Application of MsGDSL gene in improving salt tolerance

Abstract

The application discloses MsGDSL Application of a gene in improving salt tolerance belongs to the technical field of biotechnology. MsGDSL The gene is located in the nucleus and cytoplasm. It is found through yeast self-activation detection that the gene has no transcriptional self-activation activity. MsGDSL After overexpression of the gene, MsGDSL The expression amount is significantly up-regulated, the root length of a transgenic strain is significantly higher than that of WT, the degree of wilting is lower than that of WT, the chlorophyll content is higher than that of WT, the conductivity and mortality are lower than those of WT, and the salt tolerance function is enhanced. MsGDSL After silencing of the gene, MsGDSL The expression amount is reduced, the chlorophyll content and survival rate of the transgenic strain are lower than those of CK, the color is darker after DAB and NBT staining, and the oxidative damage is serious. MsGDSL The application discloses the positive regulation effect of the gene in plant salt tolerance for the first time, and has great significance in the field of breeding of new varieties (strains) of salt-tolerant plants.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and particularly relates to... MsGDSL Application of genes in improving salt tolerance. Background Technology

[0002] Soil salinization is a significant factor limiting global agricultural development. Statistics show that there are over 10 billion mu (approximately 6.67 million hectares) of saline-alkali land worldwide, and a large amount of farmland is subjected to secondary salinization every year. Therefore, developing and utilizing saline-alkali land is an important way to increase my country's land reserve resources and ensure national food security.

[0003] Alfalfa is an important high-protein, high-quality forage for developing dairy and other forage industries. It is also the most widely distributed and cultivated perennial legume forage in the world. Due to its high nutritional value, rich in protein, carbohydrates, and various vitamins, it is known as the "King of Forages." Furthermore, alfalfa's root nodules can fix atmospheric nitrogen, playing a vital role in improving saline-alkali land and enhancing agricultural production. Therefore, improving alfalfa's salt tolerance can not only increase the yield of high-quality forage and alleviate the shortage of protein forage in my country, but also significantly improve the utilization rate of saline-alkali land. Currently, a number of salt-tolerant alfalfa varieties have been bred using conventional breeding methods. However, genetic improvement of salt tolerance in alfalfa remains challenging, mainly because alfalfa has high heterozygosity and severe self-pollination depression, making it difficult to generate inbred lines. In addition, its response to salt stress is physiologically and genetically complex; salt tolerance is controlled by multiple genes and involves various biochemical and physiological mechanisms, which also contributes to the slow progress of alfalfa breeding.

[0004] Traditional breeding methods are time-consuming and inefficient, thus genome-wide association studies (GWAS) have opened up new avenues for breeding. GWAS uses natural populations, accumulating rich information on genetic recombination and variation, and offers high mapping accuracy, capable of simultaneously detecting multiple alleles. However, current research on its application to alfalfa salt tolerance is still very limited. Summary of the Invention

[0005] To address the aforementioned shortcomings in existing technologies, this invention, for the first time, employs GWAS analysis to analyze the genetic structure of traits related to salt tolerance at the seedling and early flowering stages in the field, identifying several significant SNP loci. Simultaneously, through transcriptome analysis and genome-wide association analysis, candidate genes associated with salt tolerance traits were identified. MsGDSL This candidate gene can provide an important genetic resource for the molecular genetic improvement of salt tolerance in alfalfa.

[0006] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: One of the objectives of this invention is to provide MsGDSLThe application of genes in regulating plant salt tolerance, the aforementioned MsGDSL The nucleotide sequence of the gene is shown in SEQ ID NO.7.

[0007] Furthermore, the aforementioned MsGDSL Genes play a positive regulatory role in this application.

[0008] Furthermore, the positive regulation method includes overexpression MsGDSL Gene.

[0009] A second objective of this invention is to provide a primer combination for overexpressing the... MsGDSL The gene, whose nucleotide sequence is shown in SEQ ID NO.9~SEQ ID NO.10.

[0010] A third objective of this invention is to provide a kit containing the aforementioned primer combination.

[0011] A fourth objective of this invention is to provide a recombinant expression vector containing the aforementioned... MsGDSL Gene.

[0012] The fifth objective of this invention is to provide a host bacterium containing the aforementioned... MsGDSL The gene may contain the recombinant expression vector.

[0013] The sixth objective of this invention is to provide the application of the primer combination, the kit, the recombinant expression vector, and / or the host bacterium in improving plant salt tolerance.

[0014] Furthermore, the applications include the development of new salt-tolerant plant varieties and / or new lines.

[0015] Furthermore, the plant species mentioned include alfalfa.

[0016] Furthermore, the present invention also provides a method for silencing the aforementioned... MsGDSL Primer combinations for genes, kits, recombinant expression vectors, host bacteria, and their applications in reducing plant salt tolerance (some studies also require reducing plant salt tolerance, and this has practical application value).

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention is the first to discover MsGDSL Its role in improving plant salt tolerance. And this was clarified through subcellular localization experiments. MsGDSL It is located in the cell nucleus and cytoplasm. Yeast self-activation assays revealed no transcriptional self-activation activity. Overexpression and silencing vectors were constructed, and overexpression showed… MsGDSL After genes, MsGDSLSignificantly upregulated expression levels resulted in transgenic lines with significantly longer root lengths, lower yellowing levels, higher chlorophyll content, lower electrical conductivity and mortality rates, and enhanced salt tolerance; while silenced expression levels were significantly higher. MsGDSL After genes, MsGDSL Reduced expression levels, lower chlorophyll content and survival rate in transgenic lines compared to the control (CK), and darker staining with DAB and NBT indicating severe oxidative damage. This invention is the first to discover... MsGDSL Genes play a positive regulatory role in plant salt tolerance. This invention is of great significance in the field of breeding new salt-tolerant plant varieties (lines). Attached Figure Description

[0018] Figure 1 In Embodiment 1 of the present invention MsGDSL Gene structure, CDs sequence, amino acid sequence.

[0019] Figure 2 In Embodiment 1 of the present invention MsGDSL Results of tissue-specific gene differentiation and expression levels under different stresses.

[0020] Figure 3 In Embodiment 1 of the present invention MsGDSL Subcellular localization.

[0021] Figure 4 In Embodiment 1 of the present invention MsGDSL The self-activation verification result.

[0022] Figure 5 Overexpression in Example 1 of the present invention MsGDSL Identification results of positive Arabidopsis thaliana plants.

[0023] Figure 6 Overexpression in Example 1 of the present invention MsGDSL Identification of positive alfalfa plants, note: (A) 1 day; (B) 7 days; (C) 45 days; (D) 3 months; (E) 5 months; (F) 8 months.

[0024] Figure 7 Overexpression in Example 1 of the present invention MsGDSL Specific alfalfa positive plants MsGDSL Gene expression levels.

[0025] Figure 8 The results show the identification of the alfalfa gene-edited mutant plant in Example 1 of this invention.

[0026] Figure 9 The VIGS plant in Example 1 of this invention MsGDSL Results of expression level analysis.

[0027] Figure 10 Overexpression in Example 1 of the present invention MsGDSL Verification process of enhanced salt tolerance in Arabidopsis thaliana.

[0028] Figure 11 This describes the salt-treated phenotype and physiological indicators of the gene-silencing line in Example 1 of this invention.

[0029] Figure 12 Overexpression in Example 1 of the present invention MsGDSL Verification process of enhanced salt tolerance in alfalfa. Detailed Implementation

[0030] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the invention without departing from its spirit and essence are within the scope of the invention. The reagents, kits, and instruments used in the following examples are commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventional methods.

[0031] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0032] Example 1 1 MsGDSL Gene cloning, bioinformatics analysis, and gene expression pattern analysis 1.1 Materials and Methods 1.1.1 Test Materials “Zhongmu No. 4” alfalfa and Tobacco Benedict are from the forage breeding team of the Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences.

[0033] 1.1.2 Gene cloning and analysis of basic physicochemical properties Cloning from alfalfa (Zhongmu 4) MsGDSL The full-length cDNA of the gene was obtained, and the primers are shown in Table 1. Amplification was performed using 2×PhantaMax Master Mix (Dye Plus) (Vazyme, Nanjing, China), with a total reaction volume of 50 μL (Table 2). The reaction program was as follows: pre-denaturation at 98℃ for 30 sec; 34 cycles of denaturation at 98℃ for 10 sec; annealing at 55℃ for 5 sec; extension at 72℃ for 5 sec / kb; and final extension at 72℃ for 1 min. The PCR products were purified by gel electrophoresis (TransGen, Beijing, China). The purified products were sent to the company for sequencing and sequence comparison.

[0034] Table 1. Primers for gene cloning

[0035] Table 2 Gene Cloning PCR System

[0036] Gene structure analysis was performed using the GSDS online software, analyzing open reading frames (ORFs), 5'-UTRs, and 3'-UTRs. Subcellular localization prediction was performed using the WOLF PSORT online software. ProtParam online software was used for analysis. MsGDSL The physicochemical properties of the gene-encoded protein were determined, and its molecular weight and isoelectric point were estimated. TMHMM 2.0 software was used to analyze the protein. MsGDSL Predicting the transmembrane structure of gene-encoded proteins.

[0037] 1.1.3 Different biological stress treatments on alfalfa Alfalfa seeds were germinated in petri dishes under light for 5 days, then transferred to hydroponic boxes containing half Hoagland nutrient solution and cultured at approximately 25°C for 4 weeks. Normally growing plants were then transferred to nutrient solutions containing 200 mmol / L NaCl, 15% PEG, and normal nutrient solution for salt treatment, drought treatment, and control, respectively. Additionally, some plants were placed in a 4°C cryogenic incubator for cryogenic treatment. Samples were taken at 0 h, 6 h, 12 h, 24 h, and 6 days after treatment. Equal amounts of roots, stems, and leaves were collected, frozen in liquid nitrogen, and stored at -80°C.

[0038] 1.1.4 Real-time quantitative PCR (qRT-PCR) determination of expression levels The samples were removed from the refrigerator, and total RNA was extracted according to the RNAiso reagent manufacturer's instructions (TaKaRa, Dalian, China). Reverse transcription was performed using 1 µg of the extracted RNA (Novizan, Nanjing, China). qRT-PCR primers were designed using Primer Premier 5.0 software (Table 3). The internal reference gene was alfalfa. Actin Genes. The total reaction volume was 20 μL, as shown in Table 4 below. qRT-PCR amplification was performed using a BioRad instrument. The program was as follows: pre-denaturation at 95℃ for 5 min; 40 cycles of 95℃ for 15 sec and 60℃ for 30 sec; after cycling, a melting curve was generated at 65℃-95℃. Using 2... -ΔΔCt Relative expression levels were calculated using the method described above. Error analysis of the data used was based on standard deviation (SEM).

[0039] Table 3 qRT-PCR primers

[0040] Table 4 Real-time fluorescence quantitative system

[0041] 1.1.5 Subcellular localization experiment Prepare the resuspension: Add water to 100 mL of 1 mL MES (1 mol / L), 1 mL MgCl2 (1 mol / L), and 10 μL acetylsylgenone (1 mol / L).

[0042] A quantity of wild-type tobacco seeds were sown and cultured under 22℃ light for 12 hours. After one month of culture, the seeds were ready for experimentation. LB liquid (containing Kan and Rif) was added to a container with a pH of 1300- MsGDSL1 Agrobacterium-tuberculosis containing the recombinant plasmids pFGC-YFP (overexpression vector) and pFGC-YFP (empty vector) was cultured at 28°C until OD600. 600 =0.6; centrifuge at 4000 rpm / min for 4 min, discard the supernatant; resuspend the resuspended solution to OD. 600 =0.4, place at room temperature for 2-3 hours; select tobacco plants with good growth, inject into the lower epidermis of tobacco leaves using a 1 mL syringe with the nozzle removed, and make a label; culture the injected tobacco plants in low light for 2 days, and then observe; take the labeled tobacco leaves injected with Agrobacterium, make them into slides, observe them under a laser confocal microscope, and take pictures.

[0043] 1.1.6 Verification of Transcriptional Autoactivation 1) Take three 1.5 mL centrifuge tubes and label them "Negative Control", "Positive Control", and "Recombinant Vector (pGBKT7-)" respectively. MsGDSL Add the following to each of the following: 50 μL competent cells, 10 μL plasmid DNA (approximately 0.5-1 μg, empty vector / positive control / recombinant vector), 50 μL 2 mg / mL ssDNA (denatured by boiling in water for 5 min, then cooled in an ice bath), and 300 μL PEG3350-LiAc mixture (240 μL 50% PEG3350 + 36 μL 1 M LiAc + 24 μL sterile water). Vortex and incubate at 30°C for 30 min (gently mix every 10 min).

[0044] 2) Add 40 μL DMSO, gently invert to mix, and heat shock in a 42℃ water bath for 15 min (do not shake).

[0045] 3) Ice bath for 2 min, centrifuge at 12000×g for 15 s, and discard the supernatant.

[0046] 4) Resuspend the bacterial cells in 100 μL of sterile water and spread them onto SD / -Trp solid medium (to screen for transformants containing the vector). Incubate at 30°C upside down for 3-5 days until single colonies grow.

[0047] 5) Pick single colonies (1-2 mm in diameter) from each experimental group from the SD / -Trp plate and streak them onto the following culture media: SD / -Trp-Leu and SD / -Trp-His-Ade-Leu. Incubate at 30℃ for 3-5 days and observe the colony growth.

[0048] 1.2 Results and Analysis 1.2.1 Analysis of gene structure and basic physicochemical properties Subcellular localization prediction discovery MsGDSL The encoded proteins are mainly located in the extracellular matrix and cytoplasm. MsGDSL The coding region of the gene is 1116 bp long, with a total of 372 amino acids and 5 exons. Figure 1 Analysis using online analysis tools showed that the protein has a relative molecular weight of 41.54 kDa, a theoretical isoelectric point of 6.4, and is a neutral protein. MsGDSL The signal peptide score is greater than 0.5, indicating the presence of signal peptide distribution. It is a secreted protein and lacks transmembrane domains (Table 5).

[0049] Table 5. Analysis of Basic Physicochemical Characteristics of Genes

[0050] MsGDSL Nucleotide sequence (SEQ ID NO.7) MsGDSL Amino acid sequence (SEQ ID NO.8) MKIFILFGITFACGFFGNFISNANPLPYEAIFNFGDSTSDTGNAAFDHLKEMEKLIPYGSTYFKHPSGRMSNGRLIIDFIAEAYGLPFLPAYKNITKIPDAIKKGVNFAYAGSTALDVKYFSGISGVTAPTESLNVQFDWFKKLKPDLCKSKEECDSFFKNSLFIVGEIGANDIFYHLSKTITEL REIIPLMVESIKNTTNALIEEGAVELVVPGNPFPMGCNTDILSKKISQKKEDYDEFGCLTAYNTLIEYFNEQLKKSIETIKQKHPQAKIVYFDYYNDARRLYQAPQQYGFTSDKVEVLKACCGGSGPYHHDLYWCGTPNTTVCSDPSKLINWDGPHFTEAAYKQIAKGLIEGPFAYPSLKPAPFKIA 1.2.2 MsGDSL Gene expression pattern analysis Detected by qRT-PCR MsGDSL The expression levels of alfalfa in roots, stems, and leaves were analyzed, and found to be significantly higher in roots than in leaves and stems, with no significant difference between leaves and stems. Simultaneously, qRT-PCR was used to detect... MsGDSL The expression patterns of alfalfa under salt, drought, and cold stress were analyzed, and the results showed that under salt treatment... MsGDSL Gene expression levels initially increased and then decreased, with a significant increase at 12 hours of treatment. Under drought treatment, expression levels initially increased and then decreased, with significant changes at 6, 12, and 24 hours. Under cold treatment, expression levels decreased over time, with significant decreases at 6 hours, 12 hours, and 6 days. Therefore, MsGDSL Genes respond to salt, drought, and cold stress at the transcriptional level. Figure 2 ).

[0051] 1.2.3 MsGDSL Subcellular localization Detected by subcellular localization assay MsGDSL Location and structural discovery of expression in cells MsGDSL It is located in the nucleus and cytoplasm, which differs from the predicted location, possibly due to the presence of a free signal peptide in the gene. Figure 3 Yeast self-activation detection revealed the presence of BD-. MsGDSLYeast colonies containing the vector plasmid can grow normally on a two-deficient medium, but cannot grow on a four-deficient medium, demonstrating that it lacks transcriptional autoactivation activity. Figure 4 ).

[0052] 2. Genetic transformation of Arabidopsis thaliana and alfalfa and identification of positive plants 2.1 Materials and Methods 2.1.1 Test Materials “Zhongmu No. 4” alfalfa and Tobacco Benedict are from the forage breeding team of the Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences.

[0053] 2.1.2 Arabidopsis genetic transformation 2.1.2.1 Gene Cloning RNA was extracted from leaf tissues of 'Zhongmu 4' alfalfa and reverse transcribed into cDNA. Cloning was then performed from the cDNA. MsGDSL Gene. Cloning primers are shown in Table 6, and the reaction system and procedure are the same as in 1.1.2.

[0054] Table 6 Gene Cloning Primers

[0055] Note: In the above sequences, lowercase letters represent homologous arms of primers, and uppercase letters represent primers for gene fragments.

[0056] 2.1.2.2 Carrier Construction Using restriction endonucleases NcoI (TaKaRa, Dalian, China) The 3301 vector was digested with enzymes, and the digestion reaction system is shown in Table 7. The digestion reaction procedure was as follows: 30℃, 1 h. The reaction product was purified by gel electrophoresis.

[0057] Table 7 Enzyme digestion reaction system

[0058] Using the Basic Mix reagent provided by TransGen, the target fragment and the enzyme-digested vector were recombined by homologous recombination (Table 8). The recombination reaction procedure was as follows: 50℃, 15 min; then cooled to 4℃ or immediately placed on ice.

[0059] Table 8 Homologous recombination reaction system

[0060] The above product was transformed into *E. coli* chemically competent cells DH5α, and plasmids were extracted from successfully transformed monoclonal colonies. 500 ng of plasmid DNA was added to *Agrobacterium* competent cells EAH105, and transformation was performed to obtain cells containing... MsGDSL Agrobacterium -3301 overexpression vector.

[0061] 2.1.2.3 Infection of Arabidopsis thaliana Wild-type Arabidopsis seeds were sown into the soil and cultured under 22℃ light for 16 hours. Once the Arabidopsis reached its peak flowering period, it was ready for experiments. The preserved seeds containing... MsGDSL Agrobacterium overexpressing the vector was added to LB liquid medium (containing Kan and Rif) and cultured at 28°C until OD600 = 1.2-2.0; centrifuged at 5000×g, 4°C for 10 min, and the supernatant was discarded; the culture was resuspended in 5% sucrose solution, and the OD600 of the culture was 0.8-1.0; the culture was activated at 120 rpm for 30 min, and 0.03% Silwetl-77 surfactant was added to infect Arabidopsis thaliana; the inflorescences were completely immersed in the culture for 10 sec and cultured in the dark for 24 h, and a second infection was performed after one week. Mature Arabidopsis seeds were collected after 3-4 weeks.

[0062] After surface sterilization, T0 generation Arabidopsis seeds were spread onto 1 / 2 MS medium plates (containing 7.5 mg / L PPT) and vernalized for two days before being placed in a light incubator. Plants that were still growing normally after 10-14 days of culture were selected and transplanted into soil. Leaves were harvested 3-4 weeks later, and DNA was extracted for identification of positive seedlings. Primers used in the PCR identification are shown in Table 9.

[0063] Table 9 Primers for Vector Identification

[0064] Seeds from T1 generation positive seedlings were harvested and screened until T3 generation homozygous; homozygous lines were selected for qRT-PCR verification, using the same method as in 1.1.4. Transgenic lines OE6, OE11, and OE14 with high expression levels were selected for subsequent experiments.

[0065] 2.1.3 Genetic transformation of alfalfa Take 3-4 leaves from the top of the stem of alfalfa (Zhongmu 4), and wash them with an appropriate amount of Tween and deionized water; disinfect the leaves in a clean bench with 75% alcohol and 10% sodium hypochlorite before inoculation; add [amount of ingredients] to LB liquid medium (containing Kan and Rif) MsGDSL Agrobacterium overexpressing the vector (same as the vector in 2.1.2) was cultured at 28°C until OD600 = 0.6-0.8; centrifuged at 5000×g at 4°C for 10 min, and the supernatant was discarded; the bacterial suspension was resuspended in SH3a liquid medium until OD600 = 0.2-0.4; the sterilized leaves were placed in the resuspended bacterial suspension for sonication and vacuuming; then the leaves were dried in a clean bench and placed on co-culture medium.

[0066] Leaves were cultured in the dark on a co-culture medium for 2 days; then transferred to a selective medium for continued dark and room temperature culture until callus was induced, with subculture performed every two weeks; the callus was transferred to MSBK differentiation medium and cultured at room temperature for 16 h / 8 h light and dark alternation until green shoots differentiated, with subculture performed during this period; finally, the differentiated callus was transferred to a rooting medium, and after rooting, it was transferred to soil for culture.

[0067] Leaves of regenerated plants were taken, frozen in liquid nitrogen, ground, and DNA was extracted for identification of positive seedlings. Transgenic positive plants were selected for qRT-PCR verification, using the same method as 1.1.4. The construction of the gene editing vector for alfalfa was based on the article by Ye Qinyi et al. ([1] Ye Q, Meng X, Chen H, et al. Construction of genic male sterility system by CRISPR / Cas9 editing from model legume to alfalfa[J]. Plant Biotechnology Journal, 2022, 20(4): 613-615. [2] Zhu F, Ye Q, Chen H, et al. Multigene editing reveals that MtCEP1 / 2 / 12 redundantly control lateral root and nodule number in Medicago truncatula[J]. Journal of Experimental Botany, 2021, 72(10): 3661-3676.). The transformation method and DNA identification method for mutant plants were the same as above.

[0068] 2.1.4 Alfalfa gene silencing 2.1.4.1 Carrier Construction Will MsGDSL The CDs sequence of the gene was input into the SGN VIGS Tool (https: / / vigs.solgenomics.net / ) online website to obtain a 300 bp silent sequence. This fragment was cloned using the method described in 2.1.2, and TRV2- was constructed. MsGDSL Vector. The primer sequences for cloning the gene are as follows: TRV- MsGDSL -F: ctctagaaggcctccatgggTACTTCAAACATCCATCAGG (SEQ ID NO.13), TRV- MsGDSL-R: cgtgagctcggtaccggatcGTTTTTGAAGAAGCTGTCACA (SEQ ID NO.14). Note: In the above sequence, lowercase letters represent homologous arms of the primers, and uppercase letters represent primers for gene fragments.

[0069] 2.1.4.2 Alfalfa plant infection Pick positive Agrobacterium colonies (TRV1 and TRV2-) MsGDSL The cells were inoculated separately into LB liquid medium containing antibiotics and cultured at 28°C with shaking at 200 rpm until OD (occurrence depth) was reached. 600 The OD value was 0.6-1.0. Agrobacterium cells were collected by centrifugation, resuspended in infection buffer (10 mM MgCl2, 10 mM MES, 200 μM acetylsalicylic acid, pH 5.6), and the OD value was adjusted. 600 Adjust the concentration to 0.8-1.0 and let stand at room temperature for 3-4 hours to activate the Vir zone of Agrobacterium. Immerse the whole alfalfa plant in the infection solution and vacuum permeate (0.05-0.1 MPa) for 10-15 minutes. Place the infected plants under suitable conditions for cultivation (22-25℃, 16 h light / 8 h dark) and observe the phenotypic changes of the plants regularly.

[0070] RNA was extracted from the treated plants and reverse transcribed into cDNA. The mRNA expression level of the target gene was detected by RT-PCR. The expression level was significantly reduced compared with the control, indicating that the silencing was successful.

[0071] 2.2 Results and Analysis 2.2.1 Identification of Arabidopsis thaliana overexpression positive seedlings The selected T1 generation positive seedlings were transplanted into soil. When they grew to about four weeks old, DNA was extracted from the leaves and PCR amplified using the primers listed in Table 1.7 of the vector. The gel electrophoresis results showed that ( Figure 5 The band size of the DNA amplified from the positive seedlings was the same as that of the plasmid DNA. T1 generation positive seeds, identified by PCR, were harvested. Extraction... MsGDSL RNA from the leaves of T3 generation transgenic Arabidopsis thaliana seedlings was used to identify gene expression. Analysis of qRT-PCR data showed that ( Figure 5 ), MsGDSL The highest relative expression level of the gene in Arabidopsis thaliana can reach 800, and the lowest is 2. Therefore... MsGDSL The gene was successfully transferred into Arabidopsis thaliana, and the lines OE6, OE11, and OE14 with moderate expression levels were used for subsequent experiments.

[0072] 2.2.3 Identification of alfalfa overexpression positive seedlings and editing mutant plants A large number of overexpressing regenerated plants were obtained through stable genetic transformation of alfalfa. Forty-eight of these plants were selected for DNA and RNA level identification. qRT-PCR analysis of the alfalfa-positive plants revealed… MsGDSL Gene expression levels varied significantly among different positive seedlings. The expression levels of OE4, OE9, OE10, OE15, OE17, OE19, OE23, and OE25 alfalfa positive seedlings were significantly higher than those of the wild type. OE4, OE9, and OE10 were used in subsequent experiments. Figure 6 , Figure 7 ).

[0073] Transfer MsGDSL 46 regenerated plants using gene-editing vectors ( Figure 8 PCR amplification and sequencing were performed on sequences near the target site. The sequencing results (Table 10) showed that all lines had mutations at the target site. The mutation types at the target site included base deletions (including 1-7 base deletions), with AACTT deletions occurring most frequently. Other mutation types included base mutations (including 1-2 base mutations) and base increases (only one base increase).

[0074] Table 10 Target sequences of gene-edited regenerated plants

[0075] 2.2.4 Identification of gene-silenced alfalfa plants The expression levels of alfalfa plants after gene silencing were measured, and six silent plants with significantly lower expression levels than the root mean (WT) were selected (TRV1, TRV2, TRV4, TRV7, TRV10, and TRV11). Figure 9 ).

[0076] 3 MsGDSL Verification of the salt tolerance function of the gene 3.1 Materials and Methods 3.1.1 Test Materials Transgenic Arabidopsis thaliana, transgenic alfalfa, gene-silenced alfalfa.

[0077] 3.1.2 Overexpression of Arabidopsis thaliana salt treatment and determination of germination rate and root length Prepare 1 / 2 MS agar medium containing 100 mmol / L NaCl (pH 5.8, agar concentration 1-1.2%) and pour it into Petri dishes to solidify. Select plump, uniformly sized seeds, and after surface sterilization (75% ethanol for 30 seconds → rinse with sterile water → 2% sodium hypochlorite for 5-10 minutes → rinse with sterile water 5 times), evenly sow them on the surface of the medium (the same number per dish, at least 3 replicates). Incubate in an Arabidopsis thaliana incubator (16 hours light / 8 hours dark, temperature 22±2℃, humidity 60-70%). Observe regularly and record the germination rate (radicle breaking through the seed coat as the standard) and the taproot length (measured with a ruler or software after 3-7 days of culture).

[0078] After sterilization, seeds from three transgenic Arabidopsis thaliana lines and wild-type Arabidopsis thaliana were placed on 1 / 2 MS medium and grown for 10 days before being transplanted into soil. After four weeks of growth in the soil, plants with uniform growth (six plants per pot, three replicates) were selected and treated with 1000 mL of 250 mmol / L NaCl solution. 1000 mL of NaCl solution was added to the tray every 24 hours. A control group was also set up, and morphological changes after treatment were observed.

[0079] 3.1.3 Gene Silencing in Alfalfa: Salt Treatment and Physiological Indicator Measurement The obtained gene-silenced plants and control plants were divided into 6 replicates each and treated with nutrient solutions containing 150 mmol / L NaCl and normal nutrient solutions. Phenotypic changes (degree of leaf wilting, yellowing rate, and survival rate) were observed over two weeks.

[0080] Select leaves from the same location on the plant, wash off surface dirt, and blot dry with filter paper. Cut the leaves (avoiding the midrib) into small pieces approximately 0.5cm × 0.5cm, mix thoroughly, weigh 0.1g and place in a clean 15ml centrifuge tube, add 14ml of distilled water, and prepare a blank control. Let stand at room temperature for 24 hours, then use a conductivity meter to measure the conductivity P0 of the blank control and P1 of the experimental group. Boil the above extract along with the leaves for 30 minutes to completely rupture the cell membrane and release all intracellular electrolytes. Cool to room temperature and measure the conductivity P0' and P1' of the blank control and experimental group again. Calculate using the formula: Conductivity (%) = (P1-P0) / (P1'-P0') × 100%.

[0081] Leaves from the same location on the plant were selected, chopped, and 0.1 g were weighed and placed in a 15 ml centrifuge tube. 14 ml of 95% ethanol was added, and the mixture was left to stand for 48 h. Using 95% ethanol as a blank control, the absorbance (A) of the extract was measured at wavelengths of 665 nm (characteristic peak of chlorophyll a) and 649 nm (characteristic peak of chlorophyll b). 665 A 649). Calculate the concentrations of chlorophyll a and b (unit: mg / L) using the following formula: Chlorophyll a concentration (C a = 13.95 × A 665 - 6.88×A 649 ; Chlorophyll b concentration (C β = 24.96 × A 649 -7.32×A 665 Total chlorophyll concentration (Ctotal) = Ctotal a + C β .

[0082] Prepare 1 M NaH₂PO₄ solution, 1 M Na₂HPO₄ solution, sodium phosphate buffer (16 ml 1 M NaH₂PO₄ + 84 ml 1 M Na₂HPO₄), DAB staining solution (0.05 g DAB + 45 ml distilled water + 500 μl Na₂HPO₄, adjusted to pH 3.8), and NBT staining solution (0.1 g NBT + 50 ml sodium phosphate buffer, adjusted to pH 7.5). Take leaves from the same location on the plant, wash the leaves thoroughly with distilled water, and immerse them in DAB and NBT staining solutions. Incubate in the dark at room temperature for 12 h. Discard the staining solutions, immerse the leaves in anhydrous ethanol, and incubate in a boiling water bath for 30 min with intermittent shaking.

[0083] 3.1.4 Overexpression of alfalfa salt treatment and determination of physiological indicators The treatment methods and physiological index measurements for this experiment are the same as in 3.1.3.

[0084] 3.2 Results and Analysis 3.2.1 Analysis of Salt Tolerance Function of Genes in Arabidopsis thaliana Germination rates of transgenic Arabidopsis lines OE6, OE11, H9, and WT were measured under 150 mmol / L NaCl treatment and a control treatment. The results showed that the growth and germination of transgenic Arabidopsis were superior to WT. Root lengths of transgenic Arabidopsis lines OE6, OE11, H9, and wild-type Arabidopsis were measured under 100 mmol / L NaCl treatment and a control treatment. Under salt treatment, WT roots were shorter, with OE14 showing significantly longer roots than WT. Salt treatment in soil with both transgenic and WT Arabidopsis revealed that transgenic Arabidopsis exhibited better yellowing and wilting than WT. Therefore... MsGDSL Arabidopsis thaliana has a certain salt tolerance function. Figure 10 ).

[0085] 3.2.2 Alfalfa MsGDSL Salt tolerance function analysis of genes 3.2.2.1 Alfalfa MsGDSLSalt tolerance trait analysis after gene silencing Alfalfa was obtained through gene silencing (VIGS) technology. MsGDSL Gene-silenced plants were subjected to short-term salt treatment experiments, and the results showed that under salt treatment... MsGDSL Silent plants exhibit reduced salt tolerance; after salt treatment, silent plants showed lower chlorophyll content and survival rate compared to the control (CK), and their chlorophyll color was darker after DAB and NBT staining, indicating more severe oxidative damage. Figure 11 ).

[0086] 3.2.2.2 Alfalfa MsGDSL Salt tolerance analysis after gene overexpression Overexpression was achieved through stable genetic transformation. MsGDSL Three positive alfalfa plants with relatively high expression levels were screened and subjected to a two-week salt treatment experiment. After salt treatment, the overexpressing plants showed higher chlorophyll content, lower electrical conductivity, and lower mortality rates than the WT group. These results indicate that under salt treatment... MsGDSL Overexpression enhances salt tolerance in plants. Figure 12 ).

[0087] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. MsGDSL The application of genes in regulating plant salt tolerance is characterized by, The MsGDSL The nucleotide sequence of the gene is shown in SEQ ID NO.7, and the plant species is alfalfa.

2. The application according to claim 1, characterized in that, The MsGDSL Genes play a positive regulatory role in this application.

3. The application according to claim 2, characterized in that, The positive regulation method includes overexpression MsGDSL Gene.

4. The application of a recombinant expression vector in improving the salt tolerance of alfalfa, characterized in that, The recombinant expression vector contains the active ingredient described in claim 1. MsGDSL Gene.

5. The application of a host bacterium in improving the salt tolerance of alfalfa, characterized in that, The host bacteria contain the active ingredient described in claim 1. MsGDSL Genes or recombinant expression vectors contained in the application of claim 4.

6. The application according to any one of claims 4-5, characterized in that, The applications include those in the development of new salt-tolerant alfalfa varieties and / or lines.

7. Overexpression MsGDSL The application of genes in improving salt tolerance in Arabidopsis thaliana is characterized by, The MsGDSL The nucleotide sequence of the gene is shown in SEQ ID NO.7.

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