vector pYES2::ZosmaMGT10 is used for Mg 2+ Mg in defective yeast CM66 2+ Verification of transport activity.

[0008] This invention also provides an application of the magnesium ion transporter gene ZosmaMGT10 in *Zosma macrocarpa*, wherein the application is that gene ZosmaMGT10 regulates the magnesium ion transporter in *Zosma macrocarpa*. 2+ Application in absorbing and enhancing the stress resistance of *Zosma MGT10*; the gene ZosmaMGT10 in the cultivation of Mg 2+ Applications of the gene ZosmaMGT10 in marine plant varieties with high absorption efficiency and strong stress resistance; applications of the gene ZosmaMGT10 in coastal ecological restoration and stress-resistant crop breeding.

[0009] Furthermore, the prokaryotic expression can be performed using pET30a, pET32a, or pCold-TF; the eukaryotic expression can be performed using the pCAMBIA series vectors. Binding site verification: In addition to point mutations (299E, 300E, 303E mutations), deletion mutations can be used to verify Mg binding sites. 2+ Integration ability.

[0010] Inductively coupled plasma optical emission spectrometry (ICP-OES) was used to detect Mg in the roots and leaves of *Leptochloa macrophylla* after 14 days of treatment in both the control group and ABA-treated groups. 2+ Changes in content.

[0011] The beneficial effects of the technical solution provided by this invention are: 1. This invention is the first to confirm that Zosma MGT10 contains Mg. 2+ Transport activity, molecular docking experiments, and Biacore assays collectively confirmed that this protein specifically binds to Mg through its amino acid residues at positions E299, E300, and E303. 2+ And can complement Mg 2+ Growth defects of defective yeast strain CM66.

[0012] 2. This invention provides a stress-resistance gene resource: ZosmaMGT10. This gene can be used as a key candidate gene for cultivating Mg... 2+ Marine plant species with high absorption efficiency and strong stress resistance provide new molecular tools for coastal ecological restoration and stress-resistant crop breeding. Attached Figure Description

[0013] Figure 1 For *Echinochloa crus-galli*, Mg was added after 100 μM ABA stress. 2+ Changes; where A represents the Mg content in the leaves and roots of *Echinopsis macrocarpa* after 14 days of treatment with 100 μM ABA stress. 2+ Content; B represents the Mg content in the leaves of *Echinochloa macrophylla* after 24 h of NMT treatment with 100 μM ABA stress. 2+ Flow velocity bar chart; C represents the Mg content of leaves of *Echinochloa macrophylla* after 24 h of NMT treatment with 100 μM ABA stress. 2+ Flow velocity line graph; Figure 2 for ZosmaMGTs The expression pattern and structure of ZosmaMGT10; where A is ZosmaMGTs Expression heatmaps in early female flowers, late female flowers, male flowers, roots, and leaves; B represents the expression levels of *Echinochloa macrophylla* after treatment with 100 μM ABA for 12 h. ZosmaMGTs Expression heatmap; C represents the ZosmaMGT10 protein domain, containing Mg 2+ The CorA domain, a hallmark of the CorA family of transport proteins; D is a schematic diagram of the two transmembrane structures at the C-terminus of ZosmaMGT10 protein; E shows the subcellular localization of ZosmaMGT10. Figure 3 Zosma MGT10 and Mg 2+ The binding ability; where A is the binding of ZosmaMGT10 with Mg 2+ Molecular docking results; B represents Zosma MGT10 intra and Zosma MGT10 E299AE300AE303A intraSDS-PAGE analysis of purified fusion protein; C represents Zosma MGT10. intra With Mg 2+ Binding and dissociation curves of the interaction and global fitting curve; D is ZosmaMGT10 intra With Mg 2+ Global fitting curve of the interaction; E is ZosmaMGT10 E299AE300AE303A intra With Mg 2+ Binding and dissociation curves of the interaction and global fitting curve; F is ZosmaMGT10 E299AE300AE303A intra With Mg 2+ The global fitted curve of the interaction; Figure 4 ZosmaMGT10 in Mg 2+ Mg in defective yeast CM66 2+ Absorption capacity. Detailed Implementation

[0014] The specific embodiments of the present invention are described in detail below. These embodiments are intended to more fully demonstrate the technical content of the present invention and help to understand the specific implementation process of the present invention, but their content should not be construed as limiting the scope of the claims of the present invention in any way. For those skilled in the art, various adjustments, modifications, and substitutions made to the embodiments without departing from the spirit and scope of the present invention are all within the scope of protection sought by the present invention.

[0015] Example 1 1. ABA stress treatment of *Echinochloa macrophylla*: *Echinochloa macrophylla* plants were cultured in a light incubator with a photoperiod of 14 h light followed by 10 h darkness, a light intensity of 10,000 lux, and a temperature of 15℃. ABA (purity ≥98%) was added to artificial seawater (sea crystal, salinity approximately 30‰) to a final concentration of 100 μM to stress the *Echinochloa macrophylla* plants. The control group received no ABA, and all other culture conditions were the same as the treatment group.

[0016] 2. Mg under ABA stress 2+ Content detection: After treating 7-month-old *Leptochloa macrophylla* plants with 100 μM ABA for 14 days, the Mg content in roots and leaves was measured using inductively coupled plasma optical emission spectrometry (ICP-OES). 2+Content. Accurately weigh 0.5000 g of *Leptochloa macrophylla* samples from the control group and ABA treatment group. Place the weighed samples into a muffle furnace, set the temperature to 550℃, and start timing after the muffle furnace temperature reaches 550℃ for 5 hours of ashing. Arrange the ashing samples in order, and use a pipette to draw 10 ml of 60% aqua regia solution, place it into a crucible, and extract for 30 min. Pour the extract into prepared filter paper and a funnel. After filtering, remove the funnel, dilute the filtered solution 3 times, and perform ICP-OES to detect Mg. 2+ Quantitative analysis was performed, with each sample undergoing three biological replicate analyses. Figure 1 A in the figure indicates that 100 μM ABA stress treatment for 14 days promoted the Mg2+ resistance of *Echinopsis thunbergii*. 2+ Absorption.

[0017] After treating 7-month-old *Echinochloa macrophylla* plants with 100 μM ABA for 24 h, the Mg content in *Echinochloa macrophylla* leaves was detected in real time using non-destructive micrometabolism (NMT) while maintaining the physiological integrity of the *Echinochloa macrophylla*. 2+ Content changes. Leaves of *Leptochloa macrophylla* from both the control and ABA-treated groups were immersed in the test solution (control group: artificial seawater containing 0.05 mM KNO3; ABA-treated group: artificial seawater containing 100 μM ABA and 0.05 mM KNO3). After standing for 0.5 hours, three monitoring points were selected on the leaf surface, and data were recorded for 5 minutes at each point. Six replicates were performed for each group. The experiment was conducted under the same light conditions (10000 lux) throughout. Figure 2 B and C in the figure indicate that after 24 h of treatment with 100 μM ABA stress, the Mg content in the leaves of *Echinochloa macrophylla* decreased. 2+ The net absorption rate of ABA was significantly higher than that of the control group, indicating that ABA stress promoted the absorption of Mg in the leaves of *Echinochloa macrophylla*. 2+ Absorption.

[0018] Example 2 1. Laminaria japonica ZosmaMGTs Analysis of expression patterns in different organizations: Transcriptome data from various tissues and organs, including early female flowers, late female flowers, male flowers, roots, and leaves, were obtained from the NCBI database (accession number SRP056873). After log2 transformation, heatmaps generated using TBtools-II software were used to visually present the data. ZosmaMGTs The expression pattern. Figure 2 The result A in the data shows that, Zosma MGT10High expression was observed in the leaves of *Lycopodium clavatum*, with a relative expression level 8.3 times higher than in the roots, while low expression or no expression was observed in other tissues and organs. The results were obtained from the transcriptome data of *Lycopodium clavatum* after 12 h of treatment with 100 μM ABA (accession number PRJNA1308516) published on NCBI. ZosmaMGTs The representation, after log2 transformation, was plotted using TBtools-II software. ZosmaMGTs The heatmap of expression. Figure 2 Results B showed that after 12 h of treatment with 100 μM ABA, Zosma MGT10 The expression level in the leaves was significantly increased, with a relative expression level 3.7 times that of the control group, indicating that... Gene ZosmaMGT10 It is induced by ABA.

[0019] 2. Mg from *Echinochloa macrophylla* 2+ Basic information about the transporter Zosma MGT10: Based on published genome information of *Zosma*, we obtained the gene coding sequence of *ZosmaMGT10*, whose nucleotide sequence is shown in SEQ ID NO.1. The complete open reading frame of this gene is 1239 bp in length, encoding 412 amino acids. The predicted molecular weight (MW) of this protein is 46.47 kDa, the theoretical isoelectric point (pI) is 6.30, the hydrophilicity coefficient (GRAVY) is -0.033, and the instability index is 46.12. Conserved domain analysis of the protein sequence revealed the presence of a conserved CorA domain (…). Figure 2 (C in the middle), and has two transmembrane regions at the C end ( Figure 2 (D in the middle).

[0020] 3. ZosmaMGT10 is located in the cell membrane and cytoplasm: The fusion expression vector pBI121-EGFP-2::ZosmaMGT10 was constructed using homologous recombination. The target gene ZosmaMGT10 was amplified using primers: GFP-ZosmaMGT10-F: tagaggatccccgggggtaccATGCAATCTTTCTTCTATTCCCTCTC and GFP-ZosmaMGT10-F: gcccttgctcaccatggtaccTAATATTTTTTTTGATTTGAGATAAGAATACA, with *Zosma macrocarpa* cDNA as a template. KpnThe pBI121-EGFP-2 vector was linearized with restriction endonucleases. The amplified gene ZosmaMGT10, containing a homologous arm, was inserted into the linearized pBI121-EGFP-2 vector. Ligation was performed using the OK Clon DNA Ligation Kit II at 50°C for 10 min. The resulting vector was transformed into *E. coli* DH5α competent cells, and the recombinant vector sequence was verified to be correct by sequencing. The empty pBI121-EGFP-2 vector and the recombinant vector pBI121-EGFP-2::ZosmaMGT10 were transformed into *Agrobacterium tumefaciens* GV3101 competent cells and plated on LB agar plates containing 20 μg / mL rifampin and 100 μg / mL kanamycin. The plates were incubated upside down at 28°C for 2-3 days. Single colonies were selected for colony PCR to screen for positive *Agrobacterium* strains.

[0021] Healthy leaves from tobacco plants aged 4-6 weeks were selected, and *Agrobacterium tumefaciens* positive bacteria were injected into tobacco cells through the abaxial surface of the leaves using a 1 mL syringe (needle removed). After dark incubation for 24 h, the cells were transferred to a light incubator for 48 h. Temporary slides were prepared from the treated tobacco leaves, and GFP fluorescence signals were observed using a laser confocal microscope. The results showed that the fluorescence signal of the ZosmaMGT10 fusion protein was mainly distributed on the cell membrane and cytoplasm, indicating that ZosmaMGT10 is localized in the cell membrane and cytoplasm. Figure 2 (E in the text)

[0022] Example 3 ZosmaMGT10 and Mg 2+ The ability to combine: The MIB2 online website (https: / / combio.life.nctu.edu.tw / MIB2 / ) predicts the relationship between Zosma MGT10 and Mg. 2+ The combination of . Figure 3 The A in the figure indicates that ZosmaMGT10 is related to Mg 2+ Strong binding affinity was observed at sites E299, E300, and E303. Using the Novizan Mut Express II Fast Mutagenesis Kit and the constructed pBI121-EGFP-2::ZosmaMGT10 recombinant vector as a template, the above three sites were mutated to A299, A300, and A303, respectively, to obtain the mutant ZosmaMGT10. E299AE300AE303A The Zosma MGT10 was validated using optical surface plasmon resonance (SPR) technology via a Biacore S200. intra and its mutant ZosmaMGT10 E299AE300AE303A intra With Mg 2+The binding ability of [the gene / organism] was assessed. The fusion expression vector pGEX4T-1::ZosmaMGT10 was constructed using homologous recombination. intra and the mutant pGEX4T-1::ZosmaMGT10 E299AE300AE303A intra Amplification containing Mg 2+ The first intracellular region (1-1047 bp) of the binding site of ZosmaMGT10 was identified using primers pGEX4T-1-ZosmaMGT10-F: gatctggttccgcgtggatccATGCAATCTTTCTTCTATTCCCTCTC, and pGEX4T-1-ZosmaMGT10-R: acccgggaattccggggatccTTACTCAAATCTGCTCAATTCAAGTCG, respectively. The primers were pBI121-EGFP-2::ZosmaMGT10 and the mutant ZosmaMGT10. E299AE300AE303A The target product is obtained by amplification using a template. Restriction endonuclease. Bam The pGEX4T-1 vector was linearized using HI, and the target gene was ligated to the linearized vector using the OK Clon DNA Ligation Kit II at 50°C for 10 min. The ligation was then transformed into *E. coli* DH5α competent cells, and sequencing confirmed the correct sequence of the recombinant vector. Recombinant plasmids were constructed. These plasmids were transformed into *E. coli* BL21(DE3) competent cells. After small-scale induction, the fusion protein was found to be located in inclusion bodies after 20 h of induction at 16°C, 200 rpm, and 0.5 mM IPTG. The fusion protein was then induced extensively with 200 mL LB broth (containing 50 μg / mL ampicillin) under small-scale induction conditions. After centrifugation (10000 rpm, 4°C, 20 min), the supernatant was discarded, and the cells were resuspended in lysis buffer (PBS). The cells were then sonicated and centrifuged (10000 rpm, 4°C, 20 min), and the precipitate was collected. Inclusion bodies were resuspended in denaturing buffer containing 8 M urea (50 mM Tris-HCl pH 8.0, 0.5 mM EDTA, 50 mM NaCl, 5% glycerol, 5 mM DTT). The denatured protein was placed in a dialysis bag and dialyzed sequentially in refolding buffers containing 6 M, 4 M, 2 M, and 0 M urea (50 mM Tris-HCl pH 8.0, 0.5 mM EDTA, 50 mM NaCl, 5% glycerol, 5 mM DTT) at 4°C for 12 h at each urea concentration. Finally, the protein was dialyzed with PBS for 12 h to achieve refolding. ZosmaMGT10 was purified by glutathione affinity chromatography. intra and its mutant ZosmaMGT10 E299AE300AE303A intra Fusion protein, SDS-PAGE electrophoresis was used to detect the purification status of the fusion protein. Figure 3 (B in the middle).

[0023] The fusion protein and Mg were verified using SPR (Optical Surface Plasmon Resonance) technology via Biacore S200. 2+ The binding affinity of purified ZosmaMGT10. intra and its mutant ZosmaMGT10 E299AE300AE303A intra The fusion protein was diluted to 100 μg / mL with 10 mM sodium acetate (pH 4.0) and coupled to a CM7 chip, using different concentrations of Mg... 2+ The analytes were measured at concentrations of 1.953, 3.906, 7.8125, 15.625, 62.5, and 125 μM, and their binding kinetics were determined in 1xPBS-P+ buffer containing 5% DMSO. Figure 3 The CF display shows that the Zosma MGT10 intra Can be with Mg 2+ Specific binding, with a dissociation constant KD value of 4.958 × 10⁻⁶. -5 M binds to E299, E300, and E303 residues, and its activity depends on these residues. The mutant ZosmaMGT10E... 299AE300AE303A intra With Mg 2+ The binding affinity of E299, E300, and E303 decreased significantly, with a dissociation constant KD value of 3.72 M, indicating that E299, E300, and E303 are ZosmaMGT10 and Mg²⁺. + Key residues for binding.

[0024] Example 4 ZosmaMGT10 contains Mg 2+ Transshipment capacity: To verify the Mg content of ZosmaMGT10 2+ To assess transport capacity and functional complementarity, ZosmaMGT10 was inserted into the pYES2 expression vector using homologous recombination, as pBI121-EGFP-2::ZosmaMGT10 and the mutant ZosmaMGT10, respectively. E299AE300AE303A The amplification was performed using pYES2-ZosmMGT10-F as the template. The primers used for amplifying the target gene were pYES2-ZosmMGT10-F:gggaatattaagcttggtaccATGCAATCTTTCTTCTATTCCCTCTC and pYES2-ZosmMGT10-R:gcggccgttactagtggatccCTATAATATTTTTTTTGATTTGAGATAAGAATAC. The restriction endonuclease was [missing information]. Kpn I and BamHI, the target gene was ligated to the linearized pYES2 vector using the OK Clon DNA Ligation Kit II at 50℃ for 10 min, and then transformed into E. coli DH5α competent cells to construct the recombinant plasmid pYES2::ZosmaMGT10 and the mutant recombinant plasmid pYES2::ZosmaMGT10. E299AE300AE303A The obtained recombinant vector sequence was verified to be correct by sequencing. The recombinant plasmid and empty vector were transformed into MgCl2 using the LiAc transformation method. 2+ A yeast strain with a missing transport gene, CM66, was transformed into a wild-type yeast strain CM52 using an empty vector and cultured on SD-Ura solid medium (containing 100 mM Mg). 2+ Incubate at 30℃ upside down for 3 days. Pick positive single colonies and inoculate them into 5 mL of solution containing 100 mM Mg. 2+ SD-Ura liquid medium was incubated at 30°C with shaking at 200 rpm for 24 h until the bacterial culture OD reached its maximum. 600 =1.0-1.5, complete the bacterial activation, and take 50 μL of bacterial solution to inoculate into 5 mL of solution containing 100 mM Mg 2+ In SC-Ura liquid medium (containing 2% galactose), cultured at 30°C with shaking at 200 rpm until OD. 600 ≈1.0, adjust to the same OD using sterile water. 600 Then, a 10-fold serial dilution was performed to obtain 10 0 10 -1 10 -2 and 10 -3 Four concentration gradients were used. 5 μL of each diluted bacterial suspension was taken and inoculated onto substrates containing different concentrations of Mg, according to the gradient. 2+ (0.1, 4 and 10 mM Mg) 2+ Functional complementation experiments were conducted on SC-Ura (containing 2% galactose) solid medium. Each sample was repeated 3 times. Wild-type yeast CM52 transformed from empty pYES2 (pYES2::CM52) was used as a positive control, and CM66 transformed from empty pYES2 (pYES2::CM66) was used as a negative control. The yeast was incubated upside down at 30℃ for 3 days to observe its growth.

[0025] The results show that ( Figure 4 The positive control wild-type yeast CM52 was able to [achieve a certain effect] in the presence of 0.1 mM Mg. 2+ It grows well on Mg medium, while the negative control pYES2::CM66 cannot grow on Mg medium. 2+ It grows normally on media with a concentration less than 4 mM. pYES2::ZosmaMGT10 and pYES2::ZosmaMGT10 E299AE300AE303ACM66 yeast transformants overexpressed with the vector were subjected to treatment with 0.1, 4, and 10 mM Mg²⁺. + The growth on solid culture media was superior to that of the pYES2::CM66 yeast transformant, with the pYES2::ZosmaMGT10 transformant showing growth closer to the positive control. This indicates that ZosmaMGT10 is a Mg... 2+ Transporter genes, which supplement the missing Mg in CM66 2+ Transport function, ability to complement the growth defects of CM66 yeast mutants; while the mutant transports Mg 2+ The ability to significantly decrease further confirms that E299, E300, and E303 are the active components of ZosmaMGT10 for Mg. 2+ Key sites for transport function.

[0026] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A magnesium ion transporter gene, ZosmaMGT10, from the algae Zostera macrophylla, characterized in that... Its CDS sequence is shown in SEQ ID NO.

1.

2. The *ZosmaMGT10* magnesium ion transporter gene from *Zosma* according to claim 1, characterized in that, The protein encoded by the gene has an amino acid sequence containing 412 amino acids encoded by the sequence shown in SEQ ID NO.1; the protein encoded by the amino acid sequence contains a conserved CorA domain and has two transmembrane regions at the C-terminus.

3. A recombinant vector containing the ZosmaMGT10 magnesium ion transporter gene from *Zosma* as described in claim 1, characterized in that, The vector is a prokaryotic expression vector, a eukaryotic expression vector, or a yeast expression vector.

4. The recombinant vector according to claim 3, characterized in that, The recombinant vector was constructed using homologous recombination; the restriction endonucleases used were adjusted according to the vector type. The pBI121-EGFP-2::ZosmaMGT10 vector was constructed using... Kpn I restriction endonuclease was used to construct PGEX4T-1::ZosmaMGT10. Bam HI restriction endonuclease was used to construct pYES2::ZosmaMGT10. Kpn I and Bam HI restriction endonuclease.

5. The recombinant vector according to claim 3, characterized in that, The prokaryotic expression vector is PGEX4T-1, pET30a, pET32a or pCold-TF vector; the eukaryotic expression vector is pBI121-EGFP-2 or pCAMBIA series vector; the yeast expression vector is pYES2, pYES3 or pYES6 vector.

6. The *ZosmaMGT10* gene, a magnesium ion transporter gene from *Alternaria macrocarpa* as described in claim 1 or 2, or the recombinant vector as described in any of claims 3-5, in regulating the magnesium ion transport of *Alternaria macrocarpa*. 2+ Applications in absorption.

7. The application according to claim 6, characterized in that, The regulation of Mg in *Macrophyllum macrocarpa* 2+ Absorption is used to cultivate plants with enhanced stress resistance; said stress resistance includes enhanced tolerance to high salt stress, drought stress, or osmotic stress.

8. The application according to claim 7, characterized in that, The plants with enhanced stress resistance are used for coastal ecological restoration.

9. The application according to claim 6, characterized in that, The method for regulating magnesium ion absorption in *Zosma macrocarpa* involves introducing the magnesium ion transporter gene *ZosmaMGT10* into plant cells to overexpress the gene, thereby promoting the plant's absorption of magnesium ions. 2+ Absorption.

10. The application according to claim 6, characterized in that, Plants treated with abscisic acid can be combined to further induce the expression of the ZosmaMGT10 gene, thereby enhancing the gene's regulation of Mg. 2+ Absorption effect.

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