Alfalfa MsKTI3 gene and its application in improving plant salt tolerance

Abstract

This invention discloses the alfalfa MsKTI3 gene and its application in improving plant salt tolerance. Through transcriptome sequencing analysis, the inventors screened for a candidate gene, MsKTI3, associated with alfalfa salt tolerance. MsKTI3 was cloned, overexpressed in alfalfa and Arabidopsis, and the gene's function was identified. This invention lays the foundation for in-depth research on the function and salt tolerance regulatory mechanisms of MsKTI3 and provides a candidate gene resource for molecular improvement of alfalfa salt tolerance.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, in particular to an alfalfa MsKTI3 gene and an application thereof in improving plant salt tolerance. Background Art

[0002] Alfalfa (Medicago sativa) is a high-quality perennial legume forage, widely used as a feed additive for its high yield, rich nutritional value, and good palatability. This forage not only promotes livestock growth and improves the quality of livestock products, but also enhances production and reproductive performance, thus playing a vital role in livestock production. However, the global decline in arable land and the increasing problem of soil salinization have led to a decrease in alfalfa yield per unit area. Salt accumulation in the soil degrades the plant growth environment. Excessive salt causes osmotic stress, hindering water absorption by plant roots and causing physiological drought. Salt stress also leads to nutrient imbalances in plants, inhibiting photosynthesis and respiration, thereby affecting crop yields and undermining ecosystem stability. Furthermore, salt stress disrupts plant metabolism, generating large amounts of reactive oxygen species (ROS), such as superoxide anions and hydrogen peroxide, within plant cells. If these ROS are not promptly removed, they can cause oxidative damage to biomacromolecules such as proteins, nucleic acids, and lipids, further disrupting cellular structure and function, posing a serious challenge to plant survival. Currently, my country relies primarily on imports for forage, and the annual increase in alfalfa imports has significantly restricted the development of the alfalfa industry. Therefore, exploring the genetic resources associated with salt tolerance in alfalfa and exploring the functions of salt-tolerance genes in alfalfa during salt stress response is of great significance for the genetic improvement of alfalfa.

[0003] Protease inhibitors are an important class of bioactive substances that regulate protein metabolism and participate in various physiological and pathological processes by inhibiting protease activity. Protease inhibitors found in nature are primarily divided into four categories: cysteine protease inhibitors, serine protease inhibitors, metalloproteinase inhibitors, and aspartic acid protease inhibitors. Serine protease inhibitors can be divided into several families based on their conserved domain structure, including the Bowman-Birk family of soybean trypsin inhibitors, potato protease inhibitor families I and II, RT I / MT II, pumpkin inhibitor family, soybean trypsin inhibitor family (Kunitz type), and Serpin family. The Bowman-Birk family and potato protease inhibitor families I / II have been extensively studied, while the Kunitz trypsin inhibitor (KTI) has been relatively less studied in response to abiotic stresses. Kunitz trypsin inhibitors are globular proteins primarily found in plant storage organs that inhibit trypsin, chymotrypsin, and serine (pro)enzymes similar to elastase. These inhibitors react rapidly with serine proteases to form stable complexes. Kunitz trypsin inhibitor has a molecular weight of approximately 20 kDa and consists of approximately 180 amino acid residues. It has a highly conserved three-dimensional structure consisting of a distinct hydrophobic core, three pairs of highly conserved disulfide bridges, a three-stranded β-sheet, an N-terminal 310-helix, and a C-terminal α-helix. This highly conserved structure enables Kunitz trypsin inhibitor to specifically bind to and inhibit the activity of various serine proteases. Kunitz trypsin inhibitor was first discovered and described by Kunitz in bovine pancreas in 1947 and was later extracted from soybeans. To date, no studies have been reported on the function of MsKTI3 in alfalfa, and its mechanism of regulating alfalfa tolerance remains unclear. Summary of the Invention

[0004] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide an alfalfa MsKTI3 gene and its application in improving plant salt tolerance.

[0005] To achieve the above objectives, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides an alfalfa MsKTI3 gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] Furthermore, the amino acid sequence encoded by the MsKTI3 gene is shown in SEQ ID NO.2.

[0008] In a second aspect, the present invention provides a recombinant vector containing the alfalfa MsKTI3 gene.

[0009] Furthermore, the recombinant vector is a vector for overexpressing the alfalfa MsKTI3 gene.

[0010] In a third aspect, the present invention provides a recombinant strain or recombinant cell containing the alfalfa MsKTI3 gene.

[0011] In a fourth aspect, the present invention provides the use of the alfalfa MsKTI3 gene in improving plant salt tolerance.

[0012] Furthermore, the above application is the application of overexpressing the alfalfa MsKTI3 gene in enhancing plant salt tolerance.

[0013] Furthermore, the plant is alfalfa or Arabidopsis thaliana.

[0014] Based on the above technical solution, the embodiments of the present invention can produce at least the following technical effects:

[0015] The present invention provides an alfalfa MsKTI3 gene and its application in improving plant salt tolerance. Through transcriptome sequencing analysis, a candidate gene MsKTI3 for alfalfa salt stress response was screened out, MsKTI3 was cloned, alfalfa and Arabidopsis were overexpressed, and the function of the gene was identified. This lays the foundation for in-depth research on the function and salt tolerance regulation mechanism of MsKTI3, and also provides a candidate gene resource for molecular improvement of alfalfa salt tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. 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 the structures shown in these drawings without paying any creative work.

[0017] Figure 1 This is the electrophoresis diagram of the PCR amplification product of the coding region sequence of the MsKTI3 gene;

[0018] Figure 2 Positive identification of MsKTI3 gene-overexpressing Arabidopsis lines; A: PCR electrophoresis analysis of MsKTI3 gene-overexpressing Arabidopsis lines, +: positive control, -: negative control, =: negative plants, 1-12: positive lines; B: Fluorescence quantitative PCR analysis of wild-type Arabidopsis and MsKTI3 gene-overexpressing lines;

[0019] Figure 3Figure 2: Germination and growth of wild-type (WT) Arabidopsis and Arabidopsis overexpressing MsKTI3 on culture media with different concentrations of sodium chloride. Figure 3: Comparison of germination and growth of wild-type (WT) Arabidopsis and Arabidopsis overexpressing MsKTI3. The NaCl concentration was set at 0 mmol•L -1 (A), 100mmol•L -1 (B), 150mmol•L -1 (C) and 200mmol•L -1 (D); EH: Wild-type (WT) Arabidopsis and Arabidopsis overexpressing MsKTI3 gene at 0 mmol•L -1 (E), 100mmol•L -1 (F), 150mmol•L -1 (G) and 200mmol•L -1 (H) Germination rate at different time points on sodium chloride medium; WT: wild-type Arabidopsis, At-OE2, At-OE3, At-OE9: Arabidopsis lines overexpressing the MsKTI3 gene;

[0020] Figure 4 Comparison of the phenotypes of wild type and transgenic Arabidopsis thaliana overexpressing MsKTI3 after salt stress; AB: phenotype before NaCl stress; CD: 0 mmol•L -1 (C), 250mmol•L -1 (D) Phenotypes of wild-type and overexpressing Arabidopsis plants after 17 days of NaCl stress treatment;

[0021] Figure 5 Analysis of rosette leaf number and chlorophyll content in wild-type and MsKTI3-overexpressing transgenic Arabidopsis lines after 17 days of salt stress treatment; A: rosette leaf number analysis, B: chlorophyll content analysis;

[0022] Figure 6 Detection of oxidative damage in Arabidopsis leaves under salt stress; A: DAB staining; B: NBT staining, the darker the color, the more severe the oxidative damage;

[0023] Figure 7 Analysis of salt tolerance phenotype of plants overexpressing MsKTI3 gene in alfalfa roots; A: Phenotype of plants grown normally under hydroponic conditions; B: Phenotype of plants grown under 200mmol•L hydroponic conditions -1 Phenotype after 4 days of NaCl stress, C: CAT activity detection of plants; WT: wild-type alfalfa plants, K: alfalfa plants overexpressing the MsKTI3 gene in roots. DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the ability of ordinary technicians in this field to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0025] 1. Experimental Materials

[0026] The alfalfa (M. sativa cv. Zhongmu-4) and Arabidopsis thaliana (Col-0, WT) used in this experiment were provided by the Forage Breeding and Cultivation Team of the Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences. The experimental materials were cultivated using both greenhouse soil culture and artificial climate chamber cultivation. The greenhouse soil culture conditions were 22°C / 20°C (day / night), 50% humidity, and 14 h light / 10 h dark; the artificial climate chamber culture conditions were 24°C / 22°C (day / night), 60% humidity, and 16 h light / 8 h dark.

[0027] 2. Experimental Methods

[0028] 1. Cloning and bioinformatics analysis of the MsKTI3 gene from alfalfa

[0029] Based on previous salt-tolerance transcriptome data from alfalfa, we screened for the salt-stress-responsive MsKTI3 gene. Primers for the full-length amplification of the MsKTI3 gene were designed using DNAMAN software, as shown in Table 1. Root RNA was extracted from alfalfa according to the instructions of the RNA extraction kit and reverse-transcribed into cDNA. The reverse transcription system consisted of 4 µL of 5× All-in-one qRT SuperMix, 1 µL of Enzyme Mix, and 1 µg of template RNA (total RNA). RNase-free ddH2O was added to make up to 20 µL, the mixture was shaken and mixed, and the sample was collected by instant centrifugation. The cDNA template was synthesized by PCR with incubation at 50°C for 15 minutes and heating at 85°C for 5 seconds. The cDNA template was then stored at -80°C until ready for use. The CDS sequence of the MsKTI3 gene was amplified using cDNA as a template. The reaction mixture (25 μL) consisted of 12.5 μL of 2× Phanta MaX Master Mix, 1 μL of MsKTI3-F, 1 μL of MsKTI3-R, 2 μL of cDNA, and 8.5 μL of RNase-Free ddH2O. The reaction procedure was as follows: 98°C for 30 seconds, 98°C for 10 seconds, 52°C for 5 seconds, and 72°C for 4 seconds (28 cycles), followed by 72°C for 5 minutes. After the amplified product was correctly aligned with the gene length, the gel containing the target fragment was excised using a UV-cleaving device and transferred to a 1.5 mL centrifuge tube for gel recovery. The recovered product was cloned into the pEASY®-T5 vector and sequenced after positive results were detected by PCR in the bacterial culture.

[0030] Table 1 List of primers for this experiment

[0031]

[0032] 2. Construction of MsKTI3 gene overexpression vector and acquisition of transgenic Arabidopsis

[0033] Using restriction enzyme QuickCut TM SmaI was used to single-cut the pSmRY-YFP vector, and cloning primers Y-MsKTI3-F / R containing homology arms of the pSmRY-YFP vector were designed. Using the root cDNA of alfalfa No. 4 as a template, the full-length coding sequence of the MsKTI3 gene was amplified with a high-fidelity enzyme. The MsKTI3 gene fragment and the enzyme-cut pSmRY-YFP vector were ligated with T4 DNA ligase and transformed into DH5α Escherichia coli competent cells. Positive single colonies were picked and activated after shaking. After sequencing and alignment, the plasmid was extracted. The recombinant expression vector was then transformed into GV3101 Agrobacterium competent cells and transformed into wild-type Arabidopsis thaliana using the floral dip method. -1Homozygous T3 plants were screened on 1 / 2 MS solid medium containing glufosinate. PCR and qRT-PCR were used to identify transgenic Arabidopsis.

[0034] 3. Analysis of salt tolerance in Arabidopsis thaliana overexpressing MsKTI3

[0035] Wild-type Arabidopsis thaliana (Col-0) and harvested T3 transgenic seeds were sterilized with 75% alcohol for 5 minutes, then rinsed 4-5 times with sterile water in a clean bench, vernalized at 4°C for 2 days, and then transferred to a light incubator for culture and subjected to different treatments. -1 、100mmol•L -1 、150mmol•L -1 , 200mmol•L -1 The seeds were cultured on 1 / 2MS solid medium containing NaCl, and the seed germination rate was counted after 10 days.

[0036] To further determine the effect of MsKTI3 gene on salt tolerance of Arabidopsis, Arabidopsis plants cultured in a light incubator for 14 days were transplanted to a nutrient medium with a volume ratio of 1:1 of nutrient soil and vermiculite for 17 days and then continued with subsequent treatments. -1 The NaCl solution was used for stress. Specifically, 30 ml was evenly poured into each pot with a 5 mL syringe, once every 3 days, and the phenotype was observed and physiological indicators were tested 17 days after treatment. The determination method is as follows: relative conductivity was measured, and chlorophyll content was determined by 95% anhydrous ethanol extraction. NBT and DAB staining were performed on Arabidopsis leaves after 17 days of stress. The staining method is as follows: leaves in the same position were taken, cleaned with distilled water, and the samples were immersed in DAB and NBT staining solutions. They were stained for several hours in a dark place and at room temperature. The samples were then immersed in anhydrous ethanol and placed in a boiling water bath for 30 minutes with intermittent shaking. Finally, the samples were moved to filter paper soaked in 60% glycerol for photographic observation.

[0037] 4. Analysis of root growth and salt tolerance in MsKTI3 overexpressing alfalfa

[0038] The amplified MsKTI3 gene fragment was ligated with the linear vector pSmRY-YFP for transformation. After double identification by bacterial liquid PCR and company sequencing, the positive single clone plasmid was transformed into Ar.1193 competent cells. After sequencing analysis, the positive colonies were selected and the clones with the correct sequence were re-inoculated on TY solid medium (tryptone 5g·L -1 , yeast extract 3g•L -1 , calcium chloride 0.6g•L -1 , agar powder 6g·L -1) streak culture. Before streaking, germinate the seeds. Place Zhongmu No. 4 seeds in a 50mL centrifuge tube and sterilize with 75% alcohol for 8 minutes. Rinse five times with ddH2O, then transfer to a 10% sodium hypochlorite solution and rinse for 15 minutes. Rinse five to six times with ddH2O and place in a 9mm glass dish containing filter paper. Ensure the filter paper is completely soaked, leaving a small amount of water in the dish. Wrap in tin foil and place in a 4°C refrigerator for 2 days. Next, transfer to a 25°C light incubator (16 hours light / 8 hours dark) for 2 days. At this point, the seedlings will have two cotyledons and a hypocotyl of approximately 1-1.5 cm. Cut off 3-5 mm of the root tip of the alfalfa seedling. Gently dip the cut end into the bacteria on the TY plate and transfer to modified Fahraes medium for co-cultivation. The infected seedlings were then cultured vertically on Fahraeus medium at 22°C for 7 days with 16 h light / 8 h dark. One week later, the roots growing near the radicle were completely cut off, leaving the swollen part at the bottom of the radicle. The plants with the second root cutting were then transferred to the selection medium (mFahraeus + 2 mg·L -1 The seedlings were incubated vertically in glufosinate (PPT) at 22°C for 10 days with 16 h light / 8 h dark. After 10 days, the agar on the roots of the seedlings was carefully and thoroughly washed off under running water. The seedlings were incubated in 1 / 2 Hoagland nutrient solution for 12 days and then verified by PCR amplification. The positive hairy root lines were then amplified with 200 mmol·L -1 After 4 days of NaCl stress, the phenotypes were observed and the CAT activities were measured.

[0039] 3. Results and Analysis

[0040] 1. Cloning of the MsKTI3 gene

[0041] The MsKTI3 gene was cloned from the cDNA of alfalfa root by PCR amplification using MsKTI3-F and MsKTI3-R as primers. Figure 1 The nucleotide sequence was aligned by Blast on NCBI, and the similarity with KTI3 of Medicago truncatula was 97%. The nucleotide sequence of MsKTI3 gene is shown in SEQ ID NO.1, and the amino acid sequence encoded by it is shown in SEQ ID NO.2.

[0042] SEQ ID NO.1:

[0043] ATGCTAGCATTTCTTCTTCTCTTTGCATTGTCTTCGCAACCACTACTTGGATCAACTGAAGCTTCACCCGATCAAGTCATTGACACATCGGGCAAGAAGCTTCGAGCTGATACCAATTACTACATCATTCCGGCTAAGCCATTTACAATATGTGGCTTTGTTAGTTGTTTCAATAGTGGAGGCATTGCTCTTGAAACCGTCGGAGAATCTTGCCCTCTTGATGTAGTAGTTGTGAAACATAATCAAGGTTTACCGTTGAGGTTCACACCCGTTAACAACAAGAAAGGCGTTATTCGTGTCTCCACTGATCTCAACATAAAGTTCTCTAATGATGCTTATGATTCTAGATGTCCTAACCATTCCTTAGTGTGGAAGATTGATCCTTTTTCTAAAGAAGAAACATTTGTGACTACTAATGGTGTATTGGGAAACCCGGGTTCAAATACAATCCACAATTGGTTCCAAATTGAGAAGTATGAAGATGCTTATAAGTTGGTCTATTGTCCTAATGTGTGCCCTACTTGCAACCATGTATGCAAAGATATTGGAATTTACGTGTATAAGACTAGGGAAATGCGTTTGGCTCTCACTAATGTTCCCTTTAAAGTTAAGTTTCAGAAGGTTTGA。

[0044] SEQ ID NO.2:

[0045] MLAFLLLFALSSQPLLGSTEASPDQVIDTSGKKLRADTNYYIIPAKPFTICGFVSCFNSGGIALETVGESCPLDVVVVKHNQGLPLRFTPVNNKKGVIRVSTDLNIKFSNDAYDSRCPNHSLVWKIDPFSKEETFVTTNGVLGNPGSNTIHNWFQIEKYEDAYKLVYCPNVCPTCNHVCKDIGIYVYKTREMRLALTNVPFKVKFQKV*。

[0046] 2. Identification and Expression Level Detection of MsKTI3 Transgenic Arabidopsis thaliana Positive Seedlings

[0047] Agrobacterium (GV3101) containing the MsKTI3 gene was transformed into wild-type Arabidopsis thaliana (Col-0, WT) by inflorescence infection and then treated with PPT (2 mg·L -1 ) were screened to obtain T3 generation homozygous seeds, and a total of 12 transgenic lines were screened, such as Figure 2 As shown in A. Three transgenic lines (At-OE2, At-OE3, and At-OE9) were randomly selected, and the expression levels of the MsKTI3 gene in wild-type and transgenic Arabidopsis were detected by qRT-PCR. The results showed that the MsKTI3 gene was successfully expressed in transgenic plants, while its expression level was not detected in wild-type Arabidopsis. Figure 2 As shown in B.

[0048] 3. Analysis of the germination rate of Arabidopsis thaliana under salt stress

[0049] Wild-type Arabidopsis and overexpression lines (At-OE2, At-OE3, and At-OE9) were treated with 0 mmo L -1 、100mmo•L -1 、150mmo•L -1 and 200mmo•L -1 The germination results showed that under 0mmo•L -1 In the control group, there was no significant difference in germination rate between wild type and overexpressed Arabidopsis. -1 Under NaCl stress, there was no significant difference in germination rate between the overexpression strain and the wild type; while under 150mmo•L -1 Under NaCl stress, the germination rate of the overexpression strain was higher than that of the wild type. -1 Under NaCl stress, Arabidopsis thaliana began to germinate on the 2nd day. On the 6th day, the germination rate of the overexpression strain and the wild type reached the highest, and the germination rate of the overexpression strain was higher than that of the wild type. The germination rate of each strain was ranked as At-OE3>At-OE9>At-OE2>WT. Figure 3 shown.

[0050] 4. Overexpression of the MsKTI3 gene promotes salt tolerance in Arabidopsis seedlings in soil

[0051] In order to further determine the salt tolerance of transgenic Arabidopsis seedlings, the overexpression plants and wild-type plants were transplanted into soil (vermiculite: nutrient soil = 1:1) and grown for 3 weeks. At this time, there was no significant difference in the size of the overexpression plants and wild-type plants. Figure 4 As shown, there were no significant differences in leaf color and leaf size between unstressed wild-type plants and transgenic plants.

[0052] Use 250mmol•L-1 After NaCl stress, the growth of wild-type and transgenic lines was significantly inhibited, manifested as short plants and wilting and partial yellowing of leaves, but the degree of inhibition of transgenic lines was less; Figure 5 As shown in the figure, the number of rosette leaves of overexpressed Arabidopsis thaliana treated with salt stress was significantly higher than that of wild-type plants. The relevant physiological indicators of salt-stressed Arabidopsis thaliana were measured and found that at 0mmol•L -1 Under NaCl stress, there was no significant difference in chlorophyll content between the overexpression strain and the wild-type strain. -1 Under NaCl stress, the chlorophyll content of the overexpressing strain was significantly higher than that of the wild type (p<0.05). These results indicate that the leaves of the overexpressing strain were less damaged and had stronger photosynthesis and osmotic regulation capabilities.

[0053] 5. Analysis of the antioxidant capacity of Arabidopsis thaliana under salt stress by overexpressing MsKTI3

[0054] In the natural environment, when plants encounter salt stress, high concentration of salt environment will cause serious oxidative damage to plants. -1 ) treated Arabidopsis thaliana for 17 days, and leaves from the same position of wild-type plants and overexpression plants were stained with NBT (appears blue) and DAB (appears brown). The results showed that under normal culture conditions, there was no obvious difference in the color of Arabidopsis leaves between wild-type (WT) and overexpression (OE) lines; however, under salt stress conditions, compared with wild-type, overexpression lines showed less blue and brown leaves, such as Figure 6 This indicates that less peroxide accumulated in the leaves of Arabidopsis thaliana overexpressing MsKTI3 and the degree of leaf oxidative damage was relatively lower.

[0055] 6. Analysis of Phenotypic and Physiological Indicators of Medicago sativa Overexpressing MsKTI3 in Roots Under Salt Stress

[0056] The recombinant expression vector was transferred into Ar.1193 competent cells. After the colony was identified by PCR, a single clone was picked and streaked on TY solid medium. The sterilized Zhongmu No. 4 seeds were placed on moist filter paper for germination. After 7 days, the seedlings grew two cotyledons and were cut into roots in a clean bench. Then, the roots containing the wound were dipped in Agrobacterium and co-cultured on the culture medium. After 7 days of culture, the cut part began to swell and grow hairy roots. The roots grown from the swollen part were cut a second time (the root cutting at this time was to prevent the occurrence of false positives, but the swollen part at the bottom of the radicle of the plant could not be cut off), and the seedlings were transferred to the selection medium for further culture. After 7 days, hairy roots can be observed growing from the cut of the seedlings. After continuing to culture on the selection medium for 10 days, they were transferred to 1 / 2 Hoagland hydroponic nutrient solution for 10 days. Transgenic hairy root plants were obtained by PCR identification and 200mmol·L -1 After 4 days of NaCl stress, it was found that compared with alfalfa plants overexpressing MsKTI3 in roots, the leaves of wild-type plants turned yellow and wilted more significantly, and their growth was more inhibited. The CAT activity of wild-type plants and overexpressing plants was measured, and the results showed that there was no significant difference in CAT activity between wild-type plants and overexpressing plants without salt stress. -1 After NaCl stress, the CAT activity of the overexpression plants was significantly higher than that of the wild-type plants (p < 0.05). Figure 7 The above results showed that under salt stress, compared with the wild type, overexpression of MsKTI3 in the roots of alfalfa could maintain a relatively stronger antioxidant capacity, which ensured the normal growth of the plant to a certain extent.

[0057] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Use of a vector for overexpressing the alfalfa MsKTI3 gene in improving salt tolerance in alfalfa or Arabidopsis thaliana, characterized in that: The nucleotide sequence of the MsKTI3 gene is shown in SEQ ID NO. 1.

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