Application of taSCAMP17 and recombinant overexpression vector thereof in improving salt tolerance of plants

Abstract

This invention belongs to the field of genetic engineering technology, specifically involving TaSCAMP17 The application of its recombinant overexpression vector in improving plant salt tolerance, the TaSCAMP17 The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. This invention will... TaSCAMP17 Functional validation was performed in Arabidopsis thaliana after overexpression, and the results showed that overexpression... TaSCAMP17 The salt tolerance of wild-type Arabidopsis thaliana is significantly improved, therefore TaSCAMP17 It plays a positive regulatory role in wheat salt tolerance, providing a theoretical basis and gene resources for wheat molecular breeding, and is of great significance for obtaining salt-tolerant and high-yielding lines and improving wheat salt tolerance in the future.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a... TaSCAMP17 The application of its recombinant overexpression vector in improving plant salt tolerance. Background Technology

[0002] Wheat is a staple food for more than one-third of the world's population and is widely cultivated worldwide. However, abiotic stresses such as salinity, coupled with environmental changes in recent years, have severely hampered the further development of wheat production. Therefore, there is an urgent need to provide a new strategy for plants to cope with soil salinity stress. Summary of the Invention

[0003] To address the harmful effects of soil salinity on plants, this invention provides a solution. TaSCAMP17 Applications in improving plant salt tolerance.

[0004] The technical solution adopted in this invention is:

[0005] This invention provides TaSCAMP17 Application in improving plant salt tolerance, the aforementioned TaSCAMP17 The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0006] A second aspect of the present invention provides a recombinant overexpression vector, the recombinant overexpression vector comprising the... TaSCAMP17 .

[0007] A third aspect of the present invention provides a method for preparing the recombinant overexpression vector, comprising the following steps:

[0008] Wheat RNA was extracted, reverse transcribed into cDNA, and then amplified using the cDNA as a template. TaSCAMP17 The overexpression vector was digested with enzymes; TaSCAMP17 The recombinant overexpression vector was ligated with the enzyme-digested overexpression vector to obtain the recombinant overexpression vector.

[0009] Preferably, the overexpression vector is pSuper1300.

[0010] Preferably, the restriction endonuclease used when digesting the overexpression vector is [insert restriction enzyme name here]. Pst I and Kpn I.

[0011] The present invention also provides an application of the recombinant overexpression vector, which improves the salt tolerance of plants by constructing overexpression plants.

[0012] Preferably, the method for constructing the overexpression plant is as follows:

[0013] The recombinant overexpression vector was transformed into Agrobacterium competent cells; the cells were cultured on a large scale to prepare an infection solution; wild-type plants were infected with the infection solution and cultured to obtain overexpressing plants.

[0014] Preferably, the Agrobacterium competent cells are EHA105.

[0015] Preferably, the OD600 value of the infiltration solution is 1.

[0016] Preferably, the conditions for the expanded culture are 28°C, 220 rpm / min dark culture until the OD600 value is 2.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] This invention provides TaSCAMP17 Application in improving plant salt tolerance, the aforementioned TaSCAMP17 The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. This invention will... TaSCAMP17 Functional validation was performed in Arabidopsis thaliana after overexpression, and the results showed that overexpression... TaSCAMP17 The salt tolerance of wild-type Arabidopsis thaliana is significantly improved, therefore TaSCAMP17 It plays a positive regulatory role in wheat salt tolerance, providing a theoretical basis and gene resources for wheat molecular breeding, and is of great significance for obtaining salt-tolerant and high-yielding lines and improving wheat salt tolerance in the future.

[0019] This invention constructs TaSCAMP17 A recombinant overexpression vector of the gene was used to transform Arabidopsis thaliana, resulting in stable overexpression plants. In the Arabidopsis transgenic experiment, the biomass of the overexpression plants after salt stress was significantly higher than that of the wild type. Furthermore, the content of free radicals H2O2 and plasma membrane peroxide MDA, which cause cell damage in the overexpression plants, was significantly reduced after salt stress, while the content of CAT, SOD, POD, APX protective enzymes that can scavenge reactive oxygen species and soluble sugars and soluble proteins Pro, which have osmotic regulatory functions, was significantly increased. This ultimately enhanced the salt tolerance of Arabidopsis thaliana. Attached Figure Description

[0020] Figure 1 for TaSCAMP17 Subcellular localization results: a: Expression results of 35S-GFP in Arabidopsis thaliana; b: Expression results of 35S-GFP in Arabidopsis thaliana. TaSCAMP1 c: Expression results of 7-GFP in tobacco; d: Expression results of 35S-GFP in tobacco TaSCAMP1 7-GFP expression results.

[0021] Figure 2Phenotypic diagrams for salt stress tests on Arabidopsis thaliana overexpression: a: normal treatment group; b: salt treatment group.

[0022] Figure 3 The graphs show the physiological parameters of MDA, reactive oxygen species, and macromolecular osmotic regulators in Arabidopsis leaves before and after salt treatment. In the graphs, a represents MDA content, b represents H2O2 content, and c represents O2 content. - Content graphs: d represents proline content, e represents soluble sugar content, and f represents soluble protein content.

[0023] Figure 4 The graphs show the physiological indicators of antioxidant enzymes in Arabidopsis thaliana leaves before and after salt treatment. In the graphs, a represents POD activity, b represents SOD activity, c represents CAT activity, and d represents APX activity. Detailed Implementation

[0024] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0025] The inventive concept of this invention is as follows:

[0026] SCAMP, or secretory carrier membrane protein, is mainly involved in the endocytic pathway, shuttling between the plasma membrane, endosomes, and the trans-Golgi complex. SCAMP mainly interacts with Na+. ＋ and Cl - Transportation-related, SCAMP for Na + / H + Transporters are of great significance in improving the salt tolerance of plants.

[0027] Currently, in Arabidopsis thaliana and soybean, the effects of... SCAMPs Detailed analysis of the SCAMP gene family has been conducted; however, few reports exist on salt tolerance in wheat. Therefore, to identify salt-tolerant SCAMP genes in wheat, this invention performs a systematic analysis of the wheat SCAMP family at the whole-genome level, screening for candidate salt-tolerant genes. TaSCAMP17 Further functional identification will be conducted to provide candidate gene resources for the genetic improvement of wheat salt tolerance.

[0028] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0029] Example 1

[0030] TaSCAMP17The application of its recombinant overexpression vector in improving plant salt tolerance is as follows:

[0031] 1. TaSCAMP17 Subcellular localization.

[0032] Based on wheat SCAMP Gene family analysis and qRT-PCR expression analysis identified a salt stress-responsive gene. SCAMP Member. Therefore, the full-length CDS region sequence of this gene was cloned from the wild-type wheat variety Fielder. The CDS region sequence is shown in SEQ ID NO.1, and it was named... TaSCAMP17 Its amino acid composition is shown in SEQ ID NO.2.

[0033] SEQ ID NO.1:

[0034] ATGTACCGAGATCCCAACCCCTTCGACGAGGGCGCCGACGACAACGCCTTCTCCAATGGGGGAGGACGCGGTGGTGCTGGTGGTGGCGGCGGCGGCGGCAAGTCGCAGTTCCAGTTTCGCCCGACGGAGCCCGTCGGATTCGGCGCCGGCGGCAACGGGGACGCCGCCGTCGACATCCCCCTTGACAACATGAACGGTTCGAATGGCAAGGAAAGTGAGCTCTCACAGTGGCAGGCAGATCTCAAGAGGCGAGAGGCGGACATCAAGAGGAGGGAGGAAGCTCTCAAGAGCGCTGGGGTGCCCATGGAGGACAAGAACTGGCCCCCGTTCTTCCCGATCATCCACCACGACATTGCCAATGAAATACCAGCCAACGCGCAGCGGTTGCAGTATCTTGCTTTTGCTAGCTGGCTCGGCATCGTGCTTTGCCTCGTTTGGAATTTCATTGCTGTCACAGTCTGTTGGATCAGAGGGGGAGACTCTAAACTGTTTTTCCTGGCTACTATTTATGGTATGCTTGGAGTACCTTTGTCCTACTTGATGTGGTATAGGCCTCTGTACCGTGCAATGAGAACTGACAGCGCATTCAGCTTTGGGTGGTTTTTCCTTTGTTACATGCTCCACATTGGCTTTTGTATAATTGCTGCCATTGCTCCGCCAATCGTATTTCGTGGGAAGTCATTAACGGGTATACTGGCTGCAATCGATACCTTCTCTGATCATGCATTAGTTGGGATACTTTACTTTGTCGGATTTGCCTTGTTTTCATTGGAGACAGTTGTGAGCATTTGGGTTCTTCAGAGAGTATACATGTATTTCAGAGGGCACAAGTGA。

[0035] SEQ ID NO.2:

[0036] MYRDPNPFDEGADDNAFSNGGGRGGAGGGGGGGGKSQFQFRPTEPVGFGAGGNGDAAVDIPLDNMNGSNGKESELSQWQADLKRREADIKRREEALKSAGVPMEDKNWPPFFPIIHHDIANEIPANAQRLQYLAFASWLG IVLCLVWNFIAVTVCWIRGGDSKLFFLATIYGMLGVPLSYLMWYRPLYRAMRTDSAFSFGWFFLCYMLHIGFCIIAAIAPPIVFRGKSLTGILAAIDTFSDHALVGILYFVGFALFSLETVVSIWVLQRVYMYFRGHK*. Where "*" represents the stop codon.

[0037] Using Fielder wheat as the material, when the wheat reached the two-leaf-one-heart stage, 1g of wheat leaves were removed with tweezers, and RNA was extracted using a plant RNA extraction kit purchased from Aikerui Biotechnology Co., Ltd. The specific method for RNA extraction is as follows:

[0038] 1.1 Wheat leaf rupture.

[0039] (1) Transfer fresh plant tissue samples of wheat leaves to a mortar pre-cooled with liquid nitrogen, and grind the wheat leaves with the mortar until they are ground into powder, while continuously adding liquid nitrogen during the grinding process.

[0040] (2) Take 100 mg of wheat leaves that have been ground into powder and transfer them to a 1.5 mL sterile, enzyme-free centrifuge tube containing 500 μL of lysis buffer, ensuring that there are no visible particles. Before use, confirm that 50× reducing agent has been added to the lysis buffer.

[0041] (3) After the previous step is completed, let the above lysis solution stand at room temperature for 2 minutes and then centrifuge at 12000 rpm and 4℃ for 5 minutes.

[0042] (4) Carefully aspirate the supernatant into a new 1.5 mL sterile enzyme-free centrifuge tube.

[0043] 1.2 Purification.

[0044] (1) Add 250 μL of anhydrous ethanol to the supernatant above and mix it with a pipette. If obvious viscous substances or precipitates appear, mix with a pipette several times to disperse the precipitate.

[0045] (2) Transfer all of the above liquid to a plant RNA filter column, centrifuge at 12000 rpm at room temperature for 2 min, and discard the filtrate.

[0046] (3) Add 600 μL of buffer RWA to the plant RNA filter column, centrifuge at 12000 rpm at room temperature for 1 min, and discard the filtrate.

[0047] (4) Add 750 μL of buffer RWB to the plant RNA filter column, centrifuge at 12000 rpm for 1 min at room temperature, and discard the filtrate.

[0048] (5) Deoxyribonuclease I digestion: Prepare the deoxyribonuclease I reaction solution according to Table 1 and mix well. The deoxyribonuclease I reaction solution is the same as the DNase I reaction solution. Add 50 μL of DNase I reaction solution to the center of the membrane of the plant RNA filtration column and let it stand at room temperature for 15 min. Add 350 μL of buffer RWB to the center of the above plant RNA filtration column membrane, centrifuge at 12000 rpm at room temperature for 1 min, and discard the filtrate.

[0049] Table 1. Reaction system of deoxyribonuclease I reaction solution

[0050]

[0051] (6) Add 750 μL of buffer RWB to the plant RNA filter column, centrifuge at 12000 rpm for 1 min at room temperature, and discard the filtrate.

[0052] (7) Place the adsorption column of the plant RNA filter column onto a new 2 mL collection tube and centrifuge at 12000 rpm for 2 min at room temperature.

[0053] (8) Place the adsorption column of the plant RNA filter column onto the new filter column, add 70 μL of sterile water to the center of the adsorption column membrane, let stand at room temperature for 5 min, and then centrifuge at 12000 rpm at room temperature for 2 min to elute the RNA, which can be used for subsequent experiments. If subsequent experiments are not to be performed immediately, the dissolved RNA can be stored at -80℃.

[0054] 1.3. RNA was reverse transcribed into cDNA using a cDNA synthesis kit purchased from Aikerui Biotechnology Co., Ltd. The specific steps for reverse transcription into cDNA are as follows.

[0055] (1) Prepare RNA template solution on ice according to Table 2, and place it in a PCR instrument for denaturation and annealing reaction; denaturation and annealing reaction conditions: 65℃, 5min; store at 4℃.

[0056] Table 2 RNA template solutions

[0057]

[0058] (2) Prepare the reverse transcription reaction system according to Table 3 and synthesize cDNA.

[0059] Table 3 Reverse transcription reaction system

[0060]

[0061] The reaction conditions for cDNA synthesis are shown in Table 4.

[0062] Table 4. cDNA Synthesis Reaction Conditions

[0063]

[0064] 1.4 Design gene-specific primers as shown in Table 5 below, and amplify cDNA using cDNA as a template. TaSCAMP17 The full-length coding frame is linked into the intermediate carrier p-EASY, as follows:

[0065] Table 5 Gene-specific primers

[0066]

[0067] TaSCAMP17 The connection reaction system with the intermediate carrier p-EASY is shown in Table 6.

[0068] Table 6 Connection Reaction System

[0069]

[0070] Mix gently and react at 37°C for 15 minutes. After the reaction is complete, place the centrifuge tube on ice to obtain the ligation product.

[0071] 1.5. Transformation.

[0072] S1. Add the ligation product to 100 μL of E. coli competent cells. Add the ligation product when the E. coli competent cells have just thawed, gently mix, and incubate on ice for 30 min.

[0073] S2, heat shock in a 42℃ water bath for 45 seconds, then immediately place on ice for 3 minutes.

[0074] S3. Add 700 mL of LB liquid medium and incubate at 37°C for 1 h at 200 rpm / min.

[0075] Centrifuge at 5000 rpm for 3 min, discard 700 mL of supernatant, and plate 100 μL of bacterial culture. Incubate at 37 °C for 16 h. Pick 10 single clones from the plate and transfer to 700 mL of LB liquid medium. Incubate at 200 rpm and 37 °C for 6 h. After successful sequencing, pEASY is obtained. TaSCAMP17 Vector, extract plasmid for later use.

[0076] Saved pEASY: TaSCAMP17 Using this as a template, design a pair of primers 35S-TaSCAMP17 -GFP-F and 35S- TaSCAMP1 7-GFP-R, amplification TaSCAMP17 ORF fragments of genes are used for subcellular localization analysis. 35S- TaSCAMP17 -GFP-F and 35S- TaSCAMP17 The sequence of -GFP-R is shown in SEQ ID NO.5 and SEQ ID NO.6.

[0077] SEQ ID NO.5, 35S- TaSCAMP1 7-GFP-F:

[0078] 5'-ACTCTAGAAAGCTT CTGCAG ATGTACCGAGATCCCAACCCC-3'.

[0079] SEQ ID NO.6, 35S- TaSCAMP17 -GFP-R:

[0080] 5'-GCCCTTGCTCACCAT GGTACC CTTGTGCCCTCTGAAATACATGTATA-3'. The underlined portions in SEQ ID NO.5 and SEQ ID NO.6 are the restriction enzyme sites.

[0081] The gel was purified using the In-Fusion® HD Cloning Kit homologous recombination reagent. TaSCAMP17 The ORF fragment was homologously recombinated with the super1300-35S-GFP vector. The reaction product was transformed into E. coli DH5α, and positive clones were sent to a sequencing company. Single clones of the GFP fusion protein that were correctly sequenced, ensured no frameshift, and could be translated into the complete gene were extracted and preserved as plasmids. The resulting plasmid was 35S- TaSCAMP17 -GFP. The In-Fusion® HD Cloning Kit homologous recombination kit was purchased from Takara Corporation, Japan.

[0082] 1.6 TaSCAMP17 Subcellular localization of genes.

[0083] Leaves from 4-week-old Arabidopsis thaliana seedlings were placed in 50 mL Erlenmeyer flasks, and 10 mL of enzymatic hydrolysis solution was added to completely immerse the leaves. After vacuum sealing, the flasks were wrapped with sealing film and enzymatically hydrolyzed at 23°C under weak light for 3 hours. The hydrolysis product was filtered through 40 μm nylon gauze, and the protoplasts were collected into 2 mL centrifuge tubes. The tubes were centrifuged at 400 rpm for 5 minutes at 4°C to collect the precipitate. The precipitate was washed twice with pre-chilled W5 solution, resuspended, and incubated on ice for 30 minutes. The resuspended solution was centrifuged at 400 rpm for 5 minutes, and the precipitate was resuspended in MMG solution. 35S- TaSCAMP17 - GFP plasmid, cell membrane localization marker plasmid, and 40% PEG4000 solution were slowly inverted to mix, and incubated at 22.5°C for 20 min. The protoplasts were diluted with W5 solution to terminate the reaction. The mixture was centrifuged at 400 rpm for 5 min, and the precipitate was washed twice with W5 solution and resuspended. The mixture was then incubated overnight at 23°C under low light. The protoplast suspension was centrifuged at 400 rpm for 5 min to collect the protoplasts, and the fluorescence distribution was observed under a laser confocal microscope.

[0084] The formula for W5 solution is: 154mM NaCl, 125mM CaCl2, 2mM KH2PO4, 2mM MES.

[0085] The MMG solution is formulated as follows: 0.4M Mannitol, 15mM MgCl2·6H2O, 4mM MES.

[0086] The enzymatic hydrolysate was formulated as follows: 1.5% cellulase R-10 by mass, 0.75% dissociative enzyme R-10 by mass, 0.6M mannitol, 10mM MES, and 40% 2-mercaptoethanol by volume.

[0087] Cellulase R-10: Enzyme activity 10 U / mg, purchased from Solarbio.

[0088] Isolase R-10: Enzyme activity is 3000 U / g, purchased from Solarbio.

[0089] At the same time, observations were also made in tobacco leaves. TaSCAMP17 The subcellular localization of genes showed that... TaSCAMP17 Genes are located on the cell membrane, overlapping with the signals of cell membrane localization markers, such as... Figure 1 As shown.

[0090] 2. TaSCAMP17 Application of recombinant overexpression vectors in improving plant salt tolerance.

[0091] 2.1 Construction of recombinant overexpression vectors.

[0092] Saved pEASY:TaSCAMP17 Using plasmid as a template, design a pair of primers. TaSCAMP17 -OE-F1 and TaSCAMP17 -OE-R1, amplification TaSCAMP17 ORF fragments of genes. TaSCAMP17 -OE-F1 and TaSCAMP17 The primer sequences for -OE-R1 are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively.

[0093] SEQ ID NO.7, TaSCAMP17 -OE-F1:

[0094] 5'-GACTCTAGAAAGCTT CTGCAG ATGTACCGAGATCCCAACCCC-3'.

[0095] SEQ ID NO.8, TaSCAMP17 -OE-R1:

[0096] 5'-GCCCTTGCTCACCAT GGTACC TCACTTGTGCCCTCTGAAATACA-3'.

[0097] The underlined part in SEQ ID NO.7 is Pst The restriction enzyme site of I, underlined in SEQ ID NO. 8 Kpn I. Enzyme cleavage site.

[0098] The gel was purified using the In-Fusion® HD Cloning Kit homologous recombination reagent. of TaSCAMP17 The ORF fragment was subjected to homologous recombination with the enzyme-digested overexpression vector pSuper1300. The reaction product was transformed into E. coli DH5α, and positive clones were sent to the company for sequencing. Plasmids were extracted and preserved from correctly sequenced single clones to obtain the recombinant overexpression vector, pSuper1300. TaSCAMP17 The specific steps are as follows:

[0099] (1) The pSuper1300 enzyme digestion system is shown in Table 7. TaSCAMP17 The amplification system is shown in Table 8.

[0100] Table 7. Enzyme digestion system for overexpression vector pSuper1300

[0101]

[0102] Table 8 TaSCAMP17 Amplification System

[0103]

[0104] The overexpression vector pSuper1300 after enzyme digestion and the amplified TaSCAMP17 Perform glue recycling and verify the accuracy of the recycling.

[0105] (2) Using seamless cloning, the enzyme-digested overexpression vector pSuper1300 and the amplified... TaSCAMP17 For ligation, please refer to the integrated ultra-rapid cloning kit from Nanjing Novizan Biotechnology Co., Ltd. The cloning reaction system is shown in Table 9, and the specific steps are as follows:

[0106] Table 9 Cloning reaction system

[0107]

[0108] Mix gently and react at 50°C for 15 minutes. After the reaction is complete, place the centrifuge tube on ice to obtain the ligation product.

[0109] The conversion process is as follows:

[0110] S1. Add the ligation product to 100 μL of E. coli competent cells. Add the ligation product when the E. coli competent cells are just thawed, gently mix, and incubate on ice for 30 min. The E. coli competent cells were purchased from Qingke Biotechnology Co., Ltd.

[0111] S2, heat shock in a 42℃ water bath for 45 seconds, then immediately place on ice for 3 minutes.

[0112] S3. Add 700 mL of LB liquid medium and incubate at 37°C for 1 h at 200 rpm / min.

[0113] Centrifuge at 5000 rpm for 3 min, discard 700 mL of supernatant, take 100 μL of bacterial culture, plate it, and incubate at 37 ℃ for 16 h.

[0114] After selecting single colonies and identifying positive colonies by PCR, send them for testing. After amplifying and shaking the correctly sequenced bacterial culture, extract the plasmid to obtain the recombinant overexpression vector for later use.

[0115] The recombinant overexpression vector was transformed into Agrobacterium competent cells EHA105, plated on YEB plates containing rifampicin + kanamycin resistance, and incubated at 28°C for approximately 48 hours. Positive clones were verified by PCR. The positive clone culture was mixed with an equal volume of 30% sterile glycerol, rapidly frozen in liquid nitrogen, and stored at -80°C for later use.

[0116] 2.2 Construction and validation of overexpression plants.

[0117] Agrobacterium containing the recombinant overexpression vector was inoculated into 5 mL of liquid LB medium containing rifampicin and kanamycin antibiotics and cultured in the dark at 28°C with shaking at 220 rpm / min for approximately 18 h. At a ratio of 1:100, 1 mL of the bacterial culture was inoculated into 100 mL of liquid LB medium containing rifampicin and kanamycin antibiotics and cultured in the dark at 28°C until the OD600 value reached 2. The culture was then transferred to sterile 50 mL centrifuge tubes and centrifuged at 5000 rpm / min at 28°C for 8 min to collect the bacterial cells, discarding the supernatant. The collected bacterial cells were resuspended in transformation osmotic buffer until the OD600 value reached 1, yielding the infection solution. Flower buds of wild-type Arabidopsis thaliana in the flowering stage were immersed in the above infection solution for 2 h for infection. After infection, the plants were placed sideways in a moist tray and kept in darkness for 48 hours before being placed upright for further cultivation at 23°C with a photoperiod of 16h / 8h. Seeds were collected as much as possible as possible; the collected seeds were the T1 generation. After sterilization, the T1 seeds were spread extensively on culture plates containing 25 mg / L hygromycin. Healthy seedlings with well-developed root systems were selected and transferred to seedling pots for further cultivation. The mass ratio of nutrient soil, vermiculite, and perlite in the seedling pots was 6:3:1. Before bolting, leaf DNA was extracted to verify positive plants, using a recombinant overexpression vector as a positive control and wild-type Arabidopsis as a negative control. RNA was extracted from leaves of the verified positive plants and reverse transcribed for expression level analysis, using Arabidopsis as the basis for further analysis. Actin As an internal reference gene, the primer sequences for positive plant validation and expression level analysis are as follows:

[0118] SEQ ID NO. 9, super1300-F: 5'-GCCATTCGCCTTTTCAG-3'.

[0119] SEQ ID NO.10, TaSCAMP17 -R1: 5'-TCACTTGTGCCCTCTGAAATACA-3'.

[0120] SEQ ID NO.11, AtActin -F: 5'-AGGAAGGATCTGTACGGTAAC-3'.

[0121] SEQ ID NO.12, AtActin- R: 5'-TTCTGTGAACGATTCCTGGAC-3'.

[0122] SEQ ID NO. 9 and SEQ ID NO. 10 are primers for verifying positive plants; SEQ ID NO. 11 and SEQ ID NO. 12 are... Actin Amplification primers.

[0123] Select TaSCAMP17 Individual positive plants with high gene expression levels were harvested to obtain T2 seeds. T2 seeds were spread on culture plates containing 25 mg / L hygromycin, and resistance performance was recorded based on seedling survival rate. Lines with a positive:negative segregation ratio of 3:1 were retained. Healthy seedlings from the retained T2 lines were transferred to nutrient pots for further cultivation. Upon maturity, T3 seeds were harvested from individual plants. 100 seeds from each T3 line were spread on culture plates containing 25 mg / L hygromycin, and resistance performance was recorded based on seedling survival rate. Lines with a chi-square test result of 95:5 were retained. The three overexpressing plants were designated as SCAMP. - OE1, SCAMP - OE2 and SCAMP - OE3.

[0124] The formulation of the conversion osmotic solution is as follows: 1 / 2 MS, 5% sucrose by mass, 20% silwet L-77 by volume, 0.05% MES by mass, 1% AS by mass, and 1% 6-BA by volume.

[0125] 2.3 TaSCAMP17 Identification of salt tolerance phenotype in plants with overexpressed genes.

[0126] SCAMP was obtained from wild-type Arabidopsis thaliana and overexpression plants. - OE1, SCAMP - OE2 and SCAMP - OE3 seeds were sterilized with 75% alcohol (v / v) for 2 min, then sterilized with 10% sodium hypochlorite (v / v) for 5 min, and rinsed 5 times with sterile water for 2 min each time. The sterilized seeds were evenly spread on 1 / 2 MS solid medium containing 1.5% sucrose and 0.4% plant gel (pH 5.8). Excess moisture on the surface of the medium was removed. After vernalization at 4°C for 3 days, the seeds were placed in an Arabidopsis thaliana culture chamber at 23°C for 16 h / 8 h incubation. At the four-leaf stage, the seeds were transplanted into nutrient pots. After two weeks of cultivation, when the seeds reached 6 leaves, a salt treatment group and a normal treatment group were established for the experiment. The salt treatment group was subjected to 250 mM NaCl stress, while the normal treatment group was not subjected to NaCl stress. Phenotypic changes were observed after 10 days. Each experiment was repeated three times to ensure data accuracy.

[0127] After 10 days of treatment, the phenotype of Arabidopsis thaliana was observed and recorded. Figure 2In the normal treatment group, both wild-type and overexpression lines grew normally without significant differences. In the salt treatment group, however, a significant difference emerged between the wild-type and overexpression lines. Wild-type plants exhibited weaker growth and severe leaf wilting, while the overexpression plants maintained normal growth, demonstrating… TaSCAMP17 The overexpressing plants exhibit better salt tolerance compared to the wild-type plants.

[0128] Salt stress can damage biofilms, causing selective biofilm destruction, accumulation of free radicals (H2O2), and increased content of plasma membrane peroxides (MDA). Under salt stress conditions, plant cells accumulate reactive oxygen species (ROS) and produce excess O2. ⁻ This can trigger oxidative damage, destroying membrane lipids, proteins, and DNA, ultimately leading to cell death. The overexpressing plants of this invention were subjected to MDA, H2O2, and O2 tests with wild-type Arabidopsis. - Content determination showed that, under normal conditions, the levels of MDA, H2O2, and O2 in overexpressed plants and wild-type Arabidopsis thaliana were significantly higher. - There was no significant difference in the content of MDA, H2O2, and O2 in plants overexpressing under salt stress treatment. - The content was significantly reduced. Experiments showed that overexpression... TaSCAMP17 Following gene modification, plants were able to reduce the damage to biofilm caused by salt stress and improve their adaptability to high-salt environments. The above results are shown in [reference needed]. Figure 3 a~c.

[0129] Under salt stress, plants can enhance their osmotic regulation capacity and improve salt tolerance by upregulating proline, soluble sugar, and soluble protein levels. The contents of proline, soluble sugar, and soluble protein (Pro) in overexpressing plants and wild-type Arabidopsis were measured. The results showed that under normal conditions, there was no significant difference in the contents of proline, soluble sugar, and soluble protein between the overexpressing plants and wild-type Arabidopsis; however, under salt stress treatment, the contents of proline, soluble sugar, and soluble protein in the overexpressing plants were significantly increased. This experiment demonstrates that... TaSCAMP17 Following gene modification, plants can enhance their osmotic regulation capacity and salt tolerance by increasing the content of proline, soluble sugars, and soluble proteins. (See above results.) Figure 3 d~f.

[0130] CAT, SOD, POD, and APX are protective enzyme systems in plants. Their coordinated action can scavenge H2O2, reactive oxygen species, and MDA, thereby protecting the membrane system from damage. The contents of CAT, SOD, POD, and APX in overexpressing plants and wild-type Arabidopsis were measured. The results showed that under normal conditions, there was no significant difference in the content of protective enzymes between the two plants, but the content of protective enzymes in the overexpressing plants was significantly increased under salt stress treatment. This experiment demonstrates that overexpression... TaSCAMP17 Following gene modification, plants can enhance their ability to scavenge H2O2, reactive oxygen species, and MDA by increasing the levels of CAT, SOD, POD, and APX, thereby improving their salt tolerance. These results are shown in [link to relevant documentation]. Figure 4 .

[0131] The above results show that TaSCAMP17 Overexpression of genes can improve the salt tolerance of plants.

[0132] In production practice, the above can be used as a reference. TaSCAMP17 Wheat explant cells were transformed, and the resulting callus tissue was then cultured into plants. Salt-tolerant wheat was bred by transforming explant cells using plant expression vectors via transgenic methods.

[0133] In production practice, the above can also be applied to... TaSCAMP17 Marker-assisted selection (MAG) in breeding methods enhances the efficiency and accuracy of breeding objectives. For example, molecular markers can be used to select target genes... TaSCAMP17 This invention relates to the salt tolerance trait in wheat, and by detecting the presence of the target gene, aims to select for the target trait. TaSCAMP17 The study of genes regulating salt tolerance in plants and their specific applications provides new genetic resources for wheat salt tolerance breeding and also lays the foundation for further analysis. TaSCAMP17 This lays the foundation for understanding the molecular mechanisms regulating salt tolerance in plants.

[0134] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0135] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. TaSCAMP17 The application of genes in improving plant salt tolerance is characterized by, The TaSCAMP17 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2; Improving plant salt tolerance refers to: utilizing the above-mentioned... TaSCAMP17 Recombinant overexpression vectors of genes, construction TaSCAMP17 Gene overexpression in plants can improve their salt tolerance. The plant in question is either Arabidopsis thaliana or wheat.

2. The application as described in claim 1, characterized in that, The preparation method of the recombinant overexpression vector is as follows: Wheat RNA was extracted, reverse transcribed into cDNA, and then amplified using the cDNA as a template. TaSCAMP17 Gene sequence; The overexpression vector was digested with enzymes; Will TaSCAMP17 The gene sequence is ligated with the enzyme-digested overexpression vector to obtain a recombinant overexpression vector.

3. The application as described in claim 2, characterized in that, The overexpression vector is pSuper1300.

4. The application as described in claim 3, characterized in that, The restriction endonuclease used for digesting the overexpression vector is: Pst I and Kpn I.

5. The application as described in claim 1, characterized in that, The method for constructing the overexpression plant is as follows: The recombinant overexpression vector was transformed into Agrobacterium competent cells; Expand the culture and prepare the infection solution; Wild-type plants were infected with an infection solution and cultured to obtain overexpressing plants.

6. The application as described in claim 5, characterized in that, The Agrobacterium competent cells were EHA105.

7. The application as described in claim 5, characterized in that, The OD600 value of the infiltration solution is 1.

8. The application as described in claim 5, characterized in that, The conditions for the expanded culture were 28°C and 220 rpm in the dark until the OD600 value was 2.

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