Application of halophyte hordeum marinum HmSTG1 gene in improving plant salt tolerance

Abstract

This invention discloses the application of the HmSTG1 gene in improving the salt tolerance of the halophyte barley, belonging to the fields of molecular biology and plant genetic engineering. The coding region sequence of the HmSTG1 gene is shown in SEQ ID NO.1; the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2. This invention isolated a Na+ gene from the halophyte barley. + / H + The antitransporter protein encoding gene HmSTG1 was discovered, and for the first time, it was found that gene HmSTG1 has a strong Na+ binding capacity. + Transport capacity, and maintain low Na levels in plants such as rice. + Concentration and high K + / Na + This gene plays an important role in resisting high salt stress. The HmSTG1 gene can be transformed into crops such as rice, corn, wheat, and soybeans, or woody plants such as apples and pears, to improve the salt tolerance of transgenic plants, thereby increasing their yield and quality, which has significant economic value and social benefits.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and plant genetic engineering, and in particular to the application of the HmSTG1 gene in the halophyte barley in improving plant salt tolerance. Background Technology

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

[0003] Soil salinization is one of the major abiotic stresses facing global agricultural production. Currently, the world has a large and widespread saline-alkali soil area, with over 424 million hectares of topsoil (0-30 cm) and 833 million hectares of subsoil (30-100 cm) already salinized, accounting for 4.4% and 8.7% of the total land area, respectively. Furthermore, irrational farming practices, irrigation methods, and excessive industrial pollutant emissions further exacerbate the salinization of arable land. Developing salt-tolerant crop varieties is one of the most effective ways to utilize saline-alkali soils, improve crop productivity, and ensure food security.

[0004] Through long-term natural domestication and artificial selection, the genetic polymorphism of grass crops such as rice and wheat has sharply decreased, and the narrow genetic background of cultivated varieties has become a bottleneck for breeding improvement. However, the abundant genetic diversity of wild grass resources can provide a valuable gene pool to break through this bottleneck. Introducing superior exogenous traits / genes from wild relatives into cultivated crops can breed new varieties with breakthrough improvements in specific traits. For example, introducing Na+ from a wild relative of wheat, such as wheat grains, can improve the genetic diversity of cultivated varieties. + The introduction of the transporter protein encoding gene TmHKT1;5-A into the durum wheat variety Tamaroi increased yields by nearly 25% when grown in saline soils.

[0005] Maintain K + / Na + Ion balance is one of the main physiological mechanisms of salt tolerance in plants. Plants maintain ion balance through the action of related proteins such as ion channels, transport proteins, and proton pumps, including the SOS, HKT, and NHX protein families. + and K + The transport and Na + They play an important role in differentiation. For example, halophytes such as Suaeda salsa and Acer palmatum differentiate themselves at the molecular level through Na+. + Transport proteins such as SOS1 (located in the plasma membrane) and NHX1 (located in the vacuolar membrane), and the proton pump (H) that provides them with energy. + Two types of genes (-ATPase) respond to excessive Na+ in the body. + .

[0006] Therefore, it is crucial to provide more genes that can improve crop salt tolerance traits. Summary of the Invention

[0007] The purpose of this invention is to provide the application of the HmSTG1 gene in halophyte barley in improving plant salt tolerance, so as to solve the problems existing in the prior art.

[0008] To achieve the above objectives, the present invention provides the following solution:

[0009] One of the technical solutions of this invention is the application of the HmSTG1 gene in improving plant salt tolerance.

[0010] The second technical solution of this invention is the application of HmSTG1 protein in improving plant salt tolerance.

[0011] The third technical solution of the present invention is the application of recombinant expression vectors, overexpression vectors, interference vectors, recombinant viruses, recombinant bacteria or recombinant gene expression cassettes containing the HmSTG1 gene in improving plant salt tolerance.

[0012] The fourth technical solution of this invention is the application of the HmSTG1 gene, HmSTG1 protein, recombinant expression vector containing the HmSTG1 gene, overexpression vector, interference vector, recombinant virus, recombinant bacteria or recombinant gene expression cassette in salt-tolerant plant breeding.

[0013] The fifth technical solution of the present invention is a product for improving the salt tolerance of plants, comprising the HmSTG1 gene, HmSTG1 protein, a recombinant expression vector containing the HmSTG1 gene, an overexpression vector, an interference vector, a recombinant virus, a recombinant bacterium, or a recombinant gene expression cassette.

[0014] The sixth technical solution of the present invention is a method for improving the salt tolerance of plants by exogenously transferring the HmSTG1 gene into the plant or upregulating the expression of the HmSTG1 gene in the plant genome.

[0015] The seventh technical solution of the present invention is a method for breeding salt-tolerant plants, including the method for improving the salt tolerance of plants.

[0016] Based on the above technical solution, the present invention has the following technical effects:

[0017] This invention isolates a Na group from the halophyte barley (Haloxylon ammodendron), a member of the Poaceae family. + / H + The antitransporter protein encoding gene HmSTG1 was discovered, and for the first time, it was found that gene HmSTG1 has a strong Na+ binding capacity. + Transport capacity, and maintain low Na levels in the plant + Concentration and high K + / Na +This gene plays an important role in resisting high salt stress. It can be transformed into annual crops (herbaceous plants) such as rice, corn, wheat, barley, and soybeans, or woody plants such as apples and pears, to improve the salt tolerance of transgenic plants, thereby increasing their yield and quality, which has significant economic value and social benefits. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1This study presents the gene structure, phylogenetic analysis, and expression levels in roots and shoots of the halophyte *H. sarcodactylon* HmSTG1 under salt stress. A represents a schematic diagram of the *H. sarcodactylon* HmSTG1 gene structure; B represents agarose gel electrophoresis images of PCR-amplified fragments of the promoter (Pro) and coding sequence (CDS) sequences of the *H. sarcodactylon* HmSTG1 gene and the rice OsSTG1 gene; C represents a phylogenetic tree analysis of the protein encoded by the *H. sarcodactylon* HmSTG1 gene; D and E represent the expression levels of *H. sarcodactylon* HmSTG1 and its three homologous genes in the roots (D) and shoots (E) of plants under normal (0 mM NaCl) and salt stress treatments (150 and 300 mM NaCl). RNA-seq data from *H. sarcodactylon* plants under control and salt stress conditions were processed to obtain the FPKM values ​​of gene expression. M: DL5000 DNA Marker; L1: HmSTG1 gene promoter fragment; L2: HmSTG1 gene CDS sequence fragment; L3: OsSTG1 gene promoter fragment; L4: OsSTG1 gene CDS sequence fragment. HmSTG1: SEQ ID NO.2; HmSTG1_L1: Barley HORMACH03G01231600; HmSTG1_L2: Barley HORMACH07G03884300; HmSTG1_L3: Barley HORMACH07G03986800; HvSTG1: Barley HORVU3Hr1G003150; PtSTG1: Imperata cylindrica Pt_Chr0401412; OsSTG1: Rice LOC_Os12g44360; SvSTG1: Barley LOC_Chr0401412; OsSTG1: Rice LOC_Os12g44360; SvSTG1: Barley LOC_Chr0401412; OsSTG1: Rice LOC_Chr0401412; SvSTG1: Barley LOC_Chr0401412; OsSTG1: Rice LOC_Chr0401412; OsSTG1: Barley ... G1: Foxtail grass SEVIR_3G427000v2; BdSTG1: Two-spike short-stalked grass BRADI_4g00290v3; CqSTG1: Quinoa XP_021727954.1; SsSTG1: Suaeda salsa AHJ14584.1; GmSTG1: Soybean GLYMA_08G092000; AtSTG1: Arabidopsis thaliana AT2G01980; EsSTG1: Salt mustard XM_006395762.2; *: Significant difference (P<0.05).

[0020] Figure 2 This is a map of the p416-GPD yeast expression vector and the gene sequence insertion sites. The black boxes indicate the restriction enzyme sites used in the construction of the recombinant vector.

[0021] Figure 3 To confirm the Na+ of HmSTG1 protein through yeast heterologous complementation assay + Transport capacity. HmSTG1 and OsSTG1 proteins, as well as their truncated forms, were transported in yeast Na... +The target protein was expressed in the transport-deficient strain axt3k. The growth of the negative control (transformed with an empty vector) and the recombinant yeast strain on salt-containing (NaCl) AP medium was compared to determine whether the target protein possessed NaCl. + Transport capacity. In the figure, Empty vector: the axt3k yeast strain transformed with an empty vector; HmSTG1-FL and OsSTG1-FL: the full-length HmSTG1 and OsSTG1 proteins; HmSTG1 981 and OsSTG1 976 The truncated forms of HmSTG1 and OsSTG1 proteins with the self-inhibition domain removed.

[0022] Figure 4 This is a map of the pCAMBIA2300 plant expression vector and the gene sequence insertion sites. The black boxes indicate the restriction enzyme sites used in the construction of the recombinant vector.

[0023] Figure 5 This study evaluates the salt tolerance of transgenic rice lines inoculated with the HmSTG1 gene from the halophyte barley. In the diagram, A represents the expression vectors using the promoters of the rice and barley STG1 genes to drive the HmSTG1 gene, respectively; B represents the semi-quantitative RT-PCR detection of wild-type and transgenic rice lines; C and D represent the growth of three-week-old wild-type and transgenic rice lines after 20 days of treatment in normal nutrient solution (C) and culture medium containing 200 mM NaCl (D). M: DL2000 DNA marker; WT: wild-type control; HmPro-1, -2: transgenic rice lines using the barley HmSTG1 gene promoter to drive the HmSTG1 gene; OsPro-1, -2: transgenic rice lines using the rice OsSTG1 gene promoter to drive the HmSTG1 gene.

[0024] Figure 6 This study investigated the dry weight of roots and shoots, as well as the Na and K content, of transgenic rice lines inoculated with the HmSTG1 gene from the halophyte *Haloxylon ammodendron* under salt stress. A and B represent the dry weight of roots and shoots of three-week-old wild-type and transgenic rice lines after 20 days of treatment in normal nutrient solution (A) and culture solution containing 200 mM NaCl (B). C and D represent the Na content of roots and shoots of three-week-old wild-type and transgenic rice lines after 20 days of treatment in culture solution containing 200 mM NaCl. EG represents the K content of shoots of three-week-old wild-type and transgenic rice lines after 20 days of treatment in culture solution containing 200 mM NaCl. + / Na +Ratio (E), root Na uptake (F), and Na translocation from roots to aboveground parts (G). WT: wild-type control; HmPro-1, -2: transgenic rice lines with the HmSTG1 gene promoter driven by the barley HmSTG1 gene; OsPro-1, -2: transgenic rice lines with the HmSTG1 gene promoter driven by the rice OsSTG1 gene; RDW: root dry weight. Detailed Implementation

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0028] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0029] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0030] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0031] This invention provides the application of the HmSTG1 gene in improving plant salt tolerance.

[0032] In some specific embodiments, the nucleotide sequence of the HmSTG1 gene is as shown in SEQ ID NO.1 or has 95% or more of the same or identical nucleotide sequence as shown in SEQ ID NO.1 and expresses the same functional protein.

[0033] The nucleotide sequence may be DNA, such as cDNA, coding region sequence (CDS), genomic DNA, or recombinant DNA; the nucleotide sequence may also be RNA, such as mRNA or hnRNA.

[0034] The term “identity” or “sameness” as used here refers to sequence similarity to a natural nucleic acid sequence. Identity or identity can be evaluated using computer software, such as the BLAST algorithm (Karlin and Altschul. 1993. Proc. Natl. Acad. Sci. USA 90: 5873-5877).

[0035] In the above nucleotide sequences, the 95% or more consistency or identity can be at least 95%, 96%, 97%, 98%, or 99% consistency or identity.

[0036] This invention also provides the application of HmSTG1 protein in improving plant salt tolerance.

[0037] In some specific embodiments, the HmSTG1 protein is a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the sequence shown in SEQ ID NO.2 or the sequence shown in SEQ ID NO.2.

[0038] The HmSTG1 protein can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically.

[0039] In some specific implementations, the protein tag can be FLAG (DYKDDDDK), Poly-His (6 HHHHHH), c-myc (EQKLISEEDL), Poly-Arg (6 RRRRR), or Strep-tagII (WSHPQFEK).

[0040] This invention also provides the application of recombinant expression vectors, overexpression vectors, interference vectors, recombinant viruses, recombinant bacteria, or recombinant gene expression cassettes containing the HmSTG1 gene in improving plant salt tolerance.

[0041] The recombinant expression vector can be constructed using existing plant expression vectors. These plant expression vectors include binary Agrobacterium vectors and vectors suitable for plant microbombardment, such as pCAMBIA1300, pCAMBIA1301, pCAMBIA2300, pCAMBIA2301, pCAMBIA3301, pGreen0029, pBI121, pBin19, or other derived plant expression vectors. When constructing a recombinant expression vector using the gene, a promoter of the gene or its homologous gene, or any enhancing, constitutive, tissue-specific, or inducible promoter can be added before its transcription initiation nucleotide, such as the cauliflower mosaic virus (CaMV) 35S promoter, the ubiquitin gene Ubiquitin promoter (pUbi), the stress-inducible promoter rd29A, etc. These can be used alone or in combination with other plant promoters. Furthermore, when constructing a recombinant expression vector using the gene of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used.

[0042] The embodiments of the present invention also provide the application of HmSTG1 gene, HmSTG1 protein, recombinant expression vector containing HmSTG1 gene, overexpression vector, interference vector, recombinant virus, recombinant bacteria or recombinant gene expression cassette in salt-tolerant plant breeding.

[0043] In some specific implementations, the plants include rice, corn, wheat, barley, soybeans, apples, and pears.

[0044] This invention also provides a product for improving plant salt tolerance, including the HmSTG1 gene, HmSTG1 protein, a recombinant expression vector containing the HmSTG1 gene, an overexpression vector, an interference vector, a recombinant virus, a recombinant bacterium, or a recombinant gene expression cassette.

[0045] This invention also provides a method for improving plant salt tolerance by exogenously transferring the HmSTG1 gene into the plant or upregulating the expression of the HmSTG1 gene in the plant genome.

[0046] In some specific implementations, the method for exogenously transferring the HmSTG1 gene into the plant is selected from any one of Agrobacterium-mediated transformation, gene gun transformation, polyethylene glycol (PEG)-mediated transformation, electroporation transformation, or pollen tube pathway transformation.

[0047] One method for exogenously introducing the HmSTG1 gene is to transform plant cells or tissues with a plant expression vector carrying the HmSTG1 gene using conventional biological methods such as Ti plasmid, Ri plasmid, direct DNA transformation, microinjection, electrocoagulation, or Agrobacterium-mediated transformation, and then cultivate the transformed plant cells or tissues into complete plants.

[0048] Methods for upregulating the expression of the HmSTG1 gene in the plant genome may include: introducing DNA fragments that can activate or enhance the transcriptional or translational levels or protein activity of the HmSTG1 gene; or controlling the synthesis of specific small RNA molecules to upregulate the accumulation of HmSTG1 mRNA.

[0049] The specific small RNA molecule mentioned can be: microRNA (miRNA), small interfering RNA (siRNA), or artificial microRNA (amiRNA), etc.

[0050] This invention also provides a method for breeding salt-tolerant plants, including the method for improving the salt tolerance of plants.

[0051] Includes the following steps:

[0052] The HmSTG1 gene was transferred into the starting plant to induce HmSTG1 gene expression, resulting in transgenic plants; the transgenic plants exhibited higher salt tolerance than the starting plants.

[0053] Halophytic barley (Hordeum marinum Huds.) is a wild relative of barley and wheat, and is one of the most salt-tolerant species known in the Poaceae family. It also has some interbreeding compatibility with common wheat, making it a potential gene donor for improving salt tolerance in crops such as wheat. Studies have found that the expression level of the HmSTG1 gene in Halophyum marinum Huds. is significantly upregulated after salt stress, but its function remains unclear. Related transgenic research has not yet been conducted, and whether transgenic plants with increased gene expression can grow and develop normally, or whether salt tolerance can be improved, are all unknown.

[0054] The inventors of this invention specialize in the molecular mechanisms and genetic improvement of salt tolerance in gramineous crops. During their research on the transcriptional profiles of halophyte *Barley spp.* and non-halophytes barley and wheat under salt stress, they discovered that many ion transport-related genes, including STG1, are significantly induced in *Barley spp.*, while they are expressed at low levels or not at all in barley and wheat. This suggests that the promoters of *Barley spp.* genes may possess higher activity or specific regulatory sequences.

[0055] This invention first utilizes transcriptome data analysis and reverse transcription PCR technology to obtain the HmSTG1 gene from the halophyte barley, and then uses yeast Na... + Heterologous complementation assays of the transport-deficient strain axt3k confirmed that the HmSTG1 protein possesses strong Na+ ionization. + Transshipment capacity.

[0056] Based on the high economic value, moderate salt sensitivity, and great potential for salt tolerance improvement of rice, this invention uses rice as the target plant and utilizes the promoter of the barley gene HmSTG1 and the promoter of the rice homolog OsSTG1 to drive the expression of the HmSTG1 gene in rice. Salt tolerance evaluation of T2 generation transgenic rice obtained using Agrobacterium-mediated transformation revealed that the expression of the HmSTG1 gene in transgenic rice significantly improved its salt tolerance.

[0057] Thus, this invention obtains a halophyte, sea barley Na + / H + The reverse transporter gene, which can significantly improve the salt tolerance of rice, is named Salt Tolerant Gene 1 (HmSTG1) based on its functional characteristics.

[0058] The coding region sequence and amino acid sequence of the HmSTG1 gene are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0059] Example 1

[0060] Cloning of coding sequence (CDS) and promoter fragment of barley HmSTG1 and rice OsSTG1 genes

[0061] (I) Total RNA was extracted using an RNA extraction kit (RC411, Novizan, Nanjing). The specific method is as follows:

[0062] (1) Weigh approximately 100 mg of root tissue from barley H559 and rice Zhonghua 11 (ZH11), grind them with liquid nitrogen, and immediately add 600 μL of Buffer EL. Vortex vigorously for 30 seconds to ensure that the sample and lysis buffer are thoroughly mixed. Centrifuge at 12,000 rpm for 5 min and immediately proceed with subsequent operations.

[0063] (2) Take about 500 μL of the supernatant into FastPure gDNA-Filter Columns III, centrifuge at 12,000 rpm for 30 seconds, discard FastPure gDNA-Filter Columns III, and collect the filtrate;

[0064] (3) Add 0.5 times the volume of filtrate (about 250 μL) of anhydrous ethanol to the collection tube and shake to mix for 15 seconds;

[0065] (4) Transfer the above mixture to FastPure RNAColumns V, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate;

[0066] (5) Add 700 μL Buffer RWA to FastPure RNAColumns V, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate;

[0067] (6) Add 500 μL of Buffer RWB to FastPure RNAColumns V, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate;

[0068] (7) Repeat step (6);

[0069] (8) Place FastPure RNAColumns V back into the collection tube and centrifuge at 12,000 rpm for 2 min;

[0070] (9) Transfer FastPure RNA Columns V to a new RNase-free Collection Tubes 1.5mL centrifuge tube, add 30-100μL of RNase-free ddH2O preheated at 65℃ to the center of the adsorption column membrane, let stand at room temperature for 5min, and centrifuge at 12,000rpm for 1min.

[0071] (10) Determine RNA concentration. The concentration of RNA and the ratios of A260 / 280 and A260 / 230 were determined using a NanoDrop 2000 micro-spectrophotometer.

[0072] (11) Store at -80℃, or use directly for the following reverse transcription experiments.

[0073] (II) First-strand cDNA synthesis was performed using a reverse transcription kit (R412, Novizan, Nanjing). The specific method is as follows:

[0074] (1) Prepare the mixture shown in Table 1 in a 0.2 mL RNase-free centrifuge tube (where the total RNA is obtained from the extraction in step (I); if the RNA is stored at -80℃, it must be placed on ice to thaw slowly).

[0075] Table 1

[0076]

[0077] (2) Gently pipette and mix well, place in a PCR instrument and heat at 65°C for 5 min, then quickly place on ice to cool, and let stand on ice for 2 min to complete the denaturation and annealing reaction;

[0078] (3) Genomic DNA removal. Add 2 μL of 5×g DNAwiper Mix to the centrifuge tubes, gently mix with a pipette, and place in a PCR instrument at 42℃ for 2 min;

[0079] (4) Prepare the first-strand cDNA synthesis reaction solution according to Table 2;

[0080] Table 2

[0081]

[0082] (5) Gently mix with a pipette. Perform the first-strand cDNA synthesis reaction on a PCR instrument according to the conditions in Table 3;

[0083] Table 3

[0084]

[0085] (6) The synthesized reverse transcription product cDNA is used for subsequent PCR reactions or stored at -20℃ for later use.

[0086] (III) Genomic DNA was extracted from barley and rice using the Edwards method, as follows:

[0087] (1) Place two 3mm grinding beads in a 2mL centrifuge tube. Take about 50mg of tender leaves of barley H559 and rice ZH11, cut them into small pieces and place them in the tube. Grind the samples using a grinder with the following parameters: 40Hz, 60s, twice. After grinding, add 400μL Edwards buffer (200mM Tris-HCl pH 7.5, 250mM NaCl, 25mM EDTA and 0.5% SDS).

[0088] (2) Mix well by inverting the metal bath at 65℃ for 10 minutes (or oven at 65℃ for 15 minutes) with a 5-minute interval.

[0089] (3) Centrifuge at 13,000 rpm for 5 min. Transfer 200 μL of the supernatant to a new 1.5 mL centrifuge tube, being careful not to aspirate any precipitate;

[0090] (4) Add an equal amount of 200 μL isopropanol, mix by inversion, and let stand in a -20℃ refrigerator for 10 min;

[0091] (5) Centrifuge at 13,000 rpm for 5 min. Discard the supernatant and air dry in a clean bench.

[0092] (6) Add 60-100 μL ddH2O, vortex to dissolve DNA, and store at 4°C for later use.

[0093] (iv) Obtaining CDS and promoter sequences

[0094] The full-length cDNA of the HmSTG1 gene from barley and the OsSTG1 homolog from rice, along with the reference sequence of the 2kb upstream ATG promoter, were obtained from the NGDC and NCBI databases. For CDS cloning, gene-specific primers (Hm-TF and Hm-TR; Os-TF and Os-TR) were designed, and high-fidelity enzymes (P510, Novizan, Nanjing) were used to amplify the cDNA synthesized by reverse transcription in step (II) as templates. For promoter cloning, gene-specific primers (HmPro-TF and HmPro-TR; OsPro-TF and OsPro-TR) were designed, and high-fidelity enzymes were used to amplify the genomic DNA extracted in step (III) as templates.

[0095] Hm-TF:5'- GGATCC ATGGAGGCGGGGGCGGAG-3' (SEQ ID NO.3); The underlined part is the BamHI restriction site;

[0096] Hm-TR:5'- CCCGGG TCAGTTGCCTCGCGGCGG-3' (SEQ ID NO.4); The underlined part is the XmaI restriction site;

[0097] Os-TF:5'- GGATCC ATGGACAATCCCGAGGCG-3' (SEQ ID NO.5); The underlined part is the BamHI restriction site;

[0098] Os-TR:5'- CCCGGG TCATCGATCAGCAGCGCTG-3'(SEQ ID NO.6); The underlined part is the XmaⅠ restriction site;

[0099] HmPro-TF: 5'- GGATCC CGCCAATTCTTTCTCGCCC-3' (SEQ ID NO.7); The underlined part is the BamHI restriction site;

[0100] HmPro-TR: 5'- CCCGGG CGACGGCCGGCCGGCGAG-3' (SEQ ID NO.8); The underlined part is the XmaI restriction site;

[0101] OsPro-TF: 5'- GGATCC TGTACTCCACGCAGCATTCG-3' (SEQ ID NO. 9); The underlined part is the BamHI restriction site;

[0102] OsPro-TR: 5'- CCCGGG GGATTACTGATGAGGAGGAGGAAG-3'(SEQ ID NO.10); the underlined part is the XmaⅠ restriction site.

[0103] The PCR amplification system is shown in Table 4 (this system was used for all the double primer PCR reactions performed below).

[0104] Table 4

[0105]

[0106] PCR reaction program: 98℃ pre-denaturation for 30 seconds, cycling parameters are 98℃ denaturation for 10 seconds, 58℃ annealing for 5 seconds, 72℃ extension for 20 seconds, for 35 cycles; 72℃ complete extension for 1 min.

[0107] After the PCR reaction, 1% agarose gel electrophoresis was performed to detect the presence of bands of appropriate size. The results are as follows: Figure 1 As shown in Figure B, the CDS band size of barley HmSTG1 and rice OsSTG1 is around 3.4 kb, and the promoter fragment band size of both is around 2.1 kb, which is consistent with the expected size.

[0108] The product was then subjected to gel extraction (following the TaKaRa "MiniBEST Agarose Gel DNA Extraction Kit" procedure), vector ligation (4 μL of the gel-extracted product was ligated to the pMD19-T vector, following the instructions of the "pMD19-T Vector Cloning Kit"), transformation (the ligation product was transformed into E. coli competent cells DH5α, and cultured upside down at 37°C for 12-14 h on LB agar plates containing ampicillin; single colonies were picked and cultured in LB liquid medium for 3-5 h), plasmid DNA was extracted using alkaline lysis, and enzyme digestion was performed (BamHI and XmaI double digestion for identification). Sequencing was then performed (the plasmids with correct enzyme digestion were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing). After sequencing, the sequences were aligned using DNAMAN software, yielding the genes HmSTG1 and OsSTG1, with their CDS sequences shown in SEQ ID NO.1 and SEQ ID NO.11; their promoter sequences are shown in SEQ ID NO.12 and SEQ ID NO.13.

[0109]

[0110]

[0111]

[0112]

[0113] Preserve the plasmid DNA of correctly sequenced single clones pMD19-T-HmSTG1, pMD19-T-OsSTG1, pMD19-T-HmSTG1Pro, and pMD19-T-OsSTG1Pro at -20℃ for subsequent functional verification experiments.

[0114] Example 2

[0115] Amino acid sequence analysis and cluster analysis of HmSTG1 protein from barley.

[0116] (1) The CDS length of the HmSTG1 gene is 3,420 bp. Sequence analysis using VectorNTI software shows that it encodes 1,139 amino acids, and the amino acid sequence is shown in SEQ ID NO.2. The predicted molecular weight is approximately 125.92 kDa. The isoelectric point pI is 6.27.

[0117]

[0118] Using the database and analysis software of the InterProScan website, the functional and conserved domains of the HmSTG1 protein were analyzed. The results showed that the HmSTG1 protein contains the IPR006153 / PF00999 domain (annotated as Na / HExchanger, i.e., Na). + / H + (Reverse transporter) and IPR000595 / PF00027 domain (annotated as cNMP binding), such as Figure 1 As shown in Figure A.

[0119] (2) Searching databases such as NCBI, NGDC, and Ensembl yielded the following results: HmSTG1(HORMACH01G00017900), HmSTG1_L1(HORMACH03G01231600), HmSTG1_L2(HORMACH07G03884300), HmSTG1_L3(HORMACH07G03986800), HvSTG1(HORVU3Hr1G003150), and PtSTG1(Pt_Chr0401412). The amino acid sequences of OsSTG1 (rice LOC_Os12g44360), SvSTG1 (foxtail grass SEVIR_3G427000v2), BdSTG1 (braperis lanceolata BRADI_4g00290v3), CqSTG1 (quinoa XP_021727954.1), SsSTG1 (Suaeda salsa AHJ14584.1), GmSTG1 (soybean GLYMA_08G092000), AtSTG1 (Arabidopsis thaliana AT2G01980), and EsSTG1 (Salt mustard XM_006395762.2) were determined.

[0120] (3) The amino acid sequences were compared using MUSCLE software (https: / / www.ebi.ac.uk / Tools / msa / muscle / ), and then phylogenetic trees of STG1 proteins from different species were constructed using the Neighbor-Joining Algorithm (NJ) using MEGA software (https: / / www.megasoftware.net / ). It was found that HmSTG1 in *Hygrophila spp.* was most closely related to HvSTG1 in barley, PtSTG1 in *Imperata cylindrica*, and HmSTG1_L1 and HmSTG1_L3 in *Hygrophila spp.* Figure 1 (C)

[0121] Example 3

[0122] Expression levels of HmSTG1 and its three homologs in barley under salt stress

[0123] (1) The data used here were downloaded from NCBI, accession number PRJNA639318. The experimental procedure was as follows: Barley H559 seedlings were cultured in 1 / 5 Hoagland nutrient solution until the four-leaf stage. NaCl was added to the nutrient solution daily at concentrations of 50 and 100 mM, respectively, to final concentrations of 150 and 300 mM NaCl. The control group did not receive any NaCl. After 4 days of treatment, RNA was extracted from roots and aboveground tissues. After passing quality inspection, library construction and sequencing were performed.

[0124] (2) The sequencing reads were processed using the RNA-seq data analysis software HTSeq (https: / / htseq.readthedocs.io / en / ) to obtain the expression level of each gene (expressed as FPKM value, i.e., the number of fragments per kilobase length from a gene per million fragments). The expression levels of HmSTG1 and its three homologous genes HmSTG1_L1, HmSTG1_L2, and HmSTG1_L3 were extracted and plotted as bar charts. The results are as follows: Figure 1 As shown in D and E, only the expression level of the HmSTG1 gene was significantly upregulated in the roots and shoots under salt stress.

[0125] Example 4

[0126] Na of HmSTG1 protein + Transshipment capacity analysis

[0127] To verify whether the HmSTG1 protein of barley contains Na... + Transport capacity, transporting the full-length HmSTG1 protein and its truncated form (with the self-inhibition domain removed) in yeast Na... + The target protein was expressed in the transport-deficient strain axt3k. The growth of the negative control (transformed with an empty vector) and the recombinant yeast strain on salt (NaCl) medium was compared to determine whether the target protein possessed Na+. + Transport capacity. Rice OsSTG1 protein was used as a positive control.

[0128] (I) Expression vector construction and yeast transformation

[0129] (1) Specific primers were designed and PCR amplification was performed using the pMD19-T-HmSTG1 plasmid as a template with the Novozymes P510 high-fidelity enzyme. After electrophoresis, the PCR purified products of the full-length CDS (Hm-p416-F and Hm-p416-R) and the truncated CDS (Hm-p416-F and HmS-p416-R) of HmSTG1 were obtained by gel recovery. Specific primers were designed and PCR amplification was performed using the pMD19-T-OsSTG1 plasmid as a template with the above high-fidelity enzyme. After electrophoresis, the PCR purified products of the full-length CDS (Os-p416-F and Os-p416-R) and the truncated CDS (Os-p416-F and OsS-p416-R) of OsSTG1 were obtained by gel recovery.

[0130] Hm-p416-F:5'-caggaattcgatatc AAGCTT ATGGAGGCGGGGGCGGAG-3' (SEQ ID NO. 14); lowercase letters are homologous arms, and the underlined part is the HindIII restriction site;

[0131] Hm-p416-R:5'-gtcgacggtatcgat AAGCTT TCAGTTGCCTCGCGGCGG-3' (SEQ ID NO. 15); lowercase letters are homologous arms, and the underlined part is the HindIII restriction site;

[0132] HmS-p416-R:5'-gtcgacggtatcgat AAGCTT TCAGCTCTGCGTGCGTGGCGG-3' (SEQ ID NO. 16); lowercase letters are homologous arms, and the underlined part is the HindIII restriction site;

[0133] Os-p416-F:5'-caggaattcgatatc AAGCTT ATGGACAATCCCGAGGCG-3' (SEQ ID NO. 17); lowercase letters are homologous arms, and the underlined part is the HindIII restriction site;

[0134] Os-p416-R:5'-gtcgacggtatcgat AAGCTT TCATCGATCAGCAGCGCTG-3' (SEQ ID NO. 18); lowercase letters are homologous arms, and the underlined part is the HindIII restriction site;

[0135] OsS-p416-R:5'-gtcgacggtatcgat AAGCTTTCAGCTTTGTGTCCGAGGCAG-3' (SEQ ID NO. 19); lowercase letters are homologous arms, and the underlined part is the HindIII restriction site.

[0136] (2) Single enzyme digestion of the p416-GPD empty vector plasmid was performed using HindIII restriction enzyme. Figure 2 After electrophoresis, the gel was recovered to obtain the linearized carrier.

[0137] (3) Homologous recombination reaction was carried out using a seamless cloning kit (C116, Novizan, Nanjing). The reaction solution was prepared according to Table 5.

[0138] Table 5

[0139]

[0140] (4) Gently mix with a pipette and place in a PCR instrument at 50°C for 5 min; then cool to 4°C or immediately place on ice to cool.

[0141] (5) The recombinant product was transformed into Escherichia coli DH5α and cultured on LB plates containing ampicillin for 12-16 h. Single clones were selected and plasmids were extracted using the alkaline lysis method. The plasmids of the correctly sequenced single clones were used to transform yeast.

[0142] (6) The above plasmids were transformed into yeast Na using the PEG / LiAc method. + The defective strain axt3k competent cells (CC323, Coolplay, Beijing) were transported, and the procedures were performed according to the instructions provided with the kit.

[0143] (II) Growth of recombinant yeast and wild-type defective yeast strains on salt (NaCl) medium

[0144] Recombinant yeast and yeast strains transformed with empty vectors were pre-cultured in liquid AP medium (PM4050L, Cooler Master, Beijing) until OD500. 600 =0.4. Perform three orders of magnitude 10-fold serial dilutions, dropping 10 μL onto AP medium plates containing 0, 25, 50, and 75 mM NaCl, and incubate upside down at 28°C for 3–5 days before photographing. Results are as follows. Figure 3 As shown, the recombinant yeast transformed with full-length HmSTG1 and OsSTG1 proteins showed no difference in growth status compared to the negative control (transformed with empty vector); however, the recombinant yeast transformed with truncated HmSTG1 proteins exhibited significantly stronger growth than the negative control, with the recombinant yeast transformed with truncated HmSTG1 protein showing superior growth compared to the yeast transformed with truncated OsSTG1 protein. This indicates that the HmSTG1 protein from barley has strong Na+ resistance. + Transshipment capacity.

[0145] Example 5

[0146] Construction of HmSTG1 gene expression vector

[0147] (1) Specific primers were designed to perform PCR amplification using pMD19-T-HmSTG1Pro plasmids (HmP-2300-F and HmP-2300-R) and pMD19-T-HmSTG1 plasmids (HmC-2300-F and HmC-2300-R) as templates with P510 high-fidelity enzyme. The purified PCR products containing the HmSTG1 promoter and full-length CDS were obtained after gel electrophoresis. Using specific primers, PCR amplification was performed with pMD19-T-OsSTG1Pro plasmids (OsP-2300-F and OsP-2300-R) and pMD19-T-HmSTG1 plasmids (OsC-2300-F and HmC-2300-R) as templates using P510 high-fidelity enzyme. After electrophoresis, the PCR purified products of the OsSTG1 promoter and the full-length CDS of HmSTG1 were obtained by gel recovery.

[0148] HmP-2300-F:5'-tacgaattcgagctc GGTACC CGCCAATTCTTTCTCGCCC-3' (SEQ ID NO. 20); lowercase letters are homologous arms, and the underlined part is the KpnI restriction site;

[0149] HmP-2300-R: 5'-tccgcccccgcctccatCGACGGCCGGCCGGCGAG-3' (SEQ ID NO. 21);

[0150] HmC-2300-F: 5'-tcgATGGAGGCGGGGGCGGAG-3' (SEQ ID NO. 22);

[0151] HmC-2300-R:5'-tctagaggatccccg GGTACC TCAGTTGCCTCGCGGCGG-3' (SEQ ID NO. 23); lowercase letters are homologous arms, and the underlined part is the KpnI restriction site.

[0152] OsP-2300-F:5'-tacgaattcgagctc GGTACC TGTACTCCACGCAGCATTCG-3'(SEQ ID NO.24); lowercase letters are homologous arms, and the underlined part is the KpnI restriction site.

[0153] OsP-2300-R: 5'-cgcctccatGGATTACTGATGAGGAGGAGGAAG-3' (SEQ ID NO. 25);

[0154] OsC-2300-F: 5'-atcagtaatccATGGAGGCGGGGGCGGAG-3' (SEQ ID NO. 26).

[0155] (2) Single enzyme digestion of the empty vector pCAMBIA2300 plasmid was performed using KpnI restriction enzyme. Figure 4 After electrophoresis, the gel was recovered to obtain the linearized carrier.

[0156] (3) Homologous recombination with two-fragment insertion was performed using a seamless cloning kit (C116, Novizan, Nanjing); the recombination product was transformed into E. coli DH5α and cultured on LB plates containing kanamycin for 12-16 h. Single clones were picked and plasmids were extracted using the alkaline lysis method. The plasmids of the correctly sequenced single clones were stored at -20℃; finally, the expression vector HmSTG1, which drives the HmSTG1 gene promoter of barley, was obtained. Pro The expression vector Os, which drives the HmSTG1 gene expression vector driven by the rice OsSTG1 gene promoter. Pro ( Figure 5 (A)

[0157] (4) The carrier Hm Pro and Os Pro The plasmid was introduced into Agrobacterium strain EHA105 using the freeze-thaw method, and positive single clones were selected for subsequent rice genetic transformation.

[0158] Example 6

[0159] The acquisition of genetically modified rice

[0160] The genetic transformation of rice was outsourced to Wuhan Boyuan Biotechnology Co., Ltd., and the main operational steps are as follows:

[0161] (1) The rice cultivar ZH11 was used as the baseline material. Rice seeds were dehulled and soaked in a 5% sodium hypochlorite solution containing 1 drop of Tween 20 for 15 minutes for disinfection, then rinsed 5 times with sterile water. They were then disinfected again with a 5% sodium hypochlorite solution without Tween for 15 minutes and rinsed 10 times with sterile water to thoroughly remove any sodium hypochlorite residue. After blotting the seeds with sterile filter paper, the embryos were inserted obliquely onto NBD medium, with 30 seeds per dish. The medium was incubated at 28℃ for 16 hours of light / 8 hours of darkness.

[0162] (2) Take 10 μL of Agrobacterium tumefaciens bacterial culture and add it to AAM liquid medium containing 50 μg / mL kanamycin and 20 μg / mL rifampin. Incubate at 28°C in the dark with shaking at 200 r / min until OD. 600 The concentration was 0.4–0.6. The bacterial cells were collected by centrifugation and resuspended in liquid NBD-As to OD. 600=0.1. Select callus tissue of suitable size and good condition, immerse it in Agrobacterium resuspension for 30 min, blot off excess bacterial solution on sterile filter paper, and transfer the callus tissue to solid NBD-As medium with a layer of sterile filter paper, and incubate in the dark for 3 days. Wash the callus three times with sterile water, then soak it in sterile water containing 150 mg / L Timentin for 10 min, blot off excess water, and transfer it to NBD-As medium for recovery culture for 4 days. Then transfer it to NBD-TK ​​medium containing the screening antibiotic G418 for continued selection culture, and subculture every 2 weeks. G418 resistant callus tissue is transferred to differentiation medium RE1. When the catalyzed adventitious shoots grow to 3-5 cm seedlings, the seedlings are cut and transferred to rooting medium RE2 for rooting induction to obtain T0 generation seedlings.

[0163] (3) The formulations of the culture media used for rice tissue culture and transformation are shown in Table 6.

[0164] Table 6

[0165]

[0166] Example 7

[0167] Molecular identification of genomic DNA in transgenic rice (positive detection)

[0168] Hm was extracted using the Edwards method in step (iii) of Example 1. Pro and Os Pro Genomic DNA from different lines of transgenic rice and wild-type rice was used as a template. PCR amplification was performed using upstream primers (HmPro-F; OsPro-F) on the HmSTG1 and OsSTG1 promoter sequences, and the specific downstream primer HmSTG1-R on the HmSTG1 gene CDS. Transgenic plants were identified by amplification of clear bands.

[0169] HmPro-F: 5'-CCGACGCTCGATGGAGGTTT-3' (SEQ ID NO. 27);

[0170] OsPro-F: 5'-GGAGCTCAAGTGAGGAGGCTG-3' (SEQ ID NO. 28);

[0171] HmSTG1-R: 5'-AGCTTGCCGAGGCCATGCTT-3' (SEQ ID NO. 29).

[0172] Example 8

[0173] Evaluation of salt tolerance in genetically modified rice

[0174] To determine the function of transgenic rice plants, the salt tolerance of the T2 generation transgenic rice lines was evaluated.

[0175] (1) Rice seed germination and pre-culture

[0176] From the two types of transgenic rice lines obtained (Hm) Pro and Os Pro Two T2 generation lines were randomly selected from each of the four lines. Seeds of uniform size and plumpness from these four lines and the wild-type ZH11 were selected, sterilized with 3% hydrogen peroxide, soaked at 30℃ for germination, and treated with 0.5mM calcium chloride to promote root growth. The seeds were then transferred to 1 / 2 Kimura B rice nutrient solution (pH 5.6) and cultured in the dark at 28℃. Once the sprouts reached 2-3cm in length, they were introduced into the light environment under the following conditions: 10h light / 14h darkness, 28℃ / 22℃. The nutrient solution was changed every three days.

[0177] (2) Relative expression level of HmSTG1 gene in transgenic rice

[0178] Genomic DNA molecular identification was performed according to the method in Example 7. Total RNA was extracted from the roots of four transgenic lines and wild-type plants under normal growth (Control) and 200 mM NaCl treatment conditions, and reverse transcribed into cDNA, using the same methods as steps one and two in Example 1. Specific primers for the HmSTG1 gene, HmSTG1-qF and HmSTG1-qR, and internal control primers for rice OsActin, OsActin-F and OsActin-R, were designed. Semi-quantitative RT-PCR detection was performed using the above primers.

[0179] HmSTG1-qF: 5'-GCTGCGGGGGAAGAAGCTAA-3' (SEQ ID NO. 30);

[0180] HmSTG1-qR: 5'-CGAGCAACCCAGTCTTCCCG-3' (SEQ ID NO. 31);

[0181] OsActin-F: 5'-TGCTATGTACGTCGCCATCCAG-3' (SEQ ID NO. 32);

[0182] OsActin-R: 5'-AATGAGTAACCACGCTCCGTCA-3' (SEQ ID NO. 33).

[0183] The reaction program was as follows: PCR reaction program: 98°C pre-denaturation for 30 seconds, cycling parameters: 98°C denaturation for 10 seconds, 58°C annealing for 5 seconds, 72°C extension for 5 seconds, for 30 cycles; 72°C final extension for 1 min. The amplified products were analyzed by electrophoresis on a 1.0% agarose gel, and band brightness was detected using a Bio-Rad Gel Doc XR gel imaging system to determine the expression level of the HmSTG1 gene in these transgenic lines.

[0184] The results showed that the expression levels of the internal reference gene OsActin in different lines did not differ significantly under normal and salt treatment conditions; the expression level of the HmSTG1 gene in Hm Pro and Os Pro The transgenic rice was successfully expressed in all lines, but the expression levels varied among different lines, with Hm showing the highest expression level. Pro -1 and Os Pro -1 was expressed at a higher level ( Figure 5 (B)

[0185] (3) Growth of HmSTG1 gene-transformed rice seedlings under normal conditions and salt stress treatment

[0186] Three-week-old wild-type and transgenic rice lines were treated for 20 days in normal nutrient solution and culture solution containing 200 mM NaCl, and root and aboveground dry weights were measured. Results showed no significant difference in growth and dry weight between transgenic and wild-type rice under normal conditions. Figure 5 C; Figure 6 (A); Hm under 200mM salt treatment Pro and Os Pro The genetically modified rice lines showed significantly stronger growth than the wild type, and the root and aboveground dry weight of the genetically modified rice was on average more than twice that of the wild type. Figure 5 D; Figure 6 (B)

[0187] (4) Na and K content in tissues of HmSTG1 gene-transformed rice seedlings under salt stress treatment

[0188] The dried root and aerial samples were digested using concentrated nitric acid. The procedure was as follows: the sample was placed at the bottom of a 20 mL digestion tube, 3 mL of concentrated HNO3 was added, and then the tube was placed in a constant temperature metal bath (DTU-2CN, TAITEC, Japan). The temperature was first raised to 80 °C, and after the sample dissolved to a liquid state, the temperature was raised to 120 °C until the sample was completely dissolved to a liquid state (generally requiring 2-3 hours). The acid was then removed by heating until 1-1.5 mL of liquid remained in the test tube. After cooling, the volume was adjusted to 20 mL with deionized water. Elemental content was determined using inductively coupled plasma mass spectrometry (iCAP-RQ, Thermo Fisher Scientific, USA). The results showed that Hm under 200 mM salt treatment... Pro and Os Pro Aboveground Na of transgenic rice lines + The concentrations of K in both roots and aboveground parts were significantly lower than those in the wild type. + The concentration was significantly higher than that of the wild type. Further analysis revealed that, compared to the wild type, the transgenic line K... + / Na + Higher than, the root system has a higher affinity for Na. + The absorption and translocation to the above-ground parts are lower. Figure 6 Medium CG).

[0189] The above results indicate that the HmSTG1 gene from barley, when transferred into rice, reduces the plant's root system's susceptibility to Na+. + It increases the absorption and translocation of K in the body to the terrestrial parts, while also increasing the amount of K absorbed. + Concentration and K + / Na + This increased the salt tolerance of the transgenic plants.

[0190] In summary, this invention has isolated a Na group from the halophyte barley. + / H + The antitransporter protein encoding gene HmSTG1 was discovered, and for the first time, it was found that gene HmSTG1 has a strong Na+ binding capacity. + Transport capacity, and maintain low Na levels in the plant + Concentration and high K + / Na + This gene plays an important role in resisting high salt stress. It can be transformed into annual crops (herbaceous plants) such as rice, corn, wheat, barley, and soybeans, or woody plants such as apples and pears, to improve the salt tolerance of transgenic plants, thereby increasing their yield and quality, which has significant economic value and social benefits.

[0191] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. 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 claims of the present invention.

Claims

1. The application of the HmSTG1 gene in improving plant salt tolerance, characterized in that, The nucleotide sequence of the HmSTG1 gene is shown in SEQ ID NO.1, and the plant is rice.

2. The application of HmSTG1 protein in improving plant salt tolerance, characterized in that, The amino acid sequence of the HmSTG1 protein is shown in SEQ ID NO.2, and the plant is rice.

3. The application of recombinant expression vectors or recombinant gene expression cassettes containing the HmSTG1 gene in improving plant salt tolerance, characterized in that... The nucleotide sequence of the HmSTG1 gene is shown in SEQ ID NO.1, and the plant is rice.

4. The application of the HmSTG1 gene, HmSTG1 protein, recombinant expression vector containing the HmSTG1 gene, or recombinant gene expression cassette containing the HmSTG1 gene in salt-tolerant plant breeding, characterized in that... The nucleotide sequence of the HmSTG1 gene is shown in SEQ ID NO.1, the amino acid sequence of the HmSTG1 protein is shown in SEQ ID NO.2, and the plant is rice.

5. A method for improving the salt tolerance of plants, characterized in that, The HmSTG1 gene was introduced into a plant to upregulate its expression. The nucleotide sequence of the HmSTG1 gene is shown in SEQ ID NO.

1. The plant is rice.

6. A method for breeding salt-tolerant plants, characterized in that, The HmSTG1 gene was introduced into a plant to upregulate its expression. The nucleotide sequence of the HmSTG1 gene is shown in SEQ ID NO.

1. The plant is rice.

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