Application of halophyte sea barley HmSTG1 gene in improvement of plant salt tolerance

Abstract

The invention discloses an application of a halophyte sea barley HmSTG1 gene in improving the salt tolerance of plants, and belongs to the technical field of molecular biology and plant genetic engineering. The sequence of a coding region of the HmSTG1 gene is as shown in SEQ ID NO. 1; the amino acid sequence of the protein coded by the gene is shown as SEQ ID NO. 2. According to the invention, a Na < + > / H < + > antitransport protein coding gene HmSTG1 is separated from halophyte sea barley, and the first research finds that the gene HmSTG1 has strong Na < + > transport capability, and plays an important role in maintaining low Na < + > concentration and high K < + > / Na < + > ratio in plants such as rice and the like so as to resist external high salt stress. The gene HmSTG1 can be used for transforming crops such as rice, corn, wheat and soybeans or woody plants such as apples and pears, so that the salt tolerance of transgenic plants is improved, the yield and quality of the transgenic plants are further improved, and the gene HmSTG1 has great economic value and social benefit.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology and plant gene engineering, and in particular to the application of a halophyte sea barley HmSTG1 gene in improving the salt tolerance of the plant. Background Art

[0002] The information disclosed in this section is only intended to enhance understanding of the general background of the invention and is not necessarily considered to be an admission or any form of suggestion that this information constitutes the prior art known to a person of ordinary skill in the art.

[0003] Soil salinization is one of the main abiotic stresses facing global agricultural production. At present, the world's saline soil area is large and widely distributed, with more than 424 million hectares of topsoil (0-30 cm) and 833 million hectares of subsoil (30-100 cm) having been salinized, accounting for 4.4% and 8.7% of the total land area, respectively. In addition, factors such as unreasonable farming systems, irrigation patterns, and excessive emissions of industrial pollutants have further aggravated the degree of salinization of cultivated land. Breeding salt-tolerant crop varieties is one of the most effective ways to utilize salinized soils, improve crop productivity, and ensure food security.

[0004] Under the influence of 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 and improvement. The rich genetic diversity of wild resources of the grass family can provide a valuable gene pool to break this bottleneck. Introducing excellent exogenous traits / genes from wild relatives into cultivated crops can breed new varieties with breakthrough improvements in specific traits. For example, introducing Na from the wild relative of wheat, Echinops triticum + The transporter encoding gene TmHKT1;5-A was introduced into the durum wheat variety Tamaroi, which increased the yield of wheat grown on saline soil by nearly 25%.

[0005] Maintain K + / Na + Balance is one of the main physiological mechanisms of plant salt tolerance. Plants achieve ion balance through the action of related proteins such as ion channels, transporters and proton pumps. For example, the SOS, HKT and NHX protein families play an important role in Na + and K + Transport and Na + For example, halophytes such as Suaeda salsa and Atriplex cannabinoides play an important role in compartmentalization through Na + Transport proteins such as SOS1 (localized in the plasma membrane) and NHX1 (localized in the vacuole membrane) and proton pumps (H + -ATPase) genes to respond to excessive Na + .

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

[0007] The purpose of the present invention is to provide an application of the halophyte sea barley HmSTG1 gene in improving the salt tolerance of plants, so as to solve the problems existing in the above-mentioned prior art.

[0008] To achieve the above object, the present invention provides the following solutions:

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

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

[0011] A third technical solution of the present invention is the use of a recombinant expression vector, an overexpression vector, an interference vector, a recombinant virus, a recombinant bacterium or a recombinant gene expression cassette containing the HmSTG1 gene in improving the salt tolerance of plants.

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

[0013] A fifth technical solution of the present invention is a product for improving plant salt tolerance, comprising an HmSTG1 gene, an HmSTG1 protein, a recombinant expression vector comprising 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, which comprises 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, comprising 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] The present invention isolated a Na + / H + The antiporter encoding gene HmSTG1 was first found to have a strong Na + transport capacity and maintain low Na + Concentration and high K + / Na +The gene can be transformed into annual crops (herbs) 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 improving their yield and quality, which has great economic value and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1The structure and phylogenetic tree analysis of the HmSTG1 gene of the halophyte Hordeum marinum, as well as the expression levels in the roots and aerial parts under salt stress; A represents the schematic diagram of the structure of the HmSTG1 gene of Hordeum marinum; B represents the agarose gel electrophoresis of the promoter (Pro) and coding sequence (CDS) PCR amplification fragments of the HmSTG1 gene of Hordeum marinum and the OsSTG1 gene of rice; C represents the phylogenetic tree analysis of the protein encoded by the HmSTG1 gene of Hordeum marinum; D and E represent the expression levels of HmSTG1 and its three homologous genes in the roots (D) and aerial parts (E) of Hordeum marinum plants under normal (0 mM NaCl) and salt stress treatment (150 and 300 mM NaCl). The RNA-seq data of Hordeum marinum plants under control and salt stress conditions were processed to obtain the FPKM value of gene expression. M: DL5000 DNAMarker; 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: Hordeum vulgare HORMACH03G01231600; HmSTG1_L2: Hordeum vulgare HORMACH07G03884300; HmSTG1_L3: Hordeum vulgare HORMACH07G03986800; HvSTG1: Hordeum vulgare HORVU3Hr1G003150; PtSTG1: Puccinellia tenuifolia Pt_Chr0401412; OsSTG1: Rice LOC_Os12g44360; SvST G1: Setaria viridis SEVIR_3G427000v2; BdSTG1: Brachypodium distachyon BRADI_4g00290v3; CqSTG1: Quinoa quinoa XP_021727954.1; SsSTG1: Suaeda salsa AHJ14584.1; GmSTG1: Soybean GLYMA_08G092000; AtSTG1: Arabidopsis thaliana AT2G01980; EsSTG1: Halona salsa XM_006395762.2; *: significant difference (P<0.05).

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

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

[0022] Figure 4 The map of pCAMBIA2300 plant expression vector and the insertion position of gene sequence. The black boxes indicate the restriction sites used for the construction of recombinant vector.

[0023] Figure 5 The salt tolerance evaluation of transgenic rice lines introduced with the HmSTG1 gene of the halophyte Hordeum vulgare at the seedling stage. A represents the schematic diagram of the expression vectors driving the HmSTG1 gene using the promoters of the rice and Hordeum vulgare STG1 genes respectively; B represents the semi-quantitative RT-PCR detection of wild-type rice and transgenic rice lines; C and D represent the growth of three-week-old wild-type rice and transgenic rice lines after being treated in normal nutrient solution (C) and culture solution containing 200mM NaCl (D) for 20 days. M: DL2000 DNA marker; WT: wild-type control; HmPro-1, -2: transgenic rice lines driven by the promoter of the Hordeum vulgare HmSTG1 gene; OsPro-1, -2: transgenic rice lines driven by the promoter of the rice OsSTG1 gene.

[0024] Figure 6 The dry weight of roots and aboveground parts and the content of Na and K in transgenic rice lines introduced with the HmSTG1 gene of the halophyte Hordeum vulgare under salt stress. A and B represent the dry weight of roots and aboveground parts of three-week-old wild-type rice and transgenic rice lines treated in normal nutrient solution (A) and culture solution containing 200 mM NaCl (B) for 20 days; C and D represent the Na content in roots and aboveground parts of three-week-old wild-type rice and transgenic rice lines treated in culture solution containing 200 mM NaCl for 20 days; EG represents the K content in aboveground parts of three-week-old wild-type rice and transgenic rice lines treated in culture solution containing 200 mM NaCl for 20 days. + / Na +Ratio (E), root Na uptake (F) and Na transport from roots to shoots (G). WT: wild type control; HmPro-1, -2: transgenic rice lines with the HmSTG1 gene promoter driving the HmSTG1 gene of Hordeum vulgare; OsPro-1, -2: transgenic rice lines with the HmSTG1 gene promoter driving the OsSTG1 gene of rice; RDW: root dry weight. DETAILED DESCRIPTION

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

[0026] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

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

[0030] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0031] The embodiment of the present invention provides the use of the HmSTG1 gene in improving the salt tolerance of plants.

[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 consistency or identity with the nucleotide sequence shown in SEQ ID NO.1 and expresses the same functional protein.

[0033] The nucleotide sequence may be DNA, such as cDNA, gene 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 "identity" used herein refers to sequence similarity to a natural nucleic acid sequence. The identity or consistency can be evaluated using computer software, for example, the BLAST algorithm can be used for determination (Karlin and Altschul. 1993. Proc. Natl. Acad. Sci. USA 90: 5873-5877).

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

[0036] The embodiments of the present invention also provide the use of HmSTG1 protein in improving the salt tolerance of plants.

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

[0038] The HmSTG1 protein can be artificially synthesized, or its encoding gene can be synthesized first and then obtained by biological expression.

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

[0040] The embodiments of the present invention also provide the use of a recombinant expression vector, an overexpression vector, an interference vector, a recombinant virus, a recombinant bacterium or a recombinant gene expression cassette containing the HmSTG1 gene in improving the salt tolerance of plants.

[0041] The recombinant expression vector can be constructed with existing plant expression vectors. The plant expression vector includes binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, such as pCAMBIA1300, pCAMBIA1301, pCAMBIA2300, pCAMBIA2301, pCAMBIA3301, pGreen0029, pBI121, pBin19 or other derived plant expression vectors. When the gene is used to construct a recombinant expression vector, a promoter of the gene or its homologous gene can be added before its transcription initiation nucleotide, or any enhanced, constitutive, tissue-specific or inducible promoter, such as cauliflower mosaic virus (CaMV) 35S promoter, ubiquitin gene Ubiquitin promoter (pUbi), stress-induced promoter rd29A, etc., which can be used alone or in combination with other plant promoters; in addition, when the gene of the present invention is used to construct a recombinant expression vector, an enhancer can also be used, including a translation enhancer or a transcription enhancer.

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

[0043] In some specific embodiments, the plants include rice, corn, wheat, barley, soybean, apple and pear.

[0044] The embodiment of the present invention also provides a product for improving plant salt tolerance, including HmSTG1 gene, HmSTG1 protein, recombinant expression vector, overexpression vector, interference vector, recombinant virus, recombinant bacteria or recombinant gene expression cassette containing HmSTG1 gene.

[0045] The embodiment of the present invention also provides a method for improving the salt tolerance of plants, which comprises exogenously transferring the HmSTG1 gene into the plant or up-regulating the expression of the HmSTG1 gene in the plant genome.

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

[0047] Among them, the method of exogenously transferring the HmSTG1 gene can be: transforming plant cells or tissues with a plant expression vector carrying the gene HmSTG1 by using conventional biological methods such as Ti plasmid, Ri plasmid, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated, etc., and cultivating the transformed plant cells or tissues into complete plants.

[0048] The method for up-regulating the expression of gene HmSTG1 in the plant genome may include: introducing a DNA fragment that can activate or increase the transcription level, translation level or protein activity of gene HmSTG1; or controlling the synthesis of specific small RNA molecules to up-regulate the accumulation of gene HmSTG1 mRNA.

[0049] The specific small RNA molecules can be: microRNA molecules (microRNA, miRNA), small interfering RNA (smal1interfering RNA, siRNA) or artificial miRNA (artificial microRNA, amiRNA) and the like.

[0050] The embodiment of the present invention also provides a salt-tolerant plant breeding method, including the method for improving plant salt tolerance.

[0051] The following steps are involved:

[0052] The HmSTG1 gene is transferred into the starting plant to express the HmSTG1 gene, thereby obtaining a transgenic plant; the transgenic plant has a higher salt tolerance than the starting plant.

[0053] The halophyte Hordeum marinum Huds. is a wild relative of barley and wheat. It is one of the most salt-tolerant species known in the Poaceae family and has a certain degree of interbreeding with common wheat. Therefore, it is a potential gene donor for improving the salt tolerance of crops such as wheat. Studies have found that the expression level of the HmSTG1 gene in Hordeum marinum Huds. is significantly increased after salt stress, but its function has not yet been clarified, and related transgenic research has not yet been carried out. It is unknown whether transgenic plants with increased expression of this gene can grow and develop normally and whether their salt tolerance can be improved.

[0054] The inventor of the present invention specializes in the molecular mechanism of salt tolerance and genetic improvement of gramineous crops. In the process of studying the transcriptional profiles of the halophyte sea barley and the non-halophyte barley and wheat under salt stress, it was found that many ion transport-related genes including STG1 were significantly induced in sea barley, but were lowly expressed or non-induced in barley and wheat. It is believed that the promoter of the sea barley gene may have higher activity or specific regulatory sequences.

[0055] The present invention firstly obtains the halophyte sea barley gene HmSTG1 by using transcriptome data analysis and reverse transcription PCR technology, and then uses yeast Na + Heterologous complementation experiments with the transport-deficient strain axt3k confirmed that the HmSTG1 protein has a strong Na + Transfer capacity.

[0056] Based on the characteristics of rice with high economic value, moderate salt sensitivity and great potential for salt tolerance improvement. The present invention uses rice as the target plant, and uses the promoter of the sea barley gene HmSTG1 and the promoter of the rice homologous gene OsSTG1 to drive the gene HmSTG1 to express in rice. The salt tolerance of the T2 generation transgenic rice obtained by Agrobacterium-mediated method was evaluated and it was found that the expression of the gene HmSTG1 in the transgenic rice can significantly improve its salt tolerance.

[0057] Thus, the present invention obtains a halophyte sea barley Na + / H + The antiporter gene can significantly improve the salt tolerance of rice and was named Salt Tolerant Gene 1 (HmSTG1) according to 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 Fragments of Hordeum vulgare HmSTG1 and Rice OsSTG1 Genes

[0061] (I) Total RNA was extracted using an RNA extraction kit (RC411, Novozymes, Nanjing) as follows:

[0062] (1) Weigh approximately 100 mg of root tissues of H559 and ZH11, respectively, grind with liquid nitrogen, and immediately add 600 μL of Buffer EL. Vortex vigorously for 30 seconds to mix the sample and lysate thoroughly. Centrifuge at 12,000 rpm for 5 minutes, and proceed to the subsequent operation immediately.

[0063] (2) Take about 500 μL of the supernatant and transfer it to 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 the filtrate (about 250 μL) of anhydrous ethanol to the collection tube and shake to mix for 15 seconds;

[0065] (4) Transfer the mixed solution to FastPure RNA Columns V, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate;

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

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

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

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

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

[0071] (10) Determine RNA concentration. Measure RNA concentration, A260 / 280 and A260 / 230 ratios using the NanoDrop2000 micro-spectrophotometer.

[0072] (11) Store at -80°C or use directly in the following reverse transcription experiment.

[0073] (ii) The first-strand cDNA was synthesized using a reverse transcription kit (R412, Novozymes, Nanjing) as follows:

[0074] (1) Prepare the mixed solution shown in Table 1 in a 0.2 mL RNase-free centrifuge tube (the total RNA is obtained by extraction in step (1); if the RNA is stored at -80°C, it must be placed on ice and slowly dissolved);

[0075] Table 1

[0076]

[0077] (2) Mix by gently pipetting, heat at 65°C for 5 min in a PCR instrument, quickly cool on ice, and leave on ice for 2 min to complete the denaturation and annealing reactions;

[0078] (3) Genomic DNA removal: Add 2 μL of 5×g DNA wiper mix to the above centrifuge tube, mix gently with a pipette, and place in a PCR machine at 42°C for 2 min;

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

[0080] Table 2

[0081]

[0082] (5) Mix by gently pipetting. 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°C for future use.

[0086] (III) Extracting genomic DNA of sea barley and rice using the Edwards method, the specific method is as follows:

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

[0088] (2) Place in a metal bath at 65°C for 10 min (or in an oven at 65°C for 15 min), mixing by inverting every 5 min;

[0089] (3) Centrifuge at 13,000 rpm for 5 min. Pipette 200 μL of the supernatant into a new 1.5 mL centrifuge tube, avoiding the precipitate;

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

[0091] (5) Centrifuge at 13,000 rpm for 5 min. Discard the supernatant and place on a clean bench to dry;

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

[0093] (IV) Obtaining CDS and promoter sequences

[0094] According to the NGDC and NCBI database query, the full-length cDNA of the sea barley HmSTG1 gene and the homologous gene OsSTG1 in rice and the 2kb promoter reference sequence upstream of ATG were obtained. For the cloning of CDS, gene-specific primers (Hm-TF and Hm-TR; Os-TF and Os-TR) were designed, and the sea barley and rice cDNA synthesized by reverse transcription in step (ii) were used as templates and PCR amplification was performed using high-fidelity enzymes (P510, Novozymes, Nanjing); for the cloning of promoters, gene-specific primers (HmPro-TF and HmPro-TR; OsPro-TF and OsPro-TR) were designed, and the sea barley and rice genomic DNA extracted in step (iii) were used as templates and PCR amplification was performed using the above-mentioned high-fidelity enzymes.

[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 (the following double-primer PCR reactions all use this system).

[0104] Table 4

[0105]

[0106] PCR reaction program: pre-denaturation at 98°C for 30 seconds, cycle parameters: denaturation at 98°C for 10 seconds, annealing at 58°C for 5 seconds, extension at 72°C for 20 seconds, for 35 cycles; complete extension at 72°C for 1 minute.

[0107] After the PCR reaction, 1% agarose gel electrophoresis was performed to detect whether there were bands of appropriate size. Figure 1 As shown in B. The CDS band size of HmSTG1 and OsSTG1 is about 3.4 kb, and the promoter fragment band size of both is about 2.1 kb, which is consistent with the expected size.

[0108] The product was then subjected to gel recovery (recovery according to the "MiniBEST Agarose Gel DNA Extraction Kit" of TaKaRa Company), vector connection (4 μL of gel recovery product was connected to the pMD19-T vector, and the operation steps were carried out according to the instructions of the "pMD19-T Vector Cloning Kit"), transformation (the connection product was transformed into E. coli competent cells DH5α, and inverted cultured at 37°C for 12-14 hours on LB plate culture medium containing ampicillin; a single colony was picked and cultured in LB liquid culture medium for 3-5 hours), plasmid DNA was extracted by alkaline lysis method, enzyme digestion identification (BamHⅠ and XmaⅠ double enzyme digestion identification), and sequence determination (the plasmid with correct enzyme digestion identification was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing). After sequencing, DNAMAN software was used for sequence alignment to obtain genes HmSTG1 and OsSTG1, whose CDS sequences are shown in SEQ ID NO.1 and SEQ ID NO.11; and their promoter sequences are shown in SEQ ID NO.12 and SEQ ID NO.13.

[0109]

[0110]

[0111]

[0112]

[0113] The plasmid DNA of the correctly sequenced monoclonal pMD19-T-HmSTG1, pMD19-T-OsSTG1, pMD19-T-HmSTG1Pro, and pMD19-T-OsSTG1Pro were saved and stored at -20°C for subsequent functional verification experiments.

[0114] Example 2

[0115] Amino acid sequence analysis and cluster analysis of HmSTG1 protein from Hordeum vulgare

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

[0117]

[0118] The functional domains and conserved domains of HmSTG1 protein were analyzed using the database and analysis software of InterProScan website. The results showed that HmSTG1 protein contained IPR006153 / PF00999 domain (annotated as Na / HExchanger, i.e. Na + / H + antiporter) and IPR000595 / PF00027 domain (annotated as cNMPbinding), e.g. Figure 1 As shown in A.

[0119] (2) HmSTG1 (HormacH01G00017900), HmSTG1_L1 (HormacH03G01231600), HmSTG1_L2 (HormacH07G03884300), HmSTG1_L3 (HormacH07G03986800), HvSTG1 (HormacH0RVU3Hr1G003150), PtSTG1 (Puccinellia parviflora Pt_Chr0401412) were found in databases such as NCBI, NGDC, and Ensembl. , OsSTG1 (rice LOC_Os12g44360), SvSTG1 (Setaria viridis SEVIR_3G427000v2), BdSTG1 (Brachys distachyon BRADI_4g00290v3), CqSTG1 (Chenopodium quinoa XP_021727954.1), SsSTG1 (Suaeda salsa AHJ14584.1), GmSTG1 (Soybean GLYMA_08G092000), AtSTG1 (Arabidopsis thaliana AT2G01980), and EsSTG1 (Halostium halophilum XM_006395762.2).

[0120] (3) The amino acid sequences were aligned using MUSCLE software (https: / / www.ebi.ac.uk / Tools / msa / muscle / ), and a phylogenetic tree was constructed using MEGA software (https: / / www.megasoftware.net / ) using the Neighbor-Joining Algorithm (NJ) method for STG1 proteins in different species. It was found that HmSTG1 in Hordeum vulgare was most closely related to HvSTG1 in barley, PtSTG1 in Puccinellia tenuifolia, and HmSTG1_L1 and HmSTG1_L3 in Hordeum vulgare. Figure 1 Middle C).

[0121] Example 3

[0122] Expression levels of HmSTG1 and its three homologous genes in Hordeum vulgare under salt stress

[0123] (1) The data used here were downloaded from NCBI, with the accession number PRJNA639318. The experimental process was as follows: Sea barley H559 seedlings were cultured in 1 / 5 Hoagland nutrient solution until the four-leaf and one-heart stage, and NaCl was added to the nutrient solution at a concentration of 50 and 100 mM per day, respectively, to a final concentration of 150 and 300 mM NaCl. No NaCl was added to the control group (Control). After 4 days of treatment, RNA was extracted from roots and aerial tissues, and library construction and sequencing were performed after passing quality inspection.

[0124] (2) The reads obtained by sequencing 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, that is, 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 a bar graph was drawn. The results are shown in Figure 2. Figure 1 As shown in D and E, only the expression level of HmSTG1 gene was significantly upregulated in roots and shoots induced by salt stress.

[0125] Example 4

[0126] Na of HmSTG1 protein + Transshipment Capacity Analysis

[0127] In order to verify whether the HmSTG1 protein of sea barley has Na + The full-length HmSTG1 protein and its truncated form (without the autoinhibitory domain) were expressed in yeast Na + The target protein was expressed in the transport-deficient strain axt3k, and the growth of the negative control (transfected with empty vector) and the recombinant yeast strain on a salt (NaCl) medium was compared to determine whether the target protein had NaCl. + 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 using the Norwegian P510 high-fidelity enzyme. After electrophoresis, the PCR purified products of the full-length CDS (Hm-p416-F and Hm-p416-R) and truncated CDS (Hm-p416-F and HmS-p416-R) of HmSTG1 were recovered from the gel; specific primers were designed and PCR amplification was performed using the pMD19-T-OsSTG1 plasmid as a template using the above high-fidelity enzyme. After electrophoresis, the PCR purified products of the full-length CDS (Os-p416-F and Os-p416-R) and truncated CDS (Os-p416-F and OsS-p416-R) of OsSTG1 were recovered from the gel.

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

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

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

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

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

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

[0136] (2) Use HindⅢ endonuclease to perform single restriction enzyme digestion on the p416-GPD empty vector plasmid ( Figure 2 ), and the linearized vector was recovered from the gel after electrophoresis.

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

[0138] Table 5

[0139]

[0140] (4) Mix by gently pipetting and place in a PCR machine at 50°C for 5 min; then cool to 4°C or immediately place on ice.

[0141] (5) The recombinant product was transformed into Escherichia coli DH5α and cultured on LB plates containing ampicillin for 12-16 h. A single clone was selected and the plasmid was extracted using the alkaline lysis method. The single clone plasmid with correct sequencing was used to transform yeast.

[0142] (6) The above plasmid was transformed into yeast Na using the PEG / LiAc method. + The transport-deficient strain axt3k competent cells (CC323, Coolbo, Beijing) were used, and the operation steps were carried out according to the instructions provided by the kit.

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

[0144] The recombinant yeast and the yeast strains without vector were pre-cultured in liquid AP medium (PM4050L, Coolbo, Beijing) to an OD of 600 =0.4. Perform 10-fold gradient dilutions in three orders of magnitude, pipette 10 μL and drop it on AP medium plates containing 0, 25, 50 and 75 mM NaCl, invert and culture at 28°C for 3-5 days and take pictures. Figure 3 As shown in the figure, there is no difference in the growth state between the recombinant yeast transformed with the full-length proteins of HmSTG1 and OsSTG1 and the negative control (transformed empty vector); the growth state of the recombinant yeast transformed with the truncated proteins of both is significantly stronger than the negative control, and the growth of the recombinant yeast transformed with the truncated protein of HmSTG1 is better than that of the transformed yeast with the truncated protein of OsSTG1. This indicates that the HmSTG1 protein of sea barley has a strong Na + Transfer capacity.

[0145] Example 5

[0146] Construction of HmSTG1 gene expression vector

[0147] (1) Design specific primers and use 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 for PCR amplification using P510 high-fidelity enzyme. After electrophoresis, the PCR purified products of HmSTG1 promoter and full-length CDS were recovered from the gel; Specific primers were used to perform PCR amplification using 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 OsSTG1 promoter and HmSTG1 full-length CDS were recovered from the gel.

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

[0152] OsP-2300-F:5'-tacgaattcgagctc GGTACC TGTACTCCACGCAGCATTCG-3' (SEQ ID NO. 24); lowercase letters represent homology arms, and the underlined portion represents 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) KpnⅠ endonuclease was used to digest the pCAMBIA2300 empty plasmid ( Figure 4 ), and the linearized vector was recovered from the gel after electrophoresis.

[0156] (3) A seamless cloning kit (C116, Novozymes, Nanjing) was used to perform a homologous recombination reaction for double-fragment insertion; the recombinant product was transformed into Escherichia coli DH5α and cultured on an LB plate containing kanamycin for 12-16 h. A single clone was selected and the plasmid was extracted using the alkaline lysis method. The single clone plasmid with the correct sequencing was stored at -20°C. Finally, the expression vector HmSTG1 gene driven by the promoter of the HmSTG1 gene of Hordeum vulgare was obtained. Pro and rice OsSTG1 gene promoter driving HmSTG1 gene expression vector Os 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] Obtaining genetically modified rice

[0160] Rice genetic transformation was entrusted to Wuhan Boyuan Biological Company. The main steps are as follows:

[0161] (1) Rice background material: Japonica rice variety ZH11 was used. The rice seeds were peeled of husk and soaked in a 5% sodium hypochlorite solution containing 1 drop of Tween 20 for 15 minutes, and then washed 5 times with sterile water. Then, they were disinfected with a 5% sodium hypochlorite solution without Tween for 15 minutes and rinsed 10 times with sterile water to completely remove the residual sodium hypochlorite. After the surface moisture of the seeds was absorbed with sterile filter paper, the seeds were inserted obliquely on the NBD medium with the embryo facing upward, and 30 seeds were placed in each dish. The culture was carried out at 28°C with 16 hours of light / 8 hours of darkness.

[0162] (2) Take 10 μL of Agrobacterium culture medium and place it in AAM liquid medium containing 50 μg / mL kanamycin and 20 μg / mL rifampicin. Cultivate at 28°C in the dark with shaking at 200 rpm until OD 600 The cells were collected by centrifugation and resuspended in liquid NBD-As until the OD 600=0.1. Select callus tissue of appropriate size and good condition, immerse in Agrobacterium resuspension for 30 minutes, absorb excess bacterial liquid on sterile filter paper, and transfer the callus tissue to solid NBD-As medium covered with 1 layer of sterile filter paper for dark culture for 3 days. Wash the callus with sterile water 3 times, then soak it in sterile water containing 150mg / L Timentin for 10 minutes, absorb excess water from the callus, and transfer it to NBD-As medium for recovery culture for 4 days. Then transfer it to NBD-TK ​​medium containing screening antibiotic G418 for continued screening and culture, and subculture once every 2 weeks. Callus tissue with G418 resistance is transferred to differentiation medium RE1. When the catalyzed adventitious buds grow to 3-5cm seedlings, the seedlings are cut and transferred to rooting medium RE2 for rooting induction to obtain T0 generation seedlings.

[0163] (3) The formula of the culture medium for rice tissue culture and transformation is shown in Table 6.

[0164] Table 6

[0165]

[0166] Example 7

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

[0168] The Hm was extracted by the Edwards method in step (iii) of Example 1. Pro and Os Pro The genomic DNA of different transgenic rice lines and wild-type rice was used as a template for PCR amplification using the upstream primers (HmPro-F; OsPro-F) on the HmSTG1 and OsSTG1 promoter sequences and the specific downstream primer HmSTG1-R on the CDS of the HmSTG1 gene. The transgenic plants were those that could amplify 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 transgenic rice

[0174] To determine the functionality of the 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 ) were randomly selected from each of the 4 T2 generation strains, and the seeds of the above 4 strains and wild type ZH11 were selected to be of the same size and full. After disinfection with 3% hydrogen peroxide, soaking and germination at 30℃, and rooting promotion with 0.5mM calcium chloride, they were transferred into 1 / 2 Kimura B rice nutrient solution (pH5.6) and cultured in the dark at 28℃. When the buds grew to 2-3cm, light culture was started. The growth chamber conditions were 10h light / 14h dark, 28℃ / 22℃; the nutrient solution was changed every three days.

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

[0178] According to the method in Example 7, genomic DNA molecular identification was performed, and total RNA was extracted from the roots of the four transgenic lines and the wild-type plants under normal growth (Control) and 200 mM NaCl treatment conditions, and reverse transcribed into cDNA, the method was the same as step 1 and step 2 in Example 1. Specific primers HmSTG1-qF and HmSTG1-qR of the HmSTG1 gene and rice OsActin internal reference primers 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 pre-denaturation for 30 seconds, cycle parameters were 98°C denaturation for 10 seconds, 58°C annealing for 5 seconds, 72°C extension for 5 seconds, run for 30 cycles; 72°C complete extension for 1 minute. The amplified products were analyzed by electrophoresis on 1.0% agarose gel, and the band brightness was detected by Bio-Rad Gel Doc XR gel imager 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 strains had no significant difference under normal and salt treatment conditions; Pro and Os Pro All transgenic rice lines were successfully expressed, but the expression levels varied among different lines. Pro -1 and Os Pro -1 expression level is higher ( Figure 5 (middle B).

[0185] (3) Growth of transgenic HmSTG1 rice seedlings under normal conditions and salt stress

[0186] Three-week-old wild-type and transgenic rice lines were treated with normal nutrient solution and culture medium containing 200 mM NaCl for 20 days, and the root and aboveground dry weights were measured. The results showed that there was no significant difference in growth and dry weight between transgenic rice and wild-type under normal conditions ( Figure 5 Middle C; Figure 6 Middle A); Hm under 200mM salt treatment Pro and Os Pro The growth of transgenic rice lines was significantly stronger than that of wild-type rice, and the root and aboveground dry weight of transgenic rice was more than 2 times higher than that of wild-type rice ( Figure 5 Middle D; Figure 6 (middle B).

[0187] (4) Na and K content in tissues of HmSTG1 transgenic rice seedlings under salt stress

[0188] The root and aerial dried samples were digested with concentrated nitric acid. The operation steps were as follows: place the sample at the bottom of a 20 mL digestion tube, add 3 mL of concentrated HNO3, and place it in a constant temperature metal bath (DTU-2CN, TAITEC, Japan). First, raise the temperature to 80 ° C, and after the sample is dissolved into a fluid state, raise the temperature to 120 ° C until the sample is completely dissolved into a liquid state (usually 2-3 hours). Raise the temperature to discharge the acid until 1-1.5 mL of liquid remains in the test tube, and after cooling, dilute to 20 mL with deionized water. The element content was determined using an inductively coupled plasma mass spectrometer (iCAP-RQ, Thermo Fisher Scientific, USA). The results showed that under 200 mM salt treatment, Hm Pro and Os Pro Na in the aboveground parts of transgenic rice lines + The concentrations of K in the 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 showed that compared with the wild type, the transgenic line K + / Na + The ratio is higher, the root system has a higher + The absorption and transport to the aboveground parts are lower ( Figure 6 Medium CG).

[0189] The above results show that the HmSTG1 gene of sea barley was transferred into rice, which can reduce the root system's sensitivity to Na + The absorption and transport to the aboveground parts of the body, while increasing the body's K + Concentration and K + / Na + The salt tolerance of transgenic plants was enhanced.

[0190] In summary, the present invention has isolated a Na + / H + The antiporter encoding gene HmSTG1 was first found to have a strong Na + transport capacity and maintain low Na + Concentration and high K + / Na + The gene can be transformed into annual crops (herbs) 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 improving their yield and quality, which has great 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 methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. Application of HmSTG1 gene in improving plant salt tolerance.

2. The use according to claim 1, characterized in that: The nucleotide sequence of the HmSTG1 gene is as shown in SEQ ID NO.1 or has 95% or more consistency or identity with the nucleotide sequence shown in SEQ ID NO.1 and expresses the same functional protein.

3. Application of HmSTG1 protein in improving plant salt tolerance.

4. The use according to claim 2, characterized in that: The amino acid sequence of the HmSTG1 protein is as shown in SEQ ID NO.2 or a fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of the sequence shown in SEQ ID NO.

2.

5. Use of a recombinant expression vector, an overexpression vector, an interference vector, a recombinant virus, a recombinant bacterium or a recombinant gene expression cassette containing the HmSTG1 gene in improving plant salt tolerance.

6. Application of HmSTG1 gene, HmSTG1 protein, recombinant expression vector, overexpression vector, interference vector, recombinant virus, recombinant bacteria or recombinant gene expression cassette comprising HmSTG1 gene in salt-tolerant plant breeding.

7. The use according to any one of claims 1 to 6, characterized in that: The plants include rice, corn, wheat, barley, soybean, apple and pear.

8. A product for improving plant salt tolerance, characterized in that: It includes HmSTG1 gene, HmSTG1 protein, recombinant expression vector containing HmSTG1 gene, overexpression vector, interference vector, recombinant virus, recombinant bacteria or recombinant gene expression box.

9. A method for improving plant salt tolerance, characterized in that: The HmSTG1 gene is exogenously introduced into the plant or the expression of the HmSTG1 gene in the plant genome is upregulated.

10. A method for breeding salt-tolerant plants, characterized in that: The method for improving plant salt tolerance comprises the method as described in claim 9.

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