Spartina alterniflora saHAK65 gene and application thereof in regulating salt tolerance of rice

Abstract

This invention discloses a SaHAK65 gene derived from the halophyte Spartina alterniflora and its application in regulating salt tolerance in rice. The base sequence of the SaHAK65 gene is shown in SEQ ID NO.1; or, the CDS sequence of the SaHAK65 gene is shown in SEQ ID NO.2. In this invention, SaHAK65 was introduced into rice to construct overexpression lines, and positive plants OE-SaHAK65 were obtained after screening. Salt stress experiments showed that the growth and survival rate of rice plants overexpressing SaHAK65 were significantly better than those of wild-type plants, and the aboveground Na+ content was significantly lower. + The content decreased, and the potassium content in roots and leaves was lower. + The content increased, Na + / K + The levels of antioxidant enzymes and osmotic regulators in OE-SaHAK65 plants were significantly lower than those in wild-type plants after stress, indicating that OE-SaHAK65 plants have a stronger tolerance to salt stress. The results of this invention fully demonstrate the key positive role of the SaHAK65 gene in regulating salt tolerance in rice, providing an important gene resource for breeding salt-tolerant rice and possessing significant application value.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the SaHAK65 gene of Spartina alterniflora and its application in regulating salt tolerance in rice. Background Technology

[0002] Soil salinization is a global ecological problem. Against the backdrop of global climate change, rising sea levels and increased evaporation caused by global warming will further exacerbate soil salinization. Comprehensive utilization of saline-alkali land is of significant strategic importance. Traditional breeding methods face significant bottlenecks in the aggregation and precise improvement of salt tolerance traits. This is mainly because plant salt tolerance is regulated by multiple genes in a coordinated manner, involving complex physiological and biochemical pathways such as ion homeostasis, osmotic regulation, antioxidant defense, and hormone signaling. Therefore, systematically identifying key functional genes in plant responses to salt stress using multi-omics methods such as genomics and transcriptomics has become an important strategy for improving the efficiency of salt-tolerant breeding.

[0003] Studies have shown that *Spartina alterniflora* possesses a rich pool of salt-tolerant genes, making it an important germplasm for breeding and potentially useful for developing salt-tolerant cereal crops, especially rice. Therefore, discovering the salt-tolerant gene resources of *Spartina alterniflora* for the cultivation of salt-tolerant crops would be of great significance for the restoration and protection of saline-alkali land. However, current research on the discovery of salt-tolerant genes still falls short of practical needs, and further exploration is required in the screening and cultivation of salt-tolerant crops for near-natural restoration of severely saline-alkali land.

[0004] Accumulated K under salt stress + Used to alleviate excessive Na + Cumulative toxicity to cells is considered one of the key processes affecting plant salt tolerance. HAK / KUP / KT is the most abundant and widely distributed potassium group in plants. + The high-affinity transporter family has been found in various prokaryotic and eukaryotic organisms. Most members of the KUP / HAK / KT transporter family in plants are involved in the high-affinity uptake and transport of potassium (K) from the external environment. + Another part mediates K + Low affinity absorption, through different affinity mechanisms, ensures that plants can effectively absorb the required K under different potassium supply conditions. + This helps maintain normal growth and development. Currently, there are no reports on the role of the SaHAK65 gene in regulating salt stress resistance in rice. Summary of the Invention

[0005] Purpose of the invention: To address the existing problems in the cultivation of salt-tolerant crops, this invention provides the SaHAK65 gene of Spartina alterniflora and its application in regulating the salt tolerance of rice.

[0006] Technical solution: To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a Spartina alterniflora SaHAK65 gene for regulating salt tolerance in rice.

[0008] In a first aspect, the present invention provides a SaHAK65 gene derived from the halophyte Spartina alterniflora, the base sequence of which is shown in SEQ ID NO.1; or, the CDS sequence of which is shown in SEQ ID NO.2.

[0009] Secondly, the present invention provides a protein encoded by the SaHAK65 gene, the amino acid sequence of which is shown in SEQ ID NO.3.

[0010] Thirdly, the present invention provides a plant overexpression vector, characterized in that the plant overexpression vector contains the SaHAK65 gene.

[0011] Fourthly, the present invention provides a recombinant host cell comprising the plant overexpression vector described above.

[0012] Fifthly, the present invention provides a specific primer pair for cloning the SaHAK65 gene, the specific primer pair comprising an upstream primer and a downstream primer, the base sequence of the upstream primer being shown in SEQ ID NO.4, and the base sequence of the downstream primer being shown in SEQ ID NO.5.

[0013] In a sixth aspect, the present invention provides the application of the SaHAK65 gene, or the protein, or the plant overexpression vector, or the recombinant host cell in regulating plant salt tolerance.

[0014] As a specific implementation plan, the regulation of plant salt tolerance includes increasing the potassium content in plants under salt stress conditions. + Content, reducing the Na content in plants + / K + One or more of the following: ratio, increase plant root vigor, reduce the content of osmotic regulatory substances in plants, or reduce the activity of antioxidant enzymes in plants.

[0015] Preferably, the plant is selected from rice; more preferably, the rice is selected from japonica rice.

[0016] In a seventh aspect, the present invention provides the application of the SaHAK65 gene, the plant overexpression vector, or the recombinant host cell in the preparation of transgenic rice with improved salt tolerance.

[0017] Eighthly, the present invention provides a method for cultivating transgenic plants with improved salt tolerance, comprising introducing the SaHAK65 gene into a target plant to obtain a transgenic plant with improved salt tolerance compared to the target plant.

[0018] As a specific implementation plan, the method includes constructing a plant overexpression vector using the SaHAK65 gene and introducing it into plants through Agrobacterium tumefaciens infection to improve plant salt tolerance;

[0019] Preferably, the plant is selected from rice; more preferably, the rice is selected from japonica rice.

[0020] As a specific method, the method of overexpressing the SaHAK65 gene in rice to improve the salt tolerance of rice through genetic engineering includes the following steps:

[0021] (1) RNA extraction and target gene cloning from Spartina alterniflora: Spartina alterniflora seedlings were collected and RNA was extracted using a plant RNA extraction kit. The RNA was then reverse transcribed into cDNA, and the target gene was obtained by PCR using specific primers with cDNA as a template.

[0022] (2) Construction of overexpression vector: The PCR product was digested with KpnI restriction endonuclease and then ligated to the vector using homologous recombination.

[0023] (3) The pCambia2301-JC vector was introduced into rice using Agrobacterium-mediated transformation, and rice plants overexpressing the SaHAK65 gene were obtained by screening with hygromycin.

[0024] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0025] (1) This invention cloned the SaHAK65 gene of Spartina alterniflora for the first time using PCR technology and constructed OE-SaHAK65 rice plants. Compared with the wild type, after salt stress, the growth status and survival rate of OE-SaHAK65 rice plants were significantly higher than those of the wild type, and the root activity was also significantly higher than that of the wild type.

[0026] (2) In addition, after salt stress, the leaves and roots of OE-SaHAK65 rice plants showed K... + The content was significantly higher than that of the wild type, Na + / K + Significantly lower than wild type. This indicates that overexpression of SaHAK65 can promote K production in rice under salt stress. +Furthermore, the content of osmotic regulators (proline, soluble sugars) in the aboveground parts and roots of OE-SaHAK65 rice plants was significantly lower than that of the wild type. Similarly, the activities of antioxidant enzymes (SOD, POD, CAT) in the aboveground parts and roots of OE-SaHAK65 rice plants were significantly lower than those of the wild type. These results indicate that under the same salt stress conditions, OE-SaHAK65 rice plants suffer less stress damage and have stronger salt stress tolerance. Attached Figure Description

[0027] Figure 1 The results of agarose gel electrophoresis for PCR amplification of the SaHAK65 gene.

[0028] Figure 2 The sequencing results are for the PCR amplified SaHAK65 product.

[0029] Figure 3 The growth changes of WT and OE-SaHAK65 rice plants under 150mM NaCl stress for 6 days.

[0030] Figure 4 The survival rate of WT and OE-SaHAK65 rice plants after 6 days of 150mM NaCl stress is statistically analyzed.

[0031] Figure 5 The changes in physiological parameters of WT and OE-SaHAK65 rice plants after 6 days of 150 mM NaCl stress were observed. (A) Changes in fresh weight; (B) Changes in root length; (C) Changes in root activity. Error bars represent standard deviation (SD) (n=9). Significance: *, P<0.05; **, P<0.01; ***, P<0.005; ****, P<0.001.

[0032] Figure 6 The changes in ion content in WT and OE-SaHAK65 rice plants after 6 days of 150 mM NaCl stress. (A) Na + Content; (B)K + Content; (C)Na + / K + Error bars represent standard deviation (SD) (n=9). Significance: *, P<0.05; **, P<0.01; ***, P<0.005; ****, P<0.001.

[0033] Figure 7Changes in osmotic regulators in WT and OE-SaHAK65 rice plants after 6 days of 150 mM NaCl stress. (A) Proline content; (B) Soluble sugar content. Error bars represent standard deviation (SD) (n=9). Significance: *, P<0.05; **, P<0.01; ***, P<0.005; ****, P<0.001.

[0034] Figure 8 The changes in antioxidant enzyme activities in WT and OE-SaHAK65 rice plants after 6 days of 150 mM NaCl stress were shown. (A) SOD activity; (B) POD activity; (C) CAT activity. Error bars represent standard deviation (SD) (n=9). Significance: *, P<0.05; **, P<0.01; ***, P<0.005; ****, P<0.001. Detailed Implementation

[0035] The present invention will be further described below with reference to embodiments.

[0036] Example 1:

[0037] (1) Sources and treatment of plant materials

[0038] The Spartina alterniflora seeds used in the experiment were collected from the core area of ​​the Yancheng Wetland Rare Birds National Nature Reserve in Jiangsu Province (33.60327619°N, 120.60882581°E). Spartina alterniflora plants at seed maturity (November 1, 2023) were harvested and brought back to the laboratory for threshing. After sieving and sun-drying, the collected seeds were soaked in sterile water at 4°C for one day for vernalization. The treated seeds were then drained of water and placed on trays lined with gauze in a light incubator. The daytime temperature was set at 30°C, light intensity at 100%, for 12 hours; the nighttime temperature was set at 18°C, light intensity at 0%, for 12 hours. Germinated Spartina alterniflora seeds were then transferred to hydroponic containers containing Hoagland nutrient solution for 6 weeks of continued growth (the pH of the Hoagland nutrient solution was adjusted to 5.6-6). During the growth period, the light incubator was set with a daytime temperature of 22℃ and a light intensity of 1500 lx for 16 hours, and a nighttime temperature of 20℃ and a light intensity of 0 lx for 8 hours. Six-week-old Spartina alterniflora plants were screened, and poorly growing plants were removed. The above-ground parts and root samples were collected, washed, and quickly frozen in liquid nitrogen at -80℃ for RNA extraction.

[0039] (2) Main test reagents

[0040] Sterile water, RNase-free water, sodium chloride, Hoagland nutrient solution, nitric acid, liquid nitrogen, β-mercaptoethanol, anhydrous ethanol, LB medium, Escherichia coli DH5α, Agrobacterium tumefaciens AGL0, Taq enzyme, kanamycin, rifampin, MES, magnesium chloride, acetylsuccinone, etc.

[0041] The detailed formula for Hoagland nutrient solution is shown in Table 1.

[0042] Table 1. Hoagland Nutrient Solution Components

[0043]

[0044] LB liquid medium: 1% tryptone, 0.5% yeast extract, 1% sodium chloride, adjust pH to 7.0, autoclave at 121℃ for 20 min.

[0045] LB solid medium: Before sterilization, add 1.5% agar powder to LB liquid medium.

[0046] 1 / 2MS medium: The detailed formulation is shown in Table 2.

[0047] Table 21 / 2MS Culture Medium Components

[0048]

[0049] 1 / 2MS resistant medium: Add 50 mg / mL hygromycin B stock solution to the sterilized 1 / 2MS medium solution to make the final concentration of hygromycin B 30 ug / mL.

[0050] (3) Test instruments

[0051] Pure water system, autoclave, graduated cylinder, beaker, filter paper, light-proof growth box, aluminum foil, mortar and pestle, centrifuge tubes, centrifuge, spectrophotometer, electrophoresis apparatus, gel imaging system, dry ice, petri dishes, conical flasks, sealing film, pipettes, sterile pipette tips, constant temperature shaker, constant temperature incubator, PCR instrument, ultraviolet spectrophotometer, Tecan Spark multi-well plate analyzer, NanoDrop micro spectrophotometer, Applied Biosystems 2720 Thermal Cycler PCR instrument, Applied Biosystems multiplex temperature-controlled PCR instrument, 1mL sterile syringe, fluorescence microscope, etc.

[0052] (4) RNA extraction and reverse transcription

[0053] Plant samples were removed from -80°C and thoroughly ground with liquid nitrogen. RNA was extracted using the FastPure® Universal Plant Total RNA Isolation Kit (Vazyme). After extraction, the RNA integrity was assessed by agarose gel electrophoresis, and absorbance and concentration were measured using Nanodrop (Thermo Fisher Scientific). The RNA was then reverse transcribed into cDNA using the TSK302M kit (Qingke, Beijing, China) for target gene cloning.

[0054] (5) Cloning of the target gene

[0055] Specific primers were designed at both ends of the CDS region of the target gene using SnapGene software. PCR amplification was performed using cDNA as a template with the gold-standard Mix Ver.2 high-fidelity enzyme (TSE102, Beijing Qingke). The PCR reaction procedure is shown in Table 3.

[0056] Table 3 PCR reaction procedure

[0057]

[0058] Agarose gel electrophoresis was used to identify the amplification of the target gene, and the results are as follows: Figure 1 As shown in the image. The target product band was then recovered via gel extraction, sequenced, and used for subsequent vector construction. Sequencing results are shown in the image. Figure 2 As shown.

[0059] The base sequence of the SaHAK65 gene is shown in SEQ ID NO.1, and the CDS sequence is shown in SEQ ID NO.2; the amino acid sequence of the protein encoded by the SaHAK65 gene is shown in SEQ ID NO.3.

[0060] The SaHAK65 base sequence is as follows:

[0061]

[0062] The SaHAK65 CDS sequence is as follows:

[0063]

[0064] The amino acid sequence of the protein encoded by SaHAK65 is as follows:

[0065] MDEEIGADARQGQWKTYCKTLSLLAFQSFGVVYGDLSTSPLYVYRSALSGRLDSYRDEVTIFGLFSLIFWTLTLIPLLKYVLIVLSADDNGEGGTFALYSLLCRHAKFNLLPNQQAADEELSTYYQPGVSRTAIASPLKRFLEKHRKLRTVLLLFVLFGACMVIGDGVLTPTISVLSAVSGLQDPAPGGIPGGWVVFITCVVLVGLFSLQHRGTHRVAFMFAPVIVIWLLCIGAIGLYNIIHWNPSICRALSPYYVVRFFKTTGKDGWLSLGGVLLAVTGTEAMFADLGHFSAVSIRLAFVGVIYPCLVLQYMGQAAFLSKNMSAVDNSFYQSIPSPVFWPVFIIATLAAVVGSQAIISATFSIIKQCLSLGCFPRVKVVHTSRWIYGQIYIPEINWILMVLCLAVTLGFRDTTVIGNAYGLAYITVMFVTTWLMALVIIFVWQKNLLIALSFLLFFGSIEALYLSASVVKVPQGGWAPIALALVFMSVMYVWHYGTRRKYMFDLQNKVSMKWILNLGPSLGIMRVPGIGLIYTELVTGVPSIFSHFVTNLPAFHQVLVFVCVKSVPVPYVPTEERYLIGRIGPKEYRMYRCIVRYGYKDVQKDDENFENHLVVSIARFIQMEAEEAASSRSYDSSTDGRMAVVHTTDAVGTGLVIADDDAGASMSQQLTRSSKSDTLRSLQSIYEEEAGGSLSRRRRVRFQIAEEERIDPQVRDELSDLLQAKEAGVAYIIGHSYVKARKNSNFLKTFAINYAYTFLRKNCRGPSVTLHIPHISLIEVGMIYYV (SEQ ID NO.3) ​​​​​​​ GCGGGTCGACGGTACC ATGGACGAGGAGATCGGCGC (SEQ ID NO.4)

[0069] SaHAK65-KpnI-R:

[0070] TAGACATATGGGTACC TTATACATAGTATATCATGCCGACCTCG (SEQ ID NO.5)

[0071] (6) Carrier construction and transformation

[0072] The pCambia2301-JC plasmid was linearized by KpnI single enzyme digestion. The enzyme digestion reaction system is as follows:

[0073] Table 4 Enzyme digestion reaction procedure

[0074]

[0075] After purification, the enzyme digestion products were recombined with the PCR products of the above three genes (the recombination reaction kit was the Vazyme ClonExpress-II One Step Cloning Kit).

[0076] The recombinant ligation reaction system (total volume 10 μl) is as follows:

[0077] Table 5 Recombinant Linkage Reaction System

[0078]

[0079] The above reaction solution was gently mixed using a pipette, and the mixture was briefly centrifuged to collect the solution at the bottom of the tube. The mixture was then incubated at 37°C for 30 min, and immediately placed on ice to cool.

[0080] The recombinant product was transformed into Escherichia coli DH5α competent cells.

[0081] The conversion steps are as follows:

[0082] (1) Add 10 μL of ligation product to 100 μL of competent Escherichia coli cells.

[0083] (2) Ice bath for 30 minutes

[0084] (3) Heat shock at 42℃ for 60-90s

[0085] (4) Ice bath for 2 minutes

[0086] (5) Add 800 μl of LB liquid culture medium

[0087] (6) Incubate at 37℃ on a shaker for 30 min

[0088] (7) Centrifuge at 6000 rpm for 3 min, discard the supernatant, and spread on a plate of Kana (50 mg / L) resistance medium.

[0089] (8) After incubating upside down at 37℃ for 12-16 hours, pick out resistant colonies.

[0090] (9) Add 100 μL of LB (containing Kana) liquid medium to each well of a 96-well plate.

[0091] (10) Take 4-8 colonies from each plate and incubate at 37℃ for 2 hours at 180 rpm.

[0092] (11) Take 1 μL of bacterial culture for PCR positive detection.

[0093] PCR-positive transformants were selected and cultured in a culture medium to extract plasmids for sequencing. The detection primers were the same as the gene amplification primers, and the sequencing primers were located flanking the vector insertion site. The sequences are as follows:

[0094] M13-F:TGTAAAACGACGGCCAGT

[0095] M13-R:GAGCGGATAACAATTTCACAC

[0096] Because the genes are quite long, it is also necessary to design internal primers for each gene for additional testing:

[0097] SaHAK65-ZF:CGGTGCTCTTTCTCCATAC

[0098] Plasmids were extracted from successfully transformed *E. coli* and used to transform rice (Zhonghua 11). Rice seeds were disinfected, vernalized, and transplanted after germination. After 30 days of growth, seedlings with uniform growth were selected for *Agrobacterium* inoculation and transformation. Plasmids were added to the *Agrobacterium* culture, and the cultured colonies were prepared into a bacterial suspension for PCR verification. A portion of the positive bacterial suspension was sent for sequencing comparison, while the other portion was stored for later use. The correctly sequenced bacterial suspension was activated and a resuspension was prepared. All inflorescences of healthy, flowering rice plants were immersed in the resuspension for 45 seconds, kept in the dark overnight, and the seedlings were removed the next day, straightened, and continued to be cultured until seed formation. After seed maturity, hygromycin B was used for preliminary screening, and T0 generation positive plants with successfully transformed vectors were obtained by DNA extraction and identification. The T0 generation plants were then planted in the greenhouse of the Environmental Ecological Restoration Experimental Platform of Nanjing Normal University, and T1 generation seeds were harvested. The T1 generation seeds were further screened using hygromycin B, and RNA was extracted for PCR verification to ensure the presence of the target gene.

[0099] (7) Identification of salt tolerance traits in OE-SaHAK65 rice lines

[0100] The harvested T1 generation rice seeds were disinfected, germinated, and screened for susceptibility. The susceptible seedlings were then transplanted into 96-cell hydroponic boxes and placed in a constant temperature and light incubator (16 h (light) / 8 h (dark), 25℃ (light) / 23℃ (dark), light intensity 15000 lx, humidity 50%) and cultured in clean water for one week. Subsequently, they were cultured in 1 / 4 concentration Kimura b nutrient solution for 3 days, 1 / 2 concentration Kimura b nutrient solution for 3 days, and then in full concentration Kimura b nutrient solution for one month.

[0101] Salt stress treatment: Rice plants were screened, and those with poor growth were removed to ensure that the experimental plants had roughly the same growth rate. They were subjected to salt stress for 6 days using Kimura b nutrient solution containing 150 mM NaCl. After stress treatment, growth indicators and phenotypes were measured and evaluated, and survival rates were recorded. Whole plant samples were then immediately frozen in liquid nitrogen and stored at -80°C for long-term preservation in an ultra-low temperature freezer for the determination of physicochemical indicators. The physicochemical indicators measured included fresh weight, root length, root activity, proline, soluble sugar content, tissue extract conductivity, malondialdehyde content, antioxidant enzyme activity (POD, SOD, CAT), and NaCl content in roots, stems, and leaves. + K + concentration.

[0102] The determination of soluble sugar content, root activity, proline (PRO), malondialdehyde (MDA) content, and superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities in plants was performed using commercially available kits (Solarbio, Beijing, China). Chlorophyll content was determined according to the requirements of "Determination of Chlorophyll Content in Fruits, Vegetables and Their Products - Spectrophotometric Method" (NY / T 3082-2017). The conductivity of plant tissue extracts was determined using a conductivity meter. Na+ content in the roots, stems, and leaves of *Spartina alterniflora* was... + K + Concentration determination was performed using atomic absorption spectrometry. The specific procedure was as follows: plant samples were collected at 24, 48, and 72 hours after salt stress, thoroughly rinsed with ultrapure water, and dried to constant weight. Approximately 0.2 g of the dried tissue was weighed, digested using the HNO3-H2O2 wet digestion method, and diluted with ultrapure water. Then, the sodium concentration was measured using an atomic absorption spectrophotometer. + and K + The concentration.

[0103] The results showed that before salt stress treatment, the growth status of wild-type (WT) and SaHAK65 overexpression (OE-SaHAK65) rice plants was basically the same. After 6 days of salt stress treatment, WT plants showed obvious leaf yellowing, wilting and shrinkage, while the growth of OE-SaHAK65 plants was not significantly affected. Figure 3Survival rate statistics showed that almost no OE-SaHAK65 plants died after 6 days of stress, while the survival rate of WT plants decreased to about 60%-70% (Figure 4). Further measurements of physiological indicators of the plants after stress revealed no significant difference in fresh weight and root length between WT and OE-SaHAK65 plants. Figure 5 (A, B), however, the root activity of OE-SaHAK65 plants increased significantly by 54.5% compared to WT. Figure 5 (C) indicates that the root system of OE-SaHAK65 plants suffered less damage under the same stress conditions. Ion content analysis showed that the Na+ content in the aboveground parts of OE-SaHAK65 plants was significantly lower after stress. + The content of potassium in roots and leaves decreased by 12% compared to WT; + The content increased by 93.3% and 44.6%; Na + / K + The figures decreased by 43.6% and 39.4% respectively. Figure 6 AC), indicating that OE-SaHAK65 plants can more effectively regulate Na+. + and K + Absorption and transport, maintaining low Na+ + / K + This reduces the ion toxicity caused by salt stress. Furthermore, the osmotic regulator content of OE-SaHAK65 plants ( Figure 7 A, B) and antioxidant enzyme activity ( Figure 8 Both AC and WT were significantly lower than WT, indicating that the degree of osmotic imbalance and oxidative damage induced by salt stress was relatively mild. In summary, these results clarify the key role of the SaHAK65 gene in enhancing rice salt tolerance, providing important theoretical support for the genetic improvement of crop stress resistance.

[0104] The specific embodiments listed above are merely illustrative of the invention and are not intended to limit it. All other embodiments obtained by those skilled in the art without inventive effort are, in principle, within the scope of protection of this invention.

Claims

1. A SaHAK65 gene derived from the halophyte Spartina alterniflora, characterized in that, The base sequence of the SaHAK65 gene is shown in SEQ ID NO.1; or, the CDS sequence of the SaHAK65 gene is shown in SEQ ID NO.

2.

2. The protein encoded by the SaHAK65 gene according to claim 1, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.

3.

3. A plant overexpression vector, characterized in that, The plant overexpression vector contains the SaHAK65 gene as described in claim 1.

4. A recombinant host cell, characterized in that, The recombinant host cell comprises the plant overexpression vector of claim 3.

5. A specific primer pair for cloning the SaHAK65 gene of claim 1, characterized in that, The specific primer pair includes an upstream primer and a downstream primer, the base sequence of which is shown in SEQ ID NO.4 and the base sequence of which is shown in SEQ ID NO.

5.

6. The application of the SaHAK65 gene of claim 1, or the protein of claim 2, or the plant overexpression vector of claim 3, or the recombinant host cell of claim 4 in regulating plant salt tolerance.

7. The application according to claim 3, characterized in that, The regulation of plant salt tolerance includes increasing potassium levels in plants under salt stress conditions. + Content, reducing the Na content in plants + / K + One or more of the following: ratio, increase plant root vigor, reduce the content of osmotic regulatory substances in plants, or reduce the activity of antioxidant enzymes in plants. Preferably, the plant is selected from rice; more preferably, the rice is selected from japonica rice.

8. The use of the SaHAK65 gene of claim 1, the plant overexpression vector of claim 3, or the recombinant host cell of claim 4 in the preparation of transgenic rice with improved salt tolerance.

9. A method for cultivating transgenic plants with enhanced salt tolerance, characterized in that, This includes introducing the SaHAK65 gene as described in claim 1 into a target plant to obtain a transgenic plant with improved salt tolerance compared to the target plant.

10. The method for cultivating transgenic plants with improved salt tolerance according to claim 9, characterized in that, The method includes constructing a plant overexpression vector using the SaHAK65 gene as described in claim 1, and introducing it into plants via Agrobacterium tumefaciens infection to improve plant salt tolerance; Preferably, the plant is selected from rice; more preferably, the rice is selected from japonica rice.

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